JPH0466595A - Mammalian GAP-43 compositions and methods of use - Google Patents

Abstract

PURPOSE: To obtain the subject composition for promoting therapy in nervous tissue damaged by infarction or hypoxia, comprising a recombinant mammalian GAP-43 containing a novel membrane-targetting peptide domain capable of directing GAP-43 to the cellular membrane.

CONSTITUTION: The objective composition comprises a recombinant mammalian GAP-43 or its functional derivative. The composition is obtained from newly born rat brain RNA in conventional manner. A cDNA encoding for a mammalian GAP-43 is inserted into a vector, and prokaryotic or eukaryotic host cells are transformed with such expression vector containing the cDNA. The transformant is preferably cultivated in culture medium to yield GAP-43.

COPYRIGHT: (C)1992,JPO

Description

[Detailed description of the invention]

FIELD OF INDUSTRIAL APPLICATION The present invention relates to the fields of molecular genetics and neurology. Sara
In particular, the present invention provides mammalian G A P43,
- cDN, A sequence and correspondence of protein related to growth of Ronin
Concerning the amino acid sequence. Furthermore, the present invention provides G.A.P.
-43 expression, thereby regulating axonal growth
methods, and prokaryotic or eukaryotic host cells or
The present invention relates to a method for producing G,A P43 in animals. More specifically, the present invention provides membrane condensation of GAP-43.
growth cone enrichment can be controlled and any desired
Proteins or polypeptides can also be linked to neurons or non-neurons.
The reason for GAP43, which can be oriented to the membrane of Ron cells.
New membrane targeting
rgeting) peptides. Moreover, the present invention
, GAP-43 and biologically derived from them
Function as active peptides or internal regulatory proteins (IRPs)
and act to modulate cellular functions intracellularly.
I can do it. In addition, the present invention particularly includes
GAP-43 and
Clinical I of the regulation and membrane targeting elements
Diagnosis and treatment in vivo and in vitro
Regarding application. (Prior Art) G A P-43 specifically characterizes growing axons.
It is one of the proteins (Skcne), cell (C
el ]) 37: 697 (1984); Mates (M
eiri), B.
SA83: 3537 (1980)). transported by axons
The protein to be sent is the whole thin 11'=, the smaller of the two proteins → block
Se, 1-, this A1 et al.
It can be said that the molecule directly mediates axonal growth.
Changes like - Sukefuo 3 Ascension Lyria l” (S
kpne andζ1lliard), Nyanar O
F Cellular Bio 0/- (J, Ce11. B
iol, ) 89: 86 (] 981): Henowit
and Benowitz and Lew
is), Nyarnal Off Neuroscience (J,
Ne+)r. 5science) 3:2153 (1983);
Cell 37: 697 (1
984); Meiri, BNA
Varnish (P!1AS) US A83: 3537 (
] 986)). Any of these 41 molecules undergoes a structural change.
There is a lack of direct evidence that c A P -43 mediates
(C is particularly attractive as a complement to l\.
is firstly a growth cone membrane 1 element, where the growth circle
binds to the inner surface of the pyramidal membrane and is a substrate for protein kinase C
This is because it works as (aro 3 (he 1 oyo), gloss)
Neur Off 2” - Location Mistry (J, Neur
ochem, ) 41: 649 (1983)
Kaas and Routtenpark (Ake, rsand
Routten-4-+erg), Frein Richter
(BrainRes,) 334: 147 (+983
)). Furthermore, the Rehel of the )6 (Yun-J-expression) is life.
Axonal growth in both tianbao and in~1~0
niininiirelation-4-ru('・/(t3asi), cell(
Cell) 49: 485 (1987) Nonones (
Karns), Science 236
597 (1987); Neve, Molecular
・Plain Research (Molec, Brain Re
s,) 2: 17? (] 987); Tou・U・Mo
de IaMonte et al., unpublished
table). Mature human 1 ~ The ritual process in the central nervous system (CNS)
Deficiency is a formidable clinical problem. acute ischemia or trauma
After that, there was minimal histopathological evidence of regeneration and neurological
Recovery is usually absent or incomplete. On the other hand, the end
Neurons in the treetop nervous system are like goldfish or toads.
regeneration, as do CNS neurons of other species.
It is possible to predict what will happen (Sukefu and Willert (S
kene and Williard), /Yarnal e
Off competition Cellular Sobio o No (J, Ce11.
Biol, ) 89: 86 (1981)
Benowitz andl, (・
wis), / Yarn Off Neuroscience (1
) to (・+”rt)sicncs)3: 21;
-+ 3 (1983)). Sei 1 (8 new with mammal C)
The first explanation for Con's refractory condition is that molecules that are heavily loaded with growth.
This would be an irreversible suppression of In particular, G A P-43
It has been suggested that it is critical for regeneration (
Cell 37: 697 (1
9B 4)). Evidence for this is in growth cones
Its enrichment (Skene), Science (Sci)
ence) 233: 783 (1986);
(Meiri), BNA Nis (PNAS)
L; S, A83: 3537 (1986)) and Zhou
The perinatal CNS refers to its minimal expression in the lunar adult.
Kefu and Willi F (Ske old! and Willi
ard), / Yarnal Off Se1 Noyular Hioro
No (J, Ce11. Biol,) 89:96 (
] 981) Karns, Science (S
science) 236597 (19ε37))
. Furthermore, P743 to G is toad or color separation, visual
Nerves that can be regenerated, such as nerves or mammalian peripheral nerves.
Injured friend in Urononi, increases power to high level
or a similar injury to mammalian C\8 neurons.
The i (tehasozu) must not be
Skene and Williard, Nya
Null Off Cellular Biology (J, C
e11. Biol, ) 89: 96 (1981))
. (Invention or Problem to be Solved) The present inventors have discovered that (h) GA in CNS function and disease;
The role of P-43 was investigated. human GAP43 c DN
A. was cloned and its development and adult distribution were determined in postmortem tissues.
The results were determined by this assay. In addition to high perinatal expression, this
We found that GAP-43 expression is discontinuous in adults.
crete) and unexpectedly caused acute ischemic damage.
Wounds usually occur in areas where GAP-43 is low.
We found that it is associated with high expression. (Means for solving the problem) GAP43 latency in mammalian CNS function and disease
Recognizing the potential importance of complementary DNA, we
By (cDNA) cloning, mammalian GAP-
Successfully sequenced 43 genes. rat GAP-43
The complete nucleotide sequence and
The amino acid sequence was determined. U7 About GAP-43
Human brain stem and
for human GAP-43 from the cerebellum and cerebellum libraries.
The cDNA was identified and cloned. Human G, AP-4
The amino acid sequence of 3 was also determined. Thus, in one embodiment of the invention, substantially pure
mammalian GAP-43 protein, or a functional derivative thereof
or provided. Additionally, substantially pure forms of rat and
(b) GAP-43 proteins and the functions of these proteins
Derivatives are provided. Specific embodiments of the invention each include
, amino acids corresponding to those shown in Figures 2 and 5A.
Substantially pure rat and human GAP4 with the sequence
3 proteins and polypeptides and their functional induction
Consists of the body. Another embodiment of the invention is shown in FIG. 2 or 5A.
or a nucleotide sequence substantially similar thereto.
cDNA consisting of a leotide sequence, or a derivative thereof
I will provide a. The cL)NA of the present invention is like a plasmid.
can be incorporated into a suitable expression vector, and the vector can be
transducing prokaryotic or eukaryotic host cells using
fect, and then the host cells are incubated with a suitable in vitro
vo in vitre. Alternatively, the cDNA can be expressed under in 5 situ conditions.
The result is the GAP-43 protein produced by it.
or further embodiments of the invention together with polypeptides.
Form. Thus, yet another embodiment of the invention provides
Encoding a substance GAP-43 protein or polypeptide
Vector consisting of cDNA of prokaryotes or eukaryotes
transfecting a host cell with the mammalian GAP-4
3 under conditions that allow expression of the protein or polypeptide.
The host cells are cultured in a suitable medium, and the mammalian GAP
-43 protein or polypeptide, or its functional inducer
A mammal G characterized in that the conductor is separated from the medium.
AP-43 protein or polypeptide or its functional
Methods of producing derivatives are provided. Using the substantially pure GAP-43 antigen of the invention,
The inventors succeeded in creating an anti-GAP-43 antibody.
, such antibodies and their functional and chemical derivatives are
These consist of further embodiments of the invention. GAP-4 of the present invention
3. Antibodies may be polyclonal or preferably monoclonal.
Nuclear antibodies are suitable for a variety of preparative, diagnostic and therapeutic applications.
However, it is understood that these still form further embodiments of the invention.
It is a place to be understood. Furthermore, the GAPi3 antigen and antibodies of the present invention are
A specific preparation, diagnostic or therapeutic application is deemed necessary.
, such as with respect to detectable or therapeutic markers.
suitable (competent and pharmaceutically acceptable or acceptable)
Use with other active agents in compositions that do not require
suitable for Such Camphor-recognized GAP-43 antigen, antibody and its functions
and chemical derivatives, as well as such nail compositions.
Consists of specific examples. GAP of the invention, together with its functional and chemical derivatives
-43 antigen and, in particular, antibodies, can be used for various diagnostic tests known to those skilled in the art.
It can be used in a cutting method. Immunocytochemical methods and
and immunometric methods.
Further embodiments of the present invention include
vinegar. Thus, in one exemplary embodiment of the invention, GA, P
--43 Samples suspected of containing antigens or antibodies
GAP43 antibody or antigen each detectably labeled with
to allow the formation of a GAP-43 antigen-antibody complex.
ink the sample with the antibody or antigen to
-Bate and thus formed? Do not form a complex with one body.
The conjugated antibody is isolated from the antigen or antibody and labeled.
in a sample, characterized in that the body or antigen is detected.
to measure or test mammalian GAP-43 antigen or antibody.
Provided a way to get out. This embodiment of the invention and other
Things can be carried out in vivo or in vit as desired.
It can be done ro or in 5 itu. When used in the preparation, diagnosis, or seven therapy method of the present invention, the present invention
Compounds and compositions of the invention are conveniently provided in kits.
It is possible to make the packaging unnecessary, and such a feature is also included in the present invention;
) Give one concrete example in the form [戊. Thus, a non-limiting example and
to accommodate one or more container means in a vacant compartment.
comprising a carrier means provided with a truncation in order to
Any further said container means capable of preparation, detection or treatment
Labeled GAP-43 antigen or antibody, or
GAP-43 anti-
For the preparation, purification, isolation, measurement or detection of antigens or antibodies,
or treatment with GA, P-43 antigen or antibody
Keys 7) useful for treatment are provided. We also believe that normal as well as injury or disease C
G A P-43 expression was evaluated in XS tissues. i
n vivo GAP-43 expression occurs during central tissue development.
There are regional variations in GAP 43 expression.
Something was discovered. Furthermore, GAP-43 expression is moderately
Consolidation of damage to the cardinal tissues, undergoing considerable changes
was also found. Furthermore, we found that increasing mammalian GAP-43 expression
Find a nocanism that can be strengthened or suppressed if desired.
I put it out. In particular, GAP43 expression is divine) 2\\ growth captivity
Yo--oh, this is enhanced by certain steroids.
−・We found that this was suppressed. GAP-4
The ability to modulate the expression of 3 to the central or peripheral nervous system
A baby with an injury or disease or impairment that
Is it of great therapeutic use in treating animals, especially humans?
Nowadays, the importance of these discoveries is not easily understood.
Probably. , furthermore, the inventors found that GAP-43 or
c DN encoding its functional derivative, A non-
By introducing it into neurons, non-neuronal cells...
Surprising discovery that even growth cone-like processes can be formed
Did. Again, the potential treatment of this discovery is 2AK
It is something. Therefore, in another aspect, the invention provides a method for treating diseases or
in damaged CNS tissue, whereas in normal CNS tissue
G A I)-43 activity 11 and expression evaluated or measured.
It consists of a method of determining Furthermore, the present invention provides central or peripheral
Damage, disease, or function of peripheral nerve tissue
How to treat infected mammals, and how to treat humans.
Normal (iNS♀11 weave ζ) in mammals
3. Thus, in embodiment 1, the present invention provides a method for modulating the transverse reorganization of f.
n vivo, in vitro or in 5it
Characterized by exposure to a large amount of nerve growth factor
A method for inducing the expression of GAP-43 in said cells.
Rinaru. When exposed in vitro,
In the case of embodiment 1-), while maintaining the therapeutic effect,
Damage, disease or dysfunction of central or peripheral nerve cells
b) Introducing the cells to be treated or carefully aligning the location.
It would be possible to introduce it in the same position. In another aspect, G A P-43 is
-Attach cDNA to non-neuronal cells or
GAP-43 expression can be induced by introducing
or can be strengthened. This is Transf J Ri/
Jin, cedar quality introduction and direct microinnogoku/-3
In vivo, according to various λs including
What can be achieved in vitro or in 5 ita
This does not limit the present invention.
Give a concrete example of the intended meaning. As a criminal law, the present invention
C' D\A is a retrovirus or viral vector
or any number of
Ligands attached to cell surface receptor licants
It can also be transported into cells together with the cellulose. These methods
, Narahini GAP43 c DNA and its machine
Compositions and vectors comprising functional and chemical derivatives
The results form further embodiments of the invention. Similarly, still further embodiments of the invention provide structural rearrangements.
methods of modulating, methods of modulating Nnapus plasticity, and
, nerve growth factors, steroids and their functional induction
of one or more substances selected from the group consisting of
neuron or neuron therapy characterized by exposing the cells to an effective amount.
Alter the microenvironment of cells, including neuronal and non-neuronal cells.
It depends on the method of measurement. Surprisingly, GAP-43 has 10 amino acids.
containing an acid amino-terminal exon and that this peptide
Tide transfers GAP-43 to the cell membrane, especially in the formation of neuronal cells.
We discovered that this is the cause of the orientation toward the long conical region.
. Furthermore, this]0 amino acid membrane-Turkenoting
Pepketo, and its functional derivatives, can be used to
is attached to or near the amino terminus of the peptide.
In this case, the protein or peptide can be directed to the cell membrane.
I discovered that. This surprising finding is usually found in cytoplasmic vines.
and proteins and peptides that are not normally membrane-associated.
applied to. Thus, further embodiments of the invention provide for any desired
Proteins or peptides can also be added to neuronal cells or non-neuronal cells.
Orientation to the plasma membrane of long cells - Thake, Ting
peptide or a functional derivative thereof. of the present invention
Membrane-terkenoting pepeket, or attached thereto
Label the desired protein or peptide for diagnosis or therapy
It's going to change. The membrane-tarke, using tinogpepketo,
Neuronal or non-neuronal cells of animals including humans
medical treatment or treatment in vivo, il vitro
or in 5 situ treatment provides further embodiments of the invention.
Configure. In other embodiments, the invention provides isolated genomic G.
The intron-exo located in AP-43
It provides a transcription start site as well as a transcription start site.
. Moreover, surprisingly, the GAP-43 promoter
The structure of all (general) and the expression of other structural genes
It has been found that this can be useful. In yet other embodiments, the invention provides that GAP43 is G
. to alter the binding ability of other cellular proteins, including those of
This is based on the surprising discovery that it acts intracellularly on
It is. That is, in the present invention, the GAP=43 is external.
within the same potency and availability as cellular receptors.
provides an important new class of regional regulatory proteins (rlRPJs).
It is something that In addition, the amino terminal amino acid of GAP-43
Synthetic peptides consisting of acids are biologically active as internal regulatory proteins.
It has been discovered that these proteins exhibit activity.
The compositions and uses thereof constitute other embodiments of the invention.
Ru. Below, the accompanying drawings will be briefly explained. Figure 1 Hybrid selection of GA P-43c DNA
translation. Ricciardi et al.
PNAS 76: 492
7 (1979), EcoRI insert,
mRNA was selected using GAP43-2. abbreviate
If so, the GAP43-2 insert 05μg1 or
amount of non-specific DNA, bacterial plasmid pSP65 was
Spot L, 65% formamide,
400mM NaC (,10mM 14-pipera/naC)
solution containing polysulfonic acid (PIPES), pH 6.4
, Neonatal rat brain boriatenylation “poly(A)゛2,R
Hybridize with 7.5 μg of NA1 at 42°C for 16 hours.
It made me cry. Standard enoic acid-added physiological saline at 65°C
Pre-clean in water (S S CXX I), 05% SDS.
After that, the filter was boiled and the RNA was washed with ethanol.
Proteins were precipitated and translated with Usaki reticulocyte lysate.
135S] methionine (Pelham et al.
lham et al), Neirobian Journal
・Off-biochemistry (Eur, J, B
iochem, ) 67: 247 (] 976no. wealth
Translation product or product immunoprecipitated with anti-GAP43 antibody
were separated on a 12% SDS-polyacrylamide gel.
Ta. (A) (1) No exogenous RNA, (ii) pSP6
5-Selected RNA, (iii and 1v) GA P 43
-2- Selected RNA, and (X) poly(A) nascent
In vitro in infant brain RNA (newborn). translation product. (B) Snipes et a
l), Tsuku Neurosai Afstor (Soc, N
eurosciAbstr, ) 12: 500 (19
G A P-43 protein produced by A as in 86).
After pre-absorbing the GAP-43 antibody, the translation product is immunized.
Describes (A) except for the 4th lane showing epidemiological precipitation.
As shown, the translation product of (A) for GAP-43
Immunoprecipitation with antibodies. Figure 2 Nucleotide sequence and predicted sequence of GAP-43
amino acid sequence. Embryo 17~] Root extraction from 8 day old rats
Obtain cDNA library from ganglia.
Ta. Chirgain:) (Chirg★in etal
), Biochemistry'
) l8: 5294 (1974)
Total cellular RNA, A, was released at t15 and poly(A) RNA
4 Lico-dT cellulose was selected. Gubler and Hoff
Gublerand Hofl'man, Sea
Gene 25: 263 (1983)
Double-stranded c by the described lihonuclease H method
Obtain the DNA, connect it to EcoRI linker, and add Rum Taffer.
/cloning vectors λgt 10 and λgtll
ligated to the EcoR1 site of. Anti-GAP-43 rabbit antibody
, followed by one pair of alkaline phosphatase and one saki globulin.
G (Promega Biot
By using an ec-linked antibody, isopropyl β
-F) Induction with thiocalactopyr//to(IPTG)
After that, the longest clone identified, GA, P43-2,
λg
Identified from approximately 5xlO' plaques in the t11 library
did. The cDNA insert was added to Ml 3mp 18
Passage cloned into the Eco R1 site. 2 shorter
Initial DNA sequence analysis of the new clones revealed that they were the most
It was clarified that it is included in the long term. Inte
Oxine nucleoketo chain termination method (Sangkar et al.
er et at), Bi Nis A Nis (PNA
S) 74: 5463 (197″7)) makes the array
Using a series of overlapping limit cuts LL shown below
Insert GAP43-2 was sequenced by. The 3” end of this fragment was isolated into 3 independent λgill isolates.
Common Ec. R+ site, naturally occurring in the GAP-43 gene.
It is considered to be an E co RI site. boriatenyl
There are no clones containing inserts with
Therefore, the EcoR1 site within the cDNA is live.
It appears that it was not methylated during rally construction. G
The predicted protein sequence for AP-43 was combined with the DNA sequence.
It is shown on the -L side of the column. The first methionine in italics is
Star I/Toto in the coating area was selected for the reasons described below.
I chose. Other methionines shown here as amino acid 5
It is said that Mika does not work as a starter futon. Arkinine (R) in amino acid 7 to amino acid 20
Direct protein sequence up to isoloinone (1)
Draw a line between the amino acid residues identified by the determination.
there was. Sequencing that can determine amino acids with certainty
The first cycle is Arkinine Tea-1. Ta. The following amino acids could not be determined with certainty. The inability to sequence unfragmented proteins is an
This suggests that the mino terminus appears to be blocked. E, EcoRl ;MlMspl :VPvul;)l
, Haell; P, Pstl; 5Sau3A0 nucleus
The arrow between the lines 100 and 101 indicates the first and second
Nucleotides 664 and 665 mark the boundary between the coxons
The arrow in between indicates the start of the third exon. Figure 3 GAP-43 expression in PC12 cells
adjustment. ] RP containing 0% horse serum and 5% fetal Uno serum
PC]2 cells were passaged in MI medium. Forty hours after plating the cells, the j-H substrate was
Modified to include additives. After 4 days, photograph the cells.
(A), then RNA was isolated from each cell culture.
. RNA (sample size: 0 μg) was denatured at 1.2%.
Carousel formaldehyde Kel-H C was run, and no
Gene Screen nylon filament
transferred to a filter and bonded to the filter by UV cross-linking.
and probed with 32p-labeled GAP43-2 (
B). The final wash was carried out in S S C (x
0.2) and 0.1% SDS. The additives included are
(1) None, (ii) NGF50 per milliliter
ng, (iii) 10-3M sibutyryl cAMP,
and (iv) 50 ng of NGF per milliliter and
and 10-3M dibutyryl cAMP. last level
blower) method and hybrid motion method.
RN from neonatal brains run as a positive control for
Al0Mg. Figure 4 Developmental regulation and organization of GAP-43 gene expression
Specificity. Chirgwin et al.
), Biochemistry
) 18:5294 (1979).
Total cellular RNA was isolated from the indicated rat tissues.
. Denature each RNA (10 Mg) and add 1.2% acarose
Electrophoresis was performed using formamide gel, and Nitrocell was used.
Transferred to loin. Two filters. By translation, Deoki/Nchinn 5'
-[α-3tp]h-lyphosphate labeled λgtl
l EcoRI insert from GAP43-2 and 42
Hybridization was carried out overnight at 6C. Final wash at 65°C, 5SC(xo,2), 0.1
%SDS. RNA sample = (1) Embryo 13 days old (E
13) Heart (H), (ii) E 13 Liver (L), (
iii) E13 brain (B), (iv) E-13 dorsal root ganglion
(DRG), (v) or vii) 17-day embryonic heart
, liver, brain, and posterior plate ganglia; (ix) or x
ii) Neonatal heart, liver, brain, and posterior plate ganglia: (
xiii) or xvi) Adult heart, liver, brain and
Posterior plate ganglion. 18S and 28S lyfosomal RNA positions
The location is shown on the right. Below is the same filter and Grisel Alnahi
To-3-phosphate dehydrokenase (GAPDH)
c DNA probe encoding (Bienjaczyk et al.
(Piechacyk et al), Cell (Piechacyk et al)
) 42:589 (1985))
-7 Yon. Figure 5 (A) Nucleotide of human GAP-43 cDNA
code sequence and resulting amino acid sequence. E: EcoRI; H: HaelI [; M: Msp1
0 coding regions are shown in bold. The scale is 100bp
be. Arrows indicate overlapping restriction fragments that were sequenced. (B)
Human, rat GAP-43 and mouse P-57 amino
Arrangement of acid sequences. Vertical lines indicate identity, colons indicate conservation.
Indicates the substitution that will be made. Amino acids are IUPA (, -IUB
Represented by the single letter symbol CBN. The array of objects is the second
There is a deviation from the figure, and the mouse arrangement is from Simler et al. (Cimler et al.
er et al), Journal of Biologica
Le Chemistry (J, Biol, Chem,) 26
2:12158 (1987). (
C) Zuker and Steigler
Steigler), Nucleitsk Asisoz Lisa
(Nuc), Ac1ds Res, ) 9:
133 (1980 fold program)
The predicted stem-rule in the 3”-untranslated region
structure. Figure 6 shows regional restriction of GAP-43 expression with maturation.
North Plot. Brain area of 8 days old, 16 years old, 64 years old
Load 10 μg total RNA from the region into each lane and
human GAP-43 as described in Example ■.
Probe with probe C1a. 18S and 28S
The position of the rRNA hand is shown. Figure 7: GAP'-43 expression increases after ischemic accident.
The North Plot shows that (A) field
) 17 Differences in patients with stroke (visual cortex)
Expression in RNA10Mg0 area 17 from the brain region
increases to levels comparable to maxima in the brain (area 11)
did. (B) After complete electrophoresis, cut in lanes 2 and 3.
Pros on the same plot with unrelated hands drawn
, R from field 3.1.2.5 from three patients.
NAl0Mg0 lane 3 contains a few strokes, GA
Lanes 1 and 2 show increased P43 expression, whereas lanes 1 and 2
Histology was normal. Figure 8 in si″tu hybridization/yon
Therefore, GAP-43 expression in the area adjacent to the infarct
Increase is revealed. (A1) Lipid-loaded macropher
showing diffuse infiltration of the tissue by di- and reactive astrocytes.
High magnification of the infarcted area in B. remain in this area
There are no neurons (X160). (A2) Abundant histology
Normal adjacent cortex (x 3
00). (B) In the first trunk gyrus (arrowhead) and the adjacent trunk gyrus.
Organized ischemic infarcts (10+) containing intact cortex (arrows)
Low magnification view (x12) of the visual cortex with 4 days of age). (C) Dark-field studies show that in normal visual cortex,
, GAP-43 expression is restricted to a small number of scattered neurons.
(arrow head). (D) In the infarcted cortex, antisene
There is no specific binding of the GAP-43 probe. big light
Bright focus indicates aggregation rate of non-specific labeling of sense blobs
It is from the area of Clonus (G). in contrast,
In the adjacent intact cortex, a large number of neurons G, A
Express P-43 (labeled with antisense probe)
E-bright field and F-dark field). Special W GAP-4
labeled with a sense probe (F
() vs. illumination field and (+) dark field ((, -1x160
). Figure 9: Several days after an attack of severe hypotension and hypoxia
GAP-43 release in neurons of the cerebellar cortex in
Current augmentation. All cut surfaces were processed simultaneously. (A) Inspection
Normal adult small size showing absence of visible GAP-43 labeling
brain cortex. (B) In Burkinje cells and external granule cell layer.
Post-ischemic brain showing marked increase in GAP43 expression in
cortex. (C) Sense strand blown hyphretization as a control.
All cross-sectional views adjacent to B are X315). Figure 10 GA for protrusion formation in CHO cell line
Effect of P-43. The hollow line is 4 CDM8-transfer
It represents a cell with a process in the subject lineage, and the cut
The blank line represents 4 cell lines expressing GAl''43.
Cell lines were obtained as described in Example 2. have protrusions
The percentage of cells is CHO cells
Detected by plating on coated covers.
Established. Cells with protrusions longer than 20 microns are considered positive.
and recorded it. To ensure comparability, all
The assay was carried out over a period of 30 to 45 minutes after plating.
It was done within the time frame. The important element of this is when you choose
There was a time frame. Because after a longer plating time
Or, as the cells become denser, the protrusions become less clear.
It was because it was. as many as possible during this time frame
The cells were counted and all cells counted were included. Cell numbers counted for different systems are LA, 4061B.
, 408:2A, 287 28 3035E, 234
;4,333;12,156;14゜161゜GAP-
The percentage of cells with protrusions in the 43-expressing cell line was higher than that in the control.
(p<0.001.) Figure 11 Amino-terminal exon or GAP-43 protein
It is believed that it is responsible for the orientation towards the cystic membrane and that it
Loranoff Unicol Acetyl Transferase Membrane
Schematic of experiment to prove that targeting is directed
figure. The left column (“Construction”) shows CO5,, N + + (3T
3. Transfection of CHO or PCI2 cells
The construction of the gene used in the following is shown. Right column (,rllil)
The expressed protein or fragment is then subjected to osteocyte differentiation, followed by
Western profiling, direct immunofluorescence, or
Tested by both methods to determine if there was membrane connection (+).
or (-). Substantial part of exon 2 (GA,
P (-internal)) or G A P-43 gene cal
GAP constructs lacking the boxy-terminal region (GAP tag) also
As was the case, the intact G A P-43 gene (G
AP) was significantly membrane-associated. However, G.A.
Nucleotide encoding the first four amino acids of P43
When Otide is deleted (GAP(-1-4)), the expression is
The protein fragments obtained were not membrane-associated. At position 3 (C3) or 4 (C4) of the first exon
Introducing a point mutation in the sequence that codes for /Stin
As a result, alanine was expressed in the resulting protein. C
3(GAP*C8) also i; IC4(GAp*c,)
Either mutation results in membrane defects compared to intact GAP43.
Leher (+/-) decreased by 17. The reduction was particularly pronounced when changing C4. Mutations in both C3 and C4 (GAP*C3,,)
Total elimination of membrane connections (7). This is normally expected for cytosolic enzymes.
, chloramphenicol acetyltransfrase (C
Transient expression of the gene encoding the membrane
No linking protein was produced. Coding the 10 amino acids of the first GAP-43 exon
Add the nucleotide sequence to the amino terminus of the CAT gene.
When tied at the end (GAP(1-10)CAT), the expressed
The protein was membrane-associated. Figure 12. When transfected into CH○ cells,
Normal GAP-43 has membrane and cytosolic components (M=
membrane; C-cytosol)
plot. The third or fourth sequence of the first GAP exon
Nuc coding stain (C-3 or C-4)
Mutations in the leotide sequence interfere with membrane-bound components, whereas
Mutation of both cysteines (C-3,4) completely abolishes membrane binding.
was excluded. Control cells (CON) have GAP-43
Z: The brain membrane (BR) is derived from the transfected gene.
It had the same molecular weight of GAP-43. Figure 13. Map of GAP-43 gene A.
Linear representation of the GAP-43 gene oriented from 5° to 3°
. Display of phage insert used for mapping is shown.
. Three exons are represented as vertical lines. The indicated parts are controlled.
Limited endonuclease BamHI (B), Kpn
I (K), and 5acl (S)
be. B, intron-exon boundaries and 3' polyadenyls
cation site. best match the consensus splice site
, compared with the c DNA sequence as described herein.
The exons and flanking regions were sequenced by
Tron-exon boundaries were determined (Moun
t), Nuclitsk Acids Research (Nucl,
Ac1ds Res, ) 10: 459-472 (
1982). The major polyadenylation site is RNA5 e
Determined by protection. Polyadenylated guna utilized
The putative polyadenylene often found immediately 3' of the
synthesis signals and consensus motifs (moti
f) tandem pairs are underlined (McLaughlan et al.
McLauchlanet al), Nucleitsk A
Nno's Research (Nucl, Ac1ds Res,
) 13: 1347-1368 (1985)). FIG. 14 Sequence of GAP-43 promoter region. Nuku
Reotide + 1st position is the start ATG codon of GAP-43 protein
A is shown. This array contains variables of variable size ending in +30.
Contains 1 exon. The major transcription start site is indicated by an arrow. pudding leftover
The base is indicated by an asterisk below. The consensus Pit1 binding site is
Draw a line above. Figure 15. Restriction fragments induced by H-DNA
mobility shift. Restriction sites and major homopurines
Homopyrimidine regions (thick labeled I, I[, and
GAP-4 of -518 to +85 indicating the position of
3 Schematic diagram of the promoter region. At the bottom, coat with the following enzymes.
by digesting lasmid bs1.5RIX4.
Displaying the released GAP43 promoter fragment 1)S
spl, 603bp; 2) XbaI/5spl, 560
bp: 3) SmaI/5spI, 490bp 4) Ssp
l/Nhel, 409bp; 5) Ssp1/Ns i
I, 284 bp (contains region ■), 319 bp (region ■
and ■); 6) Sspl/Acc+, 314b
p (region 1), 289 bp (region ■ and II[);
7) Xba I/Nhe I, 360bp: and 8
) Sma I/Nhe I, 295 bp. Figure 16 Partial protein sequences for p34 and p38
Column. Figure 17 GAP-43 and GAP-43 peptide
Yoru G. Modulation of (35S)GTPuS binding to. Special
A different G. (35S)GTPuS binding to 1μ
In the presence of M GAP-43, 310+40% of control (n
=7) (A. Left). G at the same concentration. GAP-43 free is detectable (
35S) Does not bind GTPuS. GAP-43a degree
Changes indicate an EC6o of +50 nM for this effect
(A, right). For various GAP-43 preparations, E
The range of C5o probably depends on the GAP~43 part during purification.
1l50-800n resulting in partial inactivation (
Compare the 17th chart). The first two from GAP-43
1mM peptide containing 4 amino acids, i.e. MLCCM
RRTKQVEKNDEDQK I For addition of EQDG
Therefore, according to 1 μM GAP-43, the same level (2
50%), GAP-43 and 1-
24 Depending on the addition of peptides, both of them were further stimulated.
Does not occur (B, left). The other three peptides, namely G
AP(35-53)-ATK IQASFRGHITR
KKLKD,,GAP(5369)=DEKKGDAP
AAEAEAKEK and GAP (2], 0-226)
= ARQDEGKEDPEADQEHA is 1mM G
. has no effect on (35S)GTPuS binding to
stomach. Stimulation of binding by the 124 peptide of 2011M
It is saturated at EC50. c AP-43 first 1
Peptide consisting of 0 amino acids, i.e. MLCCMRR
TKQ is 1mM and 80% G. against (35
S) Inhibits GTPυS binding (C1 left). Position 3 and
and 4 to 7 peptides with threonine instead of stain.
Effect on (35S)GTPuS binding to Go
does not have. IC for 1-10 peptides. is 20
μM (C1 left). Panel Y) is 50 μM 1-
GAP-43 in the presence or absence of 10 peptides
(35S) GTPuS for Go as a function of 11 degrees
Indicates a bond. GA P-43 reverses inhibition by peptides
Turn it around, GAP-43's EC5o or this Bebuchi!
・Increases from 600%M to 12μM depending on the concentration of
Be careful. 1-10 peptide and GAP-
Consistent with a direct conflict between 4:3. Method (35S) GTPuS--England Nu
Claire (New England Nuclear),
2000 Ci/mmol) bond was previously disclosed.
As described (Huff, R, M et al.
, et al.), Journal of Biologica
Le Chemis I. Lee (J, Biol, Chem,)
260: 10864-10871 (1985);
- Norl, hrup, J.
, et al, ), / Yarn Off Biolo
Computer Chemistry (J, Biol, Chem,
) 257・11416-11423(+982))
, 6 min at 30°C, 50mM Tri 5-HC
C, pH 80, 1mM EDTA, 6
mM MgCQt, 01% Lubro I
PX, 20011971fr&un serum album
100 nol of 1M dithiothreitol (in 2
TeG. (1nM, 10r+9) and (35S)G
Incubate TPuS (2 tubes 1M) and nitrocell.
filter through a loin to remove bound radioactivity.
measured by counting. These a,
The concentration of (35S) GTPuS in
It is about one-tenth of KD ()7-R.M. et al.
luff, R, M, et al,), / Yarnal O
Biological Chemistry (J, Biol,
Chem, ) 260: 10864-10871 (
1985): Northrup/A.K. et al.
hrup, J., et at.), Journal O.
Biol Chemistry (J, Biol,
Chem, ) 257:11416-11423 (19
82)), as a result, both the apparent KD and Bmax
A change in changes the level of binding. bound radioactivity
In all cases the sum of
It was. unlabeled in the presence of 10 μM unlabeled GTPuS
Measure the binding and subtract it from the total binding to determine the specific
The binding was determined. All assays were performed in duplicate. average a
The tube contains 800,000 cpm, with a total
30.000 cpm and 1500 cp non-specific binding.
I got m. GA, Addition of P43 affects non-specific binding.
increased specific binding without any effect. GAP-43 is l-
G prepared from u b r o IPX. and one
alpha. Two different trials '#1 were activated. deer
However, the range of activity varied between preparations. G.A.
Activation by P43 is used in the purification of Go
Must be sensitive to detergent and binding buffer compositions
stomach. GAP-43 was developed by Zweier et al.
tal,) method
Mistry (l Neurochem,) 44:
108:3-1090 (1985))
Coumasieve purified by 5DS-PAGE gel
According to Roux staining, the purity was over 97%. The protein is
Dialyzed extensively against assay buffer and this
GTPuS-Go incubation in indicated concentrations
added to things. Peptides are known as no word
Dical Institute Laboratory (How
ard Hughes Medical In5tit
ute Laboratory) (Massachusetts
General Hospital (Massachusetts)
General Ho5pital), Boston, Mass.
(Chuse, Tsuzhou) and purified by reverse-phase HPLC.
The structure was confirmed by amino acid sequencing. Dissolve the peptide in assay buffer and concentrate at the indicated concentration.
GTPuSG was added to the incubation at once. Figure 18 GAP-43 amino terminus and G-coupled receptor
Homology between the cytoplasmic tails of T. The first 1 of GAP43
0 amino acids were compared with the sequence of a G-coupled receptor. G
The first seven amino acids of AP-43 and numerous G-coupled receptors
homology between the amino-terminal segments of the cytoplasmic tail of
You can see the sex. Common sequence i.e. hydrophobic-Ieu
Residues matching cys-cys-x-base single base are shaded.
Injure yourself. 1/Instein shown in the septa is the last transmembrane
Approximately 13 terminal amino acids are located for the region, which is
, β, -adrenergic receptor cys341
This is consistent with Higashijima T. et al.
ijima, T. et al.) (Journal O.
Biological Chemistry (J, BiolC)
hem,) 263:6491-6494 (1988)
aligned as disclosed in . This indicated
Sequences of GAP-4 from humans (Figure 5) and fish
3. Human β-adrenergic receptor (β1-adrenergic receptor)
Drenalinergic, Frielle et al.
, T. et al.), Proceedings Op.
National Academy of Sciences (Proc.
, Natl, Acad, Sci,) U, S, A
, 84: 46-50 (1987)),
Syn (rhodopsin, non-existence
Nathans, J.
D, & Hogness, D, S, ) procedure
Tings Off Nanyonal Academy
Jens (Proc, Natl, Acad, Sc
i,) U, S, A, 81:4851-4855 (198
4)), human blue-opsin (blue-opsin,
Nathans, J. et al.
l, ), Science 232: 1
93-202 (1986)), Human alpha-adrenergic action
sex receptor (alpha, - adrenergic, cotecha)
・Cotecchias, et al.
Proceedings Off National Academy
Off Science (Proc, Na1lAcad,
Sci, ) U, S, A, 85: 7159-7163
(1988)), human platelet α, and adrenergic levels.
Scepter (pα, - adrenergic, Cobrica bi
- Koblika, B. et al.
), Science 238: 650
-656 (1987)), human muscarinic receptor
ter subtypes 1 to 5 (Mus-ACh-1 to -5, Human
Higashijima, T, et al.
et al.), Nyanal Off Biological.
Chemistry (JBiol, Chet) 263:
6491-6494 (1988); Perruta E.G.
(Perulta, E, Getal,), EM
B○ Journal (EMBOJ, ) 6: 3923-
3929 (1987)), human serotonin receptor sa
Type IA (5HT-IA, Kobrika B.K. et al.
(Koblika, B., et al.), Fuichi
+- (Nature) 329: 75-79 (198
7)) and rat serotonin receptor subtype I
C and 2 (5HT-IC, Shreyas Day et al. (J
ulius, D. et al.), Science (Sc.
ience) 241: 473-479 (1988)
and 5HT2, Brissochet Da-B et al. (P
Ritchett D, B, et al,), EMB○
Journano Ken EMBOJ,) 7:4135-4140 (
1988)). FIG. 19 GAP-43 = stimulated G. GT against
Dose-dependent reduction of PuS binding by calmodulin. The vertical axis of both panels is GTP expressed as a percentage of control.
It is a γS bond. Addition of GAP-43 to G (line
2) is 200% over compared to Gotake (line 1)
It also increased GTPγS binding. With calmodulin
addition decreased this binding to control levels (line 5). GAP43take (line 3) or GAP-4,11 soup cal
Using modulin (line 4), in the absence of G, GT
No PγS binding was observed. Karmoji on the right panel
GTPγS binding to G
Figure 2 shows the dose dependence of calmodulin's effect on DESCRIPTION OF PREFERRED EMBODIMENTS In the following description, reference will be made to the fields of molecular genetics and neurology.
Reference is made to various methods known to those skilled in the art. Publications describing such known methods to which reference is made
and other materials should be read in their entirety as if fully written.
Unify by illumination. A standard text describing the general principles of recombinant DNA technology
Contributed by Watson J. D. et al., Molecular Biology.
・Off the 0 Gene (Molecular Biolo)
gy or the Gene), Volumes I and ■,
・Benjamin/Cumming's Publinoning Can
Punny Inc. (Benjamin/
CuCumm1n Publishing Co
mpany. Inc.), Publisher, Menlo Park
Park), Californianization (1987); Turnell
・J.E. et al. (Darnell, , 1.E,
etal), Molecular Cell Biology (Mo
iecular Ce1l Biolog. Saien
Tihook American Bunox Incorporate
Nodo (Scientific American Bo)
oks, Inc.), Publisher, New York, New
York County (1986);
win, B, M, ), Gene H,
John Willie and Thump
ey & 5ons), Publisher, =, -York, Ni
New York (1985); Old Earl Gubry
Old, R, L, et at, Bouan
Mumbles Off Scene Manipulation
・Introduction・To・N5. Unidentified En
Principles of Gene
Manipulation: An introduction+
ction to Genetic Engineer
ing), 2nd edition, University of California
Runia. Press (University of California)
nia Press, Publisher, Berkley
Eley), Californianization (1981,) and Ma
Maniatis Ding et al.
), Molecular Cloning, a Laboratory
・Manual (Molecular Cloning＾
Laboratory Manual), Cold Suspension
Pulling Harbor Laboratory (Cold Spr.
ing Harbor Laboratory), Publisher
, Cold Spring Harbor
ng Harbor), New York (1982)
include. "Cloning" refers to the production of specific genes or other DNA.
il vitr to insert the sequence into the vector molecule. Refers to the use of recombinant technology. successfully target the desired gene.
In order to make a loan, a method of generating DNA fragments,
A method for linking said fragment to a vector molecule, said composite DNA molecule
how it is introduced into a host in which it is capable of replication, and its acceptance.
Select clones containing the target gene from host cells
method must be used. 1-cDNAJ is an RNA-dependent DNA polymer
produced from an RNA template by the action of enzyme (reverse transcriptase)
complementary DNA or copy DNA. write
Then, the rcDNA clone is converted into a cloning vector.
A complementary duplex to the supported RNA molecule of interest.
means a plex DNA sequence. "rcDNA library" is a collection of living organisms.
cL') consisting of the entire genome of
refers to the collection of recombinant DNA molecules with Such cD
NA libraries are known to those skilled in the art and include, for example:
Maniatis et al.
Recular Cloning A Laboratory Manual
Molecular Cloning: A La
boratoryManual), supra,
It can be prepared by any method. Generally, from its genome
of an organism in which it is desired to clone a particular gene.
RNA is first isolated from the cells. The present invention [-1 preferable
Preferred are mammalian and especially human cell lines. A “vector” is a vector into which a DNA fragment is inserted or
is a plasmid or batatteryophore that can be cloned.
means a DNA molecule derived from One or more vectors
Contains a unique restriction site for Re-L and contains the cloned sequence.
autonomous in a host or vehicle organism defined as renewable
It is either a copy or a convex J ability. Thus, "DNA expression vectors"
Host staining after additional sequences or autonomous elements are integrated into the genome
any autonomous element that can replicate in the host independently of the body
also means Such a DNA expression vector is a bacterial plasmid.
phages and phages. Preferred for the purposes of the invention
is the lambda gtI expression vector. "Substantially pure" means that each antigen or gene is free of other antigens or genes.
substantially free of or of the potential normally found in nature
Virtually free of certain other impurities and not found in nature
Any antigen of the invention present in the form of
refers to any gene encoding such an antigen.
Ru. "Functional derivative" means a "fragment", "mutant" of a molecule.
”, “homolog”, or “chemical derivative”. of molecules such as fragments of any of the cDNA sequences of the invention.
“Fragment” means any nucleotide subunit of a molecule.
shall also refer to A “variant” of such a molecule is
Substantially similar naturally occurring molecules, either whole molecules or fragments thereof
refers to molecules that occur in What is a “homologue” of a molecule?
, substantially similar non-natural elements to either the whole molecule or its fragments.
natural molecules. If the amino acid sequences in both molecules are substantially the same
, a molecule is said to be "substantially similar" to another molecule. fruit
Qualitatively similar amino acid molecules have similar biological activities
Ru. Thus, if the two molecules have similar activities,
one side of the molecule contains additional amino acid residues not found in the other
or the sequences of amino acid residues are not the same.
They are mutants, as the term is also used in
it is conceivable that. As used herein, a molecule usually
contains additional chemical moieties that are not part of the molecule
, the molecule is a "chemical derivative" of another molecule.
It is said. Such sites are responsible for the solubility, absorption, and biological
Improve half-life, etc. The site also reduces the toxicity of the molecule and makes it more desirable.
such as eliminating or attenuating side effects. It takes
Sites that can mediate the effect include, for example, Remington's
Remington
's Pharmaceutical 5science
), 16th edition, Manok Publishing Company (M
ack Publishing Co.), Easton
(Easton), Pennsylvania (1980)
It is shown. Similarly, the “functional inducer” of any gene of the antigen of the present invention
"conductors" are those whose nucleotide sequences are "substantially similar".
”, of genes encoding molecules with similar activity.
Include “fragments,” “variants,” or “homologues.”
It's the intention. DNA encoding GAP-43 or its functional
derivatives, or membrane-targeting peptides or
Its functional derivatives have blunt ends or staggered ends for tying.
Staggered ends, supplying suitable ends
Restriction enzyme digestion to properly fill sticky ends
, alkaline phosphater to avoid unwanted binding
Normal treatment including tying with suitable ligase
It can be combined with vector DNA using this technique. Techniques for such operations are described by Maniatis Ti et al.
aniatis, T., et al), supra.
as disclosed by and well known in the field.
There is. Nucleic acid molecules such as DNA carry transcriptional and translational regulatory information.
When containing a polypeptide, it is possible to express it.
” and such a sequence encodes the polypeptide.
"operably linked" to a nucleotide sequence that Operable linkage includes the regulatory DNA sequences and expression required.
DNA sequences linked together to enable gene expression.
It is a chain of events. The exact nature of the regulatory regions required for gene expression
Although the quality can vary between organisms, it is generally the case that prokaryotes
In , the promoter (which directs the initiation of RNA transcription)
protein synthesis when transcribed into RNA.
A promoter region that together contains a DNA sequence that directs the initiation of
encompasses the area. Such areas are usually
, cap sequences, CAAT sequences, etc., transcription and
Those 5゛-non-coding involved in the initiation of translation
array. include. If desired, the gene sequence encoding the protein
The non-coding area on the 3° side is obtained by the above method.
I can do it. This region is endogenously and polyadenylated
retained for its transcriptional termination regulatory sequences, such as
I can do it. Thus, the DN that codes for the protein
By retaining the 3°-region originally adjacent to the A sequence,
can provide a transcription termination signal. From beginning to end of transcription
If the signal does not function satisfactorily in the expression host cell,
By replacing the 3” region that functions in the main cell,
Wear. (Coding promoter region sequence and GAP-43
The DNA sequence of 2 is the same as the DNA sequence of 2.
As a result of the nature of chaining between columns, (1) frame-soft sudden
(2) GAP-43 gene sequence
interfere with the ability of promoter region sequences to direct transcription of
or (3) transcribed by the promoter region sequence.
7. Does not interfere with the ability of the GAP-43 gene sequence to be
are said to be operably connected. Thus,
The promoter region refers to the promoter or transcription region of the DNA sequence.
If it is possible to perform a photocopy, then
It is possible that they are connected. Thus, for protein expression, it is necessary to
Requires recognized transcription and translation/signals. The present invention provides for
Expression of GAP-43 protein (or functional derivative thereof)
including. The preferred host is E. coli (E, col
i), Bacillus, Strephtomycete
Streptomyces, /Needomonas (P
seudomonas), Salmonella
ella), Serratia, etc.
do. The most preferred prokaryotic host is E. coli. Salmonella Typhimurium
typhimurium) or Serratia marces
Sense (Serratia marceseenc),
and various Pseudomonas
) It is also possible to use other intestinal bacteria such as seeds. mosquito
Under such conditions, GAP-43 is not glycosylated.
Will. Prokaryotic hosts replicate in expression plasmids.
Must be suitable for recon and control sequences.
stomach. (For example, E. coli, B.
Squirrel (B, 5ul)t i I is), Nyudomo
Eggplant (Pseudomonas), Streptomyces (
Streptomyces), etc.) prokaryotic cells
Expressing GAP-43 protein (or its functional derivative) in
To function, the sequence encoding GAP~43 is
Must be operably linked to a target prokaryotic promoter.
It won't happen. Such promoters may be constitutive or, more preferably,
preferably regulatable (e.g. inducible or disinhibitable).
function). An example of a constitutive promoter is
1nt promoter of bacteriophage λ pBR32
+: + la promoter of β-lactamase gene of 2
, and chloramphenicol acetate of pBR325.
CAT promoter of chilltransferase gene
etc. Examples of inducible prokaryotic promoters are
Main right- and left-hand promoters of bacteriophore/λ
(PL and pa), E-r (E, col)
i) trp,,recA. 1acZ, 1acl, and gal promoter batilla
α-amyla of B. subtilis (B, 5btilis)
(Ulmanen, 1. e
tal), Journal of Battalion (J
, Bacteriol, ) 162: 176-182
(1985) and the σ-28-MX-like promoter (
Liman Yum 7/F et al. (GIiman, M, Z,...
et al), Gene 32:11~
20 (1984)), Bacillus sp.
promoter (Gry
Czan, T, J, ), Molecular No.
Iology Off The Molecule
larBiology of the Bacilli
), Akateminok Press Incorporated (A
cademeic Press, Inc.), New
- Yoke (1982)), and Strepp I. Mrs.
(Streptomyces) promoter (word
/Axm et al. (Ward, J. M., et al.
at), Molecular &/1 Furu Che Fute
GenGenet (Mo1. GenGenet) 203
468-478 (+986)). prokaryotes
The promoter is Giick.
B, R,) (/+-Nal Off Intian Qin Ma
Microbio o'; - (J, lnd, Micr
obiol, ) 1277-282 (1987);
Cenat iempo, Y.
) (Biochimie) 68: 5
05-516 (1986)); and Kotesman Niss.
(Gottesesman, S.
Ann, Rev, Genet, 1 8
: 4 1 E+-442 (1984))
are summarized. Proper expression in prokaryotic cells requires gene-coding
The presence of a liposome binding site upstream of the binding sequence is also required.
. Such liposome binding sites may include, for example, Bold L.
(Gold, L., et al) (Anu Lev
- Microbiol (Ann, Rev, Micro
biol, ) Danshi: 365-404 (198],)
) is disclosed by. The most preferred host is in vitro or
Yeast, insects, fungi, mammals in either tissue culture
Eukaryotic hosts, including cells, especially human cells. Mammal
Mammalian cells maintain the correct folding (f) at the correct site.
protein molecules that involve glycosylation or glycolysis
Provide post-translational modification for Mammalian cells that may be useful as hosts include VERO or
cells of fibroblast origin such as CHO-KI, or
Lymphoid system such as hybridoma 5P210-AGI4
cell of origin or myeloma P3x63Sg8, and its
Including derivatives. Preferred mammalian host cells are
Bromo after translation. S P 210 or
and J 558L, Nerves like Narahi 1MR322
Includes blastoma cell lines. In addition, CO8 cells are GA
Study of regulation of P-43 expression and GAP-43 expression
A convenient eukaryotic host for this purpose.
is preferable. For mammalian hosts, many possible vector systems are
Can be used to express AP-43. Depending on the nature of the host, many
Many types of transcriptional and translational control sequences can be used. adjustment
signals or associated with specific genes that have high levels of expression
When the adenovirus is activated, transcriptional and translational regulatory signals
Rus, bovine papillomavirus, 7mian virus, etc.
It may be derived from a different virus source. alternatively
Mammals such as actin, kolaken, mionn, etc.
Promoters from product expression products can also be used. repression or activation so that gene expression can be regulated.
A transcription initiation regulatory signal that enables transcription initiation can be selected. Attention
The expression of what should be suppressed by changing the temperature
Or adjustments that are temperature sensitive so that they can be initiated
signal or chemical regulation, e.g.
It is a regulatory signal that Yeast also performs post-translational peptide modifications including glycosylation
It offers substantial advantages in that it can. Place in yeast
A powerful promoter that can be used to produce the desired protein.
A large number of
Recombinant DNA technology exists. Yeast is a cloned mammal
Recognizes the leader sequence on the gene product and extracts the leader sequence.
secretes peptides (e.g., pre-peptides) that carry
do. large amounts when growing yeast on glucose-rich media
The coding for the glycolytic enzymes produced in the
The promoter and termination elements from the expressed gene are
Which of a series of yeast gene expression systems can be used?
You can also do it. Additionally, known glycolytic genes are highly effective
Can provide transcriptional control signals. For example, phosphoglyceride
Promoter and terminus of ratekinase gene
You can use the tar signal. Production of GAP-43 or its functional derivatives in insects
, for example, by methods known to those skilled in the art.
baculovirus engineered to express AP-43.
This can be achieved by dyeing. Thus, specific example of 1
The sequence encoding GAP-43 was generated in
by operably linking to the regulatory region of the polyhedral protein.
Kiru (7 Jasny), Science (Sci)
ence) 238: 1653 (1987)). set
Insect cells infected and cultured with transformed baculovirus, or
or live insects themselves contribute 20 to 50% of total protein production.
GAP-43 protein can be produced in an amount as low as 0%. live kelp
If insects should be used, at present, large-scale treatment according to the invention is not possible.
Caterpillars are the preferred host for imitation GAP-43 production. As described above, expression of GAP-43 in eukaryotic hosts
requires eukaryotic regulatory regions. Such area is
Generally, sufficient promoters are present to direct the initiation of RNA synthesis.
includes the target area. Preferred eukaryotic promoters are
Mouse metallothionein gene (Haymer, T. et al.
(Lamer, D., etal), Journal
Off-Molecular and Applied Geography
Nox (J, Mo1. Appl, Gen, )1
: 273-288 (1982)); herpesvirus
T K Fromoter (Manovite Varnish (McKn)
light, S, ), Cell 31:3
55-365 (1982)); SV40 initial promotion
(Benoist, C., et al.)
et al), Nature (Lon)
Don) 290: 304-210 (1981);
Yeast ga14 gene promoter (Nyonston Nis
・Ei et al. (Johnston, S. A., et al.
al), pro/-tings off national a
Catame Off Scien/s (Proc, Nat
1. Acad, Sci, ) (USA) 79:
697]~] 975 (1982):/Ruba Bi
Si 1ver, P, A, , et
al), Prontings Off Su/Yonaru Aka
Temmy Off Saijuns (Proc, Natl, A
cad, Sci, ) (IJSA) 81: 5951
~1955 (1984)). As is widely known, the translation of eukaryotic mRNA is
It begins with the codon that codes for the first methionine. For this reason, eukaryotic promoters and GAP-43
DN encoding a protein (or a functional derivative thereof)
A linkage with the A sequence encodes methionine.
Contains no intervening codon (Sunawaji AUG)
It is preferable to check that. Consequences of the presence of such codons
, (D coding AtJG Fudon or GAP-43
If it is in the same reading frame as the NA sequence) fusion
to form a protein or (AU G codon or G A
, a reity identical to the sequence encoding P-43.
If the frame is not in the frame, the frame software suddenly changes.
happen. Sequences encoding GAP43 and operably linked
a linear molecule or, more preferably,
Non-replicating DNA (or R
NA) As a molecule, the receptor prokaryotic or eukaryotic cell
It can be introduced into Such molecules are capable of autonomous replication.
Therefore, the expression of GAP-43 protein depends on the introduced sequence.
Can occur through temporary manifestations. Alternatively, permanent
Expression occurs through integration of introduced sequences into the host chromosome
It can happen. In embodiment 1, the desired gene sequence is stained in the host cell.
Use vectors that can be integrated into the body. introduced
Cells that have stably integrated the DNA into their chromosomes are
, allowing selection of host cells containing the expression vector
selection by also introducing one or more markers.
You can choose. The marker is a source for auxotrophic hosts.
nutritional properties, biocide resistance, e.g. antibiotics, or copper
Heavy metals etc. may be provided. selectable marker gene
I) Directly linked to the NA gene sequence
or by cotransfection.
You can introduce it. In addition, −・main chain binding protein rn
Optimal synthesis of RNA may require additional elements.
Ru. These elements are splice knobs, as well as transcriptional
Includes promoters, enhancers, and terminal signals
It is possible. cDNA expression vectors integrating such elements
is Okayama H, More
Cellular Ant Cellular Biology (Mo1
．． Ce1. Biol, ) 3: 280 (1983)
Therefore, it includes what is described. In a preferred embodiment, the introduced sequences are
a plasmid or viral vector that can autonomously replicate in
be incorporated into. Wide variety of Hephthers - all of this order
It can be used in any purpose. Specific plasmids are important in the selection of viral vectors.
The key factor is the receptor cell containing the vector or its
from receptor cells that do not contain the vector.
Selected option 4, specific location 1: desired vector in
the number of copies of the vector, and the transfer between host cells of different species.
It includes whether it is possible, desirable, or fortune-telling to move the
Ru. Preferred prokaryotic vectors are (e.g. pBR3
22, Co1E], psc 101, pACYC184
, πVX) in E. coli (E, coli)
Includes plasmids such as those capable of replication. It takes
Plasmids are described, for example, by Maniatis Ti et al.
iatis, T., et al) (Molecular
・Cloning, A Lahoratory Manual (Mo
Regular Cloning, A Laborato
ry Manual), Cold 0 Spring Herba
-・Press (Cold Spring HarborP
ress), Cold Spring Harbor (Col
dSpring Harbor), New York (1
982)). Bacillus
i l 1us) Plasmids are pc194 and pC221
, pT127, etc. Such a plasmid is Talino
Gryczan, T.
General Biology on 0 the 0 Hachiri (The
Molecular Biology of the B
acilli), Academia i Press (Academ
icPress), New York (1982), 30
7-329). appropriate stress
Ptomyces (Streptomyces) plasmid
, plJlol (Kental K. J. et al.
al l, K, J,. et al), Journal on Bacteriology (
J, Bacteriol, ) 169: 4177-4
183 (1987)) and φC31.
Streptomyces bacteriophytes
(Chater, K.F. et al.
, F, , et al) (Sixth Inter
National Symposium on Acti/Mystery
Biology (Sixth International
al Symposium on Actinomyc
etalesBiology), Academia I Kite
(Akademiai Kaido), Budapest, Han
Gully (1986), pp. 45-54). Shu
The Pseudomonas plasmid is
John, J.F. et al.
et al) (Reviews on Nmphexious)
Daysies (Rev, Infect, Dis, )
8: 693-704 (1986)), and Izaki.
Kei (lzaki, K, X Japanese Nyana)
Le Off Piteiology (Jpn, J, Bac
Terriol, ) 33: 729-742 (197
8)). Preferred eukaryotic plasmids are BPV, Tuccinia, S
V40, 2-micron circle, etc.
or its derivatives. Such plasmids
(Bo
Stein, D., et al), Miamira
Nntr Nmbo (Miami Wntr, Symp,)
19: 265-274 (1982), Broch.
Breach, J, R,: The.
Molecular Biology on the East Sa
Tucharomyces life cycle and inherita
The Molecular Biology
f the Yeast Saccharomyces
: Life Cycle and Inherita
nce), Cold 0 Spring Barber Laborato
Lee (Cold Spring) 1arbor Lab
oratory), Cold Spring Harbor (
Cold Spring Harbor), New York
Kushu, pp. 445-470 (1981);
Broach, J, R, Cell (C
ell) 28: 203204 (1982); Poron
・Davy et al. (Bol Jon, D, P, ,
et at), Journal on Tarino Hematol.
Honful (J, CI in, Hematol, 0
nco1. ) 10:39-48 (1980); Mania
Maniat is, T, Cell
・Biologinia Conbrigenbu Treaties
(Cell Biology: AComprehe
nsive Treatise), Volume 3, Gene 9 Iku
Soubret 7 Yon (Gene Expression), A
Academic Press
, New York, 563-60B (1980)). Once the vector containing the construct or DNA sequence is expressed
Once prepared for transformation, transfection
, conjugation, protoplast fusion, calcium phosphate precipitation
biochemical means such as dimethylaminoethyl (D
EAE) Application of polycations such as text run,
and electroporation, direct microin/excunyong, and
microprojec
tile) (biolist
ic) bombardment
(Johnston et al.,
Science 240 (4858):
1538 (1988) and the like.
The vector or DNA construct by either
can be introduced into suitable host cells. After vector introduction, select for proliferation of vector-containing cells.
Recipient cells are grown in a selective medium of choice. clone
As a result of the expression of the modified gene sequence, GAP-43 protein is produced.
or fragments of this protein are produced. this is,
In transformed cells, or for example, bromothioquine.
By administering uracil to neuroblastoma cells etc.
) can occur after inducing these cells to differentiate
. The expressed protein is extracted, precipitated, chromatographed,
Such as affinity chromatography, electrophoresis, etc.
It can be isolated and purified according to conventional methods. The present invention also provides GAP-43 or its fragments and fragments.
There are detectable enzymes such as tergalactone
or any desired homologous or heterologous protein or peptide
A cloned gene encoding a fusion protein consisting of
Regarding. Methods for producing such fusion proteins have not been taught.
There is. For example, Bai, D.
tl, , et al), / Yarnal on By
Logical Chemistry (JBiol, Chem,)
26 ] : ] 2395-12399 (1986)
, or Huynh, TIJ
, et al), TNA Cloning
Techniques, A Practical Approach (DN
A Cloning Techniques:A Pr
rlgtlo during the actual approach)
and construction and screening in λgtll c
DNA Libraries (Constructio
n and Screening cDNA 1-ib
Glover
) ftm, IRL Pr
ess), Onoxfor-1', 1985.49-77
page. GAP-43, a functional derivative thereof, or GAP-43
or a fragment thereof and a detectable enzyme or desired protein.
Alternatively, fusion proteins consisting of peptides can be prepared using conventional methods known to those skilled in the art.
It can be isolated by the method. For example, cells can be harvested by centrifugation.
The protein can be collected or dissolved in a suitable buffer, e.g.
D E A, E-cellulose, phosphocellulose, polymer
Lilipo/Thyric acid-Acarose, Hitoloki/Apatite
column chromatography or electrophoresis or
Can be isolated by immunoprecipitation. Alternatively, GAP43
or a functional derivative thereof, or GAP43 and detection
consisting of possible enzymes or desired proteins or peptides
The fusion protein can also be isolated by the use of anti-GAP-43 antibodies.
to a detectable enzyme or desired protein or peptide.
It can be isolated by using antibodies against it. Kaka
Antibodies can be obtained by well-known methods,
Some of them will be mentioned later. Thus, for example, poly
Preparation of clonal rabbit anti-GAP-43 serum is described herein.
This is disclosed in the Examples section of the book. Another embodiment of the invention is for G A P -4,3
Consists of protein antibodies. [Antibody] (Ab
) or [monoclonal antibody J (Mab) is an antigen
(e.g., Fab and F (ab')
. including intact molecules (such as fragments) as well as fragments thereof.
To be. Fab and F(ab'), fragments are intact antibodies
It lacks the Fc fragment of the intact antibody, rapidly disappears from the circulation, and
may have low non-specific tissue binding (Wah et al.
l ct at), Journal on Nucleatsk.
Meticin (J, Nucl, Mecl, ) 243
16-325 (1983)). Antibodies of the invention can be prepared by any of a variety of methods.
can. For example, GAP-43 protein or its functionality
Cells expressing the derivative contain a polypeptide capable of binding GAP43.
To induce the production of serum containing clonal antibodies
Can be administered to animals. In the most preferred method, antibodies of the invention are monoclonal.
It is an antibody. Such monoclonal antibodies are hybrid
(Kohl et al.
r et al), Fuichi + - (Nature) 2
56:495 (1975); Kohler et al.
et al), Neirobian Shiny On.
Immunolo, /'-(Eur, J, 1mmunoL)
6:511 (1976): Kohle et al.
r et al), Nyobian/Yarnal On
・Immuno/- (Eurl, 1mmuno1.)6
292 (1976), Hammerling et al.
ing6t al,), monoclonal antijo
Tis and Tisel hybridomas (Mo
noclona Antibodies and TCe
ll tlybridomas), Elsevier N.
Y (Elsevier, N, Y, ), 563~
681 pages (1981)). In general, such techniques
immunization of animals with 43 antigens. Lung cells of such animals
The cells are extracted and fused with the appropriate myeloma cell line. Either
Any suitable myeloma cell line can also be used in the present invention. After fusion, the resulting Hyfritoma cells were placed in HAT medium.
selectively maintained, and then Wands, N.E.R. et al.
(Wands, J, R, etal) (Castroe
Nterolone-(Gastroenterology) 8
0:225-232 (1981)
Cloning is performed by the limiting dilution method such as next
The hybridoma cells obtained through such selection are then examined.
A clone that secretes antibodies capable of binding the GAP-43 antigen
Identify the loan. Deposit of Hybridoma Cell Lines A preferred monoclonal antibody of the invention is the MAb anti-GA
Specificity of monoclonal antibody named P-43 (HB)
It is something that has a nature. As a further embodiment, the present invention
is a hybrid that produces the monoclonal antibody of the present invention.
Consists of market stocks. Preferred hybridomas according to the invention
The cell line was named HB and the MAb anti-GAP-43 (HB
) produces a monoclonal antibody named The H5 thin
The cell line was born in Maryland, USA on December 21, 1989.
State 20581, Rockville, PA
- Clone Drive (Parklawn Drive)
) 12301, American Type Culture Co.
Rection (American Type Cu1tu)
re Co11ection) (ATCC);
Granted accession number ATCCHB 10316. The antibodies of the present invention can be used in forward antibodies.
German, reverse sandwich,
Simultaneous Sand
Immunometry such as switch assay, test assay or
are known in the art, including "sandwich" assays.
Well suited for use in standard immunodiagnostic assays
. Antibodies of the present invention can be prepared by those skilled in the art, such as GAP43 antigen or its like.
Acceptable specificity, sensitivity, and
Do not perform inconvenient experiments to perform immunoassays with accuracy.
Any combination of numbers can be used, as can be determined arbitrarily.
You can also be there. The standard text on the general principles of immunology is Klein
・Jay (Klein, J, ), Immunolone
The Science on Self-Nonself Disc
1mmunology: The 5
Science of 5elf-Nonself Dis
crime), Zion Willie &
・Sands (John Wiley & 5ons), out
Publisher, New York (1982); Kenenoto R et al.
(Kennett, R., et al.), monochrome
Null Antihodes, Hybridomania Nini
- Timmenyong in Biological Anarin
Monoclonal Antibodies,
Hybridoma: ^Neb Dimensio
n in Biological Analysis)
, Blenheim Press, published
Publisher, New York (1980); Bourdon Earl et al.
(BurdonR, et al)
Techniques in biochemistry and
Recular Hyolo/-(Laboratory T
echniques in Bi. Chemistry and Molecular B
iology), Volume 13, Elsevie
R), Publisher, Amsterdam (1984), Kan in
Campbell], A.), [Mono
Clonal Antihody Technology (Mono
clonal Antibody Technology
y). "Detect" means to measure the presence or absence of a substance.
and includes quantifying the amount of a substance.
do. Thus, the term refers to both quantitative and qualitative measurements.
Refers to the use of the materials, compositions, and methods of the invention in
. Monoclonals of the same specificity as described herein
Isolation of other hybridomas that separate anti-
This can be achieved through idiotype screening techniques.
Ru. Potocmjak, etc.
al), Science 215: 1
637 (1982). Simply put, anti-idiotypic
Antibodies are antibodies produced by the clone of interest.
It is an antibody that recognizes the only determinant present in The anti-
Idiotypic antibodies as a source of monoclonal antibodies
A monoclonal antibody that focuses on animals of the same strain as used.
It is prepared by immunizing the body. immunized
Animals possess these idiotypic determinants (anti-idiotypic determinants).
immunity by producing antibodies against
Recognizes and responds to idiotypic determinants of infectious antibodies
will. monoc produced by a single clone
a second animal anti-item specific for the local antibody;
by using the iotype antibodies used for immunization.
Other clones can be identified. 2 clone's
The itiotypic identity of the product is determined by whether the two clones are identical or
are identical with respect to their recognition of epitopic determinants of
Show that. In addition, anti-iteiotypic antibodies can be used as immunogens.
immune response in yet another animal using
is epitopically identical to the original MAb.
to produce so-called anti-iteiotype antibodies.
I can do it. Thus, the epitope of a monoclonal antibody
By using antibodies against determinants, the same epitope
Identify other clones expressing tope-specific antibodies
I can do it. In antibodies, idiotypic determinants are
Located in hypervariable regions that bind to a given epitope. Therefore, using the monoclonal antibody of the present invention, B A
, in suitable animals such as LB/c mice.
Anti-idiotypic Abs can be induced. child
Spleen cells from these animals were used to identify anti-idiotypes.
Generate hybridoma cell lines. Monoclonal anti-ite coupled to KLH
BALB/
c Immunize mice. Sera from these mice were
Binding of the original Ab specific for the occupied epitope
Contains anti-anti-iteiotypic Abs with the properties
Probably. Thus, anti-itiotypic MAbs are evaluated
Itiotypes that are structurally similar to the epitope to be treated
Contains antigenic determinants. For replication, inνitr cells. It can be cultured both in vitro and in vivo. High in vivo production makes this the preferred culture method.
Make it law. Abbreviated as L′, from each individual No.
BAL cells were subjected to initial antigen challenge with pristane.
B/c mice were administered intraperitoneally to achieve the desired model of high a.
Ascites fluid containing noclonal antibodies is produced. Eye
Monoclonal antibodies of the sotype IgM or IgG are
using column chromatography methods well known to the industry.
, on culture? It can be refined from climbing. Can the antibodies of the invention be used in liquid phase?
or immunoassays that can be coupled to a solid phase support.
Particularly suitable for use in In addition, these immune
Antibodies in assays can be detectably labeled in a variety of ways.
I can do it. There are many different labels and labeling methods in the art. Examples of the types of labels that can be used in the present invention include enzymes,
Radioactive isotopes, fluorescent compounds, chemiluminescent compounds
substances, bioluminescent compounds and metal molecules.
including but not limited to. person skilled in the art
If so, do you know of other suitable labels to attach to the antibody?
and can be confirmed by the use of routine experiments.
I'm sure you can admit it. Furthermore, these signs
Conjugation to antibodies is accomplished using standard techniques commonly known to those skilled in the art.
can be achieved. One way in which the antibodies of the invention can be detectably labeled is to
By binding the body to enzymes. later exposed to that substrate
If this enzyme is
the substrate in such a way that it produces a chemical group that can be detected by physical means.
will react. An example of an enzyme that can detectably label the antibodies of the invention is
Acid dehydrogenase, staphyloconochonuclear
se, tilter V-steroid isomerase, yeast alco
aldehydroquenase, alpha glycerin phosphate
Trochenase, triose phosphate isomerase, biotin
N-Abinon Beloqui/Targe, Hose Latino Hupel
Oquintase, alkaline phosphatase, asparaquina
-se, glucose oki/ta, heterkaract/ta
-se, lihonuclease, urea-7, catalase, g
lucose-Vl-phosphate-tihydrogenase, glucose
Contains coamylase and acetylcholinesterase
do. Additionally, the presence of the detectably labeled antibodies of the present invention
Using a microcounter or scintillation counter
Radioactive isotopes that can be measured by such means as
It can be detected by labeling the antibody with a label. of the present invention
Particularly useful isotopes for this purpose are 3) (X+25I,
32p, 35S, +4C1”Cr, 38CQ
, “Co,” “Co,” “Fe,” and 75Se.Also, by labeling the antibody with a fluorescent compound, the present invention
binding of bright, detectably labeled antibodies can be detected.
Wear. Expose the fluorescently labeled antibody to light of the appropriate wavelength.
The presence of the dye can then be detected due to its fluorescence. most
Some commonly used fluorescent labeling compounds include fluorochromes.
In, isothiocyanate, rotamine, red algae, algae
Purple, allophycocyanin, 0-phthaloartechite and
There's fluorescamine. The antibodies of the present invention may also be used for l+2Eu1 or other lanthanomes.
Can be detected using metals that emit fluorescence, such as those in the id series.
It can also be labeled. These metals are
Triaminopentaacetic acid (DTPA) or ethylene diamino
Using metal chelating groups such as tetraacetic acid (EDTA)
can be attached to body molecules. The antibodies of the present invention can also be combined with chemiluminescent compounds.
detectably labeled by coupling to
I can do that. Then, chemiluminescence-labeled anti-
The presence of the body is due to the presence of luminescence that occurs during chemical reactions.
This can be determined by detecting the presence of Particularly useful chemistry
Examples of luminescent labeled compounds are Luminal, Isol
minar, theromatic a
Clidinium ester, imidazole, acrinnium
salts and oxalate esters. Similarly, bioluminescent compounds can be used to
The body can be labeled. Bioluminescence is
mediator proteins or biomolecules that increase the efficiency of chemiluminescence reactions.
It is a type of chemiluminescence found in physics.
Ru. The presence of bioluminescent antibodies indicates that luminescence
Determined by detecting presence. purpose of the sign
An important bioluminescent compound for is luciferin
, including lumferase and aequorin. The antibodies and substantially purified antigens of the invention are ideal
The kit is suitable for preparation. It takes a cow. One or more items such as vials, test tubes, etc.
a carrier partitioned to accommodate container means in a closed compartment;
means, each of said container means containing an assay to be used.
consists of separate elements. There are many types of assays that can be integrated into kit formats.
, e.g. competitive assays and non-competitive assays. Includes Sei. Assays that can utilize the antibodies of the invention
Typical examples are Radioimmunoa, Sei (RIA), Enzyme Immuno
immunoassay (EIA), immunoenzymatic assay (ELISA),
immunometric, or sandwich, immunoassay
Yes. “Immunometric assay” or “sandwich assay”
The word "Munoa, Sei" means 7 Martinius (sim
ultaneous) sandwich, forward (f
sandwich and reverse
rse) including sandwich immunoassays.
do. These terms will be well understood by those skilled in the art. Those skilled in the art will also appreciate that the antibodies of the invention can be used in other variations and
Assay formats known or to be developed in the future
You will understand how useful it is in situations. these are
It is intended to be encompassed within the scope of the present invention. Forward sandwich assays can be used, e.g.
National Patent No. 3867517, No. 4012294 and
No. 4376110. Reverse Sun
Toi, Chi assays are described, for example, in U.S. Pat. No. 40,988.
No. 76 and No. 4376110. In a preferred embodiment of carrying out the assay, a
Species ``blocker''
presence in the application medium (usually labeled
soluble antibody). Adding a "blocker"
, non-specific proteins, proteases, or in experimental samples.
Human antibodies against mouse immunoglobulins present or
Cross-linked antibodies on a phase support or radiolabeled antibodies
or destroy it, resulting in a false positive result or a false negative result.
Make sure there are no consequences. Therefore, the “blocker”
The selection substantially increases the specificity of the assay described in this invention.
increase. Same class or subclass as used in the assay
A large number of unrelated (isotypes) i.e. non-specific
heterologous) antibodies (e.g., IgG+, 1gG2., I
gM etc.) can be used as a 1 blocker. suitable
maintains high sensitivity and detects cross-reactive proteins in human serum.
To suppress unwanted interference caused by
drug concentration (usually 1 to 100 micrograms/micro
little) or important. In addition, products containing “blockers”
The buffer system used needs to be optimized. preferred buffer
The solution is imitazole, HE in the physiological pH range.
PPS, MOPS, TES, ADA, ACES,
Weak organics such as PES, PI PES, TRI S, etc.
It uses acid as haze. Somewhat undesirable
The solution is inorganic, such as phosphates, borates or carbonates.
It is a buffer solution. Finally, known protease inhibitors
into a buffer containing a "blocker" (usually 0.01 to 1
0 micrograms/mQ). Previously used and for use in the present invention
There are many solid phase immunosorbents available. Well-known immunoadsorbents include chews, beads,
Forms of glass, polystyrene, polypropylene, textile
Tran, nylon and other materials, and such materials?
microtiter plates formed or coated with
Includes Immobilized antibodies can be amide or ester
techniques such as covalent bonding via bonding, or by adsorption.
covalently or physically attached to the in-phase immunoadsorbent.
Can be combined. Those skilled in the art will appreciate that there are many suitable
Solid-phase immunosorbents and methods for immobilizing antibodies on them
As you know and use routine experiments,
You can probably confirm that. in vivo, in vitro or in 5it
For u-diagnosis, labels such as radionuclides can be used directly
or by using a mediating functional group.
Can be attached to the body. Exists as a metal cation
often used to attach radioactive isotopes to antibodies.
The mediating group is /ethylenetriaminotetraacetic acid (DTPA)
It is. Typical examples of metal thiones bound in this way
Examples are: 99 m T c, 123■, ■11 N,
+31), 9'lRu, 87CuSl17Gao
and “G a.Also, the antibody of the present invention can be used for diagnostic purposes.
It can also be labeled with non-radioactive isotopes. using this method
Particularly useful elements are +57Gcl, Mn, +82Dy,
"Cr and 56Fe. Furthermore, the antibody of the present invention has an epitope reactive with the antibody of the present invention.
benign or cancerous expressing the GAP-43 antigen associated with
in animals, including humans, with disorders such as neoplasia.
It can be used in immunotherapy. When used in immunotherapy, the antibodies of the invention are labeled with a therapeutic agent.
You can label it even if you don't have one. Immunotherapy using antibodies of the invention
Examples of therapeutic agents that can be coupled include radioisotopes,
They are Chin and Tokin/N. Lectins bind to specific sugar levels, usually from plant materials.
This is a protein isolated from Additionally, many lectins stimulate cells.
It can aggregate and stimulate lymphocytes. S/N is
It is a toxic lectin that has been used in immunotherapy. This angel
The cause of drug toxicity and the alpha peptide chain of Narurin
Site-specific delivery of toxic defects by conjugating to antibody molecules
This is achieved by making it possible. This is for example
, Vitctta et al.
Science 238: ] 098 (198
7), and Psatan et al.
, Atoha + allergy (AdvΔallergy) 47
: 641 (] 986). Toquinones are often fatal in sufficient doses.
poisonous substance produced by plants, animals or microorganisms
It is a sexual substance. For example, Nphtheria) and quinone are non-stiff.
Corynebaeter
ium diphtheria)
It is white. This toxin can be isolated under appropriate conditions.
Consists of luca and beta subunits. the toxin
The alpha component of the antibody binds to the antibody for site-specific delivery.
Can be used. Radioisotopes capable of binding to antibodies of the invention for use in immunotherapy
Examples of elements are: ``'Um, +3'I,''Y, @'TC
U,! +7Bi, look at A t, ””P b, 47
Of course, the expressed GAP-43 is normally confined within the cell membrane.
It's locked up. Therefore, those skilled in the art will understand that the anti-inflammatory agents of the present invention
In vivo diagnostic and therapeutic methods that use the body
Mechanism that can detect GAP-43 on the intracellular membrane
You will understand that some rhythm is required. 1
Such methods involve introducing antibodies or fragments thereof into cell membranes.
or introduced into the cell itself through the cell membrane.
Is Rukoto. This means, for example, that the target cell
The antibody is bound to the receptor site containing the receptor site.
This can be achieved by Thus, the antibody is
transported into the cell membrane with the recant, or
can pass through. The choice of carrier ligand is as understood by those skilled in the art.
Depends on several factors. These include, for example, ligands and
and its receptors, and all
Ligands that are activated and transported, including transport kinetics
Or preferable. In addition, the means for binding antibodies to ligands is
Varies within limits, e.g. with covalent or ionic bonds
Can such a binding beam be used as a Gantt-receptor?
should not change the affinity of
This should be kept in mind. Examples of receptors suitable for such applications include
has been shown to contain all the necessary information about its effects.
(Davis et al.,
Nal Subcellular Biology (J, Ce1
l Biol,) 107 (6/3), Abst
Lacto No. 3112 (1988) Low-density lipoprotein (LD)
Regarding receptors for L) and dopamine
including known brain-specific receptors such as
. In this regard, Ricando itself is an antibody of the invention.
or antibodies specific for the receptors that can bind to it.
It will be understood that it could be a fragment. Furthermore, those skilled in the art will understand the ligand-receptor interaction.
Seems non-interfering and can easily cross cell membranes
(such as Fab or F(ab')2 fragments)
We have found it particularly desirable to use the antibody fragments of the invention.
It will come out. As will be understood by those skilled in the art, -
Roadway antibodies may be preferred for these and other reasons
. The antibody is transported into the cell membrane or into the cell as described above.
If the antibody is to be isolated from its antigen on the GAP'-43 protein,
The label becomes relatively more effective when attached to the site.
It is preferable to label antibodies for diagnosis or treatment of sea urchins.
It would be nice. This includes, for example, antigen-antibody complexes.
Use a label that becomes active or detectable as a result of the formation of a gas.
This can be achieved by doing. Alternatively, antigen-antibody
complex formation or changes in antibody conformation.
Inducing oxidation and also unexposed or insufficiently exposed
Exposure or sufficient exposure of unmarked marks.
It is also possible to label the sea urchin antibody itself. The above criteria and
All other matters in practicing these aspects of the invention.
will be clear to those skilled in the art. In addition, liposomes containing the antibody of the present invention can be incorporated into their membranes.
to specifically transport the antibody to the target region.
You can also do it. These liposomes contain antibodies that are
In addition, drugs and radioisotopes that will be released at the target site
immunotherapeutic agents such as topicals, lectins, and toxins.
It can be manufactured to contain. an antibody, and preferably encodes GAP-43
Nucleotide sequences (and their functional and chemical derivations)
Another method is to introduce the body) into nerve cells for diagnostic or therapeutic purposes.
One preferred method is to remove viruses, including retroviruses.
Depends on the use of vectors. An example of a suitable virus is
Mention may be made of various herpesviruses. Appropriate
Viruses include human immunodeficiency virus (HIV)
do. Other suitable viruses and retroviruses are in the art.
well known to people. Introducing genes into mammalian cells
The use of viral vectors to
Varmus, Science
e) 240 (4858): 1427 (1988):
Eglitis et al., Bio
Techniques (BioTechniques)6. 7
: 608 (1988): Jenison L (J
aenisch) Science 240
(4858): 1468 (1988): and Hern
/ Bernstein et al.
Gene
t, Eng, XN, Y,) 7: 235 (1985)
It is summarized in. For the purposes of this invention, attenuated viruses or retroviruses
It would be preferable to use a virus strain. Thus, for example, for antibodies or DNA sequences of the invention.
CD4-dependent mechanism (Funke
Funke et al.
Experimental Metinn (J, Exp, Me
d, ) 165: 1230 (1987))
HIV-2ST that invades nerve cells (Konbu et al.
Science 24
0 (4858): 1525 (1988 or HIV
-2uc, (Evans et al.
Science 240 (4858)
: 1523 (1988)).
Retrovirus with cytopathy)
You can also use The neurobiology of HIV infection is e.g.
For example, Johnson et al.
F.A.N.E.B. Journal (FASE)
BJ, ), 2(14): 2970 (1988)
Are listed. A person skilled in the art would appreciate the effort of routine skill.
Different people with known susceptibility to the virus
It would be possible to target neural populations. For example, CD4 has been shown to have mutant transcripts in the human brain.
It is known that the content is highest in the forebrain (mutton
Maddon et al., Cell
47:333 (1986). Ideally, the selection of gene purine/ri system is suitable for Mi weaving.
This method ensures that gene expression is at an appropriate level.
Effective and stable gene transfer without any adverse effects
This may be done by one of ordinary skill in the art, keeping the purpose in mind. For example, Wolfet al., Ricoh
Rheum, Di
s, Cl1n, North Am, ) 14 (2)
: 459 (1983). puri to central nervous system targets
Regarding cancer, there are many viruses including HIV.
vectors have the advantage of being able to cross the blood-brain barrier (i.e.
Johnson et al., F.E.
I Nis E B Journal (FASEB J,
) 2 (14): 2970 (1988)). For isolation of rat GAP-43 using labeled probes
Code GAP-43 according to the method described above.
Maiko uses the fragment as a DNA probe.
can be used to isolate the corresponding antigen in humans.
Wear. Next, we cloned the human antigen gene and used it in the host.
Human antigens can be obtained by expression. Then this
human antigens, corresponding autoantibodies, and human
for use in diagnostic assays for animal therapeutic procedures involving
I can be there. The present inventors have demonstrated the ability to control the expression of the G A P-43 gene.
We conducted 17 well-designed experiments to reveal the regulatory mechanisms that
It was. Modulation of GAP-43 expression can be either central or peripheral.
Involves humans with neurological tissue damage, disease or dysfunction.
A convenient and effective way to treat mammals with
provide. Furthermore, according to the present invention, mammals, including humans,
Structural reorganization in normal central or peripheral nervous tissue in animals
Methods to modulate neuronal structure and function
It is important for those skilled in the art to further clarify the mechanism.
It would be useful. Preclinical use of the invention in the treatment of neurological diseases or disorders
Therapeutic use or clinical therapeutic use is for diagnosis and treatment.
as best accomplished by one skilled in the art using acceptable principles.
There will be. Such principles are known in the art, and e.g.
Peterstorf, R.N., et al.
dorf, R, G, et al) m, harrison pr
Lin/Pull on Internal Metainin (tla
rrison's Principles or In
ternaMedicine), 10th edition, McGraw-H.
McGrawHill, Publisher, New York.
State of New York (1983), especially Part A of that document.
, Section 11 “Disorders of the Central Nervous System”
ers of theCentral Nervous
System) J. Antigens, antibodies and compositions of the invention or their functions
The derivatives are well suited for the preparation of pharmaceutical compositions. of the present invention
The pharmaceutical composition can enjoy the beneficial effects of the compounds of the present invention.
It can be administered to any animal. of such animals
The first of these is humans, and the invention is not limited as such.
It's not something you can do. The pharmaceutical composition of the present invention does not achieve its intended purpose.
It can also be administered by any of these means. For example, administration may be parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal.
It can be administered by the transdermal, transdermal, or buccal route. alternative method
Administration may be by oral route, either as a
I can do it. The dose to be administered depends on the age, health, and
and body weight, type of concurrent treatment, frequency of treatment, if any,
and on the nature of the desired effect. In addition to pharmacologically active compounds, new pharmaceutical formulations include
Processing of active compounds into pharmaceutical preparations
A suitable drug consisting of excipients and additives that facilitate
The above-mentioned acceptable carriers may be included. preferably
is used for pharmaceutical preparations, especially tablets, dragees, and capsules.
used in orally administrable formulations and preferred types of administration.
Preparations that can be administered rectally, such as herbal medicines, and herbal medicines that can be administered rectally.
Formulations suitable for administration by injection, as well as solutions suitable for administration by injection, include excipients.
With excipients, about 0.001 to about 99 percent active compound
preferably from about 0.01 to about 95 percent.
Contain. The dosage range for administering the compositions of the invention will produce the desired effect.
canine, thereby preventing e.g. neoplastic disease.
Tissues are reduced, eliminated, or improved. The dose is
, unwanted cross-reactions, adverse effects such as anaphylactic reactions, etc.
should not be large enough to cause Generally, the dose
varies depending on the patient's age, condition, sex, and severity of disease.
. counterindication, also
Immune tolerance and other variables, if any, also determine the appropriate dose.
It will have an impact. Antibodies can be administered by injection or by slowing down over time.
By repeated large injections (prof us 1on)
Can be administered parenterally. The antibodies of the present invention can also be administered intravenously, intravalently (
intraparental, intramuscular or skin
Can be administered subcutaneously. The pharmaceutical formulations of the invention can be prepared by methods known per se, for example by conventional methods.
through routine mixing, granulation, dragee-making, dissolving and freeze-drying processes.
It can be manufactured. Thus, pharmaceutical formulations for oral use
The active compound is combined with a solid excipient and, if desired, the obtained
The granulated mixture is milled and the granulated mixture is processed and granulated as desired.
tablets or, if necessary, after addition of suitable excipients.
obtain the dragee core. Suitable excipients include, in particular, fillers such as sugars, e.g.
Cutose or sucrose, mannitol or sorbitol
toll preparations and/or carunium phosphates, e.g.
Tricalcium phosphate or calcium hydrogen phosphate, then
For example, corn starch, wheat starch, rice starch,
Potato starch, seratin, tragacanth, methylcellulose
base, hydroxypropyl methylcellulose, carboxylic acid
Sodium dimethylcellulose and/or polyvinylcellulose
A good binder for starch pastes using nilpyrrolidone.
Ru. If desired, the starch and carboxymethyl starch
powder, cross-linked polyvinylpyrrolidone, agar, or algin
acids or their salts such as sodium alginate
Disintegrants can be added. Additives are as described above.
All of the flow control agents and lubricants, such as silica,
Talc, stearic acid or magnesium stearate
or its salts, such as calcium stearate, and
and/or polyethylene glycol. The dragee core may, if desired, contain a suitable material resistant to gastric juices.
Apply a suitable coating agent. For this purpose, if desired,
Gum arabic, talc, polyvinylpyrrolidone, polier
tyrene glycol and/or titanium dioxide, lacquer
– solution and a suitable organic solvent or solvent mixture.
A concentrated sugar solution can be used. against gastric juices
To obtain a resistant coach ink agent, use acetylcellulose
-suphthalate or hydroquine furovir methylcellulose
-solutions of suitable cellulose preparations such as sphthalates.
Use. dyes or pigments, e.g. for identity,
or to characterize combinations of active compound doses.
Can be added to tablets or dragee coatings
. Other pharmaceutical preparations that can be used orally include gelatin
Bunonyu Fit (push-fit) made with
capsules, ceratin and glycerol or
A flexible, sealed material made with a plasticizer such as sorbitol.
Includes capsules. The plan. Filler such as lactose,
Binders such as starch, and/or talc or starch.
Lubricants such as magnesium thearate, and optionally
Contains active compound in granular form that can be mixed with more stabilizers.
I can do U. In soft capsules, active
The compound is preferably a fatty oil or liquid paraffin.
It can be dissolved or suspended in a suitable liquid. In addition, cheap
A fixing agent can also be added. Possible pharmaceutical preparations that can be used rectally include, for example, one
or more active compounds in combination with herbal bases
It includes crude drugs consisting of: Suitable crude drug bases include, for example:
With natural or synthetic triglycerides, paraffin hydrocarbons
be. In addition, the combination of active compound and base
Seratin rectal capsules may also be used. 5J
Possible bases include, for example, liquid triglyceride, polyethylene
Contains lene glycol or paraffin hydrocarbons
. Suitable formulations for parenteral administration include water-soluble forms, e.g.
and aqueous solutions of active compounds that are salts. In addition,
Suspension of the active compounds as appropriate oily injection suspensions may be administered.
I can give something. A suitable lipophilic solvent or vehicle
Cru is fat 111], such as sesame oil, or synthetic fatty acids
Esters, such as ethyl ester or triglyceride
includes the code. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension.
For example, carboxydimethylcellulose sodium, sorbitol
Containing tall and/or text runs
I can do it. Optionally, the suspending agent can contain stabilizers.
You can also do that. The GAP-43 antigen of the present invention has a binding effect on neuron cells.
unique and thus provides a convenient and useful marker.
do. Therefore, antibodies directed to GAP-43 are
Neuron cells using various techniques well known to those skilled in the art
can be identified. Furthermore, the antibodies of the present invention
detection, measurement and
Therapeutic treatment and treatment of cancer and cancer in animals, including humans.
and for preclinical and clinical evaluation and treatment of and other disorders.
Convenient and useful in vlvo, in vitro
or in 5 situ diagnosis and treatment methods. Antigens of the invention can be obtained in substantially pure form using antibodies of the invention.
It can be isolated in a stylish form. Thus, embodiments of the invention are
provides a qualitatively pure antigen GAP-43, which is derived from the present invention.
Characteristic in that it is recognized by and binds to light antibodies
Can be attached. In another embodiment, the invention provides:
one or more antibodies directed against GAP-43;
By forming a complex with the body and the antigen
, provides a method for isolating or purifying GAP-43 antibodies.
do. Substantially pure antigen GAP-43 of the present invention can be used to treat brain cerebrospinal fluid.
, anti-GAP-43 in samples such as serum or urine.
It can be used to detect or measure the body. mosquito
Therefore, one embodiment of the present invention is directed to GAP-43 antigen.
A sample containing an antibody to detectably labeled GAP
-43 and then detecting the label.
Antibody against the G A P-43 antigen in the sample as a characteristic
The presence of or a method of measuring the nail. G.A.
Immunoreactive fraction of P43 and immunoreactive fraction of GAP-43
It is understood that reactive homologs may also be used.
Dew. ``Immunoreactive fraction-・The imperial edict is related to GAP-43.
G A showing equivalent immune response to directed antibodies
, any portion of the P-43 antigen. [The term 'immunoreactive congeners,' refers to one or more
The present invention differs from the GAP~43 protein by only amino acids of
A protein that exhibits an equivalent immune response to antibodies of
Ru. In still yet another embodiment of the invention, GAP-
43 protein transports GAP-43 protein to the cell membrane, especially
A novel membrane protein that orients cells to the region of their growth cones.
- found to contain a genomic peptide domain
did. Determining the structure of this membrane-targeting domain
However, the peptide is usually cytoplasmic (not usually membrane-bound).
It is effective in directing sol proteins to the cell membrane.
Shown. A detailed illustration of the present invention was carried out in the specification.
Examples are described in detail in ■. The compositions and methods of this aspect of the invention provide, inter alia,
Any protein of interest, including proteins that are not normally membrane-associated,
It can be directed to the cystic membrane. Additionally, the compositions and methods of this aspect of the invention may be used in animals.
to treat neurological damage and disorders in vitro, i.
Can be used in vivo, and in 5 in situ therapeutic treatments.
It is clear that it can be done. Those skilled in the art will be able to diagnose and treat
The previous description of therapeutic methods is equally applicable to this embodiment of the invention.
will understand that it is used. Furthermore, the membrane of the present invention
- The targeting peptide can be any desired protein or protein.
is also used to direct peptides to the cell membrane,
Thus, diagnosis and diagnosis in non-neurological indicators as well.
It is also clear that it has therapeutic uses. Examples of such indicators
plays an important role as a cell membrane or an immunological indicator.
including, but not limited to, any application
isn't it. For modes and methods of carrying out the invention, see the examples below.
These implementations will be well understood by those skilled in the art.
The examples in no way limit the scope of the invention or the claims.
It's not something you can do. (Example) Example I Cloning of cDNA for rat GAP-43 cD from RNA of 17-day-old embryo-derived pouch and 7-tooth dorsal root ganglion
Obtain the NA library and clone it into the lid for λgil1 expression.
[Huynh et al.
-NA Cloning a Practical App
Roach (DNA Cloning A Practical
alApproach) J, D.M. Clone
- (D, M, Glover) (I.R.L.
IRL Press, Washington Day
C., 1985) pp. 49-78]. Three estimates G
The AP-43 clone was developed by Snipes et al.
Soc, Neurosci, Abstr, 12:
GAP-43 described by 500 (1986)
It was identified using an antibody against. Longest clone, GAP
The identity of 43-2 was confirmed by hybrid selection translation method.
It became fruit (Fig. 1). GAP43-2 is a hybrid
cation, the natural GAl”43, i.e. about 4
5DS-polymer with an expected mobility of 3 kD molecular size.
Translation of polypeptides moving through acrylamide gel
To direct, select Senya RNA (mRNA).
Ta. This in vitro translation product was directed against GAP-43.
selectively immunoprecipitated with an antibody. immunoprecipitation
Specificity is determined by competition with unlabeled purified GAP-43.
certified. For further confirmation, purified GAP-43
Sequenced peptides prepared by bromide/anolysis of
Ta. The sequence, Arg-X-Lys-Gln-Val-Glu
4. ys-Asn-Asp-GluAsp-Gln-L
ys-1ie is a predicted orphan of GAP43-2
completely within the leading frame (
The X is a sequence whose amino acid identity cannot be determined with certainty.
(represents a cycle of decisions). Complete nucleotide sequence and predicted GAP43-2
The amino acid sequence obtained is shown in FIG. l/-Dingfre
The system contains the sequenced vepketophage fragments.
and is identical to the β-caract/terce gene of λgill.
in the reading frame. [Rat GAP-4
The cDNA for 3 is skim=(J, )i, P,
5kene) and his colleague (Ha/(G, Ba5i)
, Jacobson (R, Jacobson), Virag (IV)
irag), like m=<J, H, P, 5kene)
, personal communication). a copy of the array
Exchanged. The predicted amino acid sequence of the present invention is skim=(JH,
Completely identical to Sanetachi's provided by P, 5kenen.
Therefore, the nucleotide sequence +lj is
There is a difference in the position of 1. ] Oven reading leaf
The methionine that coincides with the start of the frame is the nucleotide position
In-frame stop codon at position 13
It is the first methionine after (TAA) and is the first methionine in eukaryotes.
code to give you the most favorable context to start your translation.
Kozak Cell 44:283
(1,986) suggested nine Nuku1/o
Surrounded by eight of the Cyto consensus sequences
Ru. This is the first remnant of the GAP-43 coding region.
It suggests that it is a group. However, the information
is insufficient to make it clear, therefore, the second methio
Nin (amino acid 5) may play this role. The predicted composition of G A P-43 is highly polar;
Obvious transmembrane domains or possible N-linked glyconols
It has no oxidation site. This composition is G A P-4,
3 or membrane-bound, or antibody recognition is reached without membrane permeabilization.
This is consistent with the observation that it is difficult to
i) et al., PNAS USA 83:3537 (198
6) ] i This is thus connected to the internal surface of the membrane.
It might happen. GAP) 43 protein from open reading frame
The predicted molecular size of is 24 kD, which is equivalent to 5
DS-Bo1 Noak1 Apparent fraction of molecules in Fluamidekel
Suki m = (Skene) and Lyria as child size
-F Qilliard) 4
Smaller than 3 kD [Skeen = (Skene) and
Williard, Nyanar in Se
Le Biology (J, Ce11.Biol,) 89:
86 (1981), ibid., p. 9B]. GAP-4
The apparent molecular size of 3 depends on the polyacrylamide 11 degrees.
Since the molecular size is uncertain [Jacobson
(Jacobson) et al., Journal in New
J Neurosci 6:1843
(198B)], this protein or SDS polyacrylamide
This indicates that the protein falls into the category of proteins that move irregularly on the dog gel.
L Banker et al./Yarnal
In Biological Chemistry (J, Biol
, Chem, ), 247:5856 (1972)
Persson et al., Science
ce) 225687 (1984); Smart (S
mart) et al.)<Virology
112 Near 03 (1981)]. This characteristic is 1nv
The itro-translation product has a mobility similar to that of native GAP-43.
It does not seem to be due to post-translational modification because it has high mobility.
(Figure 1). code within the putative open reading frame.
5DS-polyacrylamide gel transfer of the protein to be coded.
In order to collect information on the dynamic properties, Norton (Me
Nucleic Ac1ds Re5 by L ton) et al.
Hatate by the method of 12 Near 035 (19g4)
Using the Liophane SP6 promoter, in
GAP-43R from cDNA in vitro transcription system
NA was synthesized. 5au3A site, i.e. predicted
65 bases 3” of the end of the open reading frame
800=salt by transcribing the cDNA cut with
The base RNA was obtained (Fig. 2), and the polynucleotide at the 3' end of the cDNA was
Cutting at the HindT11 site in the linker region
1100-base RNA was obtained. 800-base
Both RNA and 11001 base RNA
Translated in vitro using sphere lysate, 15%
When analyzed on 5DS-polyacnolamide gel, the apparent
directs the synthesis of a polypeptide with a child size of 40 kD.
Ta. The 4C1-kD translation product in both cases was purified using anti-GAP43 anti-
Immunoprecipitation was performed using the body. i from neonatal rat brain RNA
l Vitro-synthesized GAP-43 inhibits these translations.
Moved with the product. GAP-43 has been shown to be important for growth cone function.
Evidence for the belief that growth cone membranes are enriched with proteins
Wealth [Meiri et al., PNAS USA83
:3537 (1986) ;Sukim=(Skene)
et al., Science 233 Near 83
(1986)] and in developing and regenerating nerves.
Increased transport of proteins [Skene and Wii]
Williard, Journal in Se
Le Biolon (J, Cel].Bio1.)
89/86 (1981), ibid., p. 961.
It will be done. GAP-43 gene expression or neurite outgrowth
In order to investigate whether the expression is regulated in the same way, P
tested in C12 cells, where neurite projections
production is promoted by neurodevelopmental factor (NGF),
3', 5' to monophosphate (cAMP)
It wasn't that much. These drugs cause neurite
Different mechanisms act in inducing protrusion.
Gunning et al., Gunning et al.
+Biology (J, Ce11.Biol,) 89:
240 (1981)]. Induced by any drug
GAP-43 mRNA levels are responsible for neurite outgrowth.
The increase was greatest in cells exposed to both drugs.
Yes (Figure 3). Pattern of GAP-43 gene expression during normal growth period
To determine the
Brains of 7-day-old fetuses, neonates, and adult rats, explanted ganglia
, heart, and liver. in a neuro-specific manner
, GAP-43 was expressed (Fig. 4). at all ages
, a major hybridization of approximately 1500 nucleotides
The bands are visible deep in the neuronal tissue. The inventors
contains the Kenomisoku GAP'-43 gene or intron sequence.
We found that it contains faint and large
Molecular-sized hands are non-springing precursors
can correspond to body molecules. GAP-43 is located in neurons.
[Meiri et al., PNAS 'J
SA 83:3537 (1986) Ko, Neuron Group
GAP-43 mRNA in the glial tissue (Dahlia)
GAP-43 probably has a more neural origin than that of GAP-43.
RNA is undetectable in the glioma cell line C6.
It wasn't served. In neural tissues, the amount of GAP-43 mRNA varies depending on development.
It changes depending on the stage. Peak concentrations occur during the perinatal period;
It is slightly delayed in the central nervous system compared to the peripheral nervous system. of expression
The timing coincides with l of axonal growth [Jacobson (
Jacobson), Tee Loaf Mental Neuro
Develop-mental Neur
(Plenum)
New York, 197g)]. However, adult neural tissue
A significant amount of GAP-43 RNA during the perinatal period
Although significantly less abundant, GAP-43 protein in adult rats
consistent with the observation that it persists in the cortex [Jacobso et al.
Jacobson et al./Yarnal iso new
Neurosci (J, Neurosci, ), 618
43 (1986)]. Sustainment of GAP-43 expression
This suggests a facilitator role in the somatic nervous system. GAP-43
in an electrophoretically and antigenically indistinguishable phosphoprotein from
Certain B-50 and Fl properties are important in adult neural tissue.
It has been evaluated as such. These proteins are protein kinases
Serves as a substrate for ZeC-like enzymes, and its phosphorylation is
Neuropeptides, neurotransmitters, and long-term synergistic periods
[Jacobson
et al., Nyanal Iso Neuroscience (J, Ne
urosci, ) 6:1843 (1986);
Aloyo et al., Journal Iso Neurochem.
Story (J, Neurochem, ) 41:
649 (1983), Zweier
s) et al., progr, BrainRes, 56:4
05 (1982)]. Adult GA-1”43 regulation or inheritance
It is unknown whether this is also caused by changes in gene expression.
Not yet. Regarding the function of GAP43 in adult animals
One model is the Nnabusu alternation [Co
tman) La, Science (Science) 22
5:1287 (+984)] and structures at nerve terminals.
Other nervous system “forms” such as the learning that accompanies plastic growth
Plastic J change [Bail
ey) and Chen, Science (Sc.
Ience) 220:91 (1983)]
Facilitator may be mentioned. Example ■ Cloning of cDNA for human GAP-43 Test method Tissue acquisition Human brain tissue obtained at autopsy or within 10 hours after death was newly obtained.
I got it. Brain size less than 2X2X05cz obtained from specific area
The strips are soaked in isopentane (2 to 4
Snap frozen in chlorobutane) and then at -90°C.
It was stored. These specimens were in 5 in situ hybrids.
used for typhoid and immunocytochemistry. difference
Additionally, from the same region obtained freshly or from the frozen sample.
For Northam plot analysis, a small portion of the tissue of
Using. Formalin solids directly adjacent to the frozen block.
normal tissue in paraffin-embedded tissues.
studied. 1-day-old brainstem and 7-day-old cerebellum c I) NA
Library [Both libraries are American type
・Culture Collection (American Type)
e Culture Co11ection)
Isolated human 1-GAP-43 cDNA from
did. These libraries can be used as a 32P-sign identification package.
G A I) Screening using 43 cDNA probe
1 Karns et al., Sci.
ence) 236:597 (1987)]. hive
Retaisay/Yon is 4x standard citric acid-added physiological saline.
(SSC), 08×Denhardt,
10% dex[ran] sulfate, 40% fortamide, In
(Nishin Seiko DNA 20μ7.7gMh squirrel per liter (
Tris) (pH 7, 6), 1% SDS, and 106
cpm/rnQ probe for 16 hours at 42°C.
went. Before auto-graphing the filter,
2XSSC at room temperature 1X5SC at 3°C then again
Washed thoroughly in 1×8 SC at 60°C. positive black
pGem3Z [Promeca Biotic (Prom
both ega Biotec) 1 and M13mp18
Cloning and chain growth termination reaction method E Sanger
(Sanger) et al., Pro/-Tinges in Su/
Jonal Academy in Science Ninisny
(Proc. Najl, Acad, Sci, USA) 74:546
3 (1977) I. uwccc
rUniversity in Wisfunn Genete
1 group of computers (Universi)
ty of%'1sconsinGenetics C
computer Group)] Software Banoke
The DNA sequence was analyzed by /. Northam plot analysis guanidine thiocyanate method [Chabwin (Ch i
Biochemistry (BiOch) et al.
emistry) 18:5294 (1979)]
Total cellular RNA was isolated from human tissues autopsied. of
-Total RNA from each tissue 10μ for Sanfronod
? Denatured with 1.2% agarose-formaltech! −
Electrophoresis was carried out in a gel column, and then a 100% screen was run in 10XSSC.
(Genescreen) New England
New England Nuclea
r)l or Nytran
transferred to Si'l and cross-linked with ultraviolet light,
Randomly prepared [Feinberg
) and Vogelstein,
Anasotetical No. 1 Iochemistry (Anal, B
iochem, ) 132:6 (1983) 1hi 1-
GAP-43c 1. :) NA clone C1a and one
I brititized it overnight at 42°C. Finally, the fi
Wash the router in 01% SDS and 1x SSC at 60°C.
Purified. After C1a probe, cut the filter into small pieces.
and as a control, lanotoglyceraltehyde 3-phos
fy--1~-tehytrokenase (GAPDH) cDNA
A Clone [Piechaczy
K) et al., Cell 42:589 (1985)
The probe was then reprobed. In 5 itu hybridization of human brain tissue
The section specimens were fixed in 4% paraform (aldehyde).
treated with 03% Triton X-100, then 1μ
y/ treated with MQ Brotainase, acetylated,
50% Polumamide/2XSSC Medium-C Brehybrid
Chair (-) 50'C and humid Channo for 5 hours
- Medium, 3L' of 2 X I O'cpm per slide
S-labeled antisense or sense receptor probe [
Melton et al., Nuclic Aci
ds Res. 12 Near 035 (1984)]
I went to hybridization. Then, the tissue is cut
The piece specimen was initially containing 50% formamide,
Next, 50% hol-t-amide + 01% (-ridone (Tr)
10mM containing
Washed in 2X SSC with /thiotre
. 5 0 11'i/mQ R N Aase A treatment
-Heavy chain RNA was removed by processing. The sections were incubated with lxMDTT for an additional 2 hours.
Wash in XSSC, then 0.3M ammonium acetate
graded alcohol containing
hols). N T F3 2 Kotano
Using Kodak Emul Noyon, radioactivity
No. was detected. Emul/-3 coated slide
Counterstaining was performed with Matoki/Rin. Results Hi-GAP-4 3 is mouse carmononili
As described in Example 1, which is homologous to the protein binding protein,
4 Clone the cDNA related to 3 and use it as a probe.
using the human brainstem and cerebellum libraries.
A cDNA clone was identified. From each library
Overlapping clones are obtained, and these are located in the overlapping region.
It was a match. Sequence of longest clone (cerebellar library)
origin C1a) is shown in Figure 5A, and in Figure 5B
A comparison with GAP-43 is shown. God called P-57
transspecific mouse calmonyulin binding protein and GAP-
The identity with 43 was confirmed by Cimler et al.
Null in Biological Chemistry (J,
Biol, Chem, )262・12158 (1
987), the arrangement is shown in FIG. 5B.
Indicates a column. P-57 increases when calcium levels rise.
An example of releasing lumosolin rather than binding it.
Neurospecific lumonulin-binding protein with extrinsic properties
disclosed as [Andrea
sen) et al., Biochemistry (Biochemistry)
try) 22:4615 (1983)'. The protein is highly conserved among humans, rats and mice.
has been done. For example, the amino acid differences between humans and mice
Identity is 89%. Furthermore, the two
3゛-nontransformable structure that effectively contains the desired stem-loop structure
Retention between translation areas is unusually high (80%), which is similar to other
Metsenger RNA stability due to similarity to genes
UjJ-Reeves helps regulate sex
Proceedings in National Academy of Sciences
- In Science Ninisny (Proc, N
at 1. Acad, Sc 1USA) 83:3
22g (1986); Nyo (Shaw) and Ka
Kamen, Ce11 46:659
(1986)]. GAP-43 expression persists in isolated regions of the adult human brain.
To minimize subsequent RNA degradation,
Brain tissue was obtained from individuals. Histopathological examination of adjacent section specimens
was tested. In two infants, 8 days old and 7 months old,
Everywhere in the brain, as assessed by Northern Bronot
GAP-43 was expressed uniformly and in a healthy manner. tested
The areas are the cerebellum, temporal cortex, temporal association cortex, frontal cortex, and eye.
Foveofrontal cortex [Area 11], hippocampus, visual cortex
(fields 17/18), and contains some pith;
An example of this is shown in FIG. In these orders, for the brain,
The level of pith is low, which is associated with early maturation of this region
[Anand and Hike]
y), New England/Yarnal in Mede
Ian (New Engl, J, Med,) 317:
1321 (1987)]. GAP-43 is suitable for newborns and
Any non-nuclear substance tested either in humans or adults
- odor of tissues (including kidneys, lungs, liver and adrenal glands)
However, it was not expressed. In normal adults, GAP
-43 expression varied significantly between different regions. for example
, in three brains, Brodmann's area 11(l]ffl
! The frontal gyrus) can be seen at a level comparable to that of a newborn, and the visual acuity is
It is expressed at a very low level in the sensory cortex (areas 17/18).
Ta. In the hippocampus, there were consistently low levels. lff1
The latter is important because the rat hippocampus is rich in GAP-43.
is interesting. Similar distribution or non-uniformity (Neve
) Mo1ec, Brain Res, 2:177
(1987) recently reported. GAP-43 expression resumed after ischemic injury antemortem lO~
A small clinically silent infarction occurred on day 14.
clinjca]ly 5ilent jnfarci
Brain tissue from two patients with s) was examined. this
Histopathological features characterizing subacute infarctions include
, the following may be mentioned: [1] Infarcted tissue and intact tissue
A clear delineator/yon [2] Nieuron between the organization
Loss of: [3] Mononuclear inflammatory cells and phosphorus 1! macrophage
[4] Infiltration of necrotic tissue due to
Activation of fibrillar astrocytes. Some patients have visual skin
There was an infarction in the parietal lobe (area 17) in another patient.
3.1.2.5) had an infarction. Nocern the organization
Used for analysis, in 5 intu hybridization/yellow
, surrounding infarcted tissue and from the same Brodmann's area.
included both normal brains. Figure 7A is in field 17.
After a stroke, GAP-43 expression or GAP
−43 or the field which is usually the richest region] is comparable to 1.
This indicates that the level increases. Figure 7B shows two
G A P in area 3, 1.2.5 from normal brain
-, -43 level (lanes 1 and 2) or small at that location.
lower compared to another patient with a small seizure (lane 3).
It shows that These observations suggest that G A p-43 increases in number after ischemic infarction.
Suggests that it will increase within days. As mentioned above, GAP-43 has a dual role in its expression.
Neurons are restricted and neurons are present in the infarcted area
(Fig. 8 A1, B), probably GAP43
Increased expression is induced from morphologically intact neurons
It must have been. To test this hypothesis, in 5i
Infarcted area using TU hybridase/Fun
We studied the distribution of GAP43 expression in. , GAP-
The detailed cellular anatomy of 43 expression is described in Example 3. of
As expected from the analysis, visual cortex (area 17)
) throughout the entire area of the adult cerebral cortex, which contains
Over time, only a few cells expressed GAP-43.
were scattered (Fig. 8C). Honestly, the island is a necrotic area.
is non-specific with both antisense and sense probes.
in the infarcted area of the visual cortex
GAP43 expression was not observed (Fig. 8D, Fig. 8G
). On the other hand, adjacent morphologically normal field 17 (Fig. 8A
In 2), essentially all neurons or GAP4
Figure 8 E, Figure 8 F) clearly shows the expression of 3 in the infarcted tissue.
The adjacent region is the origin of increased GAP-43 expression.
I was sure that. Temporary non-infarction due to GAP-4,3 expression
The effects of ischemia were also examined. Cerebral cortex and hippocampus
Brain like Summer's 5ector
Neurons in certain regions are particularly affected by laminar flow reduction.
In case of selective damage to ischemic and hypoxic insults
1 Brierley that proves easy
) and Graham, Greenfield
Greenfield sN
europathology), 4th edition, Atamus (J
, H. Adams), Corsel I.
is, J, A, N, ), and Tadeene (1
) uchen, L, L), Eds, , Nidowa
Edward Arnold, Ro
Ndon, 1984, pp, 125-1561゜This type
The damage is temporary and will not be permanent within a few weeks.
Recovery is possible. ln 5itu/・Eve J Taise
Cardiac arrest with concomitant total anoxia using
The cerebral cortex of a total of two patients was examined. However, after a few days
Death occurred and autopsy showed a form of anoxic brain damage without infarction.
There were some marks. This is due to the large number of scattered nuclear agriculturists.
) neuron or water li@i (inflated) knee □
-If Ron exists, it will be present in the cerebral cortex, hippocampus, and cerebellum.
This was evidenced by the formation of empty capsules in the neuropil. cerebellum odor
Therefore, ischemic psittacine cells are edematous and colorless.
It was. [Epsilon] As shown in Figure B, in the cerebellum, mainly the positive tile
in 5 intu hybridization in adults
Detection [accompanied by 1J-capable GAP-43 expression]
The Purkingo subsaturation layer, which is an area where it was found that there is no
There was a significant increase in GAP43 expression (Figure 9A).
. Consideration: What is the internal reflection of growth? 112 To develop and regenerate rods
3) - 1frth) is the neural tree end structure [e.g.
Ramon y Caj
al), ]-te/, xfureshinyon and rinyefu
Shi-Noyon Ab0s1 Nervous System (Dege
generation and regeneration
of the Nervous System)
Oxford University Press
dshinn1versity PresS), Rontono (+
982), Keita (Kater7 and Letouanoi)
(1, etourneau)
Nerve Growth Cone (Bioiogy or
the nerve growth cone)
, Alan 1 R.I.R.I.S.)” (
Alan R1, iss, Inc.), New York
(1985)l. These are local information interlocking and
Contains the R machine for transformation and transformation, and transports transport from somatic cells.
It has a protein composition determined by protein transport. G.A.
P-43 is one of the rapidly transported proteins.
, which is important for axial tree transport when developing and regenerating nerves.
SJ3'l is of clear quality. play trick
Defects in the mammalian CNS 24 eurono are found in the adult brain.
associated with low and uninducible levels of GAP-143.
Skene, Cel
l) 37:697 (1984) l. In other words, mochi
Of course, the problems encountered in the treatment of CNS injuries and seizures
To study the regulation of this protein in humans,
This is particularly interesting. GAP-43 and growth cone GAP-43 are highly conserved between rats and humans.
, releases calmodulin when surrounding calcium increases
A calmodulin-binding protein with unusual properties that
It is clearly comparable to a mouse protein recently identified as
[Andreasen et al., Bio
Chemistry (Biochemistry) 22:4
615 (1983); Cimler et al.
, Journal in Biological Chemistry (
JBiol, Chem, ) 260:10784 (1
985); Alexander et al.
, Journal in Biological Chemistry (
J, Biol, Chem, ) 292:6108 (1
987)]. As suggested by the above authors,
The concept of its role in the long cone is derived from Calmodyri.
regulation of tumor activity and local cellular domains.
By doing so by emitting it
be. That is, GAP-43 for calmodulin
For example, after an action potential, calcium or
Then it will decrease [Belard
etti) et al., Processinges in National
・Academy in Science (Proc, Natl.
, Acad, 5ci) 83 Near 094 (1986)]
. Therefore, the calmodulin-dependent activity of growth cones is
It can be adjusted within the vicinity of the calcium inlet.
Wear. GAP-43 in adults GAP-43 expression is highly regulated during development. one
In general, the highest level corresponds to the period of peak axonal elongation rate.
have a mutual relationship. However, in individual regions of the mature brain
High levels of expression indicate that GAP-43 or some adult
It is suggested to have a facilitator in Uron. One possibility is that GAP-43 expression or
, indicating cells actively engaged in remodeling their structures.
It is to do so. Evidence for this is that in rats,
Adult neurons or hippocampal neurons expressing GAP-43
most prominently contains mitral cells of the olfactory bulb and olfactory bulb;
In adults, these neurons modify their terminals.
It is to create. Human hippocampus contains GA only at low levels
P-43 is expressed, and GAP-43 is actually in this structure.
Various regions of the human brain are markers of structural modification.
This suggests that it retained this function. GAP
Another function proposed for -43 is that its phosphoryl
learning as the state of change changes as a result of long-term synergy.
[Nelson and Ruttenbar]
Routtenberg, Experimental
Neurology (Exp, Neurol,) 89:2
F (1985)]. In fact, structural modifications can lead to long-term learning
There is a front page of [Chang and Greenough]
(Greenough), Brain Res, 30
9:35 (1984); Gere. Goelet et al., Nature
e) 322:419 (1983) J, in the area of nerve endings.
Growth is a long-term strategy at Aplysia.
Xi [Bailey]
Chan, Science
) 220:91 (1983)]. That is, one
The possibility of increasing interest lies in the structural aspects of long-term learning.
Neurons in the adult brain that use GAP-43
It means that it is something that is expressed. This possibility is
Nelson and Ro, Tenberg
Uttenberg)'s Experimental Neuro
Rosie (Exp, Neurol,) 89:213
(1985), Jacobson et al.
Journal in Neuroscience
sci.) 6:1843 (1986), and
Molec, Brain Res of Neve et al. 2:177 (1987),
This research is consistent with these suggestions.
Ru. This proposal suggests that various disease states and GAP-43 expression may
will require close interaction between the two. GAP-43 and repair CNS damage in the adult CNS
Suppression due to recovery from is poorly understood. Mammal
The peripheral nerves of mammals or even the central nerves of amphibians are incised.
After rupture, complete recovery or, on the contrary, mammalian
Such damage to the central nervous system does not recover. central god
As long as the meridians have peripheral nerve sheaths as kites,
After axotomy, adult neurons extend over long distances.
, maintain the ability to grow - Vru [Henfei (Ber +
fey) and Aguayo 3 (Aguayo), Nature
-(Nature) 296:150 (1982) I
o Failure of repair affects the central nervous system as opposed to the peripheral nervous system.
In the group of axonal transport proteins, ('I)
This was due to regulatory inhibition. Especially regarding GAP-43.
It's about that level or normal growth and resilience.
This is because it is very comparable to [Sken
e), Cell 37:697 (1984)]
. We found that after certain types of stimulation, maturing new
Ron expresses high levels of GAP-43.
showed that it can be done. Diseases such as temporary ischemic and cerebral infarctions may require axotomy.
Disability or permanent, as opposed to clinical
Recovery is of interest because it often occurs after such lesions.
It's deep. Neurological recovery or injury 1111 Capsule II
modification (induced by U or nearby intact I'l
Whether K7+ is induced in the development of L cells depends on the clinicopathological
It cannot be identified by the +HLI relationship. High△
Cell death area such as neurons expressing GAP-43
The distance between the two suggests that germination may be tinted. other
On the other hand, the increase in GAP-43 observed in these cases is due to
derived from ischemic effects in the trunk, which are associated with axotomy.
There doesn't seem to be much in between. For example, these are
release of amino acids and the resulting ＼□-methyl
D-aspartate ifK (NMDA) receptor excitation
[Olne's experimental
And Clinical Neuro I Kinokoro/-(Ex
peripheral and C11nical N
eurotoxieology), Swan! -(P,
S, 5pence) and Nyaunnog (H
, H. Schaumberg), eds, (Harte
Baltimore, MD Williams
(!illiams) and Wilkins (Wi 1k
ins), pp. 272-294 (1980);
Rothman, glossy off-knee
Neuroscience (J, Neurosci, ) 4:1
884 (1984) I. Song - Example 1 - Directed to purified GAP-43 protein
Immunohistochemistry of detection light of GAP-43R using antibody
Research has shown that GA, especially in the growing nervous system,
This suggests that P-43 is restricted to neurites.
To 1/T 2 ノ (Beno★1tz) U, E/Ya
Naru Off゛・2-Euro → Niens (1゜Npu
rosci, ) 8:339-352 (1988):
Gispen et al., Brain Res,
328:38]-385 (1985): Mates (V
Leiri) et al., Brontinges Inn.
Le Academy Off Science Ninisny (P
roc, Natl, Acad, Sci, USA) 83
:3537-3541 (1986) :Suiichiji (S
Kene et al., Science 233
A83-786 (1986)]. In this example,
In 5 itu hybridization and nerve cells
Perikaryal staining (perikaryal staining)
) using a novel antibody that has l=1 for GAP43,
Each of the present invention provides a cell individual expressing G A P-43.
We identified 8T in the adult brain and found that it is widely distributed in the adult brain. -'i passed
GAP-43 immunoreactive axons? Nealon comparison
emanated from a small population, and this mostly occurs locally.
Be less restricted. Method in 5 itu hybridase/3 cells and immune cells
New tissue used for chemistry is obtained and tri-ice is used.
Snap frozen in 2-methylbutane cooled by
did. Immediately before use, prepare cryostat sections using an appropriate fixative.
Fixed the book. Embryo Unot□ (E) (12, 15, 18
and 20 days old), postnatally developing rats (P) (1
, 7 and 14 days old) and adult rat brains.
were studied at the same time. The tissue was fixed in 4% paraformaltehy;
．． Treated with 3% Triton XIO○, then 1μ9/v=
Treatment with Q proteinase, acetylation, and 50% protein
Rumami] / 2X standard citrated saline (SSC)
) was brehybritized inside. The probe was cAp-43 anti as indicated.
I found the 1121st base of sense RNA. 50°C
5 hours, 100000000000000000000000000000000000000000000000000000H
2× ] 006 cm ″S-labeled antisense or
Hybridize with or sense lyphoprob.
I did a session. Then, apply the book to the tissue section, initially 50%
Contains formamide, then 50% formamide
110 containing Mido+01% Triton-X100
Wash in 2X SSC with l11 antithiothreitol
did. 50μ9/x (can be treated with IRNAaseA)
-Heavy chain RNA was taken out. The section specimen
, in 2X SSC with 1mM DTT for an additional 2 hours.
Washed and then containing 0.3M ammonium acetate
to graded alcohols
So I got dehydrated. NTB2 Kodak Emulsion
was used to detect the radioactive signal. Emulsion application
Counterstained the slides with hematoxylin and eosin.
It was colored. used for in 5 itu hybridization
Use adjacent tissue sections for immunohistochemistry.
there was. Chimeric GAP43-β- generated in λgtll
Rabbit odor injected with Calacto/Tase fusion protein
The polyclonal antiserum was raised to 1 Kearns (K
arns et al., Science 236
:597-600 (1987) :Young
g) et al., Prosortinges op National Aka.
Temmy in Science Ninisny (ProcN
atl, Acad, Sci, LISA) 80:119
4-1198 (1983)]. Ammonium sulfate fraction
method and DEAE cellulose chromatography.
The crude serum was treated with [Holowi
tz) et al., Functional Techniques in High
Lolosi (Fundamental Technique
s in Virology), pp, 297-31
Using growth cone membrane particles prepared from 51° neonatal rat brain.
[Menninger et al., Cell (
Cell) 35:578584 (1983)], U
Western Proto9 analysis method [Meiri et al.
Bronty/Guess in Su/Yonal Academy
In Science Ninisny (Proc, Natl
, Acad, Sci, tiSA) 83:3537-3
541 (1986)]. cormorant
Eastern Pro, Alkaline Phosphatase/BC
IP/NBT kit “Promega
] was used to expand it. Western Pro, To test method
The specificity of the antibodies that bind to
Pre-absorption with purified GAP-43 protein
[Chan et al.
・Neuroscience (J, Neurosci,) 6
:36183627 (1986)]. GAP-43
The protein is 3 as chromagen.
−3°diaminobenzidine and hematopoietic as counterstain
Using Kinlin, Avidin-Biotin Horseradish Pel
Okintase combined method [Hsu et al., Journal
In Histochemistry and Cytochemistry
- (J, Histochem, Cytochem,)
29:577-580 (1981)]
demonstrated in CNS tissue by histochemical staining. mark
The peculiarity of recognition is that neonates. Together with purified natural GAP-43 protein derived from human brain
Confirmed by incubating the first antibody.
It was. In 12- and 15-day-old embryos, GAP-
43mRNA expression is low, or neurons in explanted root ganglia
, exhibiting extreme labeling depending on the period of peak axonal growth.
did. At E20, GAP43 levels increase throughout the brain.
were uniformly and significantly higher. First week of life after birth
High levels of expression were maintained during E20 and P
In contrast to the diffuse labeling observed with l, P7 lanot
In the brain, separate labeling on individual neurons is
, by the spread of the neuropil and the growth of neuroglial elements, positive
I was able to make a good evaluation. best tested
All neurons were labeled at this age so that
. In Pl4, GAP 43 mRNA expression is
Na/Gnar strength is low, 50-75 of cortical neurons
Only % was labeled. In the adult CNS, GAP-43 is
Throughout the cortex, only diffuse cells are labeled.
expressed in a relatively small number of neurons,
The intensity of labeling was significantly reduced compared to Pl4 brains. death
The entorhinal cortex (entorhinal cortex)
x) moderately high density of GAP-43-expressing neurons
I have no idea. Dense focal labeling of neurons appears at P14
It is also present in the spinal cord, brainstem, and cerebellum of rats.
However, in adults, GAP~43-expressing neurons
presence or absence or significant presence in these areas.
They were either present at low densities. However,
In the body, extreme labeling of neurons in pigeons
It was kept in two areas: horses and fours. in the hippocampus
The pattern of labeling is consistent throughout the interdentate, CAI and CA3 areas.
Throughout the body, neurons release GAP-43 mRNA.
It was shown that it appeared. Mainly in terms of walks, high level
Mitral cell region containing GAP-43 mRNA
It was. Observation of G A P-43 expression by immunohistochemistry λg
tl] Chimera G A P -43-β-kara born inside
X = j to the protein
Riku

[Coronal antibodies cause neonatal cancer/)・Westar of the brain
GAP)-43 was specifically labeled in the sample plot.
. Special offers on GAP-,-,-43 antigens
Epidemic staining is not detected in neuroglial cells, but in neuronal cells.
-Ron odor-C was detected. immunity throughout development
Reactive GAP-43 is associated with neuronal cell traits and neurite
existed in both. Neurite-restricted immunoreactive GAP-4
3 is the previously observed “Beno
Witz et al., Journal Ben Neuroscience
(J, Neurosci,) 8:339-352 (1
98g) Gispen et al., Brain R
es, 328:381-385 (1985):
Skene et al., Science
) 233 Near 83-786 (1986)]. Cell fractionation
Research suggests that GAP43 is also present in the cell body.
[Alexander et al.
Nar Ben Bioloncal Chemistry (J, Bi
ol, Chem, ) 262:61.08-6113
(1987)i. The immunostaining method using our antibody detects GAP-43-expression.
Cell localization and in 5 situ hybridization
This made it possible to compare with Yong's take. Immunoreactivity c A
Local distribution of di-ron containing P-43 and
The growth density was determined by in 5 intu hybridization/yellow.
The t-developmental pattern observed for its mRNA
Reflected. G A P-43 immunolabeling was carried out in E12 embryos.
Not detected in buds, slightly ↑take in E】5CNS
present (as evidenced by faint immunohistochemical staining)
Ta). E18-r shows even more GAP-43 immunoreactivity.
clearly visible, and at E20, the trunk of the nerve cell and the nerve
High on both U's of the protrusion! Detected in novel. G.A.
This degree of immunostaining for p43 protein
Well it was sustained. The results and 1. , GAP-43 immunity
The reactivity decreases in the region of Haton, thus
, a more restricted portion of G A P-43 immunoreactivity
The cloth remains the same. Is it widespread in adults?
Neural labeling
ng) was demonstrated throughout the CNS. -knee
Ron neuronal cells express high levels of GAP-43
As a result, in 5 itu hybridization / 3
- In the identified area, there were six dense markers. For example, the hippocampus includes CAI, OA4, and the entire interdentate region.
Strong in both pyramidal and granule cells across
Labeled or indicated. The antibody is isolated as described herein.
Pre-absorbed together with the separated Kell purified GAP-43.
No labeling occurred when β-Calactositase
Preabsorption had no effect on labeling. cerebellum, brain
In the stem and pulp, there are few immunolabeled cells.
frontal cortex, somatosensory cortex, visual cortex and brainstem nerves
Immunolabeled cells in the nodes are of low density, and the medial olfactory cortex
In terms of quality, the density was moderately high. 1nsitu
As I noted about Iburita Seyeon, immune response
Strong rod cytoplasmic staining for responsive GAP-43
Two areas are most prominently maintained in the hippocampus and quadrilateral.
Ta. CAL, CA3 and petrous slenderness throughout the interdentate
Both cysts and granule cells were labeled. in four balls
, labeling was performed for mitral cells and for granule cell sacs.
It is largely restricted to the inner neurites. i.e.
, the pattern of GAP-43 immunoreactivity was 1 n situ
Reflected that of Hybridize/Yon. Discussion This example is based on GAP-43 or all CNS news during the perinatal period.
It has been shown that this protein is expressed in lon. development progresses
As the population increases, its anatomical distribution becomes progressively more restricted.
In adults, GAP-43-containing neurons are disproportionately distributed.
distribution, and the highest level of expression is mainly found in two distinct
It is restricted to the hippocampus and quadrants. EMBOJ by Rosenthal et al.
6:36413646 (1987).
The publication discloses heterogeneous GAP-43 labeling.
, somewhat different from that reported here, is that the cerebellum
and their finding at high levels in the frontal cortex and
There was a lack of labeling in the dentate gyrus. In the previous report
Immunoreactive GAP-43 is an indication of its cellular origin.
was detected exclusively in neurites [henovi
Benowitz et al./Yarnal Off New
Neuroscience (J, Neurosci, ) 83
39-352 (1988); Gispe
n) et al., BrainRes, 328:381-38
5 (1985); Skene et al.
Science 233 Near 83-786
(1936) Snake. β-galactonter generated by the present inventors
Antibodies against Zee GAP-43 fusion protein
RON enabled extreme labeling of neuronal cell traits. This servant
The difference with previous reports is due to the chimeric nature of the antigen.
and probably several different to different degrees
Expose the epitope. On the other hand, the difference is that the aldehyde
This reflects the omission of neuronal trait labeling.
demonstrated by the inventors to reduce the In any case, we believe that the method of the invention
The site of AP-43 gene expression is associated with GAP43 immunoreactivity.
It showed that it reflected something. G in CNS
The distribution of AP-43 differs between rats and humans. adult
In the brain, it is higher in association cortical areas than in the hippocampus.
maintain the level [Neve et al.
NGES OFF S/YONAR ACATEMY OFF SAIE
Proc, Natl, Aca
d, Sci, USA) 85:3638-3642
(1988)], on the contrary, adult runts
Highest levels of G in horses, quadribulbar and entorhinal cortex
There is AP-43 expression. The significance of this finding is unclear.
However, regarding the regional retention of neuron formation, species
This may be related to the difference in High levels in adults
CNS neurons stably expressing Bell's GAP-43
subse'/l- share biological characteristics or not.
It remains to be decided. One possibility is that GAP-43 plays a role in structural remodeling of synapses.
By being expressed in the cells included when performing
be. Growth cones are maintained in certain areas of the adult brain.
[Sotelo et al., Labnvest 2
5:653-671 (1971) 1, by direct observation.
, the progression of single cells, at least in the peripheral nervous system.
[Burgess (Pur
ves) et al., Fuichi (--(Nature) 315:
404-406 (1985)]. In fact, such
Uron modification or long-term learning [Chan et al., Br.
ain Res, 309:35-46 (1984
) ; Goelet et al., Nature (l
322:419-422 (1986)
; Horn et al., Journal of New
Neurosci (J, Neurosci, ) 5:31
61-3168 (1985)] and sexually dimorphic behavior.
(sexually dimorphic behav
ior) [Kurz et al., Sci.
ence) 232:395-398 (+986)]
There is evidence that it is essential for Growth cones and synapiosomes
Protein supplementation in es) is qualitatively less
``Ellis et al.
・Off” 2:LD Science (J, Neuro
sci, ) 5:13931401 (1985);
Katz et al., Journal of Neurosa.
Jens (J, Neurosci,) 5:l402-
1411 (+985): Sonde power
regger et al., Science 2
2]:1294-1297 (1983)], Growth Cone
synapses-to-b
Add a marker e) ``Hume et al.
Icha (Nature) 305:632-634 (
1983): Sun et al., Proceedinges
・Off Science/Yonal Academy Off Science
Ninisny (ProcNatl, Acad, Sci,
USA) 84:2540-2544 (1987)l
. Mature neurons transfer molecular components to these steps.
We can partially modulate these structures by changing the
"Grafstei et al., Ph.
ysiol, Rev, 6Ω:1167-1283
(1980) ;McQuarri
e) et al., Journal in Neuroscience (J,
Neurosci, ) 6:1593-161)5 (
1986) 1. , such changes locally cause U race
Lasek et al., C (qll Motility,
RD Goldman, T Pollard J
Rosenbaum, Eds, Col. Spr.
ing Harbor Conference
Ce1l Proliferation 5eries
: Vol 3, Cold Spring Barber
New York, p. 1.021-1049 (1976
)l, at this level of gene expression, e.g.
[Miller et al.
Nyanar in Cell Hioro/(J, Ce11.
Biol, ) 105:3065-3073 (198
7) 1 and GAP-43 by the present inventors.
brought about by the gene expression of
It will be done. Characteristics of adult CNS neurons that demonstrate plasticity
What is unknown? 1. The onset of nubs in the precocious nervous system.
Is there a similarity between neurite growth between growth 1 and replanted trunk? cormorant
At the molecular level, is there a need for growth-related proteins?
It's not unreasonable. 4 to ensure the binding of GA, P-43 to formation properties.
- Is there a marker that is highly dependent on this in adults?
Some neurons express levels of GAP-43.
There is some evidence that Nnabusu can be revised. vinegar
That is, GAP-43 expression is associated with the olfactory nerve and its targets;
High in mitral cells of the four bulbs. Olfactory neurons last a lifetime
Continuing the cycle of death and exchange with courage, U Gracia
Graziadei 3 systems, Be
rlin, Springer-Verlag pp
, 55-83 (1978)], these naps are
Must continue to change. GAP-4 in adults
3-expressing entorhinal neurons regenerate in the absence of injury.
It is not clear to design or sprout in the nerve transection area
By doing so, you can expand the peripheral area. most
Later, it was discovered that the hippocampal circuit is functionally plastic and has a unique structure.
Benow it z et al., 7 Yana, Shi, I
hmm. Neuroscience (J, Neurosci,) 8:
339352 (1988); Cotma
n) et al. Psychol 33:3
71-401 (1982); Lee et al.
11. T. ns, Akatemi, Ri, New 3-Ri,
1981.1'ip, 189-2+2;Le
e) et al./Yarnal in Neurophy/'Oro/-
(J, Neurophysiol,) 247-258
(1980):i, long-term synergy by morphological analysis
Check for changes in the number and shape of nubs that are accompanied by an effect.
[Chang et al., Brain Res,
309:3b-t6(+9g4)iozu, adult
Restricted localization of GA P-43 in the CNS
GAP-43 expressing neurons or redesigned nerve endings
The concept of being actively involved in
Applicable. Example 1 In many cases, the expression of individual knees and feet (is its subtlety).
・J＼Depends on the environment. Such 1゛-formability-: is
, e.g., by IIR progenitors of the sympathoadrenal system.
This is proven in the selection of cell fate.
This is the neuronal surface under the influence of neurodevelopmental factor (NGF).
Endocrine chromium in the present or in the presence of corticosteroids
Hypothesized to be an affinitive cell phenotype. Furthermore, neurons
During normal development and for NNAPS use, their
Redesigning the phenomenon called napus formation [E.
EaSter et al., Science
e) 230:207-511 (1985)]. One unifying theme regarding these two types of formability is
, is dramatic in establishing the original brilliant neuron shape.
, a structural reorganization that is more subtle in the rearrangement of relationships.
It is complete. One concept is the expression of a set of genes or
It means being responsible for euro/formativeness.
. Regulation of these genes by the microenvironment is due to structural changes.
will bring about. flutifosteroids) are responsible for the normal development of the mammalian nervous system.
necessary for cell growth, neuronal structure and
1 Taube (1) oupe et al.
Neuroscience (JNeurosci)
) 5:2119-2142 (1985); Dow
Doupe, Journal in Neuroscience
Neurosci (J. Neurosci,) 5:2143-2160 (1
985): Anderson and
Axes, Cell 47:10
79-1090 (1986); Bohn
and Lauder, Dev, Neuros.
ci, l:250-266 (197g);
Scheff et al., Expt. Neurology
68: ] 95-201 (1980); Chef (Sc
heff) and Footman (Cotman), Exp
t, Neurology 76:644-654 (1
9g2) Sapolsk (Sapolsk Nora, Jana)
Le in Neuroscience
) 5:1222-1227 (1985)]. Cultivating
, small extremely fluorescent (SIF) cells and adrenal medulla
Neural crest lineage cells containing cells are
, either neuronal or chromaffin phenotype.
It can also be presented. Corticosteroids reduce their chromaffin properties.
It carries a sign. In the absence of corticosteroids, NGF
Depending on their presence, they may develop neuronal properties.
[Doupe et al.
Neurosci (J, Neurosci, ) 5:2
119-2142 (1985)]. Clos derived from rat adrenal medullary chromaffin cell tumor
Cells of strain PCI2 [Green and
Tidjler (7ischler), Prosortinge
Su in Su/Yonal Academy in Science
・Nii Ni Ni (Proc, Nat') Acad
, 5ciUSA) 13:2424-2428 (1
986); Greene and Tinni
Tischler, Advances 1nC
ellular neurobiology, Vol.
J, pp. 373-413, Academic Press,
New York (19g2)] was
becomes neuroneuronal, and upon exposure to corticosteroids
A similar bipot that maintains chromophilic properties
actual fate). Surprisingly, this study of GAP-43 expression showed that PC1
2 cells and cultured sympathetic neurons.
Therefore, the usefulness of corticosteroids or GAP43 gene expression
It was found to be a negative regulator. In addition, cortyphoid fever
The stimulatory effect of NGF on GAP43 expression in telocytes
found to be suppressed. Experimental Methods Sand enzymes were prepared by Boehringer Mann
gerMannheim), New England
New England Biolabs
), or Hesesda Research Laboratories (Be
thesda Re5earchLaboratori
es) and as specified by the supplier.
used. Tissue culture products were from Gibco.
I bought it. Radiochemicals
New England N
Purchased from Uclear-Du Pont). Agar and cesium chloride were obtained from Hecesta Research Lab.
Purchased from Latries. Spusi takes the opportunity to become pregnant.
-Kew Dawley rats are Charles River Rats.
Purchased from Charles River Rat
did. Steroids are NG from /Sigma.
F is purchased from Collab. raive Research (2, 5s type).
I entered. All other chemicals were of the highest grade available.
It was. Cell culture with 5% heat-inactivated horse serum and 10% fetal bovine serum
PC1 in a bottle of Henoko Modified Eagle Medium (DMEM)
2 cells were grown. Cells are routed when the degree of assembly is approximately 20%.
I used it exactly as I expected. Cortisol measured by RIA
Levels are 3 nM or less in serum-containing media.
It was. Humidified with 5% carbon dioxide at 37°C.
Cells were grown in an incubator. 20 day old embryo
Neurons isolated from the rat superior cervical ganglion were treated with NGF (
50% g/ff, 06% glufus and 10% g/ff
Ham's F12 medium supplemented with fetal serum
Cultivated underground. For steroid testing, usually
The compound was dissolved in 95% ethanol. As a vehicle
Pair II6 performed with tanol shows that abundant GAP-4
3 or GAPDH RNA. RNA blotting The cultured cells were analyzed using the isothionate guanine/isothionate method.
Total RNA was prepared from the cells [Ch.
Biochemistry (Bi. Chemistry) 18:5294-5299 (
1979) 'j0 20 μ7 of total RNA from each culture was
12% Aca containing 2.2M formaldehyde
Electrophoresis was carried out through a low gel.Nytran (Ny
tran) [/Jureicher a7 l' ・:l/
:s-le(Schleicher andSctu+e
l l)] by capillary, and heat the nucleic acid.
Fixed by solidification. 50% formamide, 5xS
SC (lxssc: 150mM sodium chloride, 1
5zM 1-lium citrate, pl(70),]×ten
Hart's solution, 1% Doten
sodium sulfate (SDS) and 100μ9/gQ
A hybrid that contains denatured Zumon semen DNA.
For at least 1 hour at 42°C in Year-No-3 solution.
, prehybridization was performed. D N A Polymesse I to Frenou Fragmen 1
Use it to get to your destination!・Blymink (ko)
3 above - Preparation l (Also l x ] 0 'cpm/m
Contains (!32P-labeled L)NA probe.
Hybridize in the same solution at 42°C for 10-12 hours.
[Feinberg
) and Vogelstein,
Anal, Biochem, 132:6-12(1
984)]. For GAP-43 as described here
or cI)NA cloned against GAPDH
The probe was made from “Pie
chaczyk) et al., Nuel, Ac1ds Res,
12:6951-6963 (1984)l. Professional
The kit was incubated at 65°C with 2X SSC twice for 20 minutes each.
Wash at 65°C for an additional 20 minutes in 02xSSC.
Purified. -70'C, strengthen the screen, and
Traradiography was performed. 1x Ten Hearts solution, 1
%SDS. 50mMh squirrel, pl, + 7, 4 and 0.05
In a solution containing % I. Lium birophosphate, 80
Plot hybridized probes for 2 hours at °C.
Stripped from. L, KB Scanning level with Ultrascan
- Dimensions - Measurement was performed for tenn. given professional,
1. Scan several positions of 5, and measure the image intensity over time.
plotted against. Measurement of image intensity is based on the linearity of the curve
Got it from the part. GAPDHRNA level notes @deep level
Depending on the measurements, some of these experimental conditions may vary.
I wonder if there isn't. Ta. Nuclear additional inspection (Nuclear Run-0n As5a
y) 48-60 hours before each experiment, I) C12 cultures were
Suburi, Toshita. Cells may be left untreated or N
GF (50ny/z□ or dexamethacin (1 μM)
Approximately 10 million nuclei were treated for 6 hours with either
, Green-Hague's method [Green-Hague method] with the following modifications:
Greenberg et al., J. RiolC.
hem, 260:1410144110 (1985
)] were prepared from each. Cell lysis is
], OzM thorium chloride, 101M1-lith, pH 7
, 4,3mM chloride and 200 units l-
/rtrQ RN As1n [Promecha + Biotsuk (
Promega Biotec) i was discontinued. 501
1Ml-Lis, p++ 8.3.40% glycerol,
5iM Magne Chloride/Rum, 0.1zM Ethylene/Ami
Notetracholic acid (EDTA), 2xM/CH1-Rei-,
r and 200x-=-, lzlmQ RNA5i
After washing with medium cell lysis buffer, the nuclei were resuspended. Nuclei were counted and placed in liquid nitrogen at a concentration of 50 million/ic.
It was stored in The suspended nuclei were treated with an equal volume of 10 x M l-Lis, pH 8.
0.5mM Magnesium Chloride, 03M Nonolium Chloride
and respectively], OxM's adenonone, chintin and guar.
Added to a buffer containing nonnucleotide triphosphate.
Labeling of nascent chains in the thawed nucleus by adding
. Next, urinone triphosphate (32P) 30
0.11Ci is added (3000C1/mm') mole),
Nuclei were labeled for 30 minutes at 30°C. Then 3'7
°C for 45 min, 60 di Tris, p) - (7,5
, 15zM Chloride I・Lium, IOtM Chloride Magunou
and 200−1=)l−/rrtρRNA5i
Mild containing n? 100 units in Ii liquid 600μθ
No1-RQ I DNAase: Promega Bioti
The nuclei were digested by adding ``Chicken''. Then, it was disclosed
As it is [Greenberg et al.
, J. Biol. Chem., 260:14101-
14110 (1985) I, Ten Votes Identification RN A Wofu
Roteinase K [Herinmeno Mannheim (Bo
Ehringer Mannheim)]. Several times 9 phenol, chloroform-isoamyl alcohol
After extraction, the nucleic acids were extracted with ethanol in the presence of sodium acetate.
precipitated. The recovered labeled nucleic acid also contains DNA.
RQI D at 37°C for 45 minutes.
NAase (50xM Tris, 1) H7,5, IC1m
M sodium chloride, 7.5iM magnesium chloride and
Buffer solution containing 200 units of RNA 5 in/shu
50 units of enzyme at 250 μC) then at 42 °C.
Brotainase K (5% SDS, 0.5M) for 30 minutes.
Squirrel, pH 7°4.1251M EDTA and 0,
Buffer containing 2 mg/RQ proteinase K
(by adding 100μa of liquid)
Ta. several times phenol, chloroform-isoamyl alcohol
After call extraction, the labeled RNA was dissolved in ammonium acetate.
Perform ethanol precipitation three times to remove unintegrated nuclei.
Reotide triphosphate was removed. cDNA cloned for Chiro/Nytroquincea
A [Lewis et al., J. Biol, Chem.
, 285:14632-14637 (1983)]
, ] Glyceraldehyde triphosphate dehydrokenase G
APDH), pBR322 and GAP-43 genes.
A plasmid containing an 8 kilobase genome fragment was
Linearize using appropriate restriction enzymes, phenol extraction,
Ethanol precipitate and collect the waste by centrifugation.
. DNA (250μii/rc) was diluted with alkali (05N
10 volumes of 1M vinegar
Neutralization was achieved by the addition of ammonium acid. nitrocellulo
The base filter circle removes DNA5 through gravity filtration.
A load of 0 μ9 was applied. 25IMPIPEs Sodium, p
H7, 2, 50% formamide, 0.75M sodium chloride
um, 2,5FMEDTA and 100 ttg/m (
tRNA at 45°C in buffer containing 100 ml of tRNA.
Prehybridize the filter for ~12 hours.
It was. Specific activity standards ranging from 1 to 3 x 106 cpm/mρ
in the same buffer containing labeled RNA at 45°C.
Hybridization was performed for days. has not been disclosed
[Greenberg, J.
Biol, Chem, 260°14101-1411
0 (1985)], washing and RNAse treatment.
I did it. Scintillation filter is used twice.
Counted after drying in a counter. The data is
Hackground from a vector containing a filter
After subtracting the number of copies sold per million
Expressed as Results Dual regulation of GAP-43 expression Effects of NGF and carbohydrates on GAP-43 mRNA accumulation
The effect of luticoid was determined by RNA plotting method.
. Dexamethacin significantly lowers GAP-43 mRNA levels.
However, as a result of NGF addition, the basal level
significantly increased compared to Le. Measured by tensitometry
GAP-43RNA determined and corrected for RNA load.
As a result of quantification, dexamethacin reduces the amount by 5.5 times.
However, it is clear that NGF causes a 35-fold increase.
It was. Unlike c-fos, GA in the presence of NGF
Accumulation of P-43 mRNA is stable and lasts for several hours.
reached a peak within
It declines rapidly. Steroid effects on accumulated GAP-43 mRNA levels
In order to test the specificity of the effect of the
A variety of steroids were tested over a range of concentrations.
I tried it. Each class of steroids differs in vivo.
Does it selectively affect different types of neurons?
[McWen et al., Physi
Logical Rev, 66:1121-11
88 (1986)]. In one study, a 7# change in steroids was tested for 48 hours.
during the treatment. by tensitometry
to quantify small variations in RNA input.
It was normalized. estranol, testosterone,
and fregunolone, which accumulates GAP-43mR.
It had no effect on NA levels. Dexamethacin, co
Lucicosterone, aldosterone and progesterone
The levels of GAP-43 RNA were compared with the control (GA
NGF-stimulation level of P-43RNA was 100%↓(,
C]%, 15%, 10% and 15%
decreased to Progesterone effect ψ is itself a book reviewer.
These may be brought about by the scepter
Take mineralocorticoids or glucocorticoids
This suggests activation of one of the receptors [Ali
f (Arriza) et al., Science
237:268-275 (1987); Kikele (
Gigucre et al. Cell 46:645
-652 (19861゜Corticosteroids are SI
Neuronal phenotype in both F and adrenal myeloid cells
[Doupe et al., /
YEARNAL OF 2, - 口＠GOYENS (J, Neu
rosci, ) 5:21! ! l-2142 (198
5); Taube (1) oupe) et al., Nyana/lz-
Off New 0 Masu Jens (1, Neurosci,)
5:2143-2160 (1985)1. . G.A.
Interaction between two drugs on P-43 expression
In order to check the quality, NGF (5on?・ic) and digital
Xamethason (1tt lt4) in the presence of chili r, PC]
21 [B cells were soaked in oil for 3 hours.
GAP- brought about by Sametason or Ncr”
43 mRNA. The effects of NGF and steroids are directN
Direct effects of GF and corticosteroids
The question is whether it will be affected directly or indirectly.
, by using the protein synthesis inhibitor Ncrobeki/Mi1-.
Processed. Cyclohequinmide concentration of 05Hg/mQ
24 hours without any effect on thin tla growth ability.
More than 94% of the (3H) loin uptake into the
suppress. Ncrohekinomito is derived from the GAP-43 gene.
Both the current NGF enhancement and dexamethasone suppression
without interfering with the effects of any de novo protein
Show that no synthesis is required. NGF treated cells and
and NGF and 05Hg/shuttle or 2μylmσ
Cells treated with clohekinite were compared with control cells.
. 05μyl vt (2/of treatment with clohequinmide)
Afterwards, the larger size of the transcript is shown, which indicates that the splatter
It may represent a precursor that does not have a chemical reaction. GAP-
43 expression is inhibited by dexamethasone.
(Rather than using zu, cycloheki/mil and dexamethasone
It is written by I-Imonotea-). 0,'] More powerful protein synthesis inhibition at a degree of vM
These experiments were carried out for 12 hours using the drug anisomycin.
Exposure time of 6 hours for cell death to occur - C cycles
I returned it. Ryo Nisomainno is brought to you by NGF.
did not affect the increase in GAP-43 mRNA.
. Dexamethasone inhibition is slower than NGF induction;
It is not recognized until 6 hours and therefore the effect is
It was not possible to test for insulin susceptibility. N
Pre-treatment with GF directly prevents steroid suppression
I didn't. In another study, sufficient inhibition of GAP-43 mRNA
The time required to achieve the steady state level is
, NGF-) When steroid 1- is added after pretreatment,
It turns out that the layers are getting longer. Steroid suppression involves both transcriptional NGF and steroid effects! −j key
To assess the level of nodes, nuclear addition tests (nucleus
run-on experiments)
Ta. Nuclei were treated with 50% g/mONGF or 1HM textile
PCI211 treated with Tashin for 6 hours (tlllK?
Prepared from Labeled RN from each group, A, was fixed
2) Cellulose foam containing stabilized DNA, s
I hybridized the filter. Hybridize/-
After 3 cycles and washing, the specific radiation for each filter
Calculate the activity and rate the take in units of copies per million.
Table as the number quizzed (7).NGF is the basic
There was no effect on the transfer rate of the text.
Tacin reduces the transcription rate of G A P-43 by approximately 45 times.
did. In comparison, the 1,000-ton hydroquine transcription is
GAPDH transcription is altered by dexamethasone.
It didn't change to 5. G A I)-43 A set of fruits that define a transcription unit.
In a study, runof (runof) prepared from neonatal brain nuclei
f)-labeled RNAΔ, the initial part of the first in)-ron
through, a series of neighbors spanning the 5' flanking region.
Hybridized with Φ chain M 13 clones. Result is,
Transcription or coding chain
It was shown that it was not from the component. NGF-treated PCI2 cells are highly differentiated neurons.
Although considered a model, the effects of fluticosteroids
In fact, after achieving a sufficient state of differentiation, the next neuron
Therefore, it was desirable to determine whether or not it would be demonstrated. yes
In order to
1 μM dexamethacin was added to the culture for 48 hours.
Ta. Total RNA was prepared, fractionated, and plotted as described above.
and probed. Dexamethacin is a sympathetic nerve neuron
decreased the expression of GAP-43 RNA in
. Morphology of neurons in dexamethacin-treated cultures
The appearance was not different from that of untreated cells. this is,
Short-term neurite lengthening or maintenance or GAP
43 It seems that GAP does not depend on the persistence of mRNA.
-43 gene product has a long half-life, resulting in long-term efficacy.
This suggests that there is a need to evaluate the results. Discussion In this experiment, GAP-43 gene expression was influenced by NGF.
for both positive and glucocorticoid-negative controls.
Show that you are in control. Both effects are direct;
Neither requires new protein synthesis. interesting and
Surprisingly, cyclohequinmide has GAP-43m
Further increasing dexamethacin inhibition of RNA
Do you get it. Although not intended to be bound by any particular theory, this
is also due to suppression of synthesis on mRNA stabilizing proteins.
That's probably why. In vivo, the GAP-43 gene is highly regulated.
It will be done. To date, it has only been reported in neurons.
It has been. However, from the nerve ring, the present inventors
Supports low level expression in other cells induced
I got the data. Peak levels in animals are neurites
achieved during the growth of a child and related to normal growth or regeneration
There is. Molecular regulation of its cell-specific and growth-related expression
The joints have also not been revealed. Neurodevelopmental factors are
c-fosSNGF I associated with intermediate filaments
A, NGFIB, hater actin and cloning
Directly increases the expression of some genes such as cDNA
[Greenberg et al., JB
iol, Chem, 260:14101-1411
0 (1985); Mill Plant (Milbra)
ndt), Science 238 Near
97799 (1987); Mill Plant (Milb
randt), Neuron l:18
3-188 (19813) ;Leonard
ard) et al., Journal in Cell Biology (
J, Ce11. Biol, ) 106:181-193
(1988)]. This data is based on GA, 1”43% node and these other genes.
This shows that there are some notable differences between the
are doing. c-fos and NGFIA and NGFI
For those like B, NGF induction is rapid;
It takes effect within minutes and fades after a few hours. this is
, in contrast to the NGF effect on GAP-43 expression,
The start is slow and steady. In addition, a wide range of stimuli can be used to stimulate cal/lamb entry, other formations.
Long-term factor and increase in serum withdrawal symptoms and polycythemia in cattle
These effects can be applied to various cell types.
[Greenberg
) and Schiff (Zjff), Nature
311:433-438 (1984)]. c f
Genes like os are the very earliest remains of DNA viruses.
It has been likened to a gene [Goelet et al.
, Fuichi +- (Nature) 322:419-42
2 (1986)], some of which are themselves
Transcriptional regulators that reprogram cellular machinery to specialized functions
Code. The delayed response of GAP-43 to NGF is c fo
s, NGF IASNGF IB, etc.
This applies to the NGF-regulated genes of the
This appears to play a role in long-term adaptation rather than
It suggests that. Unlike these other genes, N
GF causes a large increase in GAP-43 mRNA accumulation
However, the effect on the transfer speed is negligible. did
Therefore, its effect may also be mediated through post-transcriptional mechanisms.
It seems that it will be carried out. The effects of corticosteroids on the nervous system are widespread.
[McEwen et al., Physiol
. Gical Rev, 66:1121-1188 (
1986)]. Steroids act as transcriptional regulators
It acts through the receptors of both neurons.
Is this gene regulated by cortiph steroids?
It is important to determine Consider the number of neural crest cells
When corticosteroids and N
The antagonistic effects of GF are reflected at the gene expression level.
whether the same gene is affected by two drugs
It is of particular interest to determine whether bimodally regulated
. We demonstrated that GAP-43 transcription is associated with corticosteroids.
and the simultaneous presence of NGF prevents this inhibition.
It was shown that there is no such thing. This is a cultured chromaffin cell
against neuronal differentiation brought about by NGF.
Similar to the glucocorticoid suppressive effect [Taube (D
oupe et al./Yarnal off Neuroscience
(J, Neurosci, ) 5:2119-214
2 (1985) l. Thus, GAP-43
These modifiers of fate have little to do with the known branching effects of the raters.
Comparable methods include NGF and corticosteroids.
It is bimodally adjusted by the It is called 5CGIO.
Other neural-specific genes are bimodal in PCI2 cells.
It is interesting that it is regulated [Stein et al.
, I) evelop. Biol, 127:316-325 (1988)
]. Similar to G A P-43, SC010 expression is
Stimulated by GF and inhibited by glucocorticoids
whether or not the level of repression is somewhat different between the two genes. Cortiph steroids significantly affect the shape of PC12 cells.
It has no effect. GAP-43 is suppressed, so usually
Levels of mRNA in neurite outgrowth in PCI2 cells
It is clear that this is not necessary for the long term. However, because
These results indicate that GAP-43 is not necessary for neurite growth.
It is not clear that it can be interpreted to mean that
stomach. First, low levels of GAP-43 RNA
It also exists after id suppression. Second, the protein is long
It has a long cell half-life. Furthermore, PCI2 cells are
transformed and retained by their in vivo counterparts.
It seems that different structural restrictions will be imposed than those imposed. Example ■ In this example, the present inventors demonstrated that GAP-43 was
of unique neuronal phenotypes, presumably at the level of cellular structure.
The idea is to test the hypothesis that
I will clarify. Thus, in another aspect of the invention,
Expression vector for rat GAP-43 with code E1
into several types of non-neuronal cells.
. adenovirus major late promoter, SV40 early promoter
promoter, or cytomegavirus promoter.
Under suppression, insert into a plasmid containing the SV40 origin of replication.
An expression vector was created using the rat GAP-43 cDNA introduced into
Karns et al., who built the
Science 236:597 (1987)]. The results were similar using all of these vectors.
Ta. CO87 cells [Gluzman, Y.
), Cell 23:175 (1981)]
was transfected according to the disclosure [Zub
er, M, X, ) et al., Science (Seiene
e) 234:1258 (1986)]
AGP-43 immunoreactivity using key anti-GAP antibody
Test (-T cell specific membrane protein CI) 8
Similarly, a control l.
“N-F (SeedB,) who performed transfection
U-prontinges off Nantanal Akatemi
ー・Off・＠zuiens nynisny (Proe
, Naj 1. Acad, Sci, USA)
84:3365 (+987) io cells were plate cultured.
One hour later, the control CO8 cells were tested with anti-CD8, anti-CD8
8-1-After immunofluorescence labeling of transfected cells
It was qualitatively round. GAP-431-transfected cells
In GAP-43 immunoreactivity, approximately 5-2 of the cells
It is remarkable at 0%, and it has a significant effect on transfection efficiency.
depended on. Some GAP-43 exists in cytosolic and membrane fractions.
Confirmed by Western plot analysis to compare min.
As previously observed, it appeared to be membrane-associated. Cells that express high levels of GAP-43 are
Distinctive with many cells extending processes from the periphery
It had a structure. This is due to the anti-GAP-43 antibody.
To ensure that this was not due to good visualization of the cell surface.
In order to
Labeled by agglomerate. CD84 transfected cells
, mock transfected cells (mock transfected cells)
cted cells), or expressing GAP-43.
In the same GAP-43 transfected dish,
cells produced short or single protrusions, but these
It had no distractor process. The long thin protrusions are high
It seemed to be related to the level of GAP-43 expression in Bell.
. A similar association between high levels of GAP-43 expression and protrusion growth
3T3 cells expressing polyoma T antigen, wop
cells [Valle et al., Mo1Ce11. Bi
ol, I:417 (1981)]CDM8-
GAP-43 expression vector containing GAP1CD--ma replication initiation
When transfected by a vector, it was found that
. In these temporal transfection assays
Therefore, the efficiency of transfection and the level of expression are
co-varies, making it difficult to quantify the effect. In order to solve the problem of quantification, GAP-4
A series of stably transformed clones expressing CH3
An O cell line was obtained. The control cell line after 30 minutes of plating was
-I loved boat fishing. In the GAP43 expression system, GAP
-43 expression was clearly associated with process distraction. G.A.
Many cells expressing P-43 are narrow and long
A 75 μm filopodium was distracted. Control and GAP4
In both cell lines expressing 3, the periphery is often
, containing a wide, narrow, serrated lamellibodilum.
I was reading. CH by Ca-P○4 coprecipitation method and G418 selection method
CDM8-GAP and Omainin resistance expression in O cells
By co-transfecting the plasmids, G
We established a clonal cell line that constitutively expresses AP-43.
[Maniatis et al., Molecule
- Cloning Near Laboratory - 7 Annual (M
Olecular Cloning: Harbor Labora
Tori (Cold Spring HarborLab
oratory), Cold Spring Harborney
New York (1982)]. Control transfection
In these cells, instead of CDM8-GAP, Plasma
Sumido pCDM8 [seed (3eed, B, ), non-seed
Icha (Nature) 329:840 (1987
)] was used. After the cells are confluent, trypsinize them
Glass coverslips were subcultured with polyD-lysine and coated with polyD-lysine.
plated on top. Protrusion formation transfected by control plasmid
4 independent systems and Western blot method.
Four independent cells expressing the highest amount of GAP-43 as measured by
The system was evaluated. By using Nomarski optics,
Live cells were tested. GAP-43 immunoreactivity
The total CH○ cell line with
There was a tendency to Additionally, expresses GAP-43
Cells are compared to a control cell line (0.5% to 1%)
and often have multiple processes (6% to 11% of cells
%), the length of the process is longer than that of control cells.
It was long. Therefore, the neuronal protein GAP-43
Causes changes in the shape of neuron cells. Filopodia and laminae so that cell protrusions resemble growth cones
Melipodilum extends directly from the cell trunk. In non-neuronal cells, GAP-43 is
unregulated form taken out of its biological context
Since the changes observed here are expressed in the
- Do not simulate the effects of GAP43 in Ron situations. death
In fact, GAP-43 is found in growth cones.
It is associated with growth cone function in that it is abundant in
[Katz, F. et al., Journal In
・Neuroscience (J, Neurosci,) 5
:1402 (1185) :DeGra
an, P, N, E, et al., Neuroscience
(Neurosci,) 61:235 (1985)
;Meiri, K, F, et al.
Seedinges in National Academy Inn
・Science Ninisny (Proc, Natl,
Acad, Sei, USA) 83:3537
(1986); Skene, J.
P, ) et al., Science 233 Near
38 (1986)], in vivo or in v
highest in neurons extending their axons in vitro
level [Skene, J, H, P
) et al., Journal of Cell Biology (J
, Ce11. Biol, ) 89:86 (1981),
Benowitz (Benowitz, V, E, )
et al., Neuroscience (Neurosci,) 1°
300 (1981); Meiri, K.
F.) et al. Journal in Neuroscience (
J, Neurosci, ) 8:2571; Beno
Benowitz, L., et al., T.
, 1. N, S, 10:527 (1987) E, top.
As shown, it increases in PCI2 cells exposed to NGF,
There is evidence that it is accompanied by neurite outgrowth. The inventors try to connect by a particular theory
One interpretation of these takes is that GA
P-43, a neuron-specific molecule, is completely absent from these cells.
It is possible to create a novel neuron-like structure.
It is. Another explanation is that GAP'-43 controls cell shape.
This is because it relates to a more general mechanism of control.
Some [Bray, D., et al., Science (
Science) 239:883 (1988) Varnish
Miss (Smith, S, J, ), Science (S
science) 242 near 0g (198g)']. Many cells extend filopodia or lamellipodia
This can be determined by the phase of the cell cycle, plating conditions
, and some including the second mesocene/year level
It tends to depend on factors [Allred
ed, L, E, ) et al., Surf A/Z in No.
- Maru & Marignant Sells (Surfac
es of Normal and Mali
gnant Ce1ls) No\Ines (R,0
, Hynes) (Joe 7 Willie & Son)
(John Willey & 5ons), new
York, 1979), p. 21]. For this reason, exactly
Cells were examined under the same stringent plating conditions. to cells
The cellular mechanisms that allow projections to be distracted are complex.
It is crude and contains elements that are thought to be present in all cells.
I'm reading. Therefore, growth cone protrusions and fibroblasts
The similarities with protrusions are not surprising. In fact, growth
The conical structure □ is used as a general method to impart cell movement.
Flow and selection of cortical actin that can be
It has been suggested that cellular mechanisms such as target adhesion may be utilized.
[Bray, D. et al., Scien.
Science 239:883 (1988)
Smith, S. J., Science
(Science) 242 Near 08 (1988)]
. How GAP-43 is related to this mechanism
It remains to be judged. Example ■ Identification of a novel membrane targeting peptide
In one embodiment of the invention, the GAP-43 protein is
AP-43 protein is added to the cell membrane, especially the growth circle of neuron cells.
A novel membrane that orients to the conical region - Thaake, Teingpe
It was found that it contained petitdotomain. This membrane tar
The structure of the targeting domain is determined, and the peptide is
(usually not membrane-bound) Usually cytosolic proteins
It was shown to be effective in directing to the cell membrane. The compositions and methods of this aspect of the invention provide, inter alia,
Any protein of interest, including proteins that are not normally membrane associated.
It is also possible to direct them to the cell membrane. moreover,
The compositions and methods of this aspect of the invention provide, in an animal,
Neurological damage and disorders in vitro, in
Available for in vivo and in 5 situ therapeutic treatments
It is clear that Most membrane-associated proteins intercalate directly with the cell membrane.
It is well known that it contains a highly hydrophobic domain that
Ru. However, surprisingly, GAP)-43 is
, a neuron that is attached to the cell membrane and especially develops or regenerates
Highly hydrophobic regions connected to or associated with the cell's growth cone
was discovered to be lacking. Now, as shown in Figure 2, the GAP-43 protein has three
It was discovered that it is encoded by an exon. short
(1,0 amino acid residues) The amino-terminal exon is
Especially, the membrane-terkenoting peptide domain
It was discovered that the code can be coded. Second GAP-4
Experiments in which most of the 3 exons were removed resulted in membrane condensation of the remaining protein.
It had no effect on the Similarly, the number of GA, I”43
Replacement of the carboxy terminus was found to have no effect on membrane binding.
I made it clear. However, the first four amino acids (MET
LEU CYS CYS) and opened at 5th place MET.
The synthetic GAP-43 gene that initiates is generated in neurons or non-neurons.
does not bind to the membrane of the neuron host cell (Fig. 11);
The first exon is responsible for this membrane-tarkerating function.
. "Membrane-targeting peptide" means the following MET LEU CYS CYS MET ARG A
RG THRLYS GLN or its functional derivatives
means any amino acid sequence, which is the desired protein sequence.
or attached to or near the amino terminus of the peptide
If the protein or peptide is
It will resonate. Membrane-targeting peptides of the invention can be prepared manually
, or preferably involving direct synthesis by automated methods.
but not limited to, by well-known methods.
can be attached to the desired protein or peptide
. The membrane-targeting peptide of the present invention can be targeted to a desired protein.
or another preferred method of binding to the peptide is the
The gene encoding the desired protein or peptide is
The expressed gene product has a membrane target at its amino terminus.
including modification to include a binding peptide. child
This may be blunt ended or viscous, as described herein.
This can be achieved by tying the loose ends, but is limited to these methods.
It is not something that will be done. Thus, in another aspect, the invention provides
Otide sequence atg ctg tgc tgt atg aga a
ga acc aaa cag or a functional derivative thereof
Membrane-targeting domain code
It provides cDNA to be attached. Of course, due to the degeneracy of the genetic code, it is still difficult to achieve the desired result.
It is possible to change the nucleotide while Similarly, those skilled in the art will appreciate that in certain instances
Preferred codon usage, if considered in a particular host.
It is desirable to change the nucleotide for
you will understand. These and other such modifications are
It is considered to be within the scope of the invention. Furthermore, the GAP-43 membrane-targeting domain was clarified.
(COS cells, NIH3T3 cells,
and non-neuronal cells (including CHO cells)
Neuron cells (PC12 cells) with mutation at position 3 (
C3) and cysteine nucleotides at position 4 (C4)
Plus containing the GAP-43 gene introduced into the sequence
Alternatively, in those positions
Alanine was expressed (Figure 11). Mutations in either C3 or C4 result in membrane-bound or large
It was found that the number decreased significantly. The most noticeable effect is C4
Observed when changed. Both C3 and C4 mutations
The difference completely eliminated this phenomenon. Mechanism of this effect
Although the oxidation state at those positions is not clear,
It is clear that it does not involve pure change. I mean
, because the redox experiments did not change membrane binding.
. Furthermore, the first four amino acids (MET LEU CY
Synthetic GAP lacking SCYS) and starting with MET at position 5
43 genes were constructed. The expressed protein is transferred to neurons and
and non-neuronal host cell membranes.
(See Figure 1). This may be due to the first four amino acids or membrane binding.
Indicate that it is necessary. As discussed below, typically
In experiments with cytosolic proteins, 10 amino acid peptides
Chido clearly proved to be sufficient. preliminary day
The first four amino acids are sufficient for membrane binding.
to show that Thus, in another embodiment, the invention provides:
consisting of a amino acid sequence or a functional derivative thereof;
to enable membrane binding of the desired protein or peptide.
to the amino terminus of the desired protein or peptide.
can be combined. Additionally, the first 5, 6, 7, 8 or 9 of exon l
Peptides containing amino acids of the desired protein or
If attached to a peptide, it would allow membrane binding.
. Those skilled in the art will be able to direct membrane binding in a particular application.
The sufficiency of these intermediate length peptides is simply routine.
Understand that judgment can be made by the effort of one's skill.
Yes, this is also an advantage of the teachings of the present invention. Therefore, that
Those identical to these and equivalents thereof are included in the present invention.
is within the range of In additional experiments, the first GAP43 exo described above
protein, which is normally cytosolic and not membrane-bound.
Chloramphenicol acetyltransferase (C
AT) is attached to the ami/terminus of the gene that attaches 2-1-.
. Using a plasmid containing this sequence, neurons and
and non-neuronal cells (11th
figure). Expressed CAT protein by immunofluorescence assay
is membrane-associated in transfected cells
That became clear. This is the first GAP-43 edition.
The amino acids of Xon are the membrane targets of the desired protein or peptide.
Show that it is sufficient to achieve kenoting. Furthermore, experiments showed that the first 40 access points of GAP-43
amino acids in transfected PC12 cells.
Is it possible to orient cAT to the same position as GAP-43?
Shown. These cells have a tilted length by the growth cone.
It resembles a neuron cell in that it draws out thick projections. G.A.
P-43 is usually abundant especially in neuronal cell growth cones
and the take is the membrane of the present invention~Turkenoting Peptide
Q1- is responsible for this observed growth cone enrichment.
suggests. Furthermore, the selective growth cone accumulation mechanisms described herein
In order to clarify the situation, the present inventors used G A P −
43 and chloramphenicol acetyl transfer
Mutation tolerance analysis of a fusion protein encompassing a proteinase (CAT)
Scanning confocal microscopy was used. As a result, G.A.
A short stretch of the P-/13 amino terminus forms the growth cone membrane;
be sufficient to direct accumulation particularly in filopodia
was confirmed. reporter pepchi
Various amounts of G A P-4,3 amino terminus fused to
in CO8 and PC12 cells.
It was expressed. Chloramphenicol acetyltransf.
CATase (CAT) when expressed in eukaryotic cells
is cytosolic and very stable, so the report
was selected as the target peptide. For complete CAT protein
The first 10 of GAF) 43 fused to the amino terminus
A: Fusion protein of /acid MLCCMRRTKQ (GA
Pl 0CAT), GAP- fused to Matah cAT
43 first 40 amino acid fusion protein (GAP40
A plasmid encoding CAT) was also constructed. Fusion of immunoplot method with CAgo as follows
The chimeric protein with the amino terminus of GAP-43 is C
Connects to O8 cell membrane. CA, T, GATl 0CA
T and GAP4oCAT are transiently expressed in CO8 cells.
It was done. Each transfection was performed using anti-CAT antibody.
Immunoplasts of membrane (M) and cytosolic (C) fractions from
A lot was prepared. CAT-1-transfected
In cells, immunoreactivity was found only in the cytosolic fraction
and migrate together with purified CAT protein. immunoreactivity
All of them are membrane-connected and larger than CAT.
fusion protein with 1i4000 or 1000 molecules
As expected, it moves more slowly than CAT.
do. 116.84.58.485.356 and 26
A molecular weight standard of 6 kilocroutons was used. Membrane connections of GAP and GAP40CA, T to CP12 cells
The cells were evaluated. As described herein, CAT
, , GAP40CAT, or stably expressing GAP
Transfected PCI 2 cells were selected. film
Immunoplots of (M) and cytosolic (C) fractions
-stained with CAT or anti-GAP antibody. CAT-to
Transfected cells (CAT) are in cytoplasmic vines.
The membrane fraction contained no immunoreactivity.
, this immunoreactive CAT migrates with purified CAT.
Ta. In contrast, GAP40CAT-transfected
cells (G40CAT) that move more slowly
Contained monoligated CAT immunoreactivity. Rat brain (B
Most, but not all, fractions from R)
We show that endogenous GAP-43 immunoreactivity is membrane-associated.
Indicated. Transfected P overexpressing GAP43
In CI2 cells, most of the GAP-immunoreactivity
All are membrane bound and coexist with purified GAP-43.
Moved to. In the GAP-43 expression plasmid pGAP, GAP
The sequence encoding -43 is sorted by Seed
, B, ), Fuichi+ (Nature) 329
: 84C)-846 (1987)
Add stuffer at the Xba 1 site of the CDM8 plasmid.
(stuffer) was replaced. Described in this specification
As a whole, the inserted GAP-43 sequence acts as a translation starter.
Nla ■ 6sbp from the end codon from the site
of rat GAP-43 up to the downstream 5au3A1 site.
The entire coding sequence was included. CAT expression plus
For mid-pCAT, the CAT coding sequence and
and pSV2CAT (Bormann CM, Moffat
・L.F. and Howard B.H. (Gorm
an, c, m, , nor fat. L, F. & Hotvard, B. H.), Mo.
Recular and Serious Biology (Mo
1. Ce11. Biol, )2:1044-1
Contains polyadenylation sites from 051 (1682)
The HindIII or BamHI fragment is a stuffer.
(stuffer) and polyadenylation site.
Place the HindIIl or BamHI fragment of CDM8.
I changed it. pGAPloCAT and pGAPloCAT are
Identification of CAT in pCAT by using a relinker
The beginning of GAP-43, each fused in a frame
40 or 10 amino acids. CoS fine
For transient transfection of cells, DEAE
Dextran and chloroquine as listed
(Tuba M.X., Simpson I.
- R Oyohi Waterman M R (Zu
ber, M., X., Simpson, E.
R, & Waterman, M, R,), Scien.
Science 234: 1258-1261
(1986)). Stable transfuchsia of PCI2 cells
plasmids of interest in a 1:10 ratio for
Co-transfected neomycin resistance plasmid
It was used as described herein. PCI2 cells
During selection, 400 μg/mQ of active dienetitin (
Using Geneticjn (GIBCO)
Ta. Transient transfection of PC12 cells
00 volts and 960 microfarads.
Bio-Rad Inc.
Performed by electroporation with an electroporation system of
It was. After 8 hours, the medium was changed. 24 hours after electroporation
, 50 ng/m (poly D-lysine in the presence of INGF)
-Plate cells on coated cover glass and after 24 hours
was analyzed. For immunochemical assays, aa of rat GAP-43
l to 24, aa35 to 53, aa53 to 6
9, and four types of peptides encompassing aa212 to 228.
rabbits by immunizing them against putidos.
An anti-GAP-43 antibody was produced. anti-GAP-43
Antibodies were affinity purified on GAP peptide agar.
. Cambring the agarose using the cyanogen bromide method.
10 mg/m of each peptide (!)
Bind GAP-43 antibody and elute the antibody at pH 3.5
I let it happen. Usaki anti-CAT antibody is 5 prime (Prime
e) 3 Prime (Prime) ・Incobo Ray
I got it from Tae and Do. The second antibody is produced by Organon Techni
Organon Teknika
. Jackson Immunol
ogicals), and Vector labda (Vect
or 1 abs). For cell fractionation, CO8 or PCI2 cells were
scraped from a crowded bedpan of oornm, 2000
Pelletization was carried out for 10 minutes at xg. The pelleted cells were treated with 10mM Tris-HCρ, lmM
EDTA, pH 7,6 (300) in l (!/dish)
By Polytron
and centrifuged at 4°C, 250, OOOxg for 30 minutes.
. The supernatant was collected as the cytosolic fraction. the beret
Wash by homogenization in the same buffer and centrifugation.
and then resuspended to the same volume as the cytosolic fraction.
Ta. Rat brains were obtained from 1 day old rats and treated with 10mM Tris-
HCl 2.1mM EDTA. pH 7,6 (Pot in 10 m (!/Durham wet weight tissue)
By Polytron
Ta. by centrifugation as described for cell extracts.
, cytosolic and washed membrane fractions were prepared. You
Anderson et al.
Mistry (Biochemistry) 22:
4615-4618 (1982)).
GAP43 protein was purified from rat brain and subjected to immunostaining.
This was used as a positive control. Cytosolic or membrane fraction
of polyacrylamide gel (usually 100 μQ)
(Laemmli, NE)
Icha (Nature) 227: 680-685 (
1970)). Electrophoretically convert proteins into nitrocellulose
The excess sites were blocked with 4% BSA. Then, 40 μg/m (l affinity purified anti-GA
or at a 1:1000 dilution of anti-c A T antibody.
The membranes were incubated for 24 hours at , 4°C. manufacturing
According to the manufacturer's instructions, apply anti-corrosion hectastein O'e.
ctastain) Horse ratty/Yuber Oxyta
Bound antibodies were detected using the -se method. Bel Okinda
As a substrate for
-1” (Kirkegaard) and Perry (Pe
rry), Gaithersburg
g), Maryland) was used. Immunoproyt4 induces CO8 cells or PC12 cells.
CAT expressed in cells is present only in the cytosolic fraction
It became clear that In contrast, the chimeric protein GAP
10CAT and GAP40CAT are membrane-associated. The fusion protein is extracted by detergent or thorium chloride.
, calcium chloride, or EGTA.
do not have. Thus, the nature of this membrane binding is important in the human brain.
The properties are similar to those of natural GAP-43 (Perot
Zoseguchi N.I., Waifuru Tei, Hausa Shi
PerroneBi and Benobi, T.L.I.
zzozero, N, 1. , Weiner,
D., Hauser, G., & Benowit
z. L, 1. ), Journal in Neuroscience
Research (J, Neurosci, Res,) 20
: 346-350 (1988);
・Bii, Pang Donggon, Sii Shai, Tubirus・
Oest
reicherA,B,,Van Dongen,C,
J,, Zwiers, HJ+ G15pen, W, H,
), Journal in Two Lochemistry (JNe
urochem, ) 41: 331-340 (198
3); Xiang Nissii, Kei Murakami and Lowe
Tenbergu x Yi (Chan, S, Y, Murak
ami, ni, & Routtenberg, A,),
Journal in Neuroscience x) 7th (J, Neu
rosci,) 6: 3618-3627 (1986
) + Sukefu J.H.B. and Virag.I
(Skene, J., H.P., & Virag),
Annual in Cell Biology (J, Ce1l
Biol, ) 10B 61:3-624 (1989)
). Growth of GAP43 in amino terminal neuronal cells
To determine whether it explains cone enrichment, PCI2
GAP in NGF-treated transfectants of cells
-43 and GAP-CAT chimeric protein.
It was investigated by focus microscopy. According to this assay, GAP
)-43 is a growth cone or a markedly abundant spotted pattern,
A pattern similar to that of natural GAP-43 in euron
CAT remains in the cytosol, whereas CAT remains in the cytosol.
be. The amino terminus of GAP-43 fused to CAT is
The obtained fusion protein was used to control the distribution of GAP-43 itself.
A very similar distribution was obtained. Perinuclear labeling of both GAP-43 and the chimeric protein is low level.
This was also observed with native GAP-43.
As (Pan Ho 7 C.O.M., Horsuis)
・C.M., Estreichel.A.B., Huns
Tora/J, I, De Grand BNN and
Van Hooff'
,C,O,M,, )lolthuis, C,M
Oestreicher, A.B., Boon
straJ, , De Graan, P,N.
E & G15pen, L H, J, ), Nyanal
・In Cell Biology (J, Ce1l Bio
l,) 108: 1115+~1125 (1989)
), probably due to its localization to Corgi. glutaraldehy
Do fixation result in good tissue preservation of the thin protrusions of the growth cone?
It has been shown that the chimeric protein accumulates particularly in a filamentous orientation.
It became clear. CAT,G in transfected PC12 cells
The subcellular localization of AP-43 and fusion proteins is as follows.
Particularly performed: (A) CAT in PC12 cells,
(B) GAP-43, (C) GAP40CAT, and
(D) Confocal immunofluorescence of GAPl 0CAT revealed that GA
P-43 is patchy and localized in the membrane, with some abundance in the growth cone.
CAT labeling is diffuse and cytoplasmic, whereas CAT labeling is diffuse and cytoplasmic.
It became clear that this was the case. 40 amino acids at the amino terminal
acid (GAP40CAT) or 10 amino acids (GA
If either PloCAT) is fused to CAT, the immunity
The distribution of epifluorescence is similar to that of GAP-4, including enrichment in growth cones.
The distribution was similar to that for 3. Prior to fixation, all
Cells were treated with NGF for 24 hours. For GAP-43, anti-GAP-43 antibody was used.
On the other hand, CAT, GAP40CAT and GA, Pl
For oCAT, anti-CAT antibody was used. This mutation
Control PC12 cells had undetectable levels of GAP-
43 and CAT immunoreactivity. 50μg/m
Immunofluorescence of Q2 in the presence of nerve growth factor (NGF)
PC12 cells were placed in a poly-D-lysine coated cover for 4 hours.
- Transferred to glass. 7 minutes with 3.7% formaldehyde
Fix and infiltrate with 01% TOINOTON-X-100 for 3 minutes.
I set it. Block the samples with 4% BSA in PBS for 1 hour.
, - Incubate for 1 hour in next antibody and rinse with PBS.
Ink in 0.3% H in PBS for 15 minutes at -0°
(to reduce background) and repeat the process.
and incubated in secondary antibody for 1 hour. PBS
After washing several times with water, rinse the coverslip with water and
Kerbatol (Ge) containing % gallic Mn-propyl
lvatol) to reduce fading. cell or special
If it does not contain a foreign antigen, or - the next or secondary antibody
If omitted, immunofluorescence is above background.
could not be detected. Labeled with anti-CAT antibody, /Marskii (Nomars
ki) optics and scanning co.
Focal immunofluorescence (optics)
and scanning confocal im
mun. GAP40 observed by f 1 uorescence) method
Using a high-power comparison of PCI2 cells expressing CAT,
Localization of GAP40CAT within the growth cone of PCI2 cells
certified. Cells were treated with NGF for 7 days.
. The large growth cone of No. 1 appeared brightly labeled, but the small
The small things disappeared. Even the same cell is equivalent in different growth cones
No labeling occurs in native GAP43 in neurons (
Koslyn Kay, Schreyer T.J., Sukefu
・J.H.B. and Bunker.C. (Gos.
lin, K., 5chreyer, D.J.
. 5kene, J., H.P., & Banker,
G, ), Nature 336: 67
2-674 (1988)). Nomarsky
skj) and immunofluorescence images, filopodia were identified as
It was indicated that it would be labeled. Similar results?GAP I
Observed for 0CAT. For high-resolution confocal microscopy, the fine structure of filopodia is preserved.
The newly created 4% rose formalde is essential for the
Hyde and 0.5% glutaraldehyde, followed by 01
% Tozotone-X-100 for 3 min at 2 m in PBS.
Fix cells for 10 min with g/mσ sodium borohydride.
Established. For confocal analysis, Biorad's
MRC-500 scanning confocal imaging system and
Zeiss Akinopl
an) A microscope was used. These experiments focused on the first 10 amino acids of GAP-43.
The present finding that acid is sufficient to direct growth cone accumulation
This confirms the surprising discovery of the researchers. Inventor
and at least one other non-fine membrane that accumulates in the growth cone membrane.
Intracystic membrane protein 5CGIO (Stein R, Mori N
, Mathieuse K., Lo L. C. and Antar.
Song Thi Hsieh (Stein, R., Mori,
N., , Matthews, K., Lo.
LC, & Anderson, DJ, ), Ni Ni Ron
Despite positions 22 and 24), GAP-43 a
No other proteins are known that have sequences closely related to the amino terminus.
do not have. In polarized epithelial cells, different proteins are membrane proteins.
and directing the transport of secreted proteins to their individual destinations.
depends on selection signals within the protein similar to those that direct
protrusions that are thought to be
Wickner, L.
T. & Lodish, H.F.), Science
(Science) 230: 400-407 (19
85); Herner K. and Syahura S.I. (Ver.
ner, K. & 5c-hatz G, )Science
ce) 241: 1307-1313 (1988);
Pfeffer Niss R and Rothman Hsieh
-(Pfeffer, SR, & Rothman, J.
, E. ), Ann Lev Biochem (Ann, R.
ev, Biochem, ) 56: 829-852 (
1987 to the apical and basolateral plasma membrane (mal-1
1. Matlin, K. S.,
Journal °in cell +7'? Iology (J, C
e1l Biol, ) 1032565-2568 (]
986); Ronriqueshoran E. Shei and Saha
Teni Dei De 4 (Rodriguez-Boul
an, E., J., & 5abatini, D.
D.), Pro/-Tings in National
Acatemy in Sanensis (Proc, Nat
l, Acad, Sci, ) USA75:5071
~5075 (1978): Nmons K. and Frey
・Nis Ti (Simons. L & Fuller, S. D.) Anu Lev.
Ann Res, Ce1l Biol
) 1: 243-288 (1985). In epithelial cells, such signals are apical and basolateral.
They will also recognize different regions of the plasma membrane. In neurons, the growth cone membrane also has a protein structure.
distinguished. One interesting possibility is growth cone membrane or GA
Recognizes and binds to the palmitylated amino terminus of P-43
It has a binding site that makes it possible to The inventors are not
Do you intend to be bound by any particular theory, Palmich?
The binding residues do not directly interact with the lipid bilayer.
Yes. Because it is more uniform for GAP-43
This is because it seems to cause membrane distribution. along this line
, another fatty acid site of an unbinding protein, poliovirus
The N-myristylated VP4 of the
does it interact with specific amino acid residues in viral proteins?
It has been shown that it does not interact with lipid bilayers (see
Urz A.M., Henterman L.E.O.H.
Oroszuranes (Schultz, A, M,
, Henderson, L.E. & 0roszlan+ S, ), Anu Lev Cell
・Highol (Ann, Res, Ce1l Biol,)
4: 611-647 (19ss);/Yo M,
Newman N.F., Firman Day, Hoggle
・JM, Rowlands Day Sha and Bra
Chow, M., Newman,
J., F., Filmman, D., Hog.
le, J., M. Rotvlands, D. J., & Brown,
F, ), Nature 327/4
82-486 (1987)). GAP43 and GAP
-CAT fusion protein binds to membranes of non-neuronal cells.
, similar or identical binding sites exist in other cell types.
GAP-43 must be present in neurons.
Because of their specificity, these sites are likely non-neural.
This may be a target for different proteins in human cells. The sorting domain of GAP~43 is particularly enriched in filopodia.
It is noteworthy that it causes By electron microscopy
As proven by Pan Hof C.O.M.
, Holsuis C.L., Estreiher A.
B, Boonstrassin A, De Graan B
F.E. and Syspen W.H.
Hoohh, C,0,M. Holthuis C,M, 0estreicher
A B Boonstra JDe Graan,
P, N, E, & G15pen, W, H,
), / Yarnal in Cell Biology (J, C
el I Biol, ) 1081115-1125
(1989), which indicates that GAP-
This is the normal position of 43. transfected GA
P-43 has extended filopodia as described herein.
If there is any observation that enhances the propensity of non-neuronal cells to
, related the location of GAP-43 to specific filopod locomotor activity.
It may be interesting to attach Annex I. Thus, in another aspect, the present invention provides the desired
proteins or peptides into neuronal or non-neuronal cells.
method of introduction into the crotch region of the cell, and the method of introduction of the desired protein or
Orients putids to the growth cone region of neuronal cells
provide a method. In the illustrative embodiment of 1, (a) 1, MET LEU CYS CYS MET A
RG ARG THRLYS GLN・Il, MET
LEU CYS CYS MET ARG ARG
THRLYSIIl, MET LEU CYS CY
S MET ARG ARG THR; IV, MET
LEtl CYS CYS MET ARG ARG
;V, MET LED CYS CYS MET
ARG VT, MET LEU CYS CYS M
ET ; ■ MET LEU CYS CYS and
a membrane protein consisting of an amino acid sequence selected from the group consisting of
- Chenoting peptide of desired protein or peptide
9 then (b) the membrane-terminus
The resulting protein or peptide consisting of
The protein or peptide obtained in step (b) is introduced into the membrane target.
directed to the membrane of the cell by the tutting domain.
the desired protein or peptide characterized in that
A method of orienting to the membrane of a cell is provided. In another non-limiting exemplary embodiment, the invention
amino acid sequences or their functional or chemical derivations
Membranes made up of bodies - encode targeting peptides
nucleotide sequence, as well as by well-known methods.
proteins or peptides other than GAP'-43 (and
Nucleotide sequence encoding GAP-43 itself)
The addition of these sequences to and well known to those skilled in the art
Prokaryotic or eukaryotic host of sequences obtained by the method
Provides expression in. Of course, as described herein, the desired protein or
Peptides can be diagnostically or therapeutically labeled;
The utility of the compositions and methods of this aspect of the invention will be apparent to those skilled in the art.
It should be obvious, and with just a routine effort of skill,
In animals including humans, in vitro, i.
n vivo or in 5 situ diagnosis or
It is readily available for therapeutic purposes. Example ■ The results of the present inventors as described in this specification are GA
Strongly suggest that P-43 regulation occurs at the level of gene expression.
suggest. However, until now, it has not been possible to regulate its expression.
Nothing is known about the cis- or trans-acting elements obtained.
It wasn't. Originally, it imparts its responsiveness to growth factors.
Defining elements of the GAP-43 gene and determining cellular restriction of expression
triggers and regulates the gene during nervous system development
Very interesting to do. To identify regulatory elements,
Cloning the entire rat genomic DNA of GAP-43
did. Therefore, we isolated genomic GAP-43 and analyzed its intracellular
Map long-exon boundaries and transcription start sites.
Ta. Surprisingly, promoters have a very similar structure.
It is an abnormal reaction that can form an abnormal formation.
contains several canonical promoter components.
Lacking. Transcription can start from more than one site, and
Some of these sites are different in the central and peripheral nervous system.
It is used as such. Furthermore, the present inventors demonstrated that the GAP-43 promoter is
- whether it contains a region recognized by a specific nuclear protein;
I looked into it. The GAP promoter region was subjected to gel electrophoresis.
present in brain nuclear extracts as determined by dynamic mobility shift
or binds protein(s) that are not present in liver nuclear extract.
The domain was identified. Binding activity decreases with brain maturation
do. The binding site is limited to a stretch of approximately 20 nucleotides.
It has also been determined that DNAse
Specifically protected in protection assays, liver extracts
Not according to things. The area is known as PoU.
The binding site recognized by the class of DNA-binding proteins and
have similar sequences. These results suggest that brain-specific nuclear proteins may be upstream of GAP43.
Genomic cloning and mapping cloning are experimental methods that suggest that
All methods used in Ausubel et al.
et al)m, Currant Protocols in Mo.
Recular Biology (CurrentProto)
cols in Molecular Biology
), 7 Yon 0 Willie & Sons (John V.
(1987)
It was written as follows. All enzymes are two
Ncv England
Biolabs). Ge/Mi containing three GAP-43 exons. The clone is a size fraction of rat genomic DNA.
5aullA partial digest was bacteriophage EM.
constructed by inserting into BamHl of BJ,-3.
isolated from the constructed library. The library was first cultured in colonies as described above.
Plaque Screen Filter (DuP)
ont/NEN)), followed by the first antigen challenge to the random
using a standard protocol using GAP-43 cDNA
Screened. To find the exon ■,
Re-plate the library and remove the most 5' region of cDNA.
3 oligonucleotide complementary to the region (see Figure 14).
Well, #4. -68 or -39・#2 -38 or -
9 and #5. +1 to -20) for overlapping lift (li
ft) were sequentially probed. at least 2 oligonuks
Clones positive for leotide were selected for further analysis.
I chose. Positive due to mapping with various restriction enzymes
Input from phage into pBluescr 1p
5a of t (Stratagene)
It was subcultured into 11 sites. HDNA Kel (adjusted to pH 7.4 or pH 4.0 with acetic acid) 45
Using mM Tris base as a buffer, two 25 cm long
Pour 1.4% Akaroskel. Negative '4N opposition't
f< is 5% glycerol, 0.1% furomophenol
Electrophoresis containing Ru and 01% xylene anol
It was a dynamic buffer. Plasmids listed in the legend of Figure 15
The exon I digest containing bs1.5RIX4 was
The sample was loaded onto a pH 9 column and run at 20V for 16 hours.
Stained and decolorized with ethidium bromide in acetate,
I took a photo. The sequencer as described by the sequencing supplier (USB)
Using Sequenase, Sanger et al.
(Sanger et al), Brondings
In Nanyonal Acatemy In Sanenmes,
(Proc, Natl, Acad, Sci,) U.S.
A74: 5463-5467 (1977)
Sequencing of GAP-43 promoter by Buoki method
Established. pBluescri for double-stranded sequencing
pt, (Stratagene)
and - M13 vector (main7 vector) for waterway sequencing.
Messing, Meth
, Enzymol, ) 101: 20-7 B
(1983)), i? 71J
A succession of paitheriophers containing exons of
A substitute clone was constructed. RNA5e Manobing Kusorg and Norton CKrieg and Mel
ton), Meth, Enzy
mol, ) 155: 397-4] 5(19
87) Especially the RNA5 e-protection described here.
Analysis was carried out. Regarding this protection, the translation start site or
-475 Xba 1 site to +83 5sp1 site
Kenomi of GAP-43 leading to (first in) - Ron middle)
, the piece of pSP72 (Promega)
Cloned into the Xba I and EcoRV sites. RNA5e protection analysis 47/48 from translation start site
, -51152 and 178 bases -
43 transcripts were shown. Neonatal rat lungs, explanted root ganglia,
and tRNA and RNA prepared from cerebral cortex
protected. The clone has a single translation start site (Xba1
It extended 475 bases from the site). past
Contrary to the DRG, the surplus is quite abundant in the cerebral cortex.
Long transcripts were further shown. Markers are for MSPn digestion.
There was pBR322. Other RNA5e protection appears to be in different regions of the nervous system and in the PC
12 cells to demonstrate the heterogeneity of GAP-43 transcripts.
It was. RNA from control PCI2 cells was transferred to NGF-treated P
CI2 cells, tRNA, DRG, cerebellum, cortex, and ocean
I compared it with what I got from horses. CNS-derived RNA sample
DRG or PC] higher proportion than samples from 2 cells.
It had a long transcript. In another RNA5e protection analysis, the previously described
The sumid was digested with NdeI and HindI[I, and the DN
By filling the A polymer with secret Klenow fragments,
-233 NdeI site to +83 same 5sp1 site
A genomic fragment of GAP-43 was cloned. Hint
dll) site was re-formed. In all cases, base
After linearizing the vector with Hindll, the transfer body was
Extended with polymerase. Thus, beyond this part
All extending transcripts are collected as a single band at −234.
circle. The heart, liver, lungs, cerebellum, pulp, and skin of a newborn baby
Using RNA samples from the physis, hippocampus, and dorsal root ganglion.
. The longer upstream starting site as a group is the central nervous system group.
It constituted an important fraction of RNA in the dorsal root.
It was not composed of ganglia. Cloning of the Koujo GAP-43 genomic sequence
In particular, a probe obtained from GAP-43 cDNA was used to
・Screened a genomic isota library. radiation
An initial screen with functionally labeled full-length cDNA
Two classes of phage were obtained for subsequent analysis.
corresponds to the second and third exons of the gene.
was shown. The first exon is small and therefore the cD
found to be underrepresented by NA probes.
Therefore, 3 oligonucleotides from the most 5' region of the cDNA
Further rounds of screening using cleotide probes
ning to select a clone containing the 5” end of the gene.
It was necessary to obtain it. The map of the GAP-43 gene is shown in Figure 13a, and it
The phage used to map the map is also shown. The remains
The gene spans at least 50kb and is divided into three small
Contains xon. The first one is about 80 bp (changed at the 5' end)
(See below for the description of the opposite sex), the second is 565
bp, the third one is 672 bp, and they
has two inputs larger than 24kb and 20kb, respectively.
Separated by Tron. The first exon contains the 5° untranslated sequence of the mRNA;
Codes the first 10 amino acids of a protein. concerned
This short amino-terminal region of the protein consists of G
Attachment of AP-43 (and heterologous fusion proteins) to growth cone membranes
[contains selection sequence J]. second exon
encodes the protein bulk and the calmodulin binding site
As Alexander et al.
al), Journal in Biological Chemist
Lee (J. BioChem), 263: 754
4-7549 (198B).
Contains. The third exon contains 28 amino acids at the carboxy terminus.
encodes a amino acid and contains 587 bases of untranslated sequence and
Contains a polyA addition site. The intron-exon boundaries shown in Figure 13b are sequence determined.
It was identified using a standard method, and the consensus splat
Chair part (Mount), nuclear
Shino's Research (Nucl, Ac1ds Res,
) 10:459-472 (1982)). The polyadenylation sites shown here are protected by RNA5e.
This was confirmed by Roseincourt et al.
nthal et al), E.M.B.O. (
EMBO) 6: 3641-3646 (1987)
Therefore, it was predicted to be a major site. often
, Funsen found immediately 3” side of the polyA addition site
Tantem pair of suspension motif (motif) (YGTGT
TYY) (?McLauch 1an)
, Nuclitsk Asino's Research (Nucl, ^a
idsRes,) 13: 1347-1368 (198
5)) is underlined in the figure. The GAP-43 promoter contains H-DNA. The sequence of the 5'' region of the gene is shown in Figure 14.
Contains no ATA or CAAT box, but
The sequence TATTC is identical to the Nsus Pit-1 binding site.
Contains ATG (upper line). This octamer contains prolactin and growth hormone.
A protein that is thought to regulate the transcription of several genes at the same time.
(Bonder et al.
et al), Cell, 55:505-51
8 (1988); Ingraham et al.
etal), Cell 55: 519-5
29 (1988)). An amazing feature of promoter sequences is that over 80% of promoter sequences
The binding chain is composed of purines (the asterisk is on the bottom in the diagram).
), -118 to 188, and -238, respectively.
2 consecutive purine homopolymers ranging from -370
It's something like stretching. Just change G and A.
Some regions of these homopolymer stretches are
, containing tandem repeats with some reflection symmetry (e.g.
For example, -168 to part 18). -112,232 and
Hairs that form self-complementary sequences centered at -509 and -509
Pins are on either side of the homopolymer region and influence the secondary structure
Get 12. Blue pyrimidine homopolymer stresothi, especially mirror reflection
Things with symmetry (Mirkin, Fichi)
+ - (Nature), 330: 495-497
(1987 is the triple-stranded conformation of the word H-DNA.
(For reviews, see
Ells et al., Journal I
Biological Chemistry (J, BiolC)
hem,) 263: 1095-1098 (1988)
Htun and Dah
lberg), Science 243
: 1571-1576 (1989)). GAP-43 promoter is strong in vitro
It has been shown that it contains a primary structure.
It happened when I was deciding the line. Sequencing is a double-stranded scenario
Although this technique is routinely achieved using
Readable when applied to GAP-43 promoter
A possible sequence reaches the homopolymer region at -250.
Then it suddenly stops. - Passaging small fragments into M13 for roadway sequencing
We arrive at the sequence only after
I was able to do that. Tun and Da
hlberg), Science, 24
1:1791-1795 (1988) is H-DNA
A simple proof that introduces severe tangles into DNA
Inventing the pure Kell system. Those assays are low pH
is based on promoting H-DNA stability in
. Fragments of DNA containing the H-forming region were electrolyzed at low pi (
When subjected to pneumophoresis, H-DNA-induced tangles become H-
compared to mobility in T)H, which is not favorable for DNA formation.
, slow down movement (Ftone and Dalbelve (Htu)
(1988), supra.
). We found that -2 in the GAP-43 promoter
Purine homobomer region from 40 to -370 or in
Stable H-DNA structure in linear DNA in vitro
This mobility shift proves that it is possible to form
developed the method. Figure 15 was analyzed by gel electrophoresis as described below.
Representing a restriction digest fragment of the GAP-43 promoter
It is. Possible H-DNA formation homopurine-homopi
The limidine region is shown in bold. When performing Kel electrophoresis
In this case, a part of the digested material shown in Fig. 15 was preliminarily adjusted to pH 7,
Equilibrate with Tris-acetate at either 4 or 40 min.
Loaded onto a 14% agarose gel and run in parallel.
I set it. The band shifted at pH 4 is due to the base shift from B to I.
The patient showed a smear that may have been caused by
Htun and Dahlberg
, Science (Sc 1ence), 241: 17
91-+795 (1988)). Homopolymer area■
(i.e. upstream (-240 to -370) homogeneous
Is it only the band that contains the fragment containing the stretch?
This assay shows a change in mobility at p) 14.0.
did. Plasma from outside the marker or promoter region
Fragment of lasmid or region when separated from region I
Even in fragments containing areas ■ and ■, observable /
There was no fut. Thus, only the upstream region is
showed a corresponding shift. Fragments containing areas ■ and ■
Note that the pieces did not shift at pH 4. When DNA outside the homopolymer region is gradually removed,
The relative shift in mobility increased. Region I of similar size
region ■ than if it existed alone in a small fragment.
It is considerably larger when included in the fragment along with region I.
A significant shift in mobility was observed. In this assay, regions ■ and ■ are their own
Couldn't influence the shift, but they
It may act in concert with upstream regions. Heterogeneity of GAP-43 Transcription Initiation Another notable feature of the GAP-43 upstream sequence is the T
ATA motif is absent. Genes that lack the TATA sequence that directs the initiation of transcription are often
They often have multiple heavy RNA start sites. This was found to be true for GAP-43.
. Using RNA5e protection analysis, several tissues have shown that G.
The transcription start site for AP-43 was determined. lung
, from the dorsal root ganglion (DRG) and cerebral cortex (CTX)
RNA was extended to 1475 bases from the translation start site.
It was analyzed using a microprobe. Using this probe, -47
/-48, 51/-52, and -78 transcription start
Three major bands were protected depending on the location. The identity of these same sites was confirmed by brimer extension.
did. Furthermore, the secondary band becomes visible after long exposure.
was made into Some transcripts around 230 compared to dorsal root ganglia
To a large extent, mRNA from the cerebral cortex
exist. GAP-4 in the central and peripheral nervous system
Three genes are differentially regulated (Skene et al.)
et al), Journal in Cell Biology
(J, Ce1l Biol,) 89: 86-95;
Journal in Cell Biology (JCell
Biol, ) 89: 96-103 (1981))
This is interesting given observations that suggest that
be. Therefore, other areas of the CNS as well as (sympathetic
PCI2 cells (believed to originate from adrenal progenitor cells)
RNA from the cells was analyzed. From the hippocampus, cortex and cerebellum
RNA from DRG or PC12 cells
Percentage of transcripts starting from the region around -230
is high. However, these longer messages
The amount of each is relatively small. If you start above -234
When using a probe that pools all messages
In this case, there are significant differences between the start sites in the CNS and PNS.
It became clear. This analysis shows that longer GAP-43
The photograph is actually a complete GAP-43 transcript.
and that these transcripts are
CNS than RNA from DRG or PC12 cells.
This was shown to be common in the RNA of these individuals. in short
, the 5° end of GAP-43 mRNA is of heterologous origin;
The upstream starting site is the central nervous system compared to the peripheral nervous system.
It is usually used in the meridian system. 4. This embodiment of the present invention contains the GAP'-43 gene.
The present invention is directed to the isolation and characterization of Chemistry sequences. 1
．． The three small exons corresponding to the 5kb mRNA are
, to at least 24 and 20 kb introns, respectively.
Therefore, they are separated. The promoter region is highly unusual. Triple chain “H-D
There are several lengths in the upstream region that can potentially form "NA".
There is a long homopurine-homopyrimidine stretch.
(Veils et al., F.A.
・Nis E B Journal (FASEB J,
) 2: 2939-2949 (1988)). Here, in fact, one of these areas is
It is shown that H-DNA is formed in tro. The promoter lacks the canonical TATA port,
It has a transcription initiation site. The uses of some of these parts are
Different in different parts of the nervous system. ) The GAP-43 gene spans at least 50 kb.
A single core consisting of three exons and two introns
It is a pea gene. The present inventors have determined that the exon is
We obtained some evidence that corresponds to a functional domain in
. encodes only the first 10 amino-terminal residues
The first exon is responsible for membrane targeting of GAP-43.
Contains stretching that causes this. 3rd place for protein OJ Hi 4
The cysteine in position is acylated, and as mentioned above,
, may be involved in membrane binding. The amino terminus is the membrane of GAP-43
It is necessary for the binding and, as mentioned above, the growth of foreign proteins.
to the same membrane domains as GAP-43, including those of the cone.
Contains sufficient information to direct the user. The second exon is a carmo from amino acids 43 to 51.
Durin-binding region (Alexand et al.
er et al), Journal in Biologica
Le Chemistry (J, Biol, Chem, )
263: 7544-7549 (1988)) Nara Hini
The cell at position 41 is a substrate for protein kinase C.
Phosphorus (Coggins and Coggins a
ndZwiers), Society in Neurosa
Jens (Soc, Neurosci,) Abstract
(1988)). Exons I and ■ are
Among fish and some mammalian GAP-43 proteins
Contains well-conserved regions (Lahate and Skate
Labate and 5kene, (1989
No. The promoter of GAP”-43 has differences in sequence and structure.
Always. TATA, lack of food and its consequences
The use of multiple start sites in the GAP-43 promoter
constitutively expressed housekeeping (hou
cause similar to the promoter of the gene (seekeeping)
becomes. However, the GAP-43 promoter
housekeeping gene
Consensus Sp- that has been associated with promoters
1 binding site (GGGCGGG)
nan), Trends Gen
et.al.)2: ] 96-197 (1986)). Furthermore, the closely regulated role of GAP-43 in development
expression, its specificity for neurons, and especially in adults
Its inducibility in neurons of
This suggests that it does not belong to the Russ gene. GAP-43 is differentially regulated in the central and peripheral nervous systems.
It is stipulated. For example, axotomy of mammalian central neurons
The expression and transfusion of peripheral nerve axotomy (GAJ) 43
cause an increase in traffic vs.
(Skene and Williard
illard), Journal in Cell Biology
- (J, Cell Biol, ) 89:9
6-103 (1981). As mentioned above, GAP43
does not appear to be irreversibly suppressed in the CNS;
Although it may play a role in formability other than in growth (Beno
Benovitz an
d Routtenberg), T.I.N.φx
S (T, 1.N, S,) 10:527-532 (19
87)), differences exist in central and peripheral regulation.
It is clear that there are Therefore, different star I.
The use of sites allows for mRNA amplification from different neurons.
Different possibilities exist at the five ends that modulate somesome binding or stability.
suggest potential. Examples include, but are not limited to, genes expressed in neurons.
The gene Thy-+ is regulated at the transcriptional level along with development.
demonstrated to be expressed in a tissue-specific manner
It is interesting to note that (Svanopoulou et al. (Sp
anopoulou et al), Molecular A
Cellister Biology (Molec, Ce1
1. Biol, ) 8: 3847-3856 (198
B)). Also, like GAp-43, Thy1-promoted
The selection of the transcription start site in the transcription factor varies between expression tissues.
may vary, with upstream start sites being more prominent in the brain.
(ibid.). This is true for both GAP-43 and Thy-1.
Membrane vs. Additional Levels of Control in Other Tissues
suggests. Possible upstream regulatory elements present in the GAP-43 promoter
The element is a consensus Pit-1 binding site (TA, TTCA
TG). This sequence and related sequences collectively represent P
Recognized and bound by a transcription factor known as OU protein
Ru. This group originally belonged to Pit-1,0c in mammals.
t-1 and 0ct-2, and in C. elegans, un
c86 (Herr et al.
Genes De
velop, ) 2°1513-1516 (1988
)), proteins occupying two peptide regions that characterize this family.
The discovery of a cDNA that codes for white has recently led to the expansion of white color.
Great (He et al), Nature (N
ature) 340: 35-42 (1989)). As described in the Examples below, the inventors have demonstrated that GAP
-43 in the brain that can bind this region and regulate its transcription
- A specific protein was identified and cloned. Another notable feature of the GAP-43 promoter is that
Due to the presence of a long homopurine-homopyrimidine stretch
be. These are interesting. Because that
They combined a protein specific to the GAGA stretch (pipeline).
Biggin and Tjia
n), Cell 53: 699-711 (1
988); Gilmour et al.
, Science 245: 1487
~l 490 (1989), they are H-D N, A and
We now have the possibility of adopting the so-called triple-chain formation.
This is because that. Such homopolymer regions are found in eukaryotic genes.
ten at the 5' ends of progeny and eukaryotic viral genes.
It has been found that the transcriptional regulation is expressed in
It is speculated that it seems to be somewhat involved in (Wells)
Yells et al.
-B Journal (FASEB J, ) 2:
2939-2949 (1988);
Htun and Dahlberg, Sa.
Science 243: 1571-15
76 (1989)). For example, perhaps protein interactions
Therefore, if we adopt the stabilized H configuration, DNA
It is hypothesized that it causes kinks. This twist is
Order nucleotidemes by keeping out kinked regions.
This allows the DNA around the kink to become a transcription factor.
closer to the child (Futone and Tarbelb (H
tun and Dahlberg), Science (S
science) 241: 1791~] 795(1
98B); Han and Grunstein
dGrunstein), Cell 55:
1137-1145 (1988)). In addition, it will take
Dilation causes upstream sequences to juxtapose more closely to their downstream,
Allows interactions between sequences or proteins bound to them
(Futone and Tarbel
Htun and Dahlberg, Scien.
Science 241:1791-1795 (1
988no. Instead, the H-DNA is linked to its immediate neighbor.
repressor of transcription by directly blocking access.
(Maher et al.
et al), Science 245
725-730 (1989)). Accordingly, further embodiments of the invention provide that the internal region, the
As shown in Figure 1, the code for nomic GAP-43 is
nucleotide sequence, or its functional or chemical
derivative, and encoding the GAP43 promoter.
The nucleotide sequence shown in Figure 14 or its
Consists of functional or chemical derivatives. GAP of the present invention
-43 promoter or modify the activity of GAP-43 itself.
In addition to constructing other structures using methods well known to those skilled in the art.
as a means of achieving desired modifications in gene expression
It is understood that it is extremely useful.
Will. Even more broadly, this aspect of the invention substantially
It concerns a promoter as shown in Figure 14.
, T A T A box and common 5p-1 binding
Deleted sites or multiple start sites and common Pi
characterized by containing a t-1 binding site, and
Long homopants that have the ability to form double-stranded (H-DNA) structures.
Characterized by consisting of a phosphorus-homoprimidine stretch
do. in the phase, at its amino terminus, as described herein.
The nucleotide sequence and GAP-43 promoter of
structural genes consisting of their functional or chemical derivatives;
, or fragments thereof are also contemplated by the present invention.
Form a concrete example. In a further specific example, a DN consisting of the above structural gene
A expression vector and transfection
host cells and the proteins produced by them.
provide Example■ Neuron growth cones transmit signals from the microenvironment to axons and
has a special conversion mechanism that converts it into directed growth of dendrites.
Two. Neonatal caries abundant in growth cone membranes, subcellular
The fractions have simple and distinct protein compositions. growth cone
The two major non-cytoskeletal proteins in the depreparation are 40,000
and has a molecular weight of 35,000. electrophoretic, immunological
These are determined by scientific and partial protein sequence evaluation criteria.
The protein is GTP-binding protein G. alpha and beta
It was identified as a subunit. neuronally differentiated la
Immunohistochemical staining of pheochromocytoma cells revealed cell projections.
A high concentration of Go alpha subunits is present at the distal end of the brain.
be done. These take into account that the regulation of growth cone motility is
. Suggests that it seems to use a signal conversion mechanism
. Achieved during brain development and refined through synaptogenicity
The complex state of neuron connectivity caused by neuron axons is
requires selection of specific targets. Thereby, those
Mechanisms by which axons convert information from the extracellular environment into directed growth
Kanism is not well understood. neuron axon
The distal end has a unique superstructure called a growth cone, which
is considered to be critical for this protrusion (
Bray, D., et al.
, Ann, Rev.
Ce1l Biol, )4°43 (1988)). happiness
In particular, the membrane of the axonal growth cone and thus its transformation
The system can be fractionated from other Neelon configurations (Fe
Pfenninger, et al.
K, H,, et al), Cell 3557
3 (1983); Gordon-Weeks-Bi et al.
rdon-Weeks, P., et al),
Euroscience 13
: 119 (1984); Ellis et al.
, L., et al), Journal Off Cell By
Ology (J, Ce1l Biol,)l O1゜19
77 (1985)). It is one of several major proteins in cancer.
Some of these proteins have been identified.
°Tubulin, actin, and neuronal-specific, growth
-Related protein, GAP-43 (Feninkel, Ray, A.
Pfenninger, KH, et al., Cell
(Cell) 35: 573 (1983); Elisu
Ellis, L. et al. Off Cell Biol (J, Ce1l Biol,
) I O1: 1977 (1985); Simkovinots
・Bii et al. (Simkowitz, P., et.
al), Journal of Neuroscience (J,
Neurosci, ) 9: 1004 (1989)
; Cheung, N, et al.
al), Journal of Biological Chemists
Lee (JBiol, Chem,) 263: 3935
(1988): Meiri, K.F. et al.
K, F, et at), Prontings
Off National Academy Off Sciences (
Proc, Natl, Acad, Sci, )U
S As2・3537 (1986); Sukefu Shay
・H.B. et al. (Skene, J, H, P, ,
et al), Science 233
: 783 (1986)). Other major growth cone membrane configurations
Characterization of axonal responses to extracellular cues
It will be explained. Growth cone membrane fraction was prepared from neonatal rat brain (phenylated
Nker Kei Eichira (formerly Pfenger, KH
,, et al), Cell 35: 573
(1983) Ellis, L.
, et al), Journal of Cell H Biol.
J, Ce1l Biol,) 1011977 (1
985) Simkowitz-Bi et al.
, P, , et al), /Yarnal Off New
Curosci (J, Ncurosci, ) 9:
1004 (1989), Cheu
ng, N., et al), Journal Off.
Biological chemistry (J, Biol, che
m,) 263: 3935 (1988)). This preparation has a simple protein composition by 5DS-PAGE.
(Ellis, L., et
al), Journal of Cell Biology (j. Ce1l Biol,) I 01: 1977 (19
85): '/Simkowit
z P,, et al), Nyanal Off Neuro
Science (J, Neurosci, ) 9:1
004 (1989); Cheur
+g. N,, et al), / Yarn Off Biologica
Le Chemistry (J, Biol, Chem,) 263
: 3935 (1988)). 50-55000 tal
The most strongly stained hand that moves in the tube is the tube.
was identified as phosphorus (Simkovinot-Biy et al.
With, P. et al), Journal of Neuroscience
(J, Neurosci,) 9: 1004 (19
89); Cheug, N., e.
tal), Journal of Biological Chemistry
Story (J, Biol, Chem,) 263:
3935 (1988)). Also, p34 and p38
about 35,000 and 4, especially abundant in the growth cone membrane.
There is an important protein with an M of 0,000 (Simco
Simkowitz, et al., Journal Sub Neurosci.
(J. Neurosci.) 9: 1004 (
1989)). Two unidentified proteins were further characterized.
There is white. With the exception of cytoskeletal proteins, they are the most abundant in growth cone depreparations.
is also an important protein. p34 and p38 are GTP-binding protein G. alpha of
and have a molecular weight similar to that of the beta subunit.
(Stryer, Stryer, etc.)
L, et al.), Anu Lev Ser Haio
Ann, Rev, Ce1l Biol,) 2:
391 (1986); Kilman A./I (Gi
1man, A, G, ), Anu Rep Hiokem
(Ann, Rev. Biochem, )56:
6 ] 5 (1987)). Purified orchid grainG
By co-electrophoresis of o and growth cone membrane, p34 is G. Moves with Noheta Subuninoto, p38 is alpha
It was shown that it moved with the subunit. immune pro
p34 was detected using anti-beta subunit antiserum using the kit method.
p38 reacts with anti-alpha subunit G. anti-blood
It was shown that it reacts with pure water. Furthermore, the prominent protein species of p38 is alpha. It must be
stomach. This is because it is determined by Koman Blue staining.
and equal protein concentrations of p38 and alpha. teeth
This is because they showed the same immunoreactivity. That is p3
4 and G. This also applies to the beta subunit of
Ru. This antiserum is alpha L subuni-nod.
A. The reactivity is about 1/10 of that of Kirman A.N.
Gilman, A.G., Anu Lev Ba.
Iochem (AnnRev, Biochem,) 56:
615 (1987)), therefore Alpha. immune response
Unable to explain the main basis of gender. Confirming these immunological and electrophoretic data
In order to
Figure 16). Both p34 and p38 are blocked at their amino termini.
there was. From trypun fragments separated by HPLC
, and staphylococcus separated by 5DS-PAGE.
Coccus aureus (Staphaureus) V
Sequences were obtained from the 8 protease partially digested fragments. p38?
The sequences of each of these three peptides are alpha. Same as later
And alpha. is a major component of p38 protein
was confirmed. Other known alpha subunits are similar
However, it has its own unique arrangement. The three types of p34 proteins are G
It has a sequence identical to that of the beta subunit of the protein.
there was. The two peptides are beta and beta. The subunits are from the same region and the third
one is beta, and a mixture of sequences about beta,
It contained things. Thus, the alpha and vector of G
The data subunit is a major constituent of the subcellular fraction of the growth cone membrane.
It's a minute. Are these preparations substantially enriched in growth cone membranes?
, they are not pure (Fenninger K.H.
(Pfenninger, K. H., et al.
), Cell 35:573 (1983);
Gordon-Week
S, P, et al), Neuroscience (
Neuroscience) ] 3 : ] 19
(1984)). Previous immunohistochemistry of end-fraction tissue was performed by G.
o is concentrated in the neuropil of the adult rat brain (wo
- Worley, P.F., et al.
, etal), Proceedings Sub Nanyona
Le Academy Sub-Sciences (Proc, N
at IAcad, Sci,) USA83: 456
1 (1986)), and the related G protein G. 1. becomes an adult
located in the terminal region of primary olfactory neurons (in
Jones, T.T., et al.
, et al), Science 24
4: 790 (1989)), and Go was cultured.
Stain the entire primary neuron or areas of cell-cell contact.
(Ione T.T. et al.)
s, D, T, , et al), Science (S
science) 244: 790(] 989))
I'm talking about this. These studies indicate that G in the growth cone. It does not contradict or prove the localization of . subordinate
Therefore, immunohistological methods were applied to NGF-treated PCI2 cells.
G in intact cells using. The pores were determined. NGF
These cells extend long processes tipped with growth cones.
allow it to be lengthened. - in those of the next neuron
In general, the neuronal protein GΔP-4,3 is responsible for these formations.
Abundant in long cones (Pan Hoop C.O.M.
(Van Hoofr, C, O, M, et
al), Journal Off Cell + 7' + Iolon (
1, Cell Biol, ) 108: 1115 (1
989)). alpha. immunofluorescence of PCI2 cells.
They were found in high concentration at the growing end (exclusively found at
It's not like it's going to happen. X! i Conventional method for preparation of wide-growth conical membranes (Pfcnni K.H. et al.
nger, K. It, et al), Cell (
Cell) 35: 573 (1983), ff-Lis
・ff-le et al. (Ellis, L, etal), /ya
Null Off Cell Hiolo/-(jCell Bi
ol, ) 101: 1977 (1985);
Simkow-itz, P.
, et al), Journal of Neurology
Ens (J, Neurosci, ) 9 : ] 0
04 (1989) Cheung N et al.
et al), Journal of Biological Sciences
Chemis)-Lee (J, Biol, Chem,) 263
: 3935 (1988)) with a slight modification, the growth circle
Cone membranes were prepared. Cut off the neck of the SD under 24 hours old, 032M sc
loin, 1mM Tris-HCC, 1,mMMgC (!
, pH 7.6 in 5 times the volume, Karas/Teflon homoke
Homogenize the brain at 400 ml in 6 passages in a
Ta. The following protease inhibitors were used during the procedure:
・lQOμg/mρ soybean tribun inhibitor, 1) 1g/
m12 pepstatin A, 30 μM ipebutin (Ie
upeptin), and 1mM PMSF80.7
Stage graphs of 5M, 1.0M and 2.2M sucrose
ffl bottled homogenate was layered on top of the agent. Applicable
Centrifuge the Kura/Yunto at 250,000 x g for 40 minutes and
32/○ The 75M interface was collected as a growth cone particle fraction.
Ta. Both volumes were mixed with 5mM Tris-HC (l!, pH 7.6).
and centrifuge at 250,000 x g for 40 minutes.
The membrane was collected. , the membrane was treated with 2011 g/mQ saponi
and 0.3M Na, SO2.
and collected again by centrifugation. Lie brain G
. 1 above. (Pray-Day et al. (B
ray, D., et al), Anu Lev Cell
・Biol (Ann, Rev, Ce1l Biol,)
4:43 (1988)). Anti-bovine brain alpha in production of anti-serum. and anti-beta anti-
Serum production and characterization heights
(F.R.M. et al.) turf,
R, M, et al), glossy off-bye
Oroncal Chemistry (J, Biol, Che
m, ) 26010864 (1985)). Contains SDS
Run immunoplot samples on a 10% polyacrylamide gel.
Electrophoresis is performed using
Transferred to lurose. 10mg/nonspecific protein binding site
m (J)/blocked with serum albumin and incubated at 4°.
1400 anti-alpha antiserum or l: 10
0 anti-beta anti-natant was added to the late ink-1 (
Fu R M et al. (t(ulT, R, M, ,
et al), /Yarnal Off Biological Ke.
Mistry (J, Biol, Chem, ) 26
010864 (1985)). Beluoki/Tase substrate and
Avinone-biotin complex using tetrahen/n
physical therapy (Vectastatin, PA
-Burlingame, California
Bound antibodies were detected by A. Amino Acid Sequencing of Growth Cone Membrane Proteins Growth cone membranes were fractionated as described above. Polyvinyl protein
When transferred to Nylfluorin (PDVF) membrane, p34
and spots for p38 gave no sequence
. This is because the protein is probably blocked at the amino terminus.
This is probably because it is. Therefore, the protein sequence is p34 and
and proteolytic fragments for p38. Tripunun
For digestion, proteins are transferred to nitrocellulose and pumped.
It was stained with saw S and appropriate bands were cut out. barber
Microchemistry Funality (Harva)
rd Mierochemistry Faculty
In this case, the tribun digestion was carried out on a nitrocellulose membrane.
and the released peptides were transferred to reverse phase 1−+ PLC.
Therefore, the separation was performed using a gas-phase automated sequencer (Morss-M et al.
Moos. M,, et al), Journal Sub-Biologica
Le Chemistry (J, Biol, Chem,) 263
: 6005 (sequenced in 1988 houses. In addition,
Staphylococcus aureus (Staph, Au
reus) V 8 protease (Herlinkel (Bo
Ehringer), Mannheim (Mannheim)
The amino acid sequence was obtained for p34 by partial digestion with
Ta. V8 polyacrylamide cubes containing p34
Digested in 5 ita and fractionated by DSD-PAGE,
PVDF membrane (Millipore, BET)
Ford (Bedford, Massachusetts)
Electrically flown and visualized with Combasy Blue (not visible).
T.T.E. et al. (Kennedy T.E.,
et al), Proceedings Sub National
・Academy Sub Science (Proc, Nat
l. Acad, Sci,) USA85: 7008 (19
88)). The peptide fragments were cut out and the Columbia University researchers
To Huyukes Mechanical Institute
Rotin Chemistry Core Facility (Ho
ward) rughes Medical In5t
ituteProtein Chemistry Co
Applied ``Iosis'' at re Facility)
Thames (Applied Biosystems)
Foster City, Cali
Sequencing with a 470A gas phase sequencer (Fornia)
The sequence p34-B was obtained. PC12 cell alpha. Immunostaining] PC12 cells in the presence of 00ng/mff nerve growth factor
Cells were grown on poly D-phosphorus treated coverslips for 48 hours.
I let it grow. The cells were placed in phosphate buffered saline (PBS).
fixed with 3.7% formaldehyde in 0.01
% Triton-X-100. 5 in PBS
Incubation with mg/mρ bovine serum albumin
After lysis, the cells were incubated with 1:1000 anti-wine at 23°C.
Shi brain alpha. Incubate with antiserum for 1 hour and rinse with PBS.
, 3% H,O, for 15 minutes.
Reduced background ground. Conjugated rabbit immunoglobulin
Bryn is a Texas Red Consecutive (Texas)
s red conjugated) Donkey Tsusaki Ig
G(/Jackson Immunolo/Cals(Jackson)
Immunologicals)
I put it out. Find out the result. Identification of proteins in the axonal growth cone membrane by DSD-PAGE
did. Axon growth cone membrane, purified orchid brain Go, and crude
Two separate preparations of bottled homogenate were prepared in the presence of SDS.
Bottom, electrophoresis through a 10% polyacrylamide gel.
and stained with Comassie blue. Grow against crude bottles
2, termed p34 and p38 in cone membrane preparations.
Stratification of the bands was observed. These proteins are purified G. migrated with the alpha and beta subunits of Under these conditions, actin and GAP43 are apparently separated.
Child! I moved 143,000. Anti-Go immunoplot of growth cone membranes in G. exhibit reactivity
. Alpha coma sieve in both growth cone membrane preparations
Alpha moving in the position of Roux staining. Reveal immunoreactivity
I made it. alpha. The Comassie blue sign is P38.
Load the gel to be identical to that of the other pair as well.
Adapted for. A Comassie blue stained gel was run in parallel. As total protein increases, the pair is immunostained to a fixed degree.
, this is p38 standard alpha for this antiserum. and
The degree of fixation indicates immunoreactivity. This is Haton
Any or all I) 38 or alpha. to show that
suggest Alpha for Go samples. a small amount that moves beyond
There is some immunoreactivity, which is alpha, with slight staining of
It seems that this is due to the dye or cross-reaction with it.
. This was previously reported for beta and p34, respectively.
-Growths and plants shown to be identically stained with Massie Blue
and growth cone membrane (A) fraction were not observed, and anti-
Similar immunoreactivity with the beta antiserum was demonstrated. Immunoblot analysis of purified G. Preparation and G. of
They are exactly the same. The partial protein sequences for p34 and p38 are
As shown in the figure. The sequences of three peptides from p38 are rat
Alpha derived from the brain. harmonized with the sequence of three peptides from
Goh, J.
W, )Science 244980
(1989)). The sequences of the three peptides from p34 are
/Comparison with beta 1 and heta 2 from the brain
compared to Fing H.K. Tableu et al.
ong, H, K, 1. , et al),
Writings in Su'Yonal Academy
・Sun Enns (Proc, Natl, Acad,
Sci, ) U 5A84・3792 (+987no. The two peptides are beta and beta, or have the same region.
/i should be made to be identical to the area. Other Bepeti 1-
contains a mixture of sequences for beta, and
have Alpha, 1 immunoreactivity is present at the tip of the PCI2 process.
1, PCl differentiated by nerve growth factor, 2 cell alpha. Staining reveals high concentrations of antigen at the distal ends of cell processes.
It became clear. Also, in the area of the cytoplast around the nucleus
There was also some low-level, diffuse staining. The specificity of antibodies is
It has been proven (Huff, R.M. et al.
R, M, et al), Journal in Biology
BioChem
) 260: 10864 (1985): Wallay
・B.F. et al. (Nor!ey, P, F, , e
t al), Pro/-Tings in Su/Yonal
Academy in Science/Proc, Nat
l, Acad, Sci, )U 5A83:456
1 (1986) or, as a further control, excess purified
Taran brain G. (30tt g/mc) with antiserum.
incubation, or regular worms instead of antiserum.
Add to the same incubation and replace with Saki serum.
One sample was prepared. These controls had substantially less staining of cell processes.
. Discussion The results shown in this example are based on G proteins, especially G proteins. /J'C is a major constituent of the growth cone membrane. In fact, the growth cone membrane contains more than any other non-cytoskeletal protein.
Rimo G. There are many. first revealed in the brain
However, although the role of Go is not clear,
Generally, G proteins connect transmembrane receptors to intracellular signal systems.
Coupling Strye et al.
r, L, et al), Anu Lev Sel+Ha
Biol (Ann, Rev, Ce1l Biol,
) 2: 391 (1986); Gilman A.C.
(Gilman, A. G.), Ann, Rev.
Biochem, ) 56, 615 (1987);
Neel E./. Neer, E. J., Nat.
ure) 333: 129 (+988); Ross E
・M (Ross, E, M, ), neuron (N
Euron) 3: 141 (1989).
-seC, phosphoreverse A7, potassium channel and
Affects various intracellular signal systems including calcium channels
can be given (Sukefu・n・.−I・H・Bi et al. (S
Kene, J., IL p., et al.), Sa.
Science 233: 783 (19
86) Nistrinil L et al. (Stryer, Ll
, etal), Anu Lev Ser Paior (8nn,
Rev, Cell Biol, )2: 391 (+
986): Neer, E.
J.), Nature 333: 12
9 (1988): Ross, E.
M, ), Neuron 3・141 (
1989) Brown, A.M. et al.
A, M., et al), American Journal
Am, J, Physi
ol, ) 254H401 (1988)). Rod body
and retinal G protein trans in the cone outer segment.
Comparable to transduc in,
The surprising height of Go in the long cone membrane is due to the growth cone
G in This strongly suggests a transformation system based on . c Chemotaxis towards AMP or conversion via G protein
G proteins are important for developmental morphogenesis in Bacillus subtilis (Dicty. 5telium).
It turns out that white is very important (Snarn)
Snaar-Jagals
kaB, E., et al), F.E.B.
- Varnish Letters (F, E, B, S, Lett,) 23
2: l 48 (] 988) Snarrunyakal
Snaar Jagalska,
B, E, et al), F.E.B.
Letters (F, E, B, S, Lett,) 236
: 139 (1988): Kesbeke x Fu et al.
Beke, F., et al), Journal I
Cell Biology (J, Ce1l Biol,)
107: 521 (1988 no. Similarly, the developmental nervous system
signals from pathways or targets in the growth cone membrane.
bind to, G, chain receptor, or bind to receptor
and thereby increase the level of intracellular secondary mesosengers.
, thus changing the growth cone mobility. Generally these
The signals, receptors and secondary metsensors are currently unknown.
It is knowledge. Candidate receptors for class 1 are cell adhesion molecules.
, NCAM and Ll, localized to the neuron growth cone.
(Letour)
neau, P. C., et al), Daybelonob.
Development 105: 50
5 (1989)). Antibodies against these molecules are P
12 Calcium levels and phosphotyl in cells
Altering inositol metabolism, the effects of these antibodies are
Blocked by protein antagonist pertussis toxin (Han.
van troofT
, C, O, M, . et al), Journal in Cell Biology
(J, Ce1l Biol,) 108: l 115
(1989)). G in the adult nervous system. Persistence of expression (Walley Bi
Worley, P.F. et al.
al), Proceedings in National Academy
Temmy in Sciences (Proc, Natl,
Acad, Sci, ) U 5A83: 456
1 (1986)) is axonognes/su
enesis). Another growth cone-enriched molecule, GAP-43, also appears in the adult brain.
Exists in discontinuous regions (Benobinot L.A.I. et al. (B
enowitz, L. 1. , et al), Tren
Trends Neurosci,
) 10: 527 (1987);
SkeneH, J, P, Anu Re
Ann, Rev Neurosc
i,) 12: 127 (1989)). GAP43's
localization, the nature of its gene regulation, and especially its phosphorylation.
Relationship between visualization status and long-term potentiation in hippocampal slices
Routtenberg, A.
), New York Academy in Sciences
(N, Y, Acad, Sci,) 444: 980 (
(1989) showed that G A in synaptogenesis in adults
suggests a role for P-4,3 (Benowitz L.
Benowitz, L. 1., et al.
), Trends Neur
osci, ) 10: 527 (1987);
Skene, H, J, P,
), AnnRev, Ne
Urosci, ) 12: 127 (1989)). Pertussis toxin prevents long-term potentiation and is probably still the case.
G in the protrusion of. Note that it means
Worth it (Go J Dubliny (G o + J
, L), Science 244
: 980 (1989)). Therefore, Go is neuro
Axonal outgrowth during cell development and synapses in the adult nervous system.
modulating signals for gas formation. Example ■ GAP-43 is a novel internal regulator of protein binding. In another embodiment, the invention provides a method for treating GAP43 or cell
Inside G. Modifies the binding ability of other cellular proteins, including later
I am oriented towards the surprising discovery that it works in the same way. As far as we know, this is due to external cell receptors.
Significant new features comparable in effectiveness and availability to
class of internal regulatory proteins (rlRPJX, of which GAP
-43 is a representative example). Those skilled in the art will appreciate the IRP compositions and methods of this aspect of the invention.
The method detects proteins in neurons and non-neuronal cells.
allows for internal modulation of white activity, thereby increasing cellular activity and
and internal modulation of functions. Moreover, surprisingly, the amino terminal nucleotide of GAP-43
Synthetic peptides consisting of amino acids can be synthesized from intact GAP-43 protein.
G caused by. in GTP binding by
It was found to accurately replicate the modulation. For example, G.A.P.
The peptide consisting of the first 24 amino acids of -43 is GA
Stimulates GTPus binding to the same level as P-43 and provides sufficient
Acts as a GAP-43 agonist. short peptide
, for example, a peptide consisting of amino acids 1-10 is GT
Does not stimulate Pus binding, but GA for binding to Go
competes with P-43, resulting in a reverse aconist
It seems to act as a rse agonist). well known
It is customary by those skilled in the art to determine the specific
Such biologically active peptides whose actions are determined
forms another aspect of the invention. The biologically active synthetic IRP peptide according to this embodiment is
, the following array I Ml, CCMRRTKQVEKNDEDQK
IEQDGV;Il, MLCCMRRTKQV
EKNDEDQKIEQDGIIl, MLCC
MRRTKQVEKNDEDQKIEQDIV,
MLCCMRRTKQVEKNDEDQKIEQ;
V MLCCMRRTKQVEKNDEDQKI
E ;■MLCCMRRTKQVEKNDI':DQK
I；■、MLCCMRRTKQVEKNDEI
')QK~'l MLCCMRRTQVIKII
DIDQIX, MLCCMRRTKQVEKN
DIMiD＼ Ml, CCMRRTKQVEKNDE
Xl, ML, CCJRRTKQVEKND■
MLCCMRRTKQVEKNX IIl,
MLCCMRRTKQVEKX IV, ML
CCMRRTQVE;XV, MLCCMR
RTKQV:X Vl, MLCCMRTX
QXVll, MLCCMRRTK X~1. MLCCMRRT' XIX, MLCCMRRT'X, MLCCMR; A related embodiment of the invention provides synthetic IRP peptides as described above.
nucleotide sequence to be attached;
The columns can be readily determined by one of ordinary skill in the art after understanding the teachings of the present invention.
will be done. A further aspect of the invention is that the consensus amino acid sequence
GAP-43 and beta-adrenalinergic receptors
was directed to the discovery that the sequence is found in the hydrophobic-river-eu-cys-cys-x-basic-
Consisting of basic or functional derivatives thereof. In addition, the main
Bright IRPs and IRP peptides contain
It will also be appreciated that it is susceptible to lumitylation. Also, those skilled in the art will be able to understand the IRP proteins and peptides of the present invention.
Target protein activity can be improved by changing the structure of tides.
or enhanced and, if desired, suppressed, which is unprecedented.
You will understand that it can be done. Thus, one more
In a specific example, the desired protein and its binding substrate are
Introducing an effective amount of TRP peptide into a different environment
A method or proposal for stimulating the binding activity of the desired protein characterized by
Served. The desired protein is preferably a G protein.
, most preferably G. It is. The environment preferably includes:
a living cell that may be a central nerve cell or a peripheral nerve cell
This is the internal environment of the vacuole. Those skilled in the art will appreciate that the method of the invention can be carried out in vitro, i.
n 5 in situ or in vivo?
well-known in the field, as discussed here.
If we keep in mind the principles of acceptable administration, the latter is the most preferred.
you will understand what is wrong with you. The compositions and methods of the invention are particularly useful for neuronal formation.
Directed to the mechanisms involved in long- and napus-forming properties.
As such, one of ordinary skill in the art having the benefit of the teachings of this invention will
The internal regulation of protein expression is important in mammals including humans.
provide important opportunities for effective treatment of disorders associated with
, and treatment for the effects of neurological disease or dysfunction.
of particular value in preventing, ameliorating or reversing
You will understand that. For a given medicinal indication,
Decrease and enhance nerve growth or formation
That would be desirable. This is, for example, the l of the present invention.
RP peptide, or IRP peptide or physiological
Administration of targeted antibodies to the site of effect
This can be achieved by In addition, by such means, the desired
modulating the activity of proteins with a significant degree of control
is Kano. Thus, in another aspect, the book
The invention provides antibodies directed to the IRP peptides of the invention.
, preferably monoclonal antibodies, and their functional
or chemical derivatives of said antibody or said derivative thereof.
The conductor is optionally detectably or treatably labeled.
Anything you like is fine. In another embodiment, the invention provides pharmaceutically acceptable
consisting of an IRP peptide of the invention together with a carrier, and where
optionally comprising one or more therapeutically effective agents.
Pharmaceutical compositions and the present invention together with pharmaceutically acceptable carriers.
consisting of an antibody directed to the TRP peptide of the invention;
and optionally one or more therapeutically effective agents.
A pharmaceutical composition comprising: Pharmaceutical composition of the present invention
The use of
Do not perform any inconvenient experiments while keeping in mind the principles of administration given.
This may be accomplished by one of ordinary skill in the art. In another aspect, the present invention provides a composition of the present invention.
The neural cell is characterized in that an effective amount is administered to the nerve cell.
The present invention is directed to methods of modulating structural rearrangements in cells. In yet another embodiment of the invention, the G of the invention
Isolating G-like proteins from neurons using the AP-43 sequence
Ta. Protein of MW39000 was measured using GAP-43 column.
specifically binds GAP43 with high affinity.
It turned out that. 50mM Tris, 1mMCa (Jj
, , and 1mM IV1gcQt in a buffer consisting of
Cell extracts were introduced onto the column containing AP'43. Proteins were eluted in a single batch with equimolar EDTA buffer.
I let it happen. The protein is a polyclonal antibody against G protein.
no response. Thus, it is associated with growing neurons
It is a unique and novel protein that
form. Moreover, the IRP peptide of the present invention can be prepared by any known means,
For example, it can be produced by the above-mentioned genetic recombination method;
Nucleotide sequences encoding IRP peptides of the invention
The sequence is simply an effort of routine skill to locate the desired host source.
The current system is inferred and optimized. The novel approach of the present invention in the modification of cell transformation systems such as Go
The importance of the products and methods is that Go is a neuronal growth cone.
Combined with proof that it is a major non-cytoskeletal protein present in
If you think about it, it will increase. GAP-43, whose function was previously unknown, is G0
The discovery that GAP-43 modulates the activity of cellular
The long-standing relationship between the first and second metsengers in the system
This is proof that this is the ``missing part'' that has been considered.
Ru. Although I do not intend to be bound by any particular theory,
Sustained activation of G. by GAP”43 causes cells to
It ignores boundaries and thus causes it to multiply constitutively.
Dew. In addition, this result shows that the amino terminus of GAP-43
contains a sequence similar to that of G. is controlled from inside the cell.
This suggests that there is a group of other molecules that interact. G.A.
The amino terminus of P”43 is connected to several (beta receptors)
Cytosolic domain of G protein-coupled receptors
It is very interesting that it has a great similarity to
. This is the cytosolic domain of G protein-coupled receptors.
GAP-43 has a similar structure in the molecule, as does GAP-43.
G in places. This suggests that they can interact with each other. Internal regulatory proteins and internal regulatory peptides (in this specification, TE refers to
The novel modulating effect of ``rlRP peptide'') is
For example, stimulating or inhibiting
It will be more understood. In other words, IRPs
and IRP peptides, or such internal regulatory effects.
Other substances with
For example, Remington's Pharmacy.
Pharmaceutical Sciences
cal 5sciences), 16th edition, Mark Pav
Rishing Company (MarkPublishi)
ng Co, ), Easton, Pennsylvania (+9
80) Goodman and Gi
Gilman's Pharmacological Base
The Phar
macrofical Ba5is of Ther
apeutics), 7th edition, Macmillan Publishing
MacmillanPublic is
hing Co, ), New York (+985) or
are disclosed in standard reference works, including their current editions.
What are the possible pharmacological effects of J, Una, etc.
may have. Thus, these are aconists, partial aconists,
Created as a conist, inverse agonist, antagonist, etc.
use Those skilled in the art will not be able to characterize such effects or
For any binding protein with a mind, G (for 1
As described herein for G'rP binding,
This can be done using well-known standard assay methods such as
will be recognized. According to the present invention, from a desired protein and its binding substrate,
Introducing the substance to be screened into the environment
substrate binding in the absence of the substance.
Desired protein characterized by measuring increase or decrease
Screening for substances that have the ability to modulate the binding activity of
A method is provided for The desired protein is G protein.
Yes, preferably G. It is. Result GAP-43 is G. stimulates GTPγS binding to. Regarding the identified major growth cone membrane proteins, the present inventors
These components determine whether or not they can form intermolecular complexes.
I pursued what I wanted to do. Especially G. and GAP-43. Because they are
This is because it is a major non-cytoskeletal protein in the growth cone membrane.
. Gel exclusion chromatography, immunoprecipitation and affix
GAP-43/G by Nity chromatography
Attempts to physically isolate the o complex were unsuccessful. Transient G A P-43/G o interaction in solution
In order to identify the purified G. (35S)G for
The binding of TPuS was determined by the presence of purified GAP43 at various concentrations.
Measured in Japan (F.R.M. et al. 01uff, R.
, M., et al.),
Ro/Cal Chemistry (J, Biol, Che
m, ) 260: 10864-10871 (198
5); Northrup Noei Kay et al.
p., J., et al.
Ioloncal Chemist (J, Biol, Che
m,)257: 114]f3-]1423(19
82)). GAP-43 itself is (35S) GT PuS
GAP-43 is not bound to G(, for
specific (355) GT P u S binding as a control]
, stimulate to O+ 40% (n==7) level (first
(See Figure 7A). This effect saturates at 150,800 nM
In the presence of GAP-43 it is half optimal. G.O.
and GAP-43 is not included in the solution and in v
If they are membrane-bound iv, their If is
It is approximately 2 μM in the brain. Therefore, as described in this specification,
GA, P- for Go measured in the assay
The affinity of 43 is inconsistent with the in vivo state [7]. vinegar
The assay is 200μg, /'TnρBSA presence.
The non-specific detection by G A P-43 was
Protein effects cannot explain the stimulation of GTPuS binding. ,G
o is in 3(')'C without G T P u S
Partially inactivated during incubation and incubation
It is known that
Dowd, B,'F, ej al), Sharnall
Fu6bu Bioloncal Chemistry (JRial,
CI-+0m, )263: ]59ε35-1
5992 (1988)), cAp-43 is G. Hane
Does not affect the degree of stability. The effects of compatibility with G proteins have been evaluated under various conditions.
The ruricanth receptor complex is GAl)-43?
Stimulates GTPuS binding to a nearly fixed degree (flow
Rio Buie Aan 1- Sternways P
−/−(Florio, V., A. & Sternwei
se P, C,), / Yarnal Inφ Nokioroshi
Cal Chemistry (1, Biol, Chem,)
2643909-3915 (1989)). GAP-43 decapeptide is G. interact with The amino-terminal tecabebutite is critical for membrane tethering.
Is nostein also required for G0 regulation?
In order to determine the
A decapeptide substituted with onine was synthesized. This pep
Tide has the characteristics of 1-10 peptide effects and/or stability.
(35S) GTPuS results for Go indicating the need for
(Figure 17c). is known to stimulate GTPuS binding to G proteins.
Another group of proteins that are involved are hormone and neurotransmitter receptors.
- is. Whole hormone and neurotransmitter receptor structure
structures (e.g., a large extracellular region and seven transmembrane domains)
In) is very different from the structure of GAP43. Therefore, we believe that
By focusing on these domains that appear to be
I have been pursuing the homology between proteins. Regarding receptors
and position-directed mutagenesis and peptide competition studies.
, in G-protein canobling, the third cytoplasmic loop
The carboxyl terminus and the vicinity of the cytoplasmic tail are related.
(Konig, B. et al.
,), Pro/-Dinges in National Akate
Me in science nee nis nee (Proc
, Natl, Acad, Sci, U, S, A,
) 86, 6878-6882 (1989);
0'Dowd B, F, et al.
), Nyanal in Bioloncal Chemistry
(JBiol, Chem,) 263: ] 598
5-] 5992 (1988)
Tee et al.
/ Yarnal in Biological Chemistry (J
, Biol, Chem,) 262: 16439
-16443 (1987)). Gs and β, - adrenaline
The binding of agonist receptor receptor 7 occurs in the cytoplasmic tail of the protein.
By point mutations in the valmitylated protein of
most specifically interfered with (O'Dowd B.F. et al.
'Dowd, B.F. et al.), Journal
In Biological Chemistry (JBiol,
Chem, ) 264: 7564-7569 (198
9)). G, access of GAP43 required for regulation.
Cystine at the mino-terminus also in viv. Palmitylated with
- Anto Vilag Eye (Skene, J, H,
P & Virag, 1. ),/Yarnal In Se
Le Biology (J, Ce1l Biol,) 108
2613-624 (]989)). series of receptors
The amino-terminal part of the cytoplasmic tail derived from GA, P-43
By comparison with the amino terminus, the hydrophobic -] e u-c
y s-c y sX- Base Determine whether the single base has a common sequence
(Figure 18). Within this array, the studied system
is valmitylated (skim:・JH・
Skene, J, H
, P., &Virag, 1. ), Journal in
Cell Biology (J, Ce1l Biol,) 1
08: 613-624 (1989); O'Dowd B.
-xFura (0' Dowd, B, Feta+,)
, Journal in Biological Chemistry (
J. Biol. Chem.) 264: 7564
-7569 (1989); Inkinikov Y.A. et al.
(Ovchinnikov, Y.A., et al.)
, FEBS [, etts. 230: 1-5 (1988)). Other endogenous regulators of GTPuS binding to G proteins are unknown.
It has not been done. However, it activates G protein in the opposite way.
There is a peptide toxin called mastoparan (Hikasojima te
Eera (Higashijima, T, etal,)
, Journal in Biological Chemistry (
J. Biol. Chem.) 263:64
91-6494 (1988)). GAP"43 is this
Not peptide homology. It has a large extracellular region and seven transmembrane domains.
Their overall structure is quite different from that of GAP-43.
However, regardless of these, the present inventors
We have been pursuing the homology between the amino terminus of GAP~43 and GAP~43.
. β, - Interaction between adrenergic receptors and Gs
The action is on valmitylated cysteine in the cytoplasmic tail of proteins.
most specifically disturbed by point mutations in . Cysteine at the amino terminus of GAP-43 is also valuminum.
Chilled. Hydrophobic cysteine is easily valmitylated
-leu-cys-cys-xbasic-more basic
have both GAP-43 and these receptors.
Is there a common array? GAP-43 binds to calmodulin and its interaction
It has long been known that calcium is enhanced in the absence of calcium.
(Alexander K.A. et al. (AIex)
ander, to, A, et at,) journal i
Biological Chemistry (J, BiolC)
hem, ) 262:6] 08-6113 (1987))
. The major physiological issues in this regard are not clear. G
Low grade Cd-calmodulin for o
c A by the addition of EGTA-calmodulin
P-43 (35S) in its ability to stimulate GTPuS binding.
There is no change. However, higher concentrations of carmodia
GAP-43-stimulated (355) GT P
Decreases uS binding in a dose-dependent manner (Figure 19)
. The author's research shows that intracellular formation occurs during neuron morphogenesis.
Growth in the cooperation of extracellular signals on long-related expression
Provides a conical mechanism. G in the growth cone membrane. of
Surprisingly high levels of alpha and beta sub units
is G in the neurite XfM] node. major about
Suggest a role. G in the growth cone membrane. 21 fjt is
Highly specialized outer segment of retinal photoreceptor cells
Another G protein, tra
nsducin) over 7a degrees. In a system where G protein function is clearly defined, part 1] is
Binding of extracellular signals to transmembrane receptors and intracellular secondary mechanisms
Regulation of enzymes or ion channels that modulate sosenya
It is a linkage between the clauses. Second its alpha subunit
Rimmer's alpha beta (many heterogeneous)
There is a comma G protein. Generally, alpha polypeptides are
, agonist receptor complex or GTP instead of GDP.
It exists in a GDP-bound state until an exchange occurs. Next
The GTP-binding activated alpha subunit is a secondary
It exerts its effect on the metusene/ya system. endogenous alpha
Subunion hGTPa se activity is responsible for all signal transduction.
let Go is prominently expressed in the brain, where it is expressed as G
It is the main form of protein. In adults, the neural network
The aim of the present invention is that it is a major non-cytoskeletal protein.
G at the tips of neurites in proliferating cells. with position
Let's go. Go can respond to a variety of receptors,
potassium channel, phospholiper
Numerous intracellular systems including enzyme G and phospholipase A
Prepare. Some bases for growth cones and can be resolved
There is evidence that the effect involves G-protein conversion. growth cone
against cell adhesion proteins N-CAM and L+ localized in
The antibody stimulates calcium levels and protein in PCI2 cells.
Changes suhotin linonthole metabolism. These anti
The effect on the body is due to G o/G + suppressor pertussis toxin.
(Schuch,
[1,, et al), Neuron
3:13 (1989)). Certain heliosomes (
hel iosome) In neurons, there is a canton in G.
Serotonin, which acts through receptors that have been activated,
It is an excellent inhibitor of neurite outgrowth. The restricted localization of Go may be due to protein regulation or action.
mediated by two or more molecules, especially
suggests that it will be done. GAP43 is found in neurons
The protein is enriched in growth cones. Therefore
, we showed that G A P -43 or 6° interacts with
I was wondering if he would do it or not. GAP)-43 is G. fart
enhances GTPγS binding of In addition, synthetic tecapebuti
This small region of GAP-43 defined by
exerts its effect. GTPγS binding by GAP-43
The stimulation is similar to that by the agonist-receptor complex.
The tecabebutide sequence has these 1-septer and 1,000
Same sex. I don't intend to be bound by any particular theory.
Or, the most likely interpretation is that in vivo, G
AP-43 mimics transmembrane receptors and activates G.
sexualizes to give rise to GTP-binding alpha subunits, and then
Either that or it triggers the intracellular secondary mensesia system.
That's true. Alternatively, GAP-43 binding of Go/\
Masu G. - receptor or G. -Effector interaction
It may function to destroy the purpose. Also, G,
1) 43, by some unknown method,
G by receptors. It is said to be an effector of activation.
It is also possible. G by the growth-related protein GAP-43. Modulation of conic transformation system
connect extracellular signals to intracellular processes about neuron growth.
gram and provides a mechanism to control it. Furthermore, the system
is regulated by receptor kinases such as BARK.
phosphorylation of receptors, or protein kinase C
mediated other modifications such as phosphorylation of GAP-43 by
It can happen. In this model, GAP-43 is
G for extravesicular ligands. synergistically enhance the response of
, or G. overturning the dependence on receptors of
would reduce responsiveness to ligand by
. In the latter case, what happens during synapse formation
In addition, removal of GAP-43 reduces sensitivity to extracellular ligands.
It will restore receptivity. The effect of GAP”43 is
The net effect on the efficiency of the printer is the relative concentration of the components.
It will depend. GAP-43 is a known protein that directly responds to extracellular ligands.
G-
Unique among protein regulators. However, intracellular regulation of membrane-bound GTPase proteins
have an advantage. Normal RAS proteins are widely distributed
It is stimulated by the 120KD intracellular protein GAP. that
Despite the similarity of their names, GAP- and GAP-
43 is an unrelated protein. Region of GAP-43 that stimulates GTPγS binding to Go
and palmitylation of cysteine in receptors.
attach In the experiments of the present invention, in the non-valmitylated state,
The amino-terminal peptide and possibly (membrane pH
GAP-43 (prepared by II extraction) is present. Valmitylated vs. non-valmitylated GAP-43, and
The relative ability of G-linked receptors to stimulate G proteins is unknown.
Not yet. Rapid Valmitylation - Theory Palmitylation is this.
It is possible that these proteins play a regulatory role.
be. G in the adult nervous system. Sustainability of expression (Umeley Bi
Worley, p. F. et al.
al), Pro 7-Chings in Nanyonal Aka
Demi-in-Sciences (Proc, Na1l)
Acad, Sci, )U 5A83:4561(1
(986) for the preparation of neurite outgrowth during development and regeneration.
means a role other than In addition, GAP-43 is present in discontinuous areas of the adult brain, and
Immunohistochemical analysis of two types of proteins, excluding the brain.
are surprisingly similar (Benouzotsu, A.I., et al.
(Benowitz, l, 1., et al), training
Trends Neurosci
) 10: 527 (1987);
Skene, H, J, P, )
, AnnRev, Neur
osci, ) 12: 127 (1989)). G.A.
Localization of P-43, its gene regulatory properties, and characteristics
its phosphorylation status and its long-term increase in hippocampal slatus
Relationship with strong people (Routtenb)
erg, A, ), ANU New York Academy
・In Sciences (Ann, N, Y, Acad,
Sci.) 444: 203 (1985))
The role of GAP-43 in synaptogenesis in
(Benowitz et al.)
1.1. , etal), Torrens 1 Neurosci
(Trends Neuroscj,) 10.527 (
1987); Sken
e, H, J, P, ) + Anu Lev Neurosci
(Ann, Rev. Neurosci,) 12:1
27 (1989)). Go/Gl antagonists and pertussis toxins induce long-term potentiation.
This probably prevents Go in this process as well.
(Goh. J, W., et al.), Science
) 244:980 (1989)). Therefore, growth
on neurite extension between and in the adult nervous system.
Intracellular and extracellular signals for synaptogenesis
Can be converted. From the foregoing, those skilled in the art will be able to explain specific embodiments of the invention.
Although described herein for clarity, the meaning of this specification
Various modifications may be made without departing from the illustrations and scope.
You will recognize that. Therefore, the present invention is not limited except by the scope of the claims.
Not limited.

[Brief explanation of drawings]

Figure 1 shows a hybrid of GA P-43c DNA.
It is a figure showing selective translation. Figure 2 shows the nucleus of GAP-43! - Figure showing the sequence and predicted amino acid sequence. FIG. 3 shows the regulation of GAP-43 expression in PC]2 cells. FIG. 4 is a diagram showing the developmental regulation of GAP-43 gene expression and tissue characteristics. FIG. 5 shows A the nucleotide sequence and resulting amino acid sequence of human GAP-43 cDNA, and B the human, rat GAP-43 and mouse P57 amino acid sequences.
FIG. 2 is a schematic diagram showing the predicted stem-loop structure in the 3′-untranslated region of C. FIG. 6 is a Northun plot showing the regional restriction of GAP-43 expression with maturation. FIG. 7 is a Northun plot showing that GAP-43 expression increases after ischemic events. (Figure 8 is a photomicrograph showing the increase in GAP-43 expression in the area adjacent to the high velocity by in 5 in situ hybridization. Figure 9 shows the hypotension and hypoxia episodes of number[
(cAp in neurons of the cerebellar cortex later
43 is a micrograph showing enhanced expression of 43. FIG. 10 is a graph showing the influence of GA, P-4,3 on protrusion formation in the CI-10 cell line. Figure 11 is a schematic diagram of an experiment demonstrating that the amino-terminal exon is responsible for constant force assimilation of the GAP-43 protein into the cell membrane and that it directs membrane tethering of chloramphenicol acetyltransferase. be. Figure 12 shows that when CHO cells are transfected,
Conventional GAPi3 is a Western protein that has been shown to have both membrane and cytosolic components. FIG. 13 is a schematic diagram showing a map of the rough 1-GAP-43 gene. FIG. 14 is a schematic diagram showing the sequence of the GAP-43 promoter region. FIG. 15 shows mobility rats in restriction fragments induced by H-DNA. FIG. 16 is a schematic diagram showing partial protein sequences for p34 and p38. FIG. 17: G with GAP-43 and GAP-43 peptide. FIG. 3 is a graph showing the modulation of (35S) GTPuS coupling to FIG. FIG. 18 is a schematic diagram showing the respective sequences for comparing the homology between the GAPi3 amino terminus and the cytoplasmic tail of a G-coupled receptor. FIG. 19. GAP-43-stimulated G. G to sip
1 is a graph showing the dose-dependent decrease in TPuS binding by calmodulin. Patent Applicant: The General Hospital Two 5F Resicon

Claims (1)

[Scope of Claims] (1) Recombinant mammalian GAP-43 or a functional derivative thereof. (2) The composition according to claim 1, wherein the mammalian GAP-43 is rat GAP-43. (3) The mammal GA
The composition according to claim (1), wherein P-43 is human GAP-43. (4) The composition according to claim 2, wherein the rat GAP-43 has the amino acid sequence shown in FIG. 2 or an amino acid sequence substantially similar to that shown in FIG. (5) The composition according to claim 3, wherein the human GAP-43 has the amino acid sequence shown in FIG. 5A or an amino acid sequence substantially similar to that shown in FIG. 5A. (6) A polypeptide consisting of the amino acid sequence shown in FIG. 2, or an amino acid sequence substantially similar to that shown in FIG. 2, or a functional derivative thereof. (7) A polypeptide consisting of the amino acid sequence shown in Figure 5A, or an amino acid sequence substantially similar to that shown in Figure 5A, or a functional derivative thereof. (8) A cDNA consisting of the nucleotide sequence shown in FIG. 2 or a nucleotide sequence substantially similar to that shown in FIG. 2, or a functional derivative thereof. (9) The nucleotide sequence shown in Figure 5A, or
A cDNA consisting of a nucleotide sequence substantially similar to that shown in Figure A, or a functional derivative thereof. (10) A DNA expression vector comprising the cDNA according to claim (8). (11) A DNA expression vector comprising the cDNA according to claim (9). (12) A host cell transformed with the vector according to claim (10) or (11). (13) The host cell according to claim (12), wherein the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells. (14) GAP-43 produced by the cell according to claim (13), or a functional derivative thereof. (15) cDNA encoding mammalian GAP-43
transfecting a prokaryotic or eukaryotic host cell with a vector consisting of A, culturing the host cell in a suitable medium under conditions that allow expression of the mammalian GAP-43, and then transfecting the host cell with a vector comprising the mammalian GAP-43. A method for producing mammalian GAP-43 or a functional derivative thereof, comprising separating GAP-43 from the medium. (16) Hybridoma strain H5 having accession number ATCC HB 10316, or a functional or chemical derivative thereof. (17) A hybridoma producing a monoclonal antibody having substantially the specificity of the monoclonal antibody produced by hybridoma strain H5 having accession number ATCC HB 10316. (18) Monoclonal antibody MAb anti-GAP-43 (H5) produced by hybridoma strain H5, which hybridoma strain has accession number ATCC HB 1
Monoclonal antibody MAb anti-GAP-43 (H5) characterized in that it has 0316. (19) A monoclonal antibody or a functional or chemical derivative thereof having substantially the specificity of MAb anti-GAP-43 (H5), said MAb anti-GAP-43
(H5) is produced by hybridoma strain H5, which hybridoma strain has accession number ATCC HB 1031.
6 or a functional or chemical derivative thereof. (20) Claim (19) wherein the antibody is detectably labeled
Antibodies described. (21) Claim (19) wherein said antibody is therapeutically labeled.
Antibodies described. (22) Claim (1) together with a pharmaceutically acceptable carrier;
A pharmaceutical composition comprising the composition according to any one of (2), (3), (4), (5), (6), (7), or (14). (23) The composition according to claim (22), further comprising one or more therapeutically effective agents. (24) Contacting a suspected sample containing GAP-43 with the antibody of claim (20) and incubating the sample with the antibody to enable the formation of a GAP-43 antibody complex, thereby forming a complex. A method for measuring or detecting mammalian GAP-43 in a sample, comprising separating the antibody from uncomplexed antibody and then detecting the labeled complexed antibody. (25) comprising a carrier means partitioned to accommodate one or more container means in a closed compartment, the one or more container means being detectably labeled with a GAP-
1. A kit useful for measuring or detecting GAP-43, comprising a GAP-43 antibody. (26) The kit according to claim (25), wherein the antibody is selected from the group consisting of polyclonal and monoclonal antibodies. (27) A method for inducing GAP-43 expression in cells, which comprises exposing the cells to an effective amount of neurodevelopmental factors. (28) The method according to claim (27), wherein the cells are nerve cells. (29) The method according to claim (28), wherein the cells are exposed to the nerve growth factor in situ. (30) A method for enhancing the expression of GAP-43 in a cell, which comprises introducing into the cell a DNA expression vector consisting of a cDNA encoding GAP-43. (31) The method according to claim (30), wherein the vector is introduced into the cell by transfection, transduction, or direct microinjection. (32) Claims (28), (29), (30) or (
31) inducing GAP-43 expression in nerve cells by the method according to any one of paragraphs 31) and 33) promoting structural reorganization in nerve cells in or around damaged nerve tissue; A method for promoting healing of damaged nerve tissue, characterized by inducing -43 expression. (34) Claim (33) wherein the nerve damage is caused by infarction with ischemia, temporary ischemia without infarction, hypoxia, anoxia, anoxic encephalopathy, hypoperfusion, or stroke. ) method described. (35) Structural rearrangement in neuronal cells characterized by exposing the neuronal cells to an effective amount of one or more substances selected from the group consisting of neurodevelopmental factors, steroids, and functional derivatives thereof. How to modulate. (36) modulating synaptic plasticity in neuronal cells, characterized by exposing the neuronal cells to an effective amount of one or more substances selected from the group consisting of neurodevelopmental factors, steroids, and functional derivatives thereof; how to. (37) Modulating the microenvironment of neuronal cells by exposing them to an effective amount of one or more substances selected from the group consisting of neurodevelopmental factors, steroids, and functional derivatives thereof. how to. (38) A method for suppressing GAP-43 expression in mammalian neuron cells, the method comprising exposing the mammalian neuron cells to an effective amount of one or more steroids. (39) exposing fully differentiated mammalian neuronal cells to an effective amount of one or more steroids;
Methods of modulating -43 expression. (40) Claim (35), (36), (37), (38) or (3) wherein the steroid is a corticosteroid.
9) The method according to any one of 9). (41) Claim in which the corticosteroid is selected from the group consisting of mineralocorticoids and glucocorticoids (
40) The method described. (42) The method according to claim (41), wherein the mineralocorticoid is selected from the group consisting of dexamethasone, corticosterone, aldosterone, and progesterone. (43) A method of increasing steroid suppression of GAP-43 expression in steroid-exposed mammalian neuronal cells, comprising exposing the cells to an effective amount of cycloheximide. (44) Claim (30) wherein said cell is a non-neuronal cell.
) or the method described in (31). (45) Nucleotide sequence: [There is a gene sequence] or a cDNA encoding a membrane-targeting peptide consisting of a functional derivative thereof. (46) I, [There is a gene sequence]; II, [There is a gene sequence]; III, [There is a gene sequence]; IV, [There is a gene sequence]; V, [There is a gene sequence]; VI [ A membrane-targeting peptide comprising an amino acid sequence selected from the group consisting of the gene sequence; VII, the gene sequence; and VIII, a functional derivative thereof. (47) I, [There is a gene sequence]; II, [There is a gene sequence]; III, [There is a gene sequence]; IV, [There is a gene sequence]; V, [There is a gene sequence]; VI, A DNA sequence encoding a membrane-targeting peptide consisting of nucleotides encoding an amino acid sequence selected from the group consisting of VII, VII, and VIII, a functional derivative thereof. (48) At its amino terminus, in the same phase, claim (
46) A structural gene or fragment thereof consisting of nucleotides encoding a membrane-targeting peptide having the described sequence. (49) A protein or peptide consisting of a membrane-targeting peptide consisting of the sequence according to claim (46) at its amino terminus. (50) (a) A membrane-targeting peptide consisting of the amino acid sequence according to claim (46) is attached to the amino terminal of a desired protein or peptide; then (b) a protein or peptide consisting of the obtained membrane-targeting domain. into a cell, wherein the resulting protein or peptide of step (b) is directed to the membrane of the cell by the membrane-targeting domain. A method for orienting cells to the cell membrane. (51) The method according to claim (50), wherein the cells are selected from the group consisting of neurons and non-neuronal cells. 52. The method of claim 51, wherein in the neuron cell, the resulting protein or peptide of step (b) is oriented to the growth cone region of the cell. (53) An antibody directed against the peptide according to claim (46) or against the protein or peptide according to claim (49), or a functional or chemical derivative thereof, the antibody or An antibody or a functional or chemical derivative thereof, characterized in that the derivative is optionally detectably or therapeutically labeled. (54) Claim (53) wherein the antibody is a monoclonal antibody.
) described antibodies. (55) has substantially the specificity of a MAb anti-GAP-43 (H5) or a functional or chemical derivative thereof, wherein the MAb anti-GAP-43 (H5) is
5, and the hybridoma strain has accession number A
Monoclonal antibody with TCC HB 10316. (56) Hybridoma strain H5 with accession number ATCC HB 10316, or a functional or chemical derivative thereof. (57) The first encoding genomic GAP-43
3. The nucleotide sequence shown in Figure 3, or a functional or chemical derivative thereof. (58) The nucleotide sequence shown in FIG. 14 encoding the GAP-43 promoter, or a functional or chemical derivative thereof. (59) Multiple start sites and consensus Pit-
1 binding site, but lacks the TATA box and the consensus Sp-1 binding site, and furthermore consists of a long homopurine-homopyrimidine stretch that can adopt a triplex (H-DNA) conformation. Promoter (60) substantially as shown in FIG. 14, in phase with the structural gene or fragment thereof, or its functional Chemical derivatives. (61) DN consisting of the structural gene according to claim (60)
A expression vector. (62) A host cell transformed with the vector according to claim (61). (63) Internal regulatory proteins (IRPs). (64) I, MLCCMRRTKQVEKNDEDQKIEQ
DGV;II, MLCCMRRTKQVEKNDEDQK
IEQDG;III, MLCCMRRTKQVEKNDE
DQKIEQD;IV, MLCCMRRTKQVEKND
EDQKIEQ;V, MLCCMRRTKQVEKND
EDQKIE;VI, MLCCMRRTKQVEKNDE
DQKI;VII, MLCCMRRTKQVEKNDED
QK;VIII, MLCCMRRTQVEKNDEDQ;I
X,MLCCMRRTKQVEKNDED;X,MLC
CMRRTKQVEKNDE; X I, MLCCMRRTKQVEKND; XII, MLCCMRRTKQVEKN; , MLCCMRRTK; XVIII, MLCCMRRT; XIX, MLCCMRR; XX, MLCCMR; XX I, MLCCM; XXII, an IRP consisting of an amino acid sequence selected from the group consisting of MLCC; and XXIII, a functional derivative thereof;
peptide. (65) A nucleotide sequence encoding the IRP peptide according to claim (64). (66) Consensus amino acid sequence: IRP peptide having hydrophobic-leu-cys-cys-x-basic-basic or a functional derivative thereof. (67) Claim (6) wherein the cysteine is easily palmitylated.
4) or the IRP peptide described in (66). (68) A method for modulating the binding activity of a desired protein, which comprises introducing an effective amount of an IRP peptide into an environment consisting of the desired protein and its binding substrate. (69) The method according to claim (68), wherein the desired protein is a G protein. (70) The method according to claim (69), wherein the G protein is G_0. (71) The IRP peptide is claimed in claims (64) and (66).
or the method according to claim (68), which is the peptide according to (67). (72) Claim (68) wherein said environment is inside a living cell.
) method described. (73) The method according to claim (72), wherein the cells are central nerve cells or peripheral nerve cells. (74) The method according to claim (69), wherein the binding substrate is GTP. (75) An antibody directed against the IRP peptide of claim (64) or (66), or a functional or chemical derivative thereof, wherein said antibody or said derivative thereof is optionally detectably or The antibody, or a functional or chemical derivative thereof, characterized in that it is therapeutically labeled. (76) Claim (75) wherein the antibody is a monoclonal antibody.
) described antibodies. (77) Along with a pharmaceutically acceptable carrier, claim (64)
or consisting of the IRP peptide described in (66), and
A pharmaceutical composition optionally comprising one or more therapeutically effective agents. (78) Along with a pharmaceutically acceptable carrier, claim (75)
A pharmaceutical composition comprising an antibody as described and optionally one or more therapeutically effective agents. (79) A method for modulating structural reorganization in nerve cells, comprising administering to the nerve cells an effective amount of the composition according to claim (77) or (78). (80) Introducing a substance to be screened into an environment consisting of a desired protein and its binding substrate, and measuring an increase or decrease in substrate binding relative to substrate binding in the absence of the substance. A screening method for substances having the ability to modulate the binding activity of. (81) The method according to claim (80), wherein the desired protein is a G protein. (82) The method according to claim (81), wherein the G protein is G_0. (83) specifically binds to GAP-43, has a molecular weight of 39000, is eluted in a single hand with application of EDTA in 50 mM Tris buffer, and is non-reactive with anti-G_0 polyclonal antibodies. A nerve growth-related protein characterized by: