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WO1988001646A1 - Systeme universel de mutagenese de transposons - Google Patents

Systeme universel de mutagenese de transposons Download PDF

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Publication number
WO1988001646A1
WO1988001646A1 PCT/GB1987/000598 GB8700598W WO8801646A1 WO 1988001646 A1 WO1988001646 A1 WO 1988001646A1 GB 8700598 W GB8700598 W GB 8700598W WO 8801646 A1 WO8801646 A1 WO 8801646A1
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gene
transposase
transposable element
dna
sequence
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PCT/GB1987/000598
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English (en)
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Maya Kozlowski
Roger Wayne Glasse-Davies
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Allelix Inc.
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Publication of WO1988001646A1 publication Critical patent/WO1988001646A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • the invention pertains to a process for achieving genetic transposition in eukaryotic or prokaryotic cells.
  • the invention also pertains to plasmids capable of mediating such transposition.
  • the invention further pertains to novel uses of transposable elements.
  • Transposable elements are double stranded DNA molecules which possess the capacity to insert themselves into other DNA molecules.
  • the process by which a transposable element inserts itself, termed “transposition,” requires a protein known as a “transposase” (Berg, D.E. et al., Bio/Technology 1:417-435 (1983); Kleckner, N., Ann. Rev. Genet. 15:341-404 (1981)).
  • transposition process results in the insertion of the transposable element into a particular site in a second DNA molecule.
  • This insertion has four significant consequences.
  • the original DNA sequence of the second (recipient) DNA molecule is disrupted. This disruption extends not only to the nucleotide sequence of the second DNA molecule, but also to the loss of the capacity to produce the functional gene product of the previously undisrupted gene.
  • One characteristic of transposition is that it may involve any DNA sequence of the recipient DNA molecule (i.e. transposition may be random with respect to the recipient DNA sequence being disrupted).
  • transposition provides a powerful mechanism for mutagenizing DNA sequences.
  • transposition results in the incorporation of new DNA into a second DNA molecule, it provides a means of introducing heterologous DNA into a particular DNA sequence, and thus provides an alternative to cloning strategies which employ extrachromo- somal plasmids.
  • the insertion of a transposon may disrupt mRNA transcription, and hence can be used to study the transcriptional control of gene expression.
  • transposable element so that its insertion into a DNA sequence can provide one with information regarding the expression and organization of the DNA sequences which flank the site of insertion. For example, it is possible to insert a gene which encodes a non-excreted protein near to the end of a transposable element.
  • a transposable element provides a probe for promoters, and secretion signal sequences (Casadaban, M., et al., Proc. Natl. Acad. Sci (USA), 76:4530-4533 (1979); Mansil, et al., Proc. Natl. Acad. Sci (USA), 82: 8129-8133 (1975)).
  • transposable elements places a gene whose expression can be readily monitored near to the junction between the transposable element and the disrupted DNA molecule. Since the gene lacks a promoter region it cannot be expressed unless the insertion of the transposable element is such that it causes a promoter region, present on the disrupted molecule to become operably linked to the gene. Hence expression of the gene indicates the existence and location of a foreign promoter. The secretion of the gene product from the cell indicates the presence of a foreign secretion signal sequence at the junction site. By varying the cu-.uring conditions it is possible to identify transposable elements which express the gene product only under certain circumstances (such as, for example, at elevated temperatures, in the presence of a particular molecule, etc.).
  • conditional promoters The promoters which direct such expression are termed "conditional promoters.
  • the identification of conditional, promoters is a major present use of transposable elements.
  • a fourth, consequence of transposition is the ability to probe the genetic expression and organization of the recipient DNA molecule.
  • Transposable elements are diverse in both size and functional organization. Simple transposable elements, termed "insertion sequences,” encode no functions unrelated to their own movement and are generally shorter than about 2 kb. Like all transposable elements, insertion sequences possess specialized termini which contain complementary sequences which are inverted repeats of one another. The presence of these inverted repeat sequences appears to be essential for transposition. (Cohen, S.N., Nature, 263:731-738 (1976)). Transposase enzymes are thought to mediate transposition by binding to DNA sequences at both ends of the transposable element.
  • Transposons are transposable elements which are larger than insertion sequences and which encode several gene products ⁇ such as proteins which confer cellular resistance to antibiotics or other selectable determinants), in addition to the transposase enzyme.
  • Certain viruses, such as mu, lambda, or SV40 which are capable of integrating into chromosomal DNA may also be considered as extremely large (greater than 20 kb) and complex transposable elements (Cohen, S.N., Nature, 263:731-738 (1976); Cornells, G., Bull. Inst. Pasteur, 80:3-60 (1982); Howe, M.M., Virol., 55:103- 117 (1973); Nash, H.A., Ann. Rev. Genet.
  • Tn5 The bacterial transposon Tn5 has been widely studied (see, Berg, D.E., et al., supra which is herein incorporated by reference).
  • Tn5 contains a unique central DNA sequence of approximately 2,600 bp. This central region contains genes which confer cellular resistance to streptomycin/spectinomycin, as well as an aminoglycoside phosphotransferase gene, whose product confers cellular resistance to antibiotics such as Kanamycin or Neomycin in bacteria, or G418, in eu- karyotes (Berg, D.E., et al.).
  • the presence of detectable determinants on transposons enormously facilitates their application to problems of molecular biology (Harayama, S., et al., J.
  • a 1,535 bp terminal region which contains the complementary inverted repeat sequences necessary for transposition.
  • a single base pair mutation in the left-hand terminal region of Tn5 has resulted in the inactivation of the transposase gene present in the left-hand terminal region.
  • the transposase gene present in the right-hand terminal region directs the synthesis of the transposase enzyme needed for the insertion of the transposable element.
  • Prokaryotic transposable elements have been identified in only a small number of bacterial strains. Although the large bulk of prokaryotic microorganisms are not known to contain transposable elements it is probable that many of them will ultimately be found to contain them. It is recognized that there is a need to study the genetic organization and expression as well as the identification of genes in many bacterial genera which do not have developed genetics. This is specially important for biotechnology and for industrially important bacteria. It is widely believed that the use of transposable elements would enormously facilitate the study of uncharacterized microorganisms. However, in many cases, it may be a difficult process to isolate and characterize transposable elements from a microorganism of interest. It would therefore be desirable to adapt well characterized transposable elements to function in other genera of bacteria.
  • the prototrophic bacteria such as the Rhodospirillaes
  • the gliding bacteria such as the Myxobacterales and the Cytophagales
  • the sheathed bacteria such as Sphaerotilus
  • the budding and appendaged bacteria such as Caulobacter
  • the Spirochetes such as the Spirochaeteles
  • the spiral and curved bacteria such as the Spirillaceae
  • the gram-negative aerobic rods and cocci such as the Azotobacteraceae, the Rhizobiaceae, the Methyl- omonadaceae, the Balobacteriaceae
  • the gram negative, facultatively anerobic rods such as the Vibrionaceae, the Flavobacterium, and the Zymomonas
  • the gram-negative aerobic rods and cocci such as the Azotobacteraceae, the Rhizobiaceae, the Methyl- omonadaceae, the Balobacteriaceae
  • Transposable elements have been found in eukaryotes (Beanney, D.C., et al. supra; Freeling, H., Ann . Rev. Plant Physiol., 35:2777-298 (1984); Roeder, G.S., et al., In Mobile Genetic Elements, Shapiro, J.A. (Ed.), Acad. Press, H.Y., PP 299-328,(1983)) .
  • prokaryotic transposable elements may be present in one or only a few copies per cell
  • eukaryotic cells may contain as many as 30-40 copies of a eukaryotic transposable element.
  • These multiple copies of transposable elements significantly complicate genetic manipulations .
  • the presence of multiple copies of a transposable element may lead to recombi national events between two transposable elements resulting in the generation of additional mutations.
  • Eukaryotic transposable elements are far less well characterized than prokaryotic transposable elements .
  • eukaryotic transposable elements lack selectable determinants thus significantly complicating the selection and identification of cells that carry an engineered or recombinant transposable element.
  • prokaryotic transposable elements which have rendered these transposable elements so useful, are largely absent from eukaryotic transposable elements .
  • Natural prokaryotic transposable elements are unable to undergo transposition in a eukaryotic cell. This inability results from the requirement that the transposase be present during the transposition event. If a natural prokaryotic transposable element was introduced into the nucleus of a eukaryotic cell it would not be able to express the transposase gene since the prokaryotic promoter regions of the element would not be recognized by the eukaryotic enzymes. Even if the gene were transcribed, the RNA transcript would have to leave the nucleus in order to be translated into the transposase enzyme. Thus, even if the RNA was translated into protein, the protein would be in the cell's cytoplasm.
  • Transposition could not occur unless the enzyme was present in the cell's nucleus where the transposable element and the recipient DNA molecule are located. Since bacterial transposases have no means of identifying and entering the- cell's nucleus, they are unable to catalyze transposition in eukaroytic cells.
  • Jiminez, A., et al. (Nature, 287:869-871 (1980)) introduced the Tn5 transposable element into yeast in order to determine whether the element's aminoglyco- side phosphotransferase gene could be expressed in eukaryotic and be used as a selectable determinant in eukaryotes.
  • this work later extended by Colbere-Garapin, F., et al., (J. Mol.
  • the prior art shows the desirability of using transposons to investigate, identify, modify, and control gene expression in eukaryotes.
  • the prior art further shows the desirability of using transposons which, when integrated, permit the selection of recipient cells from the total cell population.
  • natural eukaryotic transposable elements exist they are, in general, difficult to modify.
  • the production of recombinant eukaryotic transposable elements, having the utility of prokaryotic elements is not yet possible.
  • By shuttling DNA between bacteria and a eukaryotic cell it is possible to achieve transposition of eukaryotic DNA which has already been cloned.
  • the applicability of this technique is quite limited. No technique exists which is capable of directing such transposition directly in a eukaryotic cell. Such a technique would be extremely useful for the isolation and study of gene sequences.
  • the prior art also shows the desirability of using transposons to investigate, modify, and control gene expression in those karyotic microorganisms for which no known or sui e transposition system currently exists.
  • the availability of a selectable transposition system for such microorganisms would greatly accelerate our understanding of their genetics and biochemistry. Since these prokaryotic microorganisms include the great bulk of economically important microorganisms, the ability to manipulate and control gene expression in these organisms is highly desirable.
  • Figure 1 shows the cloning strategy through which the plasraids of the present invention were derived.
  • Plasmid pMKK58 is an E. coli vector which contains an excisable DNA fragment containing a structural transposase gene.
  • Figure 3 shows the cloning strategy through which plasmid pYMK2 was constructed.
  • Figure 4 shows the functional map of plasmid pYMK2 which is an E. coli-yeast shuttle vector capable of being used to provide a functional transposase enzyme to yeast.
  • Figure 5 shows the restriction endonuclease cleavage map of plasmid pYMK3 which is an E. coli-yeast shuttle vector capable of being used to provide a functional transposase enzyme, and a transposase element to yeast.
  • Fi gure 6 shows the resul ts of hybridization experiments performed to demonstrate that the insertion of a eukaryotic transposable element can occur at different sites in the yeast chromosome.
  • Fi gure 7 shows a restriction endonuclease map of plasmid pYMK100, which contains a transposable element capable of control l ing the expression of genes which are located near to the site of its insertion.
  • Fi gure 8 shows the use of an engineered transposable element to yield conditional mutations .
  • Figure 9 shows a restriction endonuclease map of plasmid SMI which carries a transposable element and, therewithin, a Tn5 transposase gene behind a Streptoi ⁇ yces promoter;
  • Figure 10 shows a map or plasmid SM2 for Strep toi ⁇ yces transposition using a Mu-based transposable element
  • Figure 11 shows a map of pl asmid SM3, similar to pSH2 shown In Fi gure 10 but having the transposase gene and the transposable element physically separated by the plasmid;
  • Figure 12 and 13 show maps of plasmids YMK20 and YMK18, respectively, each of which Is a deletion derivative of pYMK3 ( Figure 3) and Incapable of transcribing functional transposase;
  • Figure 14 is a map of plasmid YMK12 useful in eukaryotic transposition and having an ars repl ication region rather than a 2 ⁇ repl ication region;
  • Figure 15 1s a map of plasmid YMK30 which contains uas sequences to prevent histone binding in eukaryotes and incorporate a transposase gene behind an indudble promoter, and;
  • Figure 16 is a nucleotide sequence representing the uas containing segment at one terminus of the transposable element on YMK30 (Fig. 15) .
  • a universal system is described, which is capable of mediating transposition in prokaryotic or eukaryotic cells.
  • a cell In order for genetic transposition to occur, a cell must be provided with a transposable element and a functional transposase gene.
  • the present invention provides a transposable element and a modified transposase gene which will permit transposition to occur in any organism.
  • the ability to induce genetic transposition permits the study and manipulation of prokaryotic or eukaryotic cells, and is therefore a significant advance of importance in Biology, Agriculture, and Medicine.
  • the present invention relates to a recombinant DNA molecule which comprises a transposase gene having a restriction endonuclease recognition site, such that the transposase gene is capable of being operably linked to a DNA sequence selected from the group consisting of: a heterologous promoter
  • the present invention additionally pertains to a DNA construct which comprises a transposase gene operably linked to a nuclear localization signal sequence or to a heterolo moter region.
  • the present invention also relates to a transposable element which contains an exogenous DNA sequence.
  • the invention also pertains to a DNA molecule which comprises a transposable element, and a DNA construct, the DNA construct comprising a transposase gene, the transposase gene being operably linked to a heterologous promoter region sequence and/or a heterologous nuclear localization signal sequence.
  • the invention also discloses a method for inducing genetic transposition in a prokaryotic cell which comprises:
  • a DNA construct which comprises a transposase gene operably linked to a heterologous promotor region, wherein the transposase gene expresses a transposase enzyme capable of recognizing the transposable element (i) and directing its transposition in the prokaryotic cell, and
  • the invention further discloses a method for inducing genetic transposition in a eukaryotic cell which comprises:
  • the invention also provides a method for controlling the expression of a target gene in a prokaryotic cell which comprises:
  • transposable element containing an exogenous DNA sequence which comprises a conditional and heterologous promoter region sequence capable of directing the transcription in the prokaryotic cell of an additional DNA sequence when the additional DNA sequence is linked to the transposable element
  • a DNA construct which comprises a transposase gene operably linked to a heterologous promoter region, wherein the transposase gene expresses a transposase enzyme capable of recognizing the transposable element (i) and directing its transposition in the prokaryotic cell
  • the invention further provides a method for controlling the expression of a target gene in a eukaryotic cell which comprises:
  • transposable element containing an exogenous DNA sequence which comprises a conditional and heterologous promoter region sequence capable of directing the transcription in the eukaryotic cell of an additional DNA sequence when the additional DNA sequence is linked to the transposable element
  • a DNA construct which comprises a transposase gene operably linked to a nuclear localization signal sequence, the nuclear localization signal sequence being operably linked to a promoter region; the transposase gene being heterologous to the promoter region or to the nuclear localization signal sequence, wherein the promoter region directs the synthesis of a transposase enzyme, the enzyme being linked to the amino acid sequence encoded by the nuclear localization signal sequence, and capable of entering the nucleus of the eukaryotic cell; the enzyme being capable of recognizing the transposable element (i) and directing its transposition in the eukaryotic cell, and
  • the invention relates to the plasmids pMKK58, pYMK3, and pYMK100 and their functional derivatives.
  • Saccharomyces cerevisiae strain CMY135 (trp deletion; ura3-52) containing plasmid pYMK3 was deposited with the American Type Culture Collection (ATCC), Rockville, MD on June 19, 1986, and designated ATCC No. 20800.
  • Transposition is a genetic process through which DNA sequences, termed “transposable elements,” are inserted into the DNA sequence of a second DNA molecule.
  • the process is catalyzed by an enzyme, termed a “transposase.”
  • the DNA sequence into which the transposable element is being inserted is herein termed the "recipient" DNA sequence.
  • transposition does not require DNA replication of the plasmid vector and thus may occur in a cell which contains a non-replicating plasmid having a transposable element and a functional transposase gene.
  • a “transposase” gene is one which encodes a "transposase enzyme.”
  • a transposase enzyme is capable of catalyzing the transposition of a transposable element into a recipient DNA sequence.
  • a transposase gene is said to be capable of directing the expression or synthesis of a transposase enzyme if, upon introduction into a cell, the transposase gene provides sufficient information to permit the cell to synthesize a transposase enzyme.
  • some transposable elements such as , Tn3
  • require the expression of additional genes such as a "Resolvase”
  • Transposable elements which require such additional genes are disclosed in Mobile Genetic Elements , Shapiro , J .A. , et al . , eds . (Acad. Press , NY, ( 1983 ) ) . In employing such transposable elements in accord with the present invention, it would, of course , be necessary to additionally provide the cell with functional genes capable of expressing these enzymes .
  • a DNA sequence requires that the DNA sequence be "operably linked" to DNA sequences which contain transcriptional and translational regulatory information.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequences sought to be expressed are connected in such a way as to permit gene expression.
  • the precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which , in prokaryotes , contains both the promoter (which directs the initiation of RNA transcription ) as well as the DNA sequences which, when transcribed into RNA, signal the initiation of protein synthesis .
  • Regulatory regions in eukoytic cells will in general include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Two DNA sequences are said to be operably linked if the nature of the connection between the two DNA sequences does not result in either the introduction of a frame shift mutation, or interfere with the ability of the promoter region sequence to direct the transcription of the gene sequence, or of the gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a gene if the promoter were capable of transcribing the gene.
  • a nuclear localization signal sequence is said to be operably linked to a gene if the linkage results in the formation of a gene product which contains the amino acid sequence encoded by the nuclear localization signal sequence.
  • a promoter region is said to be operably linked to a nuclear localization signal sequence if transcription from that promoter results in the formation of an mRNA transcript which is translatable into the amino acid sequence of the nuclear localization signal sequence.
  • transposase gene to a heterologous promoter region and/or nuclear localization signal sequence requires, in general, the existence of a restriction endonuclease recognition site between the transposase gene and the heterologous DNA sequence with which linkage is desired. As is well known in the art, the presence of such a site need have no effect on the transcription or translation of a DNA sequence which contains it.
  • any portion of the gene which originally preceded the locus of the site will be separated from the remainder of the gene by the inserted region.
  • the restriction site may precede the first codon of the gene but, according to the present invention, must be positioned after the end of that critical DNA sequence of the homologous promoter region which prevents the natural transposase gene from being expressed. Examples of such critical DNA sequences are the promoter sequence or the protein initiation sequence of the promoter region.
  • the restriction endonuclease. site between the end of the promoter region and the beginning codons of the transposase gene it is preferable to position this site between the end of the promoter region and the first codon of the transposase gene.
  • this site it is important that no DNA sequence be introduced which interfere with the transcription or translation of the transposase gene. Examples of DNA sequences which would interfere with the transcription or translation of a gene are those sequences causing frameshift or termination mutations, or those which result in the loss of the initiation codon (ATG) from the transposon gene.
  • the introduction of the restriction site permit the transposase gene to be operably linked to a second DNA molecule containing a heterologous promoter or nuclear localization signal sequence.
  • restriction site described above may be either lost or retained after the promoter region and heterologous transposase gene have been operably linked.
  • transposase enzyme catalyzes transposition
  • Potential mechanisms for transposition are discussed in Berg, D.E., et al. supra. It is, however, widely appreciated that for transposition to occur a transposase enzyme must recognize the terminal sequences of a transposable element.
  • a DNA sequence is said to be "recognized" by a transposase enzyme if the transposase enzyme is capable of specifically interacting with, or binding to, it.
  • Transposition may occur either at a specific site, a preferential site, or at a substantially random site in the recipient DNA molecule.
  • site specific transposition is the integration of the bacterial virus lambda into the E. coli chromosome (Nash, H.A., Ann. Rev. Genet., 15:143-167 (1981)).
  • An example of transposition which results in insertion into preferential sites is the transposition of the Tn3 transposable element (Tu, CD., et al., Cell, 19:151-160 (1980)).
  • Examples of transposition which occur in a manner which is substantially independent of the DNA sequence of the recipient DNA molecule are the transposition of the E.
  • coli transposable element Tn5
  • insertion is said to be "substantially independent" of the DNA sequence of a recipient DNA molecule if either no insertion site sequence preference is di scernible , or if transposition can be observed to occur into substantially any recipient DNA sequence .
  • the terms " integrate, " "insert , “ or “disrupt” are meant to be interchangable.
  • central DNA sequence refers to a double stranded DNA sequence which is flanked on both sides by additional DNA sequences . Those DNA sequences which flank a central DNA sequence are referred to interchangeably as either " termini sequences" or “terminal sequences. " Two termini sequences of DNA are said to be “mutually complementary and inverted, " if the termini sequence to the lef t of a central DNA sequence is capable of annealing to the termini sequence of DNA present on the right side of a central DNA sequence.
  • An example of a DNA sequence which is mutually complementary and inverted are the DNA sequences at the termini of Tn5 (Berg , D.E. et al . ) .
  • flanking complementary and inverted termini sequences are represented by the flanking 13 base sequences .
  • flanking complementary and inverted termini sequences are represented as a 13 base sequence in the above example, it is to be understood that the actual mutually complementary and inverted termini of a transposable element are considerably larger and differ from the illustrative 13 base sequences shown above.
  • the mutually complementary and inverted termini sequences of the transposable element Tn5 are, for example, greater than 1,500 base pairs in length.
  • the 13 base sequence shown above is provided solely to serve as an example of a mutually complementary and inverted termini sequence.
  • termini sequences need not be completely mutually complementary and inverted; a DNA sequence is considered to be mutually complementary and inverted to another DNA sequence if a substantial number of bases in the DNA sequences are mutually complementary and inverted.
  • a transposable element may contain a "detectable marker gene” or “detectable determinant” which enables or facilitates the identification of cells which contain the transposable element.
  • marker genes enable the selection of cells which carry the transposable element by conferring, for example, cellular resistance to antibotics or by complementing an auxotrophic deficiency.
  • detectable marker genes are the aminoglyoside phosphotransferase gene of Tn5, the ura3 gene of Saccharomyces cerevisiae, or the Beta-lactamase gene of pBR322, etc.).
  • a detectable marker gene may merely facilitate the screening of desired cells by expressing a product which can be readily detected.
  • a transposable element may also contain additional DNA sequences within the central region of the transposable element. These additional DNA sequences, if not normally found within a transposable element, are termed "exogenous" sequences.
  • the DNA sequence of a transposable element or a transposase gene may contain either homologous or heterologous DNA. A DNA sequence is said to be homologous to a second DNA sequence, if both sequences are normally or naturally linked to one another.
  • a DNA sequence is said to be "heterologous" with respect to a second DNA sequence if the two DNA sequences are not normally, or naturally found to be operably linked to one another.
  • An example of a transposable element which contains an exogenous sequence is a Tn5 transposable element whose central region carries the ura-3 gene of Saccharomyces cerevisiae.
  • Such sequences may be either cryptic (i.e. incapable of encoding the amino acid sequence of a protein) or "expressible” (i.e. capable of encoding the amino acid sequence of a protein).
  • Examples of a cryptic DNA sequence are: sequences which lack transcriptional or translational regulatory regions, gene fragments, and DNA sequences which contain only transcriptional and translational regulatory regions but lack transcribable DNA sequences.
  • Examples of expressible sequences include transcribable sequences which contain transcriptional and translational regulatory regions, such as intact functional genes. It is possible to convert an expressible DNA sequence into a cryptic DNA sequence. and vice versa, as for example by removing a promoter region from a DNA sequence which contains such a region operably linked to ah intact gene, or by adding a promoter region to a DNA molecule which had previously been cryptic.
  • transposition requires the simultaneous presence of three elements: a transposable element, a recipient DNA molecule, and a transposase enzyme.
  • a transposable element as used in this invention, may be any DNA sequence which comprises a central DNA region bounded on both ends by termini regions which are recognizable by a transposase enzyme. It is possible for this central region to be of substantial size and to include several genes. It is desirable that at least one of these genes encodes a protein whose presence can be readily detected, or which confers a survival advantage to recipient cell.
  • the central region may additionally contain a transposase gene which may either be similar to or different from the transposase gene present in the right termini region.
  • the transposable element will be capable of inserting itself into a recipient DNA molecule. If the central region does not contain a transposase gene, or if the transposase gene is not expressed or expresses a product which is incapable of recognizing the terminal DNA regions of the transposable element, then the transposable element will not undergo transposition unless a transposase enzyme capable of being expressed and functioning is additionally supplied. It is possible to direct transposition using two discrete molecules - one containing a transposable element and. the second containing a functional transposase gene.
  • the transposable element of the invention may be present by itself (as a discrete DNA sequence) or may be associated with cellular, viral or plasmid DNA. Typically, one associates the transposable element with cellular, viral or plasmid DNA in order to facilitate its introduction into a recipient cell. Thus, for example, one may produce a recombinant plasmid- which carries the transposable element of the invention, and introduce the transposable element into a recipient cell along with the plasmid by transformation. An example of such a plasmid is pYMK3. Once introduced into a cell, the transposable element (and any associated DNA) is transported to the cell's nucleus.
  • DNA construct refers to a DNA sequence which has been deliberately created. A DNA construct may be present on a plasmid, virus or chromosomal DNA molecule.
  • a functional derivative of a plasmid is any DNA molecule which is capable of performing substantially the same functions as those which the plasmid is capable of performing.
  • a DNA sequence may be introduced into a cell by any of several means: transduction, transformation, conjugation, or microinjection, although it is most preferable to use transformation (Botstein, D., et al., The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, Cold Spring Harbor, N.Y., 11B:607-636 (1982); Struhl, K., Nature, 305:391-397 (1983); Bollon, A.P., et al., J. Clin. Hematol. Oncol., 10:39-48 ( 1980 ) ; Wigler, H. , et al. , Proc. Natl . Acad. Sci . U . S .A. , 76 :1373-1376 ( 1979 ) .
  • target gene may refer to any recipient DNA sequence.
  • target gene may refer to any DNA sequence whose study or investigation is desired.
  • the leu-2 gene would be a target gene.
  • both the transposable element, and the recipient DNA sequence act in an essentially pass ive manner while undergoing transposition.
  • the active participant in this process is the transposase enzyme (which determines , for example, the extent of insertion site specificity) .
  • One aspect of the present invention is to provide means for establishing a transposition system in such prokaryotic stains.
  • transposable element and a functional transpos ase gene In order for a transposition system to function in a prokarytic cell, it is necessary to provide to the cell a transposable element and a functional transpos ase gene and, if required by the transposable element, other functional genes , such as a resolvase gene. Since the transposable element and the recipient DNA sequences are passive participants in the transposition process, a transposition system can be developed in any prokaryotic cell capable of expressing a functional transposase. The expression of a functional transposase in a prokaryotic cell , requires that a transposase gene be operably linked to a functional promoter region. The inability to observe transposition in a transformable prokaryotic cell is therefore a reflection of the inability of that cell to direct the expression of the transposase gene.
  • a transposition system can be developed in any prokaryotic cell by providing to that cell the Tn5 transposable element and a Tn5 transposase which has been operably linked to an endogenous promoter of the microorganism in which transposition is sought.
  • the present invention fulfills this need by providing a plasmid vector in which the normal Tn5 transposase promoter has been removed from the Tn5 transposase gene in such a manner as to permit any promoter region to become operably linked the Tn5 transposase gene.
  • one aspect of the present invention provides a system of transposition which maybe employed in any prokaryotic species.
  • a transposition system it is necessary only to operably l ink a promoter from the prokaryotic species In which transposition is desired, to a functional transposase gene.
  • a recombinant transposase gene is introduced into a prokaryotic cel l with a transposabl e element, genetic transposi tion will occur.
  • this embodiment of the present invention is not limited to the narrow range of prokaryotic strains in which transposition as currently been observed, but rather is a general method appl icabl e to al l prokaryotic organisms.
  • a review of known prokaryotic promoters is provided by Hawl ey et al . 1983, Nucleic Acid Res, 11, 2237-2255.
  • transposase enzyme In order that a transposase enzyme shall be capable of identifying and entering the nucleus of a eukaryotic cell, it is necessary that the transposase gene be operably linked to a DNA sequence, termed a nuclear localization signal sequence which is itself operably linked to a functional eukaryotic promoter.
  • a nuclear localization signal sequence is a DNA sequence which, when operably linked to a second DNA sequence and a promoter region, permits the protein specified by the DNA sequence and nuclear localization signal sequence to identify and enter the nucleus of a eukaryotic cell.
  • Nuclear localization signal sequences are described in Kalderon, D., et al. (Cell, 39:499-509 (1984)); Silver, P.A., et al. (Proc. Natl. Acad Sci U.S.A., 81:5951-5955 (1984)); Hall, M.N., etal. (Cell, 36:1057-1065 (1984)); and Davey, J., et al. (Cell, 40:667-675 (1985)). Although any nuclear localization signal sequence may be used in this invention it is preferable to use the nuclear localiza
  • tion signal sequence of the large T antigen of SV40 (Kalderon, D. , et al . , supra . ) .
  • Any functional eukaryotic promoter such as the Metallothionein, silk f ibroin, insulin, SV 40 Large T promoter, etc. , may be employed, however it is preferable to use the ADHI promoter of Saccharomyces cerevisiae . Such linkage will result in the formation of a hybrid transposase enzyme .
  • the nuclear localization sequence is said to be heterologous with respect to the transposase gene if the nuclear localization sequence is linked to the transposase gene by recombinant DNA techniques or is not naturally found to be linked to the transposase gene .
  • heterologous nuclear localization sequence transposases are: the transposase resulting from the operable linkage of an SV40 nuclear local ization sequence with the Tn5 transposase gene , arid a eukaryotic promoter , or the transposase resulting from the operable linkage of a transposase gene with the nuclear localization sequence of a different gene and a eukaryotic promoter.
  • transposase enzyme in order for a transposase enzyme to be able to catalyze transposition in a eukaryotic nucleus , it must be capable of being expressed and of identifying and entering the nucleus of the eukaryotic cell. This objective is attained in the present invention through the operable (i .e . , functional) linkage of a nuclear localization sequence (which is itself operably linked to a functional promoter ) to a functional transposase gene.
  • the transposase gene may be provided on a discrete DNA molecule or may, like the transposable element, be associated with cellular, viral or plasmid DNA.
  • transpos ase gene is contained within the central region of a transposable element it will undergo transposition along with the transposable element. It is, however, possible to include a transposase gene on the same DNA molecule as that which contains the transposable element, but yet not include the transposase gene within the central DNA region of the transposable element.
  • An example of such a DNA molecule is plasmid pYMK3 in which the transposase gene is not contained within the transposable element. In such a molecule, the transposase gene would not undergo transposition along with the transposable element.
  • one aspect of the present invention provides a system for establishing transposition in any eukaryotic cell.
  • a transposition system it is necessary only to identify a promoter which will function in that eukaryotic cell and then to operably link such a promoter to a nuclear localization sequence which is linked to a functional transposase gene.
  • a recombinant transposase gene and a transposable element are introduced into the eukaryotic cell genetic transposition will occur.
  • the present invention is generally applicable to any eukaryote, such as the Cyanophyta, the Euglenophyta, the Chlorophyta, the Chrysophyta, the Pyrrophyta, the Phaeophyta, the Rhodophyta, the Myxomycophyta, the Eumycophyta, such as the Ascoraycetes or the Basidiomycetes, the Bryophyta, the Tracheophyta, as well as the Porifera, the Mesozoa, the Coelenterata, the Ctenophora, the Platyhelminthes, the Nemertina, the Acanthocepahala, the Aschelminthes, the Entoprocta, the Ectoprocta, the Phoronida, the Brachi opoda, the Mollusca, the Sipunculida, the Echiurida, the Annelida, the Onychophora, the Tardigrada,
  • the transposable elements of the present invention may be used in the same manner as natural prokaryotic transposable elements in the formation of novel cells or microorganisms. These novel cells or microorganisms may express new desired properties or may have been altered to prevent the further expression of undesirable properties.
  • the transposable elements of the invention may be used to produce derivative strains having new combinations of genes. Alternatively, they may be used to introduce foreign or "nonnatural" DNA sequences into a cell.
  • the transposable elements of the invention like natural prokaryotic transposable elements, may be properly considered to be tools for strain construction.
  • transposable elements of the invention may be employed as tools for strain construction in either of two possible and fundamentally different manners: by transposition, or by genetic recombination.
  • the transposase enzyme of the invention catalyzes the insertion of the invention's transposable element (which includes only the DNA between the termini regions of the transposable element, and no DNA beyond these regions) into the DNA sequence of a recipient DNA molecule. Concomitant with this insertion is the disruption of the gene of the recipient DNA molecule into which the insertion occurs.
  • transposition is genetic mutation.
  • transposition is substantially independent of the DNA sequence of the recipient DNA molecule, then it is possible for transposition to occur in an essentially random manner.
  • the consequence of performing a transposition experiment on a population of cells or microorganisms, using the above-described transposase and transposable element, is the generation of a collection of derivative cells or microorganisms, each of which possesses the same transposable element, but in which the precise site of integration of the transposable element is different.
  • the transposable element of the invention contains a selectable marker gene, it is possible to directly select for cells which have undergone transposition.
  • transposase gene does not undergo transposition with the transposable element
  • the transposase enzyme which is encoded by the transposase gene of the right termini region of the Tn5 is not linked to either a nuclear localization signal sequence nor a functional eukryotic promoter.
  • this transposase gene will not be functional in a eukaryotic cell.
  • the recombinant transposase gene does not undergo transposition with the transposable element, the transposase gene will not stably integrate or replicate in the eukroytic cell, and will be irretrievably eliminated from the cell.
  • the resulting cell contains an integrated transposable element, but does not contain any functional transposase gene.
  • a subsequent transposition event could not occur.
  • An example of a plasmic containing a transposable element consistant with this embodiment is the plasmid pYMK3 (discussed below).
  • the second use of the transposable elements of the present invention, as tools of strain construction, is to significantly facilitate genetic manipulations which involve genetic recombination. In such manipulations, it is desired to either alter the expression of a particular gene, or to introduce new genetic information into a particular cell or microorganism. Previously, such manipulations were often extremely tedious in that it was frequently difficult, or impossible, to select the desired cell or microorganism (which might be quite rare).
  • Transposable elements because of their ability to confer a selectable phenotype to recipient cells, have the potential for greatly facilitating such experiments (Kleckner, N., et al., J. Mol . Biol., 116:125- 159 (1977).
  • selection is initially made for insertion which occurs by transposition into a DNA sequence of a recipient DNA molecule adjacent to or within a desired target gene.
  • the transfer of the desired target gene into a new cell or microorganism, or the mutation of the desired target gene can then be accomplished by first assaying for the presence of the transposable element (by its ability to confer a selectable phenotype), and then assaying for the expression of the desired target gene.
  • the transposable element of the present invention finds additional use as a portable region of DNA sequence homology. Frequently, it is desirable to combine two DNA molecules (such as, for example, two plasmids) to form a single DNA molecule. Although this goal can often be achieved through the use of restriction endonucleases, such is not always the case. For example, if the two DNA molecules being combined are uncharacterized, or contain no convenient restriction endonuclease sites, then techniques which employ restriction endonuceleases cannot be used. In such a situtation, it is possible to insert transposable elements into both DNA molecules, thereby creat ing regions of DNA sequence homology in both DNA molecules. Various bacterial and mammalian enzymes (such as, for example, RecA (Radding, CM., supra.)) are capable of joining such DNA molecules through sequence specific recombination.
  • RecA RecA
  • the transposable elements of the present invention have significant additional uses in the fields of molecular biology and recombinant DNA technology.
  • the insertion of the transposable elements of the present invention into a eukaryotic cell will result in a disruption of transcription.
  • the insertion of the transposable elements of the present invention result in the formation of polar mutations in the recipient DNA molecule.
  • a polar mutation is- one which results in the interruption of mRNA transcription of a DNA sequence.
  • a promoter is believed to direct the transcription of a DNA sequence, and the transposable element of the present invention inserts itself between the promoter site and this DNA sequence, mRNA transcription will not proceed through the transposable element.
  • the DNA sequence which would normally have been transcribed by the oromoter will not be transcribed.
  • the transcriptional arrangement of a gene may be investigated through the use of transposable elements. In such an investigation, one would permit transposition to occur and then assess whether a particular gene is being expressed.
  • transposable element Failure of a particular gene to be expressed would indicate either that the transposable element had directly inserted .into the target gene, or that it had inserted itself between the target gene and the normal promoter of the target gene.
  • An example of the use of transposable elements to elucidate transcriptional patterns in bacteria is provided by Harayama, S., et al. (J. Bacteriol., 153:408-415 (1983)).
  • the transposable element of the present invention may be used to search for secretion, processing, terminator, or other regulatory signal sequences.
  • transposable element which has been further modified for this purpose.
  • Casadaban, M., et al. Proc. Natl. Acad. Sci. U.S.A., 76:4530-4533 (1979)
  • a transposable element which contained an exogenous gene within the left terminal repeated region.
  • any gene capable of expression may be employed, it is preferable to use a gene whose expression may be easily monitored. Examples of such genes are the beta-galactosidase gene of E. coli, or the chloramphenicol acetyl transferase gene of pBR325, etc .
  • exogenous gene product is one which can be easily monitored, its presence outside of the cell can eas ily be asertained.
  • a transposase like many biological catalysts, is capable of catalyzing both forward and reverse reactions .
  • a .transposase may operate on a transposable element and a recipient DNA sequence 'to direct transposition (the forward reaction ) , and may operate upon an integrated transposable element to direct the excision of the transposable element from the recipient DNA molecule (the reverse reaction) .
  • the excision of a transposable element when mediated by a transposase enzyme , is a precise event ( i . e. , the transposable element is excised in such a manner as to reform the exact nucleotide sequence which existed in the recipient DNA molecule prior to the insertion of the transposable element) .
  • transposable elements of the present invention may be modified so as to enable them to identify essential genes or to regulate and control gene expression.
  • An "essential gene” is one whose expression is necessary for cellular growth or viability. Examples of essential genes are DNA replication genes or genes which encode components of the cellular envelope.
  • transposable element which can be used to identify essential genes. To produce such a transposable element, it is desirable to modify the left-hand terminal region so as to create a functional promoter region. If the right hand terminal region is modified, the ability to express the transposase gene (which is in the right hand region) may be affected.
  • an additional functional transposase gene may, therefore, be provided.
  • the modification of either termini is possible when using this invention to regulate expression in a eukaryotic cell since for such cells an additional transposase gene is generally necessary.
  • This modification may be accomplished either by mutation, or through the cloning of a known promoter region into the transposable element. Regardless of the source or origin of the promoter region, or its position within the transposable element it is necessary, according to the present invention that the promoter region direct transcription toward the terminus of the transposable element and past its end. Additionally, it is desirable that the promoter region be capable of directing conditional expression of any DNA sequence to which it becomes operably linked.
  • suitable promoter regions would be those which direct transcription only at elevated temperatures, or in the presence or absence of a biological compound (i.e. a sugar, vitamin, amino acid, etc.).
  • a biological compound i.e. a sugar, vitamin, amino acid, etc.
  • promoter regions and controlling genes are the lac promoter and the lac I gene, or the pL promoter of bacteriophage lambda, and the lambda cl represser gene.
  • conditional promoter regions when working with poorly characterized prokaryotic or eukaryotic cells, no conditional promoter region will have been previously identified. In such a situation, as wpuld be obvious to one of ordinary skill, the previously described procedure for isolating conditional promoters could be employed to identify such promoter regions. Once conditional promoter regions had been identified, they could be cloned and ultimately positioned into the transposable element, as described above.
  • transposable elements When the above-described transposable elements undergo transposition, in a prokaryotic or eukaryotic cell, they will, at a detectable frequency, undergo transposition next to an essential gene. If this transposition event results in the formation of an operable linkage between the essential gene and the conditional promoter region, then the essential gene will continue to be expressed despite the presence of the transposable element. Since the expression of the essential gene is dependant upon a conditional promoter, the cell will cease to grow and die when the culturing conditions are altered so as to repress or deactivate the ability of the promoter region to direct gene expression.
  • an essential gene, operably linked to the above described transposable element could easily be detected by allowing transposition to occur and then screening the surviving cells for those capable of growth only under conditions which permit the expression of the conditional promoter. Because the above described transposable elements contain detectable markers, the essential gene operably linked to them may easily be isolated and subjected to further investigation. Alternatively, a transposable element containing a non-conditional promoter may be used in order to constitutively alter the expression of the adjacent gene.
  • transposable elements so as to enable them to direct the secretion of normally non-secreted gene products.
  • This can advantageously be accomplished by operably linking a secretory signal sequence to a promoter region which has been inserted intp the left-hand terminal region of the transposable element.
  • the degree and control of secretion may be accomplished by employing promoters having different levels of expression (i.e. strong or weak promoters) or by employing constitutive or conditional promoter regions.
  • conditional promoter permits one to replace or substitute a gene's normal promoter for a desired conditional promoter.
  • a gene is poorly expressed, or is expressed only under undesirable culturing condtions (such as subsequent to irradiation treatment, or at an extreme temperature, etc.) it is possible to construct and isolate cells in which the desired gene is controlled by conditional promoter regions which are activated or deactivated in a more desired manner.
  • novel transposable elements of the present invention can be used to identify and study essential genes.
  • the present invention provides a means for enabling any gene product to be expressed and excreted into the extracellular environment. Moreover, they may be used to alter the regulation and expression of any desired gene.
  • EXAMPLE 1 GENERAL PROKARYOTIC TRANSPOSITION SYSTEM The plasmid pMKK20 was grown in E. coli strain CSH4 which contained the transposable element Tn5. Plasmid DNA was purified from this bacterial strain and used to transform RR1 cells which lacked the transposable element, and transformants which were kanamycin resistant were isolated. These transformants were found to contain plasmids which carried the Tn5 transposable element.
  • One such plasmid, designated pMKK23 was found to contain a Tn5 element oriented so that the right hand inverted repeated termini region was adjacent to a Sal I restriction and the nucleus cleavage site.
  • Plasmid MP19 (P.L. Biochemicals) is a derivative of M13. Plasmid MP19 was subjected to digestion with Sal I and Bam HI restriction enzymes and the Sal I-Bcl I fragment which contained the right hand terminal region of the Tn5 was introduced into this plasmid. The Sal I-Bcl I fragment contains the Tn5 transposase gene along with its promoter region.
  • the MP19 derivative which contained the Tn5 transposase gene was subjected to in vitro mutagenesis.
  • Techniques of in vitro mutagenesis involving M13 or its derivatives are disclosed by Kunkel, (Proc. Natl. Acad. Sci. U.S.A., 82:488-492 (1985)) Nisbet, I.T., et al. (Gene Anal. Tech., 2:23-29 (1985)), and Hines, J.C., et al., (Gene, 11:207-218 (1980)), which are incorporated herein by reference.
  • Kunkel Proc. Natl. Acad. Sci. U.S.A., 82:488-492 (1985)
  • Nisbet, I.T., et al. Gene Anal. Tech., 2:23-29 (1985)
  • Hines J.C., et al., (Gene, 11:207-218 (1980)
  • the procedure en
  • M13 or one of its derivatives, such as MP19, is converted to its single strand form, and incubated in the presence of the synthetic oligonucleotide.
  • the DNA of the oligonucleotide is controllably defined, it is possible to construct an oligonucleotide capable of pairing with a complementary DNA sequence present on the single stranded plasmid. Once base pairing has occured between the oligonucleotide and the single stranded plasmid, it is possible to extend the oligonucleotide using DNA polymerase to create a double stranded DNA molecule which may then be sealed by DNA ligase.
  • This oligonucleotide contains a Sal I restriction endonuclease cleavage site which is bracketed by DNA sequences which separate the promoter region of the Tn5 transposase gene from the functional transposase gene itself.
  • MP19-Transposase 57 construct which contains a Sal I restriction endonuclease cleav age site separating the Tn5 transposase gene from the promoter of that gene (i.e. the promoter regions and the transposase gene are operably linked).
  • the presence of this restriction endonuclease cleavage site does not affect the ability of the normal Tn5 promoter to direct the transcription of the Tn5 transposase gene.
  • plasmid MPl9-Transposase 57 construct is capable of providing a functional transposase to E coli and related genera.
  • the cloning strategy which led to the formation of MPl9-Transposase 57 construct is shown in Figure 1.
  • Plasmid MPl9-Transposase 57 construct was incubated in the presence of the restriction endonucleases Sail and EcoRl and a 1.5 kb restriction fragment which contained the Tn5 transposase gene was isolated. This 1.5 kb fragment lacked the normal transposase promoter region. Plasmid pBR322 was incubated in the presence of the Sail and EcoRl endonucleases and then incubatedwith DNA lisase and the 1.5 kb transposase gene fragment. Through this procedure a plasmid, designated pMKK58 was constructed. This cloning strategy is shown in Figure 2.
  • Plasmid pMKX58 contains the Tn5 transposase gene. This gene is not operably linked to any promoter but is preceded by a Sal I restriction endonuclease site into which a promoter region or a nuclear localization signal sequence could be inserted to form an operable linkage with the transposase gene.
  • DNA from the microorganism in which transposition is desired is extracted and purified by means well known in the art.
  • the purified DNA is then subjected to random restriction endonuclease digestion.
  • restriction endonuclease Sal I it is preferable to use the restriction endonuclease Sal I to accomplish this digestion, any restriction endonuclease may be employed.
  • Plasmid pMKK58 is incubated with Sal I restriction endonuclease under conditions sufficient to cleave the plasmid molecule at the Sal I recognition site present between the Tn5 transposase gene and its promoter.
  • the random digestion fragments obtained by restriction endonuclease cleavage of the DNA of the prokaryotic microorganism is then incubated in the presence of the Sal I-digested plasmid pMKK58 under conditions sufficient to permit the incorporation of one or more of the random fragments to be incorporated into the Sal I site preceding the transposase gene.
  • a restriction endonucelase other then Sal I or an isoschizomeric endonuclease it will be necessary to adjust the termini of the plasmid and random fragments so as to make them complementary and ammenable to DNA ligation.
  • the above described plasmid pool is them introduced into the prokaryotic microorganism (in which transposition is desired) along with any transposable element whose terminal DNA regions are recognized by the Tn5 transposase enzyme.
  • any of the previously described methods for introducing DNA into a prokaryotic microorganism maybe employed, it is in general preferable to employ transformation. Transposition will occur at a detectable frequency in those prokaryotic microorganism which have received both a plasmid containing a transposable element and a plasmid in which the Tn5 transposase gene has been operably linked to a cellular promoter region.
  • CLONED GENES DNA from any prokaryotic microorganism may be isolated and purified by means well known in the art. Such DNA may then be subjected to restriction endonuclease cleavage and religated into a plasmid, such as, for example, pBR322, or alternatively any plasmid capable of replication in the microorganism in which transposition is desired.
  • a plasmid such as, for example, pBR322
  • any plasmid capable of replication in the microorganism in which transposition is desired.
  • Plasmids obtained by the above described procedure are screened to identify individual plasmids which contain DNA inserts which give rise to the production of protein molecules.
  • Such plasmids in general shall contain a cellular promoter which is operably linked to a prokaryotic gene.
  • a prokaryotic gene By techniques well known in the art, one can establish the location and direction of the transcription which proceeds from the cloned promoter region and construct plasmid vectors in which this cloned prokaryotic promoter region has been isolated from the DNA sequences with which it was originally associated.
  • promoter regions from any prokaryotic microorganism. Such promoter regions may be inserted, by means well known in the art, into the Sal I restriction endonuclease recognition site of plasmid, pMKK58 and the recombinant plasmid in which the prokaryotic promoter is operably linked to the Tn5 transposase gene may .then be isolated and purified.
  • a culture of a microorganism in which transposition is desired is provided with two plasmids, one of which contains a transposable element and the second of which contains the above described plasmid having a prokaryotic promoter operably linked to the Tn5 transposase gene. If one first selects for cells which have lost the transposable element containing plasmid (by, for example, using a plasmid which cannot replicate, or by treating the transformed cells with a plasmid curing agent such as acridine orange) and then screens or selects for the detectable marker of the transposable element one would isolate prokaryotic microorganisms in which transposition had occurred.
  • the invention could be performed with a single plasmid having both a trans posable element and the above-described transposase gene .
  • transposable elements could be isolated and operably linked to a heterologous promoter region.
  • a transposable element (recognized by the transposase) are introduced into a cell capable of recognizing the heterologous promoter region, genetic transposition will result.
  • some transposable elements require the expression of additional genes in order to undergo transposition (i.e. Tn3 and TnLO require a "resolvase").
  • the restriction endonuclease cleavage site which is introduced between the endogoneous transposase promoter and the transposase gene is preferably one which is known not to cleave elsewhere in the transposase gene.
  • restriction endonuclease can easily be identified by incubating a plasmid which contains the transposase promoter-transposase gene with various endonucleases and identifing a restriction endonuclease which fails to cleave the plasmid into a linear form.
  • the recognition site of such an enzyme then be synthesized and used as the oligonucleotide fragment as explained in Example 1.
  • transposabl e elements as Tn5, Tn916, Tn3 and Jn10 are unabl e to transpose in Streptomyces.
  • the transposable element STN1 is based on well known, broad host range transposon Tn5 and retains many of its valuable features including the transposase structural gene, transposase binding sites i.e. termini of Tn5, and a region which encodes kanamycin resistance (Km r ).
  • Tn5 for use in Streptomyces however the natural promoter of the Tn5 transposase gene was replaced with a Streptomyces-recognized promoter i.e. the Pc promoter from plasmid IJ101 (see Deng et al . , Gene 43, 295-300) which promoter 1 s known to function i n both E. col i and Streptoayces.
  • the natural promoter of the Km r gene of Tn5 was al so replaced by the Pc promoter.
  • Tn5 As a further modification of Tn5 to produce STN1, the termini of Tn5 were truncated to provide for the minimum transposase binding sequence at the termini of the el ement as described, for example, by Xwoh et al . , 1981, Gene 13, 37-46.
  • STN1 fragments were indi vidually synthesized, assembled in pUC18 and transferred to pMT660, providing pSM1 as shown in F i gure 9.
  • STN1 provides truncated Tn5 termini between which 1s a coding region for Tn5 transposase under Pc promoter influence and, In tandem a coding region for Km r al so under the influence of its own Pc promoter.
  • the transposable element is based on the phage Mu (see Bukham, Ann. Rev. Genet. 1976, 10, 389-412), known to be a large transposable eleaent.
  • Phage Mu is, like Tn5, bound by sites to which transposase binds, which sites may be truncated without losing their affinity for transposase binding.
  • the Mu transposase enzyaes MuA and MuB are encoded in a region between the Mu termini and are under the influence of the natural promoter for MuA.B transposases expression. Also within the central region of phage Mu are regions encoding numerous phage proteins.
  • phage Mu As a transposable element, phage Mu has the advantages that it has been shown to transpose in vitro indicating that host factors are not required; also, its transposase enzymes MuA.B act in trans i.e. are functional at a site remote from its site of synthesis, suggesting that it may be better able to function in a heterologous environment, such as in Streptomyces which is not a natural Mu host.
  • the transposable element STN3 is similar to STN2 in that both are based on the phage Mu.
  • Element STN3 is by design however, actually two separate segments one which codes for Mu transposase and another, separated on the pMT660 vector, which codes for Km r mini Mu.
  • the MuA and B transposase coding region is placed behind the promoter of the thiostrepton resistance gene of pMT660.
  • the mini-Mu/Km r segment of pSM2 was incorporated on pMT660 by substituting it for the Ts r gene of pMT660.
  • pSM3 separates the transposase enzyme producing segment and the transposable element segment. This separation distinguishes pSM3 from pSM2 in which the two segments are combined on the transposable element STN2. Plasmid pSMM, which incorporates these components separately is shown in Figure 11.
  • Streptomyces strain TK24 was transformed wi th pSMl which contains transposable element STN1. Transformants were selected for growth i n the presence of kanamycin and then screened for thiostrepton resistance at 30°C. The Km r , Ts r colonies were transferred to kanamycin containing growth medium and incubated at 42°C, a temperature at which the pl asmid pMT660 is unabl e to repl icate.
  • Colonies were recovered which exhibited the Km r , Ts s phenotype, indicating that ( 1) the plasmid was unstable at 42oC and the Ts r phenotype was coordinately lost and that (2) the Km r phenotype was retained despite the absence of functional pl asmid vector, suggesting that transposition had occurred.
  • the above oligonucleotide has three important properties. Firstly, the left most end provides a recognition, site for the Eco RI restriction endonuclease, whereas the right hand termini contains a recognition site for the Sal I restriction endonuclease (Maniatis, T., et al., supra). Secondly, the sequence contains an ATG codon which would be recognized in a eukaryotic cell as a translation initiation site. Lastly, the oligonucleotide contains a 21 base pair long sequence immediately adjacent to the ATG codon which duplicates the nuclear localization signal sequence of the T-antigen.
  • Plasmid pYCDE2 contains the origin of replication of the yeast 2 micron plasmid (Hollenberg, C.P., Curr. Topics, Microbiol. Immunol., 96:119-144 (1982)) as well as the origin of replication of plasmid pBR322 (Bolivar, F., et al., Gene, 2:95-113 (1977)). Plasmid pYCDE2 also contains the ADHI promoter region of Saccharomyces cerevisiae. The Eco RI site described above is immediately adjacent to the ADHI promoter region.
  • Plasmid pYMK2 is therefore a general eukaryotic vector capable of being used to provide a functional transposase enzyme to the nucleus of any eukaryotic cell which is capable of transcribing a gene from the least ADHI promoter.
  • the cloning strategy through which plasmid pYMK2 was constructed is shown in Figure 3.
  • Plasmid pYMK2 contains the Saccharomyces cerevisiae trpl gene (Department of Genetics, University of Washington, Washington Research Foundation).
  • a Tn5 element was modified so as to additionally contain the ura3 gene of Saccharomyces cerevisiae.
  • a plasmid containing the Tn5 element was purified and subjected to digestion with the endonuclease BamHI. This endonuclease recognizes and cleaves a site present in the central region of the Tn5 transposable element.
  • a 2.2 kb BamHI-Bgl II fragment which contained the ura3 gene of Saccharomyces cerevisiae (Rose, et al., Gene, 29:113-124 (1984) was ligated into the BamHI site and a plasmid containing a Tn5 derivative which contained the ura3 gene was isolated.
  • Plasmid pYMK2 was transformed into an E. coli strai n which contained the above described modified Tn5 transposable element . After overnight growth , the transformants were pooled and plasmid pYMX2 DNA was extracted from them and purif ied . This preparation of plasmid pYMK2 was transformed into an E. coli strain which did not contain Tn5. Among the resulting transformants were those which exhibited resistance, to kanamycin. The plasmid from these transformants were isolated and shown to contain the Tn5 transposable element.
  • Plasmids fulf illing these requirements are therefore suitable for providing both a transposable element and a functional transposase gene to a eukroyotic cell .
  • Plas mid pYMK3 may be envisioned as being composed of two connected regions .
  • the first, or “plasmid region” contains the trp-1 + , and the beta-lactamase genes as well as the both yeast and bacterial origins of replication. In addition this region also contains the hybrid transposase gene with its eukaryotic promoter.
  • the second, or “transposable element region” contains the yeast ura-3 + gene and the antibiotic resistance deterainant gene. The entire transposable element region is bracketed by terminal DNA regions which contain the mutually complementary and inverted DNA sequences of Tn5.
  • Plasaid pYMK3 was introduced into Saccharomyces cerevisiae spheroplasts of strain CMY135 according to the method of Hinnen , A. , et al . (Proc. Natl . Acad. Sci . U.S.A., 75 :1929-1933 (1978 ) ) .
  • CMY135 yeast cells are deficient in their capacity to synthesize trypto- phan (trp deletion) and uracil (ura3-52) .
  • the deficiencies caused by these genetic lesions may be complemented by the trp and ura genes carried by plasmid pYMK3. It is not however, possible to repair these lesions through homologous recombination with the plasmid-borne sequences .
  • the mixture of transformed cells and pYMK3 plasmid was plated on culture medium lacking uracil in order to select for the growth of yeast colonies from cells which had received the plasmid pYMK3.
  • Cells were grown on * C Medium.
  • C Medium contains (per 500 ml) : 3.35g yeast nutrient broth, 4.35g K 2 HPO 4 , 2.9g succinic acid, 20 ml of 50% glucose, 16.6 ml of 60% glycerol, and 25 ml * C concentrate.
  • C concentrate contains (per 300 nil): 120 mg each of adenine, arginine, histidine, isoleucine, leucine, methionine, tryptophan (unless deleted), tyrosine and uracil (unless del 180 mg of lysine; 300 mg of phenylalanine, 600 mg of threonine and 900 mg of valine. After 48 hours of incubation at 37°C, 2000 colonies/ plate had formed, indicating, a transformation frequency of 200 transformants/ug of DNA. These ura + yeast colonies were found to belong to one of two classes: ura + trp- (1%) or ura + , trp + (99%).
  • ura + colonies which were also observed to be proficient in the biosynthesis of tryptophan were found to contain the autonomously replicating plasmid pYMK3.
  • trp + tryptophan
  • ura + trp- Isolates arose as a result of an active transposase gene present on pYMK3
  • deletion derivatives of pYMK3 were constructed.
  • the ADHI promoter and about 600 bases of the 5' end of the transposase gene were removed by cleaving pYMK3 with Sai l and Xhol enzymes and then rel i gating the vector, thereby providing pYMK20 shown in Figure 12.
  • the parallel , another deletion derivative was created by restricting pYMK3 with Hind III to remove about 400 bases from the 3' end of the transposase gene, and the plasmid reli gated to provide pYMK18 shown in Figure 13.
  • yeast cells were separately transformed with pYMK3, pYMK18 and pYHK20, ura + trp- colonies were observed only for those cel l s transformed with pYMK3 but not those transformed by the deletion derivatives pYMK18 ar pYMK20 which are incapable of provi ding a functional mRNA traascript of the transposase gene.
  • the total DNA of these cells was partially digested with the EcoRl restriction endonuclease.
  • the DNA fragment obtained froa this treatment were separated according to size on a 10-40% glucose gradient. Fragments of similar size were pooled , the DNA .precipitated with ethanol, dried in vaccuo and resuspended in 100-200 ul of TE buffer (10mM Tris , 1mM EDTA pH8 .0 ) .
  • the DNA fragments free each pool were ligated into the EcoRl site of the piasamid pBR329 (Covarrubias , et al . (Gene , 17 :79-89 (1982) ) .
  • E. coli strain RRI were transformed with the ligation mixtures .
  • Transformants were selected oa agar containing kanamycin and screened for resistance to ampicillin (Ap R ), tetracycline (Tc R ) and kanamycin (Km R ). This procedure resulted in the identification of a group of plasmids in which the cloned yeast chromosomal DNA contained a kanamycin resistance determinant.
  • E. coli strain FB1009 which is ura-
  • All Km isolates were found to be able to com ⁇ lement the ura- mutation in this strain.
  • the cloned yeast DNA was then analyzed with restriction endonucleases. The patterns generated by EcoRl, Hindlll, Sphl, Clal, Pst and Smal were compared. The DNAs of 12 different isolates were examined. Clones contained 10-25 kb inserts. When treated with the same restriction endonuclease, the restriction patterns generated from the DNA of each isolate varied. The overall restriction patterns differed from the one obtained from plasmid pYMK3. Many sites which were outside of the transposable element region of plasmid pYMK3 were found to be missing from the clonned DNA.
  • plasmid pYMK3 contains a single EcoRl site, several of the clonned inserts contained more than one EcoRX site. These results suggested that while the cloned yeast DNA contained the kanamycin and ura3 genes of the transposable element of plasmid pXMK3, they did not contain other regions of that plasmid. Thus only the transposable element region, bat not the plasmid region of plasmid pYMK3 had been cloned. An isolate of the above described clones, designated as pMKK79-b, was found to contain four EcoRI fragments of 10, 5.0, 4.0, and 0.5 kb in size.
  • the 4.0 kb fragment was identified as the plasmid pBR329.
  • the plasmid DNA of ⁇ MKK79-b was digested with EcoRI and the
  • the pl asmids described previously in the examples as being useful i n obtaining transposition in eukaryotes i .e. pYMK2, pYMK3 and thei r source vector pYCDE2 compri se the 2 yeast origin of repl ication.
  • ars-based pl asmids are commonly used as cloning vectors in yeast. Both the 2 and ars ( autonomously repl icati ng sequence) regions function as repl icons in yeast.
  • the ars-based pl asmids are preferred because they are less stable i n the yeast host, a property which can facil itate detection of a transposition event, and because the lower copy number of the ars region in the yeast host decreases the occurrence of homologous recombination.
  • pl asnid pYHK12 was constructed and tested.
  • pYMK12 a 1.4 kb EcoRI fragment containing the ars region and coding for trp was extracted from pl asmid Yrp7 (see Tschumper and Carbon, 1980, Gene, 10, 157-166) and cloned into the EcoRI site of pBR322.
  • the plasmid pYMK12 represents a useful vector for introducing a transposable element i nto yeast in a manner substanti al ly as described for pYMK2 but having an ars-repl ication sequence rather than a 2 repl ication region.
  • Transposabl e element Tn5/ura was introduced within the Ampicil l in resi stance coding region of pYMK12 , providing pYMK12/Tn5/ura.
  • a control vector was created from pYMK12 by extracting a Sal l/Xhol fragment (shown as "Del /pYMK13 " , in Figure 14) from the 3 ' end of the transposase gene of pYMK12, thereby providing pYMK13 into which the Tn5/ura region was inserted as described for pYMK12.
  • pYMK13/Tn5/ura provides a construct which, apart from i ts inability to encode a functional mRNA transcript of the transposase gene, has substanti al ly the same characteristics as pYMK12/Tn5/ura.
  • cell s of yeast strains CMY135 were transformed with pYMK12/Tn5/ura and pYMK13/Tn5/ura, respectively, using procedures outl ined in Example 4. Colonies surviving on uracil were screened further for the trp) phenotype.
  • transposition of the yeast genome can be accompl ished using vectors which contain a 2 ⁇ replication origin or, preferably, an ars sequence in order to introduce the transposable element into the host according to the present Invention.
  • Pl asmid pYMK100 was constructed in order to provide a transposabl e element which, when integrated into chromosomal DNA, would be capabl e of control l ing the expression of those chromosomal genes adjacent to the site of its insertion.
  • the distinguishing features of plasmid pYMK100 are that it contains a transposable element into which a heterologous promoter has been inserted. Importantly, the promoter 1s capable of directing transcription toward and past one of the ends of the transposable element.
  • the heterologous promoter of plasmid pYMK100 is the gal 10 promoter of Saccharomyces cerevisiae (St. John, P.P., et al., J. Mol. Biol., 152: 285-316 (1981); Johnston, M., et al., Mol. Cell. Biol., 4:1440-1448 (1984); Guarente, L., et al., Proc. Natl. Acad. Sci. USA, 79:7410-7414 (1982); Fried, H.M., et al., Mol. Cell. Biol., 5:99-108 (1985)).
  • Plasmid pYMKlOO was constructed as follows. Plasmid YCDE2 was incubated in the presence of Sphl and Kpnl restriction endonucleases in order to liberate a 6.85 kb Sphl-Xpnl fragment containing the two micron origin of replication, the plasmid pBR322 origin of replication, the yeast trpl gene and the ampicillin resistance determinant of plasmid pBR322. The 6.85 kb Sphl-Kpnl fragment was isolated and purified.
  • a derivative of plasmid pYMK2 which contained a transposable element was purified and designated pYMK4.
  • This plasmid is essentially identical to that of plasmid pYMK3 except that the insertion site of the transposable element in plasmid pYMK4 is somewhat further from the two micron origin of replication than is the transposable element of plasmid pYMK3.
  • the orientations of the transposable elements in plasmids pYMK3 and plasmid pYMK4 are inverted with respect to one another.
  • Plasmid pYMK4 was incubated in the presence of Sphl restriction endonuclease, and a 4.5 kb Sphl-Sphl fragment was isolated.
  • This fragment contained the Tn5 transposase gene operably linked to a nuclear localization signal sequence region which was itself operably linked to the yeast ADHI promoter.
  • this fragment contained the left hand inverted terminal repeat region of the Tn5 transposable element.
  • Plasmid pYMK4 was additionally incubated in the presence of Sphl and BamHI restriction endonucleases, in order to isolate a 2.2 kb fragment having Sphl and BamHI termini. This 2.2 kb fragment contained the yeast ura3 gene.
  • the gal 10 promoter of Saccharomyces cerevisiae was isolated on an approximately 685 bp fragment having EcoRI and BamHI termini.
  • transposable element of plasmid pYMK100 In order for the transposable element of plasmid pYMK100 to be capable of undergoing transposition, it was necessary that it have both left and right handed inverted repeated termini regions.
  • An oligonucleotide was synthesized which contained a transposase binding site, and which could function as the right hand inverted repeated region necessary for transposition.
  • the transposase recognition site of the oligonucleotide was bracketed by synthetically derived EcoRI and Kpnl restriction endonuclease cleavage sites.
  • the nucelotide sequence of the synthetic oligonucleotide is shown below:
  • the EcoRI recognition site is located at the left end of the above oligonucleotide; the Kpnl restriction endonuclease site is located at the right hand termini.
  • the transposase recognition site lies between these two restriction endonuclease cleavage sites.
  • Plasmid pYMK100 was therefore produced by ligating the Sphl end of the 6.85 kb Sphl-Kpnl fragment (from plasmid pYCDE2) to the Sphl end nearest to the ADHI promoter region of the 4.5 kb Sphl-Sphl fragment of plasmid pYMK4.
  • the 2.2 kb Sphl-BamHI fragment (of plasmid pYMK4) was ligated to the free Sphl end (adjacent to the inverted left terminal repeated region) of the Sphl-Sphl fragment.
  • the EcoRI-BamHI fragment containing the gal 10 promoter sequence region was ligated to the free BamHI site.
  • plasmid pYMK100 contains a transposase gene capable of expressing a transposase enzyme in a eukaryotic cell.
  • the plasmid also contains a transposable element which is recognized by the transposase enzyme, and which can undergo transposition into DNA present in the nucleous of a eukaryotic cell.
  • the gal 10 promoter will direct the transcription of DNA sequences which (through transposition) are now located adjacent to the synthetic right hand terminal region of the transposable element.
  • the transcription of such DNA sequences will be dependant upon the induction (by galactose) of the gal 10 promoter.
  • the consequence. therefore, of transposition next to a particul ar DNA sequence is to render the transcription of that DNA sequence under the control of the conditional gal 10 promoter.
  • Control over transposition can be attained, for example, by l inking an inducible heterologous promoter such as the promoters of the meliblase gene, gal 1 gene or gal 10 gene of S. cerevisiae, to the transposase gene of Tn5 or Mu for example.
  • an inducible heterologous promoter such as the promoters of the meliblase gene, gal 1 gene or gal 10 gene of S. cerevisiae
  • inducer such as galactose In the cul ture medium
  • the expression of the transposase gene and, by consequence, the transposition event can be controlled.
  • Such a modification can be essential in those cel l s which cannot tolerate constitutive expression of transposase and the resul ting high frequency of transposition events.
  • Eukaryotic cells typically package DNA by shrouding it in protein known as histones.
  • histones This can be a disadvantage i f plasmid vector DNA and the transposable element contained on it become shrouded i n histones, particularly when the sites within the transposable element termini to which transposase binds are hi stone coated and therefore possibly rendered inert to transposase action.
  • transposition can be obtained within eukaryotes notwithstanding the abil ity of the host to hi stone-package DNA, it is believed that the frequency of transposition In these hosts may be enhanced by incorporating within the transposable element termini and adjacent the transposase binding sites therein, a DNA sequence which is relatively inert to histone binding.
  • sequences are known in the art as upstream activating sequences (uas) (see Ginger et al ., 1985 Cel l , 40, 767-774 and references cited therein) .
  • pl asmid YMK30 a pl asmid in which a transposable element havi ng uas sequence located appropri ately therein.
  • the pl asmid YMK30 comprises, as a transposable element, truncated (25bp) Tn5 termini each bordering the uas sequence of gal of S. cerevisiae and, between the uas sequences, the ura coding region.
  • This transposable element has been assembled on p329 liberated and then cloned into pYMK12 described previously.
  • pYMK30 comprises the heterologous gal 10 promoter linked operably to the Tn5 structural transposase gene. Plasmid pYMK30 is shown i n Figure 15 and the DNA sequence of one terminus/uas segment is provided in Figure 16. The other terminus is substantially as indicated in Figure 16 but is expressed in the reverse direction.
  • plasmid YMK30 combines two advantageous modifications; the i ncorporation of sequences to which histones do not bind, l ocated at sites sufficiently close to transposase binding sites as to i nterfere with histone binding at those sites; and an inducible heterologous promoter operably linked to the Tn5 transposase structural gene. Transformation of host cells with such vectors i s performed substanti al ly as herei nbefore described and transposed hosts similarly Isolated.

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Abstract

Le système universel décrit permet l'induction d'une transposition génétique dans des cellules prokaryotiques ou eukaryotiques. Ce système est dit universel dans le sens qu'il fournit un moyen d'induire une transposition dans n'importe quel organisme. La présente invention décrit en outre des vecteurs de plasmides capables de véhiculer une telle transposition génétique ainsi qu'un nouvel emploi d'éléments transposables.
PCT/GB1987/000598 1986-08-26 1987-08-25 Systeme universel de mutagenese de transposons WO1988001646A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0310586A1 (fr) * 1987-09-28 1989-04-05 Smithkline Biologicals S.A. Intégration génomique stable et expression d'une séquence codante étrangère dans une levure
FR2627508A1 (fr) * 1988-02-22 1989-08-25 Eurolysine Procede pour l'integration d'un gene choisi sur le chromosome d'une bacterie et bacterie obtenue par ledit procede
EP0485701A1 (fr) * 1990-09-28 1992-05-20 American Cyanamid Company Insertion d'ADN au moyen de transposones modifiés
EP0496027A1 (fr) * 1991-01-25 1992-07-29 R.O.B.I.T. RESEARCH AND DEVELOPMENT COMPANY LTD., c/o EFRATI, GALILI & CO. Essai de bioluminescence pour expression génétique dans des cellules mammifères en culture
WO1998014614A1 (fr) * 1996-10-04 1998-04-09 Lexicon Genetics Incorporated Banque indexee de cellules contenant des modifications genomiques et son procede d'elaboration et d'utilisation
US6051430A (en) * 1996-02-09 2000-04-18 Het Nederlands Kanker Instituut Vectors and methods for providing cells with additional nucleic acid material integrated in the genome of said cells
US6136566A (en) * 1996-10-04 2000-10-24 Lexicon Graphics Incorporated Indexed library of cells containing genomic modifications and methods of making and utilizing the same
US6306625B1 (en) 1988-12-30 2001-10-23 Smithkline Beecham Biologicals, Sa Method for obtaining expression of mixed polypeptide particles in yeast
US6489458B2 (en) 1997-03-11 2002-12-03 Regents Of The University Of Minnesota DNA-based transposon system for the introduction of nucleic acid into DNA of a cell
US6776988B2 (en) 1998-03-27 2004-08-17 Lexicon Genetics Incorporated Vectors for gene mutagenesis and gene discovery
US6855545B1 (en) 1996-10-04 2005-02-15 Lexicon Genetics Inc. Indexed library of cells containing genomic modifications and methods of making and utilizing the same
US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
EP1700914A4 (fr) * 2003-11-21 2007-06-13 Osaka Ind Promotion Org Mise au point d'une technique de modification du genome d'un mammifere a l'aide d'un retrotransposon
US7332338B2 (en) 1996-10-04 2008-02-19 Lexicon Pharmaceuticals, Inc. Vectors for making genomic modifications
US8227432B2 (en) 2002-04-22 2012-07-24 Regents Of The University Of Minnesota Transposon system and methods of use

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EP0091723A2 (fr) * 1982-02-17 1983-10-19 Imperial Chemical Industries Plc Système de vecteur
EP0130047A1 (fr) * 1983-06-22 1985-01-02 Lubrizol Genetics Inc. Molécules d'ADN recombinant
US4670388A (en) * 1982-12-30 1987-06-02 Carnegie Institution Of Washington Method of incorporating DNA into genome of drosophila

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EP0091723A2 (fr) * 1982-02-17 1983-10-19 Imperial Chemical Industries Plc Système de vecteur
US4670388A (en) * 1982-12-30 1987-06-02 Carnegie Institution Of Washington Method of incorporating DNA into genome of drosophila
EP0130047A1 (fr) * 1983-06-22 1985-01-02 Lubrizol Genetics Inc. Molécules d'ADN recombinant

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CHEMICAL ABSTRACTS, Vol. 99, 1983 (Columbus, Ohio, US) G. SERMONTI et al.: "Properties of Transposon SCTnl of Streptomyces Coelicolor A3 (2)", see page 171, Abstract No. 116940e & MGG, Mol. Gen. Genet. 1983, 191 (1), 158-61 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0310586A1 (fr) * 1987-09-28 1989-04-05 Smithkline Biologicals S.A. Intégration génomique stable et expression d'une séquence codante étrangère dans une levure
FR2627508A1 (fr) * 1988-02-22 1989-08-25 Eurolysine Procede pour l'integration d'un gene choisi sur le chromosome d'une bacterie et bacterie obtenue par ledit procede
EP0332488A1 (fr) * 1988-02-22 1989-09-13 Eurolysine Procédé pour l'intégration d'un gène choisi sur le chromosome d'une bactérie et bactérie obtenue par ledit procédé
US5595889A (en) * 1988-02-22 1997-01-21 Eurolysine Process for integration of a chosen gene on the chromosome of a bacterium using Mu transposons
US6306625B1 (en) 1988-12-30 2001-10-23 Smithkline Beecham Biologicals, Sa Method for obtaining expression of mixed polypeptide particles in yeast
EP0485701A1 (fr) * 1990-09-28 1992-05-20 American Cyanamid Company Insertion d'ADN au moyen de transposones modifiés
EP0496027A1 (fr) * 1991-01-25 1992-07-29 R.O.B.I.T. RESEARCH AND DEVELOPMENT COMPANY LTD., c/o EFRATI, GALILI & CO. Essai de bioluminescence pour expression génétique dans des cellules mammifères en culture
US6051430A (en) * 1996-02-09 2000-04-18 Het Nederlands Kanker Instituut Vectors and methods for providing cells with additional nucleic acid material integrated in the genome of said cells
US6136566A (en) * 1996-10-04 2000-10-24 Lexicon Graphics Incorporated Indexed library of cells containing genomic modifications and methods of making and utilizing the same
WO1998014614A1 (fr) * 1996-10-04 1998-04-09 Lexicon Genetics Incorporated Banque indexee de cellules contenant des modifications genomiques et son procede d'elaboration et d'utilisation
US6855545B1 (en) 1996-10-04 2005-02-15 Lexicon Genetics Inc. Indexed library of cells containing genomic modifications and methods of making and utilizing the same
US7332338B2 (en) 1996-10-04 2008-02-19 Lexicon Pharmaceuticals, Inc. Vectors for making genomic modifications
US6489458B2 (en) 1997-03-11 2002-12-03 Regents Of The University Of Minnesota DNA-based transposon system for the introduction of nucleic acid into DNA of a cell
US7148203B2 (en) 1997-03-11 2006-12-12 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US6776988B2 (en) 1998-03-27 2004-08-17 Lexicon Genetics Incorporated Vectors for gene mutagenesis and gene discovery
US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US8227432B2 (en) 2002-04-22 2012-07-24 Regents Of The University Of Minnesota Transposon system and methods of use
EP1700914A4 (fr) * 2003-11-21 2007-06-13 Osaka Ind Promotion Org Mise au point d'une technique de modification du genome d'un mammifere a l'aide d'un retrotransposon

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