JPH04505254A - Frameshift signals and utilization of ribosomes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to liposome frameshifting and its utilization.

Research from a number of viruses has recently led to a phenomenon known as liposome frameshifting. We have demonstrated a novel type of translational regulation of gene expression through mechanisms. The root of this is lipo The soma responds to a single nucleotide at a specific position in the viral mRNA. The subsequent downstream R indulging in a different decoding frame due to the backward slip due to Provides deciphering of NA sequences. This frameshift effect is due to the avoidance of stop codons. It can also be harmful (also produces bacteria).

This mechanism is first explained by the retrovirus Rous sarcoma virus (RSV) (Ja cks, T., and Varmus, H. E. (1985); However, in recent years, human immunodeficiency virus (Jacks , T, , Power, M, D, , Masiarz, F, R,, Luc iw, P, A, Barr.

P., J., and Varmus, H.E. (1988a); Charac terization of rib.

331, 280-283. Wilson, W., Braddock, M., A. dams, S.B.

Rathjen, P0mouth, Kingsman, S, M, and Kings man, A. J. (1988); HIV expression str. ategies:ribosomal frameshifting 1s directed by a 5hort 5equence in both mammalian and yeast systems; Ce1l 55.1159-1169), Mouse Breast Cancer Virus (MMTV) ( Moore, R,, old xon, M,, Sm1th R,, Peters, G.

and Mouth1ckson, C. (1987); Complete nucleus otidesequence ofa milk-transmitted ouse mamma! 1tumour virus: two frame eshift 5uppression events required f or translation ofgag and pal; J, Vi rol, 61.480-490) and coronavirus IB by the present inventor. V (Brierley, 1., Boursnell, M, B, G,.

B1nn5. M,M,,Bilimoria,B,,Blok,V,C,,B rown, 70 mouths, K.

and lnglis, S.C. (1987); ibosomal frameshifting signal in the Polymerase-encoding region of theco ronavirus IBV; EMBOJ, 6.3779-3785. ) to It is detailed in detail.

In liposome frameshifting, there are two direct changes in the translation decoding frame ( (or more) allows the synthesis of a single protein from duplicated genes ( Roth, J.R. (1981): Prameshift 5uppres sion; Ce1l 24.601-602; and Craigen, W, J., and Ca5key, C.T. (1987); Transl. ational frameshifting: where will i t5top? Ce1l 50.1-2: for reference).

To date, almost all examples of this type of regulation in higher eukaryotic cells have been caused by retroviruses. The frameshift is caused by the viral RNA-dependent DNA polymerase. It has been thought to be a mechanism for the regulation of expression. R8V and HIV-1 One stop codon in MMTV and two of them in MMTV are (-1) liposomes. Alternative overlapping reading frames (openr What is later obtained by viral polymerase (eading frame) gag-po1 polyprotein is produced. Furthermore, freight shifts are A number of retrotransbones, such as yeast Ty1 (Mellor, J., Fu lton, S. M., Dobson, M. J., Wilson, W. .

Kingsman, S0M, & Kingsman, AJ, (1985) ; A retrovirus-1ike strategy for ex Pression of a fusion protein encoded by yeast transposon Tyl; Nature 31 3.243-246. Wilson.

W., Malim, M., HoMellor, J., Kingsman, A, J, and Kingsman, S, M, (1986); Bxp response strategies of the east retr otransposon Ty: a 5hort 5equence di rects ribosomal frameshifting; Nucl, Ac1ds Res, 14.7001-7015. C1are, J.

J,, Be1court, M, and Farabaugh, Pj, (1 988); fting occurs within a served sequence nce of the overlap between the two enes of a yeast Tyl transposon; Pro c, Natl, Acad, Sci, Mouth SA 85. 6816-6820,) Average and Ty912 (C1are, J. J., and Farabaugh, P. J. , (1985); Nucleotide 5equence of ay east Ty element:evicfence faran unus ual mechanism of gene expression; P roc, Natl.

Acad, Sci, LISA 82.2829-2833. ) and Shojiyo Drosophila 17.6 (Saigo, K, Kug imiya, W., Matsuo, Y.

Inouye, S-, Yoshioka, K, and Yuki, 5 (198 4);Identification of the coding 5equ nce for a reverse transcriptase-1ik.

enzyme in a transposable genetic ele ment in Drosophila melanogaster; Nat ure　312.659-661. ) and gypsy (Mar for, R, L, , Parkhurst, S, M, and Corce s, V, G.

(1986); The Drosophila melanogaster gypsy transposable element encodes p utative gene products homologousto r etroviral proteins; Mol, Cell Biol, 6.　 1129-1134. ) is thought to be necessary for the expression of reverse transcriptase. It is.

However, in recent years, the applicant has discovered that the avian coronavirus infectious bronchitis virus (I BV) describes the first non-viral, higher eukaryotic example of this phenomenon. (Brierley et al., 1987).

Interest in the precise mechanism by which liposomal frameshifting operates is strong, and multiple Research by the group suggests that the uniqueness of this phenomenon lies in the area around the site where the frameshift occurs. It is suggested that the nucleotide sequence of the RNA of The reason is that the frame Inserting cloned DNA corresponding to the shifted site into an unrelated gene It is possible to cause a frame shift even under dissimilar ambient conditions. (Brierley et al., 1987; Jack S et al., 1988), where frameshifting occurs for RSV and HIV. The positions were identified by a combination of site-directed mutagenesis and amino acid sequence analysis ( Jacks et al., 1988a, Jacks, T., Madhani, H. D,, Masiarz, F, R, and Varmus, tl, E, (1 988b) ; Signals for ribo-(e somal fr amesh tinging in the Rous sarcoma virus gag-pollregion; Ce1l 55.447-458).

These authors found that a comparison of analyzes of the majority of retroviruses left frameshifts. Considering the use as a means of regulating gene expression, we developed two pomopolymer triplets. During translation, a certain heptanucleotide RNA sequence that starts with ) We considered that it is possible to enable tRNA slippage that leads to frameshifting.

In addition to these "slippery" sequences, most retrovirus shift sites A cryptic RNA stem-loop structure located downstream of the It is considered useful (Rjce, N, R,, 5tephens, R , M., Burny, A- and G11den, R.

V, (1985); The gag and pol genes of bovineleukemia virus: nucleotide 5e quence and analysis; Virology 142.3 57-377゜Moore et al., 1987, Jacks, T., Townsl ey, K., Varmus, f. (,E.

and Majors, J. (1987); Two efficient ribosomal frameshifting events are r required for 5 synthesis of mouse mamma ry tumour virus gag-related polyprot eins; Proc, Natl.

Acad, Sci, IJSA84.4298-4302. and Jacks et al. 1988), and in fact, the R3V frame shift) stem-loop downstream of the R position. has indeed been shown to be important (Jacks et al., 1988).

It is not known how stem-loops affect frame shifting. However, the liposome slows down or stalls in the stem-loop. We believe that this increases the possibility of RNA slippage (Jacks et al., 1988). 8b).

- The applicant's recent research in IBV shows that the frame system in IBV RNA is The goal was to identify the exact sequence that gives rise to the FT signal, and they Narrowed down to 83 nucleotides of RNA. This part has high efficiency (30% ) according to various different surrounding situations (Fig. 2). Honde The applicant proposes that the position where the liposome actually slides backwards is the tltJUAAAc sequence. , has been identified as occurring to the right of the 5' end of the frameshift signal, and this has been reported by others (Jacks et al., 1988a, b). match the result. However, the applicant has determined that downstream of this "slippery" sequence, , the RNA in the frameshift signal is folded into a tertiary structure. I also made a new discovery. This tertiary structure is called a pseudoknot. (Plei j, c, W.

A., Rietveld, K., & Bosch, L. (1985) o A new principle of RNA folding based on pseudoknotting, Nucl, Ac1ds Res.

River 1717-1731).

Applicant's experiments revealed that the exact primary nucleotide sequence of this folded region is of minor importance compared to the ability to form these types of structures. Suggests. Therefore, the applicant has determined that the nucleotide sequence of this frameshift signal is The column could be changed quite a bit while retaining its functionality. The applicant exists The region with the signal, especially nucleotide 12395 marked in Figure 2. ~12419 can be expected to be unnecessary, and in fact they are the function of the signal. could be removed without affecting the . Therefore, the applicant has approximately 60 A minimal sequence of nucleotides can be designed and this can be combined with any When placed in a seed gene, it could lead to highly efficient frameshifting.

Herein, applicants describe RNA sequences useful for frameshifting in IBV. To elaborate on column analysis and cause frameshifts with high efficiency Provide information about sufficient Fl/F2 overlap. The upstream limit of this region is It has the characteristics of a “slippery” sequence as defined in (Jacks et al., 1988b). The results of mutagenesis analysis of this sequence indicate that frameshift occurs at this site. It was completely consistent with that. The rest of this area is just below this “slippery J array”. consists of approximately 80 nucleotides in sequence, and the applicant has proposed an efficient, frame The shift is based on the pseudoknot type (Studnika, G, M ,, Rahn, G, M, , CuCumm1n, 1. W, and 5alse rW, A. (1978), Computer method for pred icting the 5 secondary 5 structure of si ngle-stranded RNA, Nucl, Ac1ds Res, 5゜3 365-3387; Pleij et al., 1985) It was revealed that it is based on the shape of

The applicant also provides detailed examples of practical uses of these frameshift signals herein. Describe.

Summary of the invention The present invention relates to a nucleotide construct, which is a liposome frame shifter as detailed herein. and their production, identification and provide access to.

The present invention provides pseudoknots in which the RNA is substantially the same as the pseudoknot shown in FIG. Frameshifting of liposomes with nucleotide sequences forming part or all of Nucleotide constructs comprising a signal or a functional variant thereof are provided.

This is due to the liposome arriving at the frameshift signal of the liposome during translation. causing the some to slide backwards by at least one nucleotide This results in translation of the subsequent < RNA sequence in the reading frame.

The present invention provides a nucleotide structure in which the liposome frameshift signal is Comprising part or all of the 83 nucleotide sequences shown in FIG. 2, Or provide a functional variant thereof. These are added to the liposomes during translation. The liposome arriving at the frameshift signal is activated by one or more nucleotides. causing a backward slippage and subsequent <R Provides translation of NA sequences. This nucleotide structure consists of the 83 nucleotides mentioned above. 24 nuclei between positions 12396 and 12419 (inclusive) of the code sequence Frameshift signature of liposomes containing one or more otides It may contain nulls. The frameshift signal of this liposome is shown in Figure 2. Contains about 59 nucleotides out of the 83 nucleotide sequence shown in (e.g. 12396 and 12419 of the 83 nucleotides) 24 nucleotides between (inclusive) positions).

The frameshift signal of this liposome is the sequence UIIUAAAC or functions with it. may comprise a "slippery" array that is equivalent in terms of some functionally Equivalent "slippery" arrays are UIJUUIIUC, UUUAAAU, UUUA There are AAA and UIIIGGGC.

Frameshifting of liposomes as described above to induce frameshifting of liposomes Provide a gene containing part or all of the signal, or the above-mentioned "slippery" sequences. provide

The present invention provides a nucleotide construct comprising a sequence encoding a first polypeptide; and a subsequent sequence encoding a second polypeptide, and between these sequences Also provided is a frameshift signal located in the liposome as described above.

Here, translation of the second polypeptide sequence depends on whether or not a frameshift has occurred. .

This second polypeptide can be a reporter molecule. This second polypeptide may comprise the first component of a bonding pair, and thus the other components of this bonding pair. components. Therefore, this second polypeptide can act as a label. allows for the assay of frameshifted products and therefore allows the initial sequence to be translated. I can prove that it was done. This second polypeptide can be recognized by a specific antibody. It can be a protein or peptide sequence known for its In other examples , this second polypeptide is a reporter whose expression level can be quantitatively monitored. -Can be molecules. This reporter molecule may be an enzyme, e.g. luciferase. Ru. Therefore, cells expressing high levels of the desired target protein will Can be selected based on peptide expression.

In a third example, the second polypeptide may be a membrane-anchor peptide sequence. Ru. If the first polypeptide is destined to be secreted from the cell (e.g. soluble attachment of this second polypeptide in the form of a membrane anchor sequence This protein is secreted based on the fact that it remains on the cell surface. A certain ratio of both protein and membrane-bound protein will be created.

Additionally, recombinant cloning vectors and replicable vectors containing this nucleotide structure The current vector is also provided.

Furthermore, recombinant vectors comprising the recombinant cloning vectors and replicable expression vectors described above. Microorganisms and cell cultures; expressed from replicable expression vectors provided herein. a protein expression product for the first and second polypeptides; Contains the leotide sequence and the liposome frameshift signal located between them. Also provided is a nucleotide sequence consisting of.

The present invention provides that the nucleotide constructs provided herein are expressed and a second polypeptide The first polypeptide in a recombinant system in which the peptide (i.e., reporter protein) is detected. Also provided are methods for monitoring the production of putide. In both cases, this expression The product preparation has a reporter molecule.

In such methods, the second polypeptide may be modified by the use of antibodies or enzyme substrates. Can be detected by use.

The invention further comprises expressing the nucleotide structures provided herein. for the production of both membrane-free and membrane-bound forms of polypeptides in recombinant systems. provide a method.

The present invention provides the aforementioned microorganism or cell culture for expressing the aforementioned nucleotide structure. a method comprising culturing a nutrient and combining the resulting different polypeptides with each other. Also provided is a method comprising separating from the .

Brief description of the drawing For a better understanding of the invention, reference is made to the drawings by way of example only. As described here: Figure 1 is a diagram of plasmid pFS7, which is a protein biobottom. During translation of mRNA derived from Sma I-digested pFs7 template DNA, Fl Expected generation according to frameshift of liposomes during duplication of /F2 indicates the expected size of an object; Figure 2 shows that highly efficient frame shifting can be derived in different surrounding situations. The smallest fragment derived from the Fl/F2 binding region of IBV gene RNA. Indicates octide sequence (tested by missingness analysis); Figure 3 shows the identification of liposome sliding sites in the IBV frameshift signal. show. The mutants shown in panel A are pFS7 (pF37.1 and others) or p Fs8 (pF58°1 et al.), and Brierley et al. In vitro in the rabbit reticulocyte lysate system exactly as detailed in 1989. Analyzed by transcription and translation (Panel B) (Plasmid pFs8 was an inducer of pFS7. conductor, and the promoter for phage T7 RNA polymerase is It is inserted immediately upstream of the influenza FBI gene. But long (the coding sequences and frameshift signals are identical): Figure 4 shows a possible mechanism by which liposome slippage occurs (Jacks et al. Ce1l 55. 447-458. 1988); Figure 5 shows the analysis of the slip site. Show the results. Nucleotide changes occur within all seven defined nucleotides shown in Figure 3. was performed by site-directed mutagenesis of PFS8 at the same site. mutation Signals were analyzed by in vitro transcription and translation as described above: Figure 6 shows that a minimal frameshift signal was added to the end of the desired target gene. Protein encoding results are shown; Figure 7 shows the nature of the IBV frameshift signal. Possible structures of pseudoknots that form part of the structure are shown. Stem-loop structure The base pairing between the nucleotides in the structural loop and the downstream region (A) is shown in the figure. Forms an extensive double helix (B). This double helix territory Areas 31 and S2 are connected by Ipponchin loops L1 and L2. In this structure, S 2 overlaps Sl, thus clockwise, apparently contiguous 16 base pairs and 1 pair A mismatched pair of double helices is formed. Impression of the three-dimensional structure of this structure side shown in (C), 10 sets of base pairs and 1 set of mismatches in S1, S2 6 pairs of base pairs in the helix, and one turn contains 11 pairs of base pairs. virtual (Arnott, S; Hukins.

D., W., L., & Dover, S., D. (1972); Optim. ised parameters for RNA double-helice s, Biochem, Biophys, Res, Commun, 48. 139 2-1399). In the resulting pseudoknot, Ll (two nucleotides length) creates a deep groove, and L2 (32 nucleotides) creates a deep groove. length) to create a shallow groove. The “folding” program of Jacobson et al. Jacobson, A, B, Good, L; Simonetti, J; and Zucker, M. (1984); Some simple e computational methods to improve he folding of large RNA’s; Nucl, Ac1 ds Res, 12.54-62) has no significant RNA secondary structure in L2. I didn't expect that. These figures are from Rietveld et al. ; Pleij, C.W., A., & Bosch, L. (1983); Three-dimensional models of the tRN A-1ike 3'-termini of someplant vira l RNA's; EMBOJ, 2.1079-1085. ) and Ple ij.

(1985). pseudoknot's The exact nucleotides considered to be part of the stem and loop are shown in D. Figure 8 shows the changes in the position relative to the slip site for the RNA pseudoknot. The effect on frame shift efficiency is shown. A is a mutation that occurred in PFS7.

B is a translation product from mRNA with a mutated frameshift signal. Figure 9 shows the summary of the mutation analysis results of loops in possible pseudoknots. to show the content. Mutations were made by site-directed mutagenesis in PFS8 as shown. and analyzed for frame shifts as described above. Wild type efficiency efficiency (20-30%) is indicated by ++, intermediate efficiency (5-20%) is indicated by 10, and low efficiency (1-5%) was indicated by +/-; Figure 10 summarizes the results of stem mutation analysis in possible pseudoknots. show. Mutations were performed by site-directed mutagenesis in PFS7 and PFS8 as shown. The frame shift was analyzed as described above. Fray The efficiency of the system shift was defined using the same representation for Figure 9; Figure 11 shows that the pseudoknot stem was created by mutagenesis of PFS7 and PFS8. This is an overview of compensatory (complementary) mutations created by Inside the frame (box) For each territory of the helix, the regions associated with the possible stems and different divisions of mutate to create an unstable mutant and restabilize the helix. In order to make it more natural, we have further changed it by introducing a double mutation. frame frame The efficiency of the lift is defined by the same representation as for Figure 9; All the mutational changes that occur in the sample and their effects on frameshift efficiency. Outline the impact: Figure 13 shows the structure of pFScass5. The nucleotide sequence shown in A (boxed) Influenza A/PR8/34PB as part of plasmid pFS1 2 gene (Brierley et al., 1987). 483), thereby allowing the liposome to slide into each decoding frame. yields products of characteristic size. This sequence is the pseudonode shown in Contains a slip site connected to the downstream region that folds to form a cut . Two derivatives of this plasmid, pFscass6 and 7, are each derived from PB2. It is created by introducing or deleting a single nucleotide in the downstream portion. child The translation products of RNA containing these artificially designed frameshifts are shown in C; Figure 14 shows IBV frameshift signals in vaccinia virus-infected CVI cells. Showing Null's test. These cells were either mock-infected or infected with WT vaccinia virus. or VACFS and is interrupted by a frame shift signal. The virus is infected with influenza virus containing the PB2 gene. infected tissue Proteins in the vesicles were labeled with 3SS methionine and subjected to direct gel electrophoresis. either after immunoprecipitation with Kamata antiserum (control) with was analyzed. Immunoprecipitation was performed as detailed in Brierley et al., 1987. It was carried out in The expected size for the frameshift product is plasmid p This was demonstrated by in vitro translation of RNA transcribed from Fsl.

Figure 15 shows the BamHI site of PF58 (T7 phage RNA polymerase protein). A derivative of PFS7 in which the motor replaces the SP6 promoter) Showing the introduction of a frameshift signal immediately downstream: Figure 16 shows a plasmid obtained from the genetic manipulation shown in Figure 15; FIG. 17 shows the smallest 13amHI fragment shown in FIG. This shows a plasmid obtained by replacing it with a DNA cassette containing the gene Figure 18 shows the fusion sequence between the IBV fragment signal and the luciferase gene. Show the location of the sequence; Figure 19 shows the sequence of RNA segment 4 encoding for HA. This N end Terminal signal sequences are shown in large boxes. Glycosylation sites and systems shown in small boxes Arrows indicate the decomposition site of the GNAR peptide and the site where HA decomposes into HAI and HA2. show more; and Figure 20 shows the arrangement of artificial frameshift signals designed for membrane anchors. shows.

Detailed explanation of specific examples Fixed IBV frameshift signal Details of this study can be found in two papers, EMBOJ by Brierley et al., 6.377. 9, 1987; and Brier Iey et al. Ce1157.537.1989 is introduced in. In summary, fragments of cloned DNA and IB Concerning the linkage between Fl and F2 open reading frames (ORFS) on the V genome (Boursnell et al., J. Gen. Viro 1°68, 57.19 87) into a suitable reporter gene (inrenza virus PB2 gene). This creates an artificial message containing the putative frameshift signal. RNA is generated by in vitro transcription of cloned DNA. This recombinant Lasmid is a cell-free system (rabbit) in which the frameshift of liposomes on mRNA designed to be monitored by translation in reticulocytes (derived from reticulocytes); useful A frameshift is a decoding product of a specified size whose identity is determined by the reporter that can be confirmed through reactivity with an antiserum specific for the downstream region of the gene. bring. By using this technique, the applicant has realized that the Fl/F2 concatenation sequence It contains a frameshift signal of virtually high efficiency, and this signal is unique to eukaryotic cells. With cell liposomes, not only a part of IBV mRNA but also a new genetic surrounding situation can be obtained. It was shown that it can be recognized even when

Furthermore, Applicants have proposed that this signal can be divided into two completely different reporter genes, viz. influenza virus PB2 gene (Brierly et al., 1987) and influenza virus. When placed in the virus FBI gene (Brierly et al., 1989) also has an equivalent effect, so this works ignoring its genetic conformation. Suggests scales.

Next, the exact structure of the frameshift signal was investigated by site-directed mutagenesis. Ta. This is a plasmid vector, pF37 (Figure 1), carrying the reporter gene. In addition to the interrupting frameshift signal, -like heavy chain DNA, plasmid replication The use of sequences that allow manufacturing and packaging has been facilitated. this The single domain form of the plasmid can therefore be used directly as a template for site-directed mutagenesis. Vine used.

First, the applicant determined the length of the frame signal to be 220 nuclei in pFS7. Continuous deletion information (progressive) from the end of the IBV-derived sequence ly deleting information). It was. By using this technique, the applicant can influence the efficiency of frame shifting. We were able to delete all but 83 nucleotides so that they had no effect.

The so-called “minimal” frameshift signal of these 83 nucleotides is shown in Figure 2. show.

Identification of “slippery parts” What is known to promote frameshifting in other systems (Jac ks et al., 1988b) with the present sequence, it is clear that this signal is quite original. The 7 nucleotide sequence UUIIAAAC located at the tip is the “slippery site” , which tends to indicate a point where the liposome effectively slips backwards by one nucleotide. It suggests that it is strong. This makes the stop codon a possible “slippery” site. Experiments in introducing these seven nucleotides on either side of the confirmed by the sequence being the only overlap region between the upstream and downstream ORFs. It has been certified. In this case, the "decoding" product means that the liposome has these seven nuclei. It is only synthesized when the frame is changed within the leotide window. Figure 3 shows the frame A frame shift is created from this mutant frame shift signal on mRNA. The applicant has shown that IIUUA continues to occur effectively in We are confident that the AAc sequence is the de facto slip site.

The mechanism of liposome slippage that occurs on the UUIIAAAC sequence is, first of all, Two codons UUA and AAC (respective peptidyl sites and tRNA) is recognized by its cognate tRNA. (Figure 4).

Both tRNAs then slide back one nucleotide onto mRNA. Sometimes, the sequence alignment at the suberi site is between 3 on the tRNA and on the mRNA. The condition is such that the bond of two of the base pairs continues to be maintained after slippage. child The model for this species is based on other types of seven nucleotides where slippage occurred. Those showing similar or greater pairing in position are also considered to be equivalent to slip areas. Suggests it works.

Applicant has tested several such arrangements (Figure 5) and indeed some are effective. We have discovered that frame shifting can be done efficiently (for example, IIUUUUU c, UUUAAAAU, UUUAAA). However, other (e.g. 1 ltltlGGGc) was functional but less efficient. So these studies? Therefore, the applicant has been able to establish predictability regarding the potential "slipperiness" of various sequences. I can do it.

Based on the slippery model, the situation where there is also a slip of -2, i.e., such a shift In the case where “2 out of 3 pairs” pairing by tRNA with slippage is still possible. (for example, an array of the type U[IU[IIIUN; where N is the entire (all nucleotides). However, in this case, the liposomes are − 1 and -2 frames, thus this lack of specificity Loss leads to less practical uses of the signal.

These data have two important practical implications.

First, the minimal frameshift signal is located in a novel genetic context. The key is that the liposome changes frame almost immediately upon contact with this signal. It is possible to invite Second, change the frame in this "slippery" arrangement. Liposomes that could not be directly stopped across this sliding site (the most (via binding to a stop codon in the first reading frame). Therefore, If a minimal frameshift signal is introduced immediately adjacent to the last codon of a particular gene, (Figure 6), two different gene products can result. In other words, the original remains A frameshifted product consisting of the gene and an additional sequence fused onto its C-terminus, and and “non-frameshifted” form of the wild-type gene product and its C-terminus. They differ only by two or three additional amino acids at. Furthermore, various Since different "slippery" sequences are available in this signal, these attachments Added amino acids to some extent minimize their effect on the structure of natural proteins You can choose as follows.

Requirements for RNA pseudoknots as part of frameshift signals subject However, this "slippery" sequence itself is not efficient for the missing analysis mentioned above. This suggests that this is insufficient to promote the slippage of liposomes. The applicant must The additional sequences required form two sets of base-pairing interactions (RNA helices). The applicant understands this possibility as having the ability to The structure of the shift signal shows that the expected interaction is unstable due to complementary mutations. structure that becomes stable and is stabilized again by complementary changes on the corresponding strand. I investigated through. This analysis shows that efficient frameshifting effectively affects both helices. It clearly suggested that it needed a form.

These results indicate that the RNA sequence immediately downstream of the "slippery site" is an RNA pseudomorph. Knot (Studn 1cka et al., Nucl, Ac1ds Res.

5, 3365. 1978; Pleij et al., Nucl, Ac1ds R es, 13. 1717. (1985) to the state of RNA tertiary structure known as and this structure is heavy for efficient frame shifting. It is essential.

The best explanation for this requirement is that the presence of a folded RNA structure is essential for translation. This restricts the progress of the liposome during translation and therefore it stops at this "slippery part". This is likely to promote the slippage of the bound tRNA. This theory is based on ``slip''. This is supported by the fact that the distance between the "easy part" and the shoot knot is important.

For example, inserting or deleting only three nucleotides between two components This greatly inhibits frame shifting (Fig. 8).

Sequence restriction in RNA pseudoknots This cheater uses frameshift signals The primary structure (nucleotide sequence) of the downstream part of the file is transformed into the appropriate state of tertiary structure. This explains the above-mentioned prediction that it is less important than the accumulated ability. this presented by extensive mutational studies.

Form two knot-heavy loops (loops 1 and 2 in Figure 9) The expected sequence analysis results are that they affect the efficiency of frameshifting. This suggests that major changes can be made without causing any harm. As part of loop 1 Both possible nucleotides can be changed to complementary sequences, and Insertion of either 1 or 3 additional nucleotides without effect can do.

Loop 2 can be made longer (by 6 nucleotides) or by 32 nucleotides. It can be as short as 8 nucleotides. Furthermore, these eight nuclei Otide can be changed to a complementary sequence, and this also has no effect. do not have. The only restriction is that these loops must be of a certain minimum length. Making loop 2 shorter than 8 nucleotides is a frameshift. increased inhibition. Applicant believes that this means that the extent of the apex and base of the appropriate helix We believe that this reflects the minimum length required for

Nucleotide mutations expected to be included in the helical morphology are generally increases frameshift inhibition (Figure 10), however, helical Changes at the end of the thread had little effect. However, in all cases this The inhibitory effect of these changes is due to the corresponding inhibitory effects of further complementary base changes in It disappeared after the introduction (Figure 11). This is predicted for the morphology of the pseudoknot. This strongly supports the proposed model.

Figure 12 shows an overview of all the site mutagenesis that occurred over the pseudoknot region. It is shown in This allows the frame to move away from the "slippery" array. Each nucleotide located within the shift signal is It is clear that changes can be made without adverse effects.

Therefore, it has the ability to form an appropriate structure to cause a frame shift. In addition, one can design an RNA sequence with the desired codons. this An example of such a signal is shown in FIG. Here, any stop codon is away from immediately downstream of the "slippery" sequence (stopping the first reading frame) must not be present in any decoding frame throughout the RNA sequence. I'm designing a signal that will help. The required sequence is the 5' protruding end (sequence G ATC) was chemically synthesized as a set of complementary DNA molecules. These are Nealation and ligation of the appropriate ends to the appropriate locus (induction). Bgln site at position 483 on Frenza PB2 gene, Fields and W Inter, Ce1128 303, 1982).

The basic plasmid obtained from this cloning process is called pFScass5, And in this case, the slippage of the liposome into the "-1" frame is 19. can lead to the first product "stopped" at Figure 13b shows that this is the actual case. and that the efficiency of frameshifting is higher than that of the wild-type signal. shows. The structure of the plasmid is such that the liposome slides into a “-2” frame. If this happens, 85 proteins will be produced. Dew. However, these results (Fig. 13B) indicate that pFScass5-derived mRNA A leads to the synthesis of a protein at only a very small amount at this specific slip site. As expected, efficient frameshifting is limited to the “−1” frame. suggests that it will be done. Two additional plasmids were created by pFScass7). Created by Metsenger RNA transcribed from this plasmid is “-1” would generate 28 proteins through frameshifting. and actually Such proteins were produced very efficiently (Fig. 12B). But long In addition, this mRNA also led to the synthesis of a small amount of protein 85. child This is surprising, and the reason is that a protein of this length is capable of frameshifting. Not only does the liposome that translates through the signal not change frame; Produced only when the stop codon at the end of the upstream reading frame is ignored or suppressed It is from. Therefore, terminating the three nucleotides in the IBV pseudoknot The downstream positioning of the liposome means that the liposome sometimes has amino acids at its stop codon. inserting a amino acid and decoding its downstream sequence in the same frame. allow it to occur.

If this effect is found to be reproducible in other genetic contexts, , by inserting a pseudoknot-forming sequence immediately downstream of every gene. The protein biosediment is in its original natural form (i.e., without any added amino acids). fusion protein by linking useful downstream sequences. It can also be expressed by wiping. There is no need to express the fusion protein at high rates. In certain situations, this technique is preferred for confirming the insertion of a complete frameshift signal. suitable.

To date, the practical use of frameshift sequences in such liposomes is beyond those skilled in the art. less conceived or thought out.

Applicants have found two different types of use.

a) attaching a potentially useful reporter molecule to the terminus of a specific target gene product; , thereby allowing a concise monitoring of its production. For this, frame The system shift signals are arranged as follows. That is, it is made from artificial genes. Although liposomes that translate rnRNA produce the desired specific genetic product, However, at a certain rate, the frame shift is fused with this reporter. Arranged on rags resulting in the production of child products.

b) targeting M-anchors or other types of cellular "targeting" signals to specific gene products; and direct a certain percentage of this gene product to a specific site in the cell. To make it possible to

Examples of these uses will be detailed in more detail.

a) Immunization of proteins to the ends of proteins expressed in eukaryotic cells that are difficult to monitor. Joining marker arrays No information is available about the encoded gene product and nucleotide sequencing There are many examples of genes that were first identified. Therefore, genes are expressed in eukaryotic cells. This is important as a means to study the function of the encoded protein. . Multiple types of expression systems are available for this purpose, but the main One of the issues is finding suitable reagents (e.g. specific antibodies) for assaying gene products. ) are generally not readily available. unless high levels of expression are achieved. (which in some cases can be toxic to the cell) and release the desired protein. It is therefore often difficult to ascertain whether or not texture has been produced. especially genetics If the child product undergoes post-translational modification, it is difficult because it does not have a predicted molecular weight. . One approach to this is to express this target sequence in a heterologous system. , for example, as a bacterial fusion protein, and this protein is specifically expressed as a bacterial fusion protein. The aim is to use it as an immunogen to make antiserum. However, this is a laboratory process, takes several months, and the resulting serum is actually the desired one. There is no guarantee that the protein will be recognized.

Therefore, upon expression of an uncharacterized gene product in eukaryotic cells, the desired It is possible to confirm whether a polypeptide is actually translated in the cell and its appropriateness. It is of great value to be able to obtain some approximation of the expression level. this thing expresses a protein as a fusion with an immunologically identifiable protein sequence. However, of course this requires that this protein be There is a high possibility that it does not retain this functionality. However, frameshift between the coding sequence of the target protein and its immune marker. Inserting a liposome that translates the hybrid mRNA Although the system normally stops at the end of the target gene (producing the natural protein), However, by occasionally shifting the frame and leading to the production of fusion proteins, both achieve the objectives of simultaneously. That is, proteins made from hybrid genes The bulk of is the natural target protein, but some of it is frameshifted. The expressed proteins allow immunological monitoring of expression levels.

If a labeled form of this protein is detected, the first protein production one can be sure that the target has also been translated, and the strength of the detected signal will depend on its origin. Provides a measurement of current levels.

Plasmid pFS 1 is a clone of IBV liposome frameshift signal modified influenza virus PB2 gene (Brierly et al., 1987, supra). ), and therefore the expression of the downstream half of the gene depends on frameshifting of the liposome. dependent on Extract the complete recombinant gene as a single-stranded fragment This plasmid was digested with BamHI and this fragment Plasmid pGs 20 (Mackett, M: Sm1th, G, L. and Mo5s, B. (1982): Vaccinia virus :a 5electable eukaryotic cloning an d expression vector.

Proc, Natl, Acad, Sci, USA 79. 7415 -7419) into the BamHI site. The resulting plasmid (p FSVAC) is a vaccinia virus located next to the promoter at 7.5 of cotton cilia virus. Enables transcription into messenger RNA by virus RNA polymerase contains the recombinant gene in the correct orientation. This gene is from cotton senior virus It is also next to sequences derived from the thymidine kinase gene and is therefore can be introduced into the genome of cotton senior virus (Smith, G.L., ). Mackett, M., and Mo5s, B. (1983) Infecti .

us vaccinia virus recombinants that express hepatitisB virus 5 surface ant igen, Nature, London, 302.490-495).

Briefly, this plasmid was introduced into CVI cells by infection; Cells were co-infected with cotton senior virus, and the progeny derived from this infection were A virus lacking thymidine kinase activity by growing the virus in a selective medium, Therefore, this indicates that the influenza virus gene has been accepted through recombination. I selected a virus that seemed to be the case. A virus that accepts the DNA sequence properly will be infected. A radioactive 4W recognition probe specific for the Frenza virus PB2 gene was used. Identified by Southern blotting of viral DNA and Prepared from stock. Infect CVI cells with this virus and Ten hours after treatment, total cell saccharide was treated with 3SS-methonine for 2 hours. Immediately after labeling, cells were prepared for analysis. Next, gel electrolyte these lysate products. Tested by electrophoresis and autoradiography (Figure 15). In this experiment , detecting a PB-2 specific protein product expressed from a recombinant virus. could not be determined by direct comparison with lysates of wild-type virus-infected cells. . This method uses cotton sinus virus and other eukaryotic vectors as expression systems. The same is true for many cases of such experiments (probably due to low expression levels). eye).

However, radiolabeled lysate and PB1 gene (liposome frame sequence) immunization with an antiserum specific for the C-terminal region of the Epidemiological precipitation reveals proteins of the expected molecular weight for the frameshift product. It was precipitated by crab. Therefore, this experiment shows that the N-terminal part of the FBI gene Although these expressions have not been directly assayed, it is considered as.

In the example above, this immune marker sequence is a relatively large portion of the viral protein. fraction, and this is detected by immunoprecipitation. However, this is exempt. By the use of Western blotting as a method for the detection of infectious diseases is likewise possible, and any protein or peptide sequence can be used as an antibody. Labeled tags that can be easily detected by Can be added to protein.

Luciferase is a protein with approximately 65 molecules that contains oxygen, ATP, and magnesium. It catalyzes the dehydrogenation of the substrate luciferin in the presence of mu ions. this Throughout the process, 96% of the energy released is identified as visible light (blue). Ru. Individual cells expressing luciferase can be easily assayed by microscopy and Furthermore, the level of expression of this enzyme is indicated by the degree of fluorescence. Therefore, Lucife Attach the enzyme gene to the end of the desired target gene (e.g. influenza virus gene). In addition, a means to enable cellular production of hybrid proteins (influenza sequence N luciferase at the C-terminus). The production level of the target protein sequence can be determined solely by observing luciferase activity. provides an easy way to monitor Of course, this means that the exogenous sequence expressing the target protein in its native form, or without any Its value is limited because it is usually desired. However, the flange of liposomes Insert the system shift signal between the target gene and luciferase as described above. This means that the main single protein sequence and the ``luciferase-tagged'' version can be produced simultaneously. make it possible to give birth. In such systems, luciferase fusion proteins Since protein production is proportional to the production of major proteins, and luciferase Because activity can be detected in living cells, luciferase detection assays are as a means of identifying "highly expressive" cells from cell lineages that express the target gene. and can be used. Sorting selects suitable cell colonies for luciferase activity sample after microscopic examination or in a fluorescence activated cell sorter (FAC3) device. This can be done through the use of The sorted cells will continue to survive and produce new can be expanded in culture to produce new, more useful expressing cell lines.

Plasmid pFS8 contains the T7 phage RNA polymerase promoter. The influenza virus FBI gene inserted next to the IBV genome R NA sequence (Boursnelf, M, E, G, Brown, T, D, ,, Foulds, 1. J., Green, P.F.

Tomley, F, M, and B1nn5. M, M, (1987), (1 'completion of these sequences of the gen ome of the coronavirus avian 1nfecti ous bronchitis virus, J, Gen, Virol, 68. IBV liposome frameshift signal derived from 57-77) and an FBI component. The coded sequence is inserted at the 1,139th nucleotide from the end of the sequence. including. Create a plasmid with two BamHI restriction sites (Figure 17) In order to generate a new frameshift signal, this plasmid is Site-specific mutations to introduce restriction sites for BamHI (Figure 16) It was mutated by different induction. This new plasmid was then deleted by BamHI. The luciferase gene is transformed and bound by the “sticky” end of BamHI. Insert the DNA r cassette consisting of a child (Fig. 18), which allows the decoding frame to be inserted. The sequence is conserved from the IBV sequence to the position of fusion of the luciferase sequence (Figure 19). ). This luciferase gene cassette was extracted from plasmid pSK8 by BamHI digestion. Created by (S, M, Kerr, Department of Path ology, [supplied by InlversityofCambrldge).

This plasmid is called plasmid pD0432 (flw et al., 5science, 23 4.856.1986), the 23 nucleotides of the luciferase start codon is obtained through the introduction of a BamHI site upstream of the domain.

Next, this hybrid gene is introduced into eukaryotic cells, and (i) the T7 RNA polypeptide is Artificial mRNA was created in vitro from this lambda hybrid gene using merase. and synthesized this mRNA from the African frog (Xenopus ooc). ytes) into microinjection (thereby Determining whether ferase is expressed in active form when fused to a heterologous protein );as well as (ii) Subclone this hybrid gene using a known eukaryotic expression vector. in order to develop a stable cell lineage that expresses this hybrid gene. Infecting cells with the obtained plasmid (latent r high expression system) were selected based on luciferase activity and FBI expression rates were determined using specific antiserum. Confirmed by Western blotting): It was expressed more.

C) of proteins that are “transported” via liposome frameshift signals. Attachment of membrane anchors to the terminal proteins, such as hormones and growth factors, Easily secreted by cells, but otherwise transported across the cell membrane It remains in the membrane usually through a hydrophobic C-terminal sequence of amino acids. this is sometimes called a menplan anchor. Examples of proteins in this category include , important cell surface “marker proteins,” receptors, and numerous viral antigens. Also included. For example, immunoglobulins are included in both categories. Therefore, depending on the nature of the cells that produce them, they are synthesized as secreted or membrane-bound. There are also things that vine.

Creating eukaryotic cells that express both membrane-bound and secreted transport proteins There may be circumstances in which this is useful. For example, certain types of immunoglobulins ( Cells designed to express Ig) convert the membrane-bound form of the protein into the secreted form. Selection for cells that express high levels of the molecule if they produce the same amount directly by fluorescence activated cell sorting (FAC3). Here, at a high level Cells with the desired protein on their surface can be treated with a reagent that specifically recognizes Ig. are ``labeled'' and ``sorted'' by this device into separate containers.

Condition of superior immune response required for specific virus-specific membrane proteins There are alternative uses. What characteristics of viral proteins make it highly immunological? It is not well understood whether membrane-bound and secreted proteins provide An immune system that crosses both types is advantageous.

For example, influenza virus genes, i.e. viral hemagglutinin genes. (HA) occurs naturally as spike-like protrusions on the surface of virus particles and infected cells. encodes a well-characterized protein that occurs in a normal, sparse It remains in the membrane via an aqueous membrane anchor sequence.

This part of the membrane anchor sequence is displaced by the liposome frameshift signal. By changing the hydrophobic sequence, the new HA sequence that the liposome translates is exposed to the hydrophobic sequence. Conditions can arise that lead to production of the secreted form of HA, which normally shuts down before the production of HA. Only a The linker sequence is translated, leading to the production of the anchored form of the protein. This main problem is , the frameshift signal that is supposed to replace the membrane anchor part replaces its natural sequences encoding amino acids that are non-hydrophobic in type and therefore membrane anchors. The essential feature of the car is that it is destroyed. However, the applicant's i.e., the primary sequence of the frameshift signal is preserved while the secondary and tertiary structure is preserved. By being able to vary as much as possible, the frame encoding a hydrophobic amino acid can be system shift and therefore design one that preserves the structure of the membrane anchor. Can be done.

Influenza A/PR8/34 hem-argtinin gene (Figure 20) as a plasmid In addition, in a similar manner to pFs7, it is synthesized by phage T7 RNA polymerase protein. can be converted to single-stranded DNA at a location under the control of the promoter. The sea urchin was cloned. Artificially designed using site-directed mutagenesis The resulting frameshift signal (Figure 21) was manipulated into the HA gene, and the result shown in Figure 22 was The nucleotides (1632-1692) shown in were substituted. This vibe transcribed the de gene into artificial mRNA, and transferred its frameshift ability to RNA. In vitro translation or microinjection of A. ) was evaluated. Next, put this hybrid gene into a plasmid (pGs20). The hybrid gene was subcloned into a recombinant vaccinia virus. used for introduction into the genome.

For the ability to produce secreted and membrane-anchored forms of bacteria, immunoprecipitation, The results were evaluated by immunofluorescence using a specific anti-HA antibody.

Identification of IBV frameshift site Slip site analysis results uru’iliaryr’ili +/6 ++ 30U LIUFI FIFIG 4/6 +/-ILI UU Sun Sun U 4/6 ++ 30U ULIU UUC5/6 ++ 30U UUG GGC'! /6 +/-IU LIUG Hibisu (5)/6 0.511. Xsu leu asn a b c d 　e,,,.

frameshift product Frame-11 frame °°0″ Frame”-2” pFScass5 22K 28K 85KpF'5cass6 28K 85K 22K - Translation decoding file Reme Submission of translation of written amendment (Article 184-8 of the Patent Act) November 18, 1991

Claims (18)

[Claims] 1. a nucleotide structure substantially as shown in FIG. 7 herein; Nucleotide sequence as RNA forming part or all of a pseudoknot or a ribosomal frameshift signal having comprises a functional variant of the signal, and during translation the ribosomal frame is Ribosomes arriving at the system shift signal at least one nucleotide minute later slides in the opposite direction, bringing subsequent translation of the RNA sequence into a different reading frame Structure. 2. 8 where the ribosomal frameshift signal is shown in FIG. 2 herein. The nucleotide according to claim 1, comprising all or part of three nucleotides. de structure. 3. The ribosomal frameshift signal is linked to the 83 nucleotides. Of these, the nucleotides between positions 12396 and 12419 (inclusive) are 1 or 3. The nucleotide structure according to claim 2, comprising a plurality of deletions. 4. The ribosomal frameshift signal is shown in FIG. 2 herein. 9 nucleotides, i.e. position 1239 of the above 83 nucleotides Claim comprising the deletion of 24 nucleotides between 6 and 12419. 2. The nucleotide structure according to 2. 5. The above ribosomal frameshift signal is: [There is an array] "slippery" sequences selected from the group consisting of or their functional equivalents; 5. The nucleotide structure of claim 4, comprising: 6. A sequence encoding a first polypeptide followed by a second polypeptide a sequence to be encoded, and the ribosome frameshift sequence located between the sequences. a nucleotide structure containing a nucleotide structure, wherein translation of the second polypeptide 2. The nucleotide structure according to claim 1, which depends on whether or not a system shift has occurred. 7. 7. The nucleic acid according to claim 6, wherein said second polypeptide is a reporter molecule. Ocide structure. 8. wherein said second polypeptide comprises a first member of a binding pair; 8. The nucleosome of claim 7, which can be detected by other members of the binding pair. Tido structure. 9. 9. The method according to claim 8, wherein said binding pair comprises an antibody and an antigen or an enzyme and a substrate. nucleotide structures. 10. 10. The nucleic acid according to claim 9, wherein said second polypeptide is luciferase. Ocide structure. 11. Claim 6, wherein said second polypeptide is a membrane-anchor peptide sequence. The described nucleotide constructs. 12. A recombinant cloning vector comprising the nucleotide sequence according to claim 6. - or a replicable expression vector. 13. Recombinant cloning vector or replicable expression vector according to claim 12 recombinant microorganisms or cell cultures containing 14. The microorganism or cell culture according to claim 13, wherein the nucleotide structure cultured for the expression of the polypeptides and the resulting separate polypeptides are separated from each other. A method comprising: 15. A protein protein expressed from a replicable expression vector according to claim 12. current product. 16. A method for monitoring the production of a first polypeptide in a recombinant system, the method comprising: Lower stage: a) expressing a nucleotide construct according to claim 7; and b) detecting said second polypeptide; 17. The second polypeptide is detected using an antibody or an enzyme substrate. 17. The method of claim 16. 18. Producing both membrane-bound and non-membrane-bound forms of polypeptides in recombinant systems A method for expressing a nucleotide construct according to claim 11. A method comprising: