WO1988003169A1 - Amplification de genes utilisant de retrotransposons - Google Patents

Amplification de genes utilisant de retrotransposons Download PDF

Info

Publication number
WO1988003169A1
WO1988003169A1 PCT/US1987/002788 US8702788W WO8803169A1 WO 1988003169 A1 WO1988003169 A1 WO 1988003169A1 US 8702788 W US8702788 W US 8702788W WO 8803169 A1 WO8803169 A1 WO 8803169A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
cells
plasmid
interest
yeast
Prior art date
Application number
PCT/US1987/002788
Other languages
English (en)
Inventor
Jef D. Boeke
Gerald R. Fink
David J. Garfinkel
Original Assignee
Whitehead Institute For Biomedical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Whitehead Institute For Biomedical Research filed Critical Whitehead Institute For Biomedical Research
Publication of WO1988003169A1 publication Critical patent/WO1988003169A1/fr
Priority to DK348488A priority Critical patent/DK348488D0/da

Links

Classifications

    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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

  • genes encod ⁇ ing proteins having, for example, medical applica ⁇ tions (e.g., hormones, enzymes, growth factors, other drugs) .
  • Production or availability of such materials has historically depended on time consum ⁇ ing, expensive techniques (e.g., extraction from animal tissues, mammalian tissue culture on a large scale, etc.) which often produce small quantities of the protein of interest.
  • prokaryotic cells e.g., bacteria
  • eukaryotic cells e.g., mouse, monkey, yeast
  • DNA fragments e.g., a controllable promoter, an inducible origin of replication, a selectable marker
  • DHFR dihydrofolate reductase
  • One approach to modifying yeast cells to produce a protein of interest is to engineer derivatives of the endogenous 2 micron plasmid bearing a selectable nutritional marker (e.g., URA3, or LEU2) .
  • a selectable nutritional marker e.g., URA3, or LEU2
  • Such strains have several disadvantages.
  • the plasmids are often unstable, and are lost by mitotic segregation. This results in accumulation of plasmid-free cells in the culture, which can, in turn, lead to significant losses of yield, particularly in large fermentor batches.
  • Third, the number of copies per cell of a particular plasmid is limited by the inherent properties of the plasmid and by competition with endogenous 2 micron plas ids.
  • the present invention relates to amplification of a gene of interest in cells.
  • Amplification of a gene of interest can be brought about, through use of the present invention, in eukaryotic cells.
  • an amplification cassette comprising 1) a gene of interest and 2) a functional retrotransposon, whose transposition or movement in host cell genomic DNA is controllable by an inducible promoter, induction of which results in abundant transcription of the amplification cassette, is introduced into cells by transformation under non-inducing conditions.
  • a gene of interest is inserted into a yeast transposon, or Ty element.
  • the resulting gene-Ty cassette is in ⁇ serted into a plasmid which also carries: 1) all or part of an origin of replication whose activation results in replication of the plasmid in high copy number and 2) a gene encoding a selectable marker which allows selection of yeast cells containing (transformed with) the plasmid.
  • a gene of interest is inserted into a Ty element and the resulting cassette is introduced into a plasmid which also carries a 2 micron origin of replication fragment; a URA3 gene; and the inducible promoter GALl.
  • Ty transcrip ⁇ tion is placed under the control of the GA l promoter, which can be turned on by culturing cells containing the plasmid on galactose-containing media, or shut off by culturing such cells on glucose containing medium.
  • the GALl promoter results in increased trans ⁇ position of the gene-Ty cassette.
  • Amplification of the gene of interest thus also results and copies of the gene are located at multiple sites in the yeast genome, even after the original plasmid is lost. Expression of the increased number of copies of the gene results in increased production of the encoded protein.
  • the method of the present invention can be used to cause amplification, or increase in the number of copies of, a gene; integration of the gene copies into yeast genomic DNA; and increased expression of the gene, which in turn results in increased produc ⁇ tion of the encoded protein.
  • FIG 1 is a schematic representation of a yeast transposon or Ty element.
  • the open box represents the DNA; the boxed triangles represent LTR (delta) sequences. Represented above the Ty element is the major Ty transcript. Represented below the Ty element are the two open reading frames (ORFs) tya and tyb.
  • Figure 2 is a schematic representation of a Ty element and other retrotransposons and illustrates the similarities among them. In each case the boxed triangles represent LTR sequences; the open box represents the central coding region; and the boxes below represent the open reading frames.
  • the wavy line above Ty and copia represent RNA.
  • Figure 3 is a schematic representation of the plasmid pGTyH3-neo (also referred to as pJEF1105) .
  • the diagonally hatched box represents the yeast GALl promoter; the boxed triangle represents LTR (delta), sequences; the larger open box represents TyH3 internal sequences; the horizontally lined box represents neo gene sequences; the smaller open box represents the URA3 gene and the dotted box repre- sents 2 micron circle DNA sequences.
  • the straight lines shown in the plasmid represent sequences derived from pBR322.
  • the plasmid is circular but is represented in the figure as though it has been linearized by cutting at the EcoRI site which corresponds to the pBR322 EcoRI site.
  • FIG. 4 is a schematic representation of pGTyH3-neo (pJEF1105) showing the sites (A, B, and C) at which the neo gene was inserted to study transposition in transformants carrying pGTyH3-neo and related plasmids. Sites A and B fall within the open reading frame tyb; site C is outside the open reading frames.
  • Figure 5 is a Southern blot analysis of cells transformed with pGTyH3-neo.
  • Figure 6 is a schematic representation of a method for causing repeated cycles of Ty-neo transposition.
  • Figure 6A is a Southern blot showing results of the method represented in Figure 6.
  • the present invention makes it possible to significantly increase the level of expression of a gene of interest in cells.
  • eukaryotic cells e.g., yeast, mammalian, insect, avian, plant cells
  • viruses e.g., viruses, viruses. This capability results from amplification of the gene of interest in a stable form within genomic DNA of cells modi ⁇ fied to contain genetic material whose expression within the cells results in transposition in the cell genome of a transposable element-gene of interest construct.
  • amplifica ⁇ tion of a gene of interest is achieved by incorporating into host cell genomic DNA an amplification cassette comprising the gene of interest and a functional transposon.
  • a transposon is a mobile genetic element (DNA sequence) which "moves" within genomic DNA by self duplication, followed by insertion of the new copy (copies) at a location in the genome other than that at which the original (parent) genetic element occurs.
  • a gene of interest is- amplified in the following manner: An amplification cassette comprising: 1) the gene of interest and 2) a transpositionally functional 5.
  • transposon such as a yeast transposon, or Ty element
  • a transposon such as a yeast transposon, or Ty element
  • the amplifi ⁇ cation cassette is incorporated into a plasmid or other shuttle vector which also carries an inducible promoter, whose induction results in abundant trans ⁇ cription of the gene-Ty amplification cassette.
  • the amplification cassette is intro ⁇ quizd into a similar plasmid containing a constitu ⁇ tive (noninducible) promoter whose induction causes abundant transcription of the cassette.
  • a further possibility is the introduction of the amplification 0 cassette into host DNA in integrated form.
  • an amplification cassette comprising 1) a gene 0 of interest and 2) a transpositionally functional Ty element whose transposition, or movement into the yeast (host) cell genome, can be controlled by exogenous signals ⁇ ⁇ is introduced into a plasmid.
  • the plasmid carries a promoter whose induction causes the gene-Ty amplification module to be transcribed abundantly.
  • the plasmid also contains an origin of replication or portion thereof whose activation results in replication of the plasmid in high copy number, as well as a gene encoding a selectable marker which makes it possible to identify cells transformed with the plasmid containing the amplification cassette.
  • a gene of interest is inserted into a Ty element, such as TyH3, whose transposition can be controlled by an external signal, to form an amplification cassette.
  • the amplification cassette is incorporated into a plasmid (e.g., pGTy) which carries: 1) a 2 micron origin of replication fragment; 2) a URA3 gene as a selectable marker; and 3) the inducible (highly controllable) yeast pro ⁇ moter GALl.
  • the inducible promoter is linked to the Ty element in such a way that the resulting trans ⁇ cript has a 5' end indistinguishable from a normal Ty RNA 5 1 end.
  • the 2 micron origin of replication fragment is obtained as described below and func ⁇ tions to allow the .plasmid to replicate in high copy number.
  • the URA3 gene is a yeast gene which specifies an enzyme in the biosynthetic pathway for uracil and thus makes it possible to select for cells containing the plasmid because it enables ura3 ⁇ cells to grow on medium lacking uracil.
  • the GALl promoter is highly controllable; that is, it is "turned off” in cells grown in media containing glucose and "turned on” in cells grown in media containing galactose.
  • Yeast cells are cultured with plasmids which carry the amplification cassette and the three components described above, under conditions appropriate for transformation to occur.
  • the co-culturing of cells is carried out in glucose-containing medium (i.e., the GALl. promoter is off) .
  • the resulting cell mixture which contains transformed and untransformed yeast cells, is cultured in medium which does not contain uracil.
  • Transformants are selected by their ura phenotype (i.e., their ability to grow on the uracil-free medium) , which is a well-known selection technique. They are subsequently cultured in galactose-con- taining media, which results in induction of the GALl promoter fused to the Ty element.
  • Induction of the promoter causes frequent transposition of the gene-Ty amplification construct to multiple sites in the yeast genomic DNA. The result of this event is the amplification of the gene-Ty amplification cassette and, thus, availability of an increased number of copies of the gene encoding the protein of interest. Expression of the increased number of gene copies results in increased production of the encoded protein.
  • Genes in the second class are present in many copies, which have no characteristic location. Each member of a species may have these mobile genes in different locations. The origin of these elements is unknown, but it is known that they "move” or "hop” within the chromosome. That is, a Ty element initially found at one locus in the chromosome, is reproduced and the new copy is inserted elsewhere in the chromosome (i.e., the initial transposon does not disappear from its initial site when it appears at a new location) .
  • One of these mobile genes is the repetitive Ty element of yeast, which represents a family of transposable elements from the yeast Saccharomyces cerevisiae. Such elements are repeated about 35 times per haploid genome and constitute about 4% of the total DNA.
  • Ty elements there are two overlapping open reading frames (ORF) : tya, which appears to be equivalent to the retroviral gag gene and specifies a protein with homology to DNA binding proteins; and tyb, which encodes a protein with homology to the protease, integrase and reverse transcriptase regions of the retroviral pol gene.
  • ORF open reading frames
  • The-gene product of tyb is thought to be synthesized as a tya-tyb fusion protein resulting from a specific frameshift at the end of tya that puts tyb in frame with tya. Clare, J. and P. Farabaugh, Proceedings of the National Academy of Sciences, USA, 82:2829- 2833 (1985); Mellor, J. et al. , Nature, 313:243-246 (1985) .
  • Ty elements move about the yeast genome by both homologous (i.e., recombinational) and nonhomologous
  • the homologous events involve reciprocal recombination and gene conversion between two Ty elements at different positions in the genome. These homologous recombin- ation events can lead to chromosomal aberrations such as translocations, deletions, and duplications.
  • the nonhomologous events are transpositions in which a Ty element is inserted into a new chromosomal location unoccupied by a previously existing Ty or delta element. Transposition of a Ty element leads to insertion of a complete Ty flanked by a 5 bp duplication of the target sequence created during transposition. Farabaugh P.J. and G.R. Fink, Nature, 2_8_6. : 352-356 (1980).
  • Ty transposition occurs in one location to a DNA copy in another nonhomologous location through an RNA intermediate. That is, Ty transposition is, in • fact, retrotransposition: the flow of sequence information is DNA— RNA—*DNA; yeast transposable elements are, thus, also referred to as retrotrans- posons.
  • This transfer of information from RNA to DNA requires a reverse transcriptase activity. This activity has been found in cells containing induced pGTy plasmids. The activity exhibits pronounced temperature sensitivity; it is almost completely inactive at 37°C, possibly explaining why Ty transposition is temperature sensitive.
  • Ty elements are, for example, two subfamilies of Ty elements: Tyl and Ty2. Cameron, et.al. , Cell, 16_:739-751 (1979). Members of the two classes differ in primary sequence in certain coding domains (i.e., in two large areas of substitution), but the basic structural elements (delta, tya and tyb) are maintained. armington, J. R. et.al. , Nucleic Acids Research, 13:6679 (1986) . The functional difference between the two types of Ty element, if any exists, is unknown.
  • Tyl elements Numerous variant structural forms occur among just the Tyl elements present in the genome; insertion mutants, deletion mutants and numerous sites of poly morphism for restriction endonuclease cleavage are known to occur. Not all Ty elements are transpositionally functional (i.e. not all are capable of moving within the genome) ; functional and nonfunctional copies of Tyl and Ty2 type elements have been found. Either type (Tyl or Ty2) of Ty element can be used to amplify a gene of interest as described herein, as long as the element selected is transpositionally functional.
  • TyH3 is able to promote high frequency transposition when linked to the GALl promoter; other Ty elements may not be able to do so.
  • Tyl73 (Simchen et al. , Proceeding of the National Academy of Sciences, USA, 81:24231 1984) is nonfunctional when present in such construction, when assayed by a method described below and by Boeke et al. , in Cell 1985.
  • Tys carrying deletions, insertions and point mutations may be nonfunctional for transposition.
  • careful selection of a tranpositionally functional Ty element is essential to amplification of a gene according to the present invention.
  • yeast transposable element in place of a yeast transposable element, another type of transposon functional in yeast cells or other type of host cell into which the amplification construct is inserted can be used in a similar manner.
  • the Ty element and other retrotransposons are represented schematically in Figure 2.
  • Copia, 17.6 and gypsy are from the fruit fly Drosophila melanogaster; IAP is from the mouse.
  • a similar element, bsl, has been found in maize.
  • the copia, 17.6 or gypsy element of Drosophila melanogaster can be fused to the Drosophila heat promoter in a similar manner to that described for fusion of TyH3 to the Gall promoter.
  • the gene of interest could then be introduced into a noncoding segment of the retrotransposon in ques ⁇ tion.
  • This can be followed by introduction of this amplification cassette into insect tissue cells by transfection (a standard technique) or into whole insects by microinjection of early embryos (i.e., P elements-mediated transformation, also a standard technique) .
  • Transposition of the amplification cassette would be induced by heat shock.
  • the latter means of introduction of the amplification cassette would require prior subcloning of the amplification cassette into a P element vector.
  • a promoter site is a specific DNA sequence within a gene which acts as an initiation signal, recognized by DNA-directed RNA-polymerase, to indicate where transcription to form RNA begins.
  • Inducible promoters are those whose activity can be controlled by external conditions or signals (e.g., temperature change, ion concentration changes, presence or absence of metabolite ' s, such as sugars) .
  • GALl is a highly controllable yeast promoter whose function can be regulated by the presence or absence of galactose in culture media. That is, the GALl promoter is turned on when cells containing the promoter are cultured in galactose-containing media; conversely, the promoter is turned off when media containing glucose is used..
  • the present invention as described includes use of the GALl promoter, other promoters, whether inducible or non-inducible, can be used in its place. It is important that the promoter used result in increased transcription and reverse transcription of the gene-Ty amplification cassette and transposition of the two elements into the yeast chromosome at multiple sites. For example, in the case of a Ty-neo amplification cassette linked to a GALl promoter, it is estimated that the number of transripts produced by the plasmid is about equal to that produced by all of the estimated 30-35 copies chromosomal Ty elements. Expression.of the multiple copies of the gene results in increased production of the protein it encodes.
  • Substitution of the GALl promoter for the Ty promoter in the pGTy plasmid used in one embodiment of the present invention results in increased Ty transcription and increased Ty transposition.
  • the gene of interest inserted into the Ty element is similarly affected: transcription and reverse tran ⁇ scription of the gene occur and the gene of interest is transposed into chromosomal DNA along with the Ty sequences. This results in increased copy number of not only the Ty element, but also the accompanying gene of interest.
  • promoters which can be used include, but are not limited to, a delta promoter (the promoter for Ty itself), ADH promoter, PGK promoter, and PHO promoter. Sentenac A. and Hall, B. , JTn: Molecular Biology of the Yeast Saccharomyces (J. Strathern et al. , ed. ) 1982.
  • yeast Most strains of yeast contain a circular DNA sequence, called the 2 micron circle, which repli ⁇ cates autonomously. This plasmid is about 6300 bp in length, occurs at about 50 copies per cell and, like bacterial plasmids, has one origin of replica ⁇ tion. Broach J.R. et al. , In: Molecular Biology of the Yeast Saccharomyces (J. Strathern et al. , ed.) 445-470 (1982) .
  • a 2 micron DNA origin of replication fragment is incorporated into the ⁇ ⁇ plasmid which also contains the amplification cassette.
  • the function of this component is to allow the plasmid (and its components) to replicate to high copy number, resulting in increased avail- ability of copies of the gene-Ty cassette for subsequent transcription and transposition.
  • the 2 micron DNA origin is useful because it is known to allow replication in high copy number.
  • an origin of replication which functions in the host cell e.g., in the embodiments described above, an origin functional in yeast cells
  • an origin of replication which functions in the host cell (e.g., in the embodiments described above, an origin functional in yeast cells) can be used.
  • an ARS1 or other ARS (autonomously replicat ⁇ ing sequence) sequence can be used.
  • the origin selected should be one, however, whose activation results in replication to high copy number of a plasmid containing the gene-Ty amplification cas ⁇ sette.
  • the present invention preferably makes use of a gene encoding a selectable trait; the gene is incorporated into the amplification cas ⁇ sette-carrying plasmid and allows selection of cells transformed with the plasmid.
  • the ura3 gene which encodes an enzyme for uracil biosynthesis is used for this purpose.
  • yeast cells with the pGTy plasmid containing the gene-Ty ampli ⁇ fication cassette cells transformed with the plasmid are identified by growing the cell mixture on medium lacking uracil.
  • Cells containing the plasmid (transformants) grow on medium lacking uracil; untransformed cells do not grow.
  • genes encoding a selectable marker or conferring a selectable phenotype can be used for this purpose.
  • the present invention it is possible to significantly increase production of a protein of interest by incorporating a gene encoding that protein into host DNA as described.
  • the gene is incorporated into yeast genomic DNA, but it is also possible to amplify a gene of interest in other types of host cells, such as viral, mammalian, avian, plant and insect cells.
  • the gene of interest is incorporated into the host DNA in combination with a transposable element, which results in movement of. the gene of interest.
  • the gene of interest and the transposable element are amplified and occur in multiple sites in host DNA. Expression of the increased copies results in increased produc ⁇ tion of the encoded protein.
  • a gene encoding neomycin phospho- transferase is introduced into yeast cells in conjunction with a Ty element, such as TyH3.
  • the gene is inserted into the Ty element, which is in turn linked to an inducible promoter, such as GALl.
  • the gene-Ty amplification cassette and the inducible promoter are incorporated into a plasmid which also carries 1) an origin of replication (e.g., a 2 micron DNA origin) whose activation results in replication of the plasmid to high copy number and 2) a gene (e.g., a ura3 gene) which confers a selectable trait on transfor ants.
  • Yeast cells are cultured with the resuiting plasmid under conditions appropriate for transformation of the cells. Transformation is carried out using standard yeast transformation techniques, such as lithium acetate (LiAc) , as described by Ito et. al. Ito, H. et. al ⁇ . , Journal of Bacteriology, 153:163-168 (1983) . Transformants contain the URA3 gene and, thus, can be selected on the basis of growth on medium lacking uracil and isolated.
  • LiAc lithium acetate
  • URA cells are then cultured under conditions appropriate for induction or activation of the promoter (e.g., in galactose-containing media if GALl is used) As a result, increased transcrip ⁇ tion, reverse transcription and transposition, of the amplification cassette occurs.
  • the neo gene is then located in many sites in the yeast genome, along with the Ty element. Expression of the increased number of neo genes results in yeast cells which are resistant to the neomycin analog, G418. If the Gall promoter is used, cells contain ⁇ ing (transformed with) the plasmid can be exposed ' to (grown on) galactose-containing media more than once in order to achieve higher copy numbers than result from a single exposure to galactose.
  • neo gene with a bacterial promoter
  • yeast TRPl gene with its own yeast promoter
  • This level of expression is achieved in spite of the fact that the passenger genes are "embedded" within a Ty element, which upon integration into the chromosome is apparently itself transcribed. Such a situation may not be ideal for optimal transcription of the passenger gene. It may be possible to increase further the expression of the passenger gene by building into the U3 region of the pGTy plasmid to be used a mutation which would render the Ty's own (LTR) promoter inactive once the gene-Ty cassette has transposed into the yeast genome.
  • LTR Ty's own
  • the gene of interest can be a gene which is: 1) normally present and normally expressed at biologically significant levels in cells into which the amplification cassette is introduced; 2) normally present in but not normally expressed at biologically significant levels in cells into which the amplification cassette is introduced; or 3) is not normally present in cells into which the amplification cassette is introduced, alone or in combination.
  • genes which are also free of non-yeast introns and free of a yeast transcriptional termina ⁇ tor sequence, can be inserted into a transposable ele ent and amplified in the manner described above.
  • cDNA clones of most genes of interest to be am ⁇ plified according to the present invention are available and intron-free genes or portions thereof are available for incorporation into the amplifica ⁇ tion cassette.
  • Transposition of a gene-Ty element amplifica ⁇ tion cassette can be achieved in cells other than yeast cells (e.g., other yeast, insect cells, avian cells, mammalian cells, plant cells) .
  • yeast cells e.g., other yeast, insect cells, avian cells, mammalian cells, plant cells
  • An approach similar to that described for yeast cells can be used.
  • the Ty element is fused to an appropriate promoter derived from the organism to be used as host.
  • pGTyH3-neo Plasmid.
  • pGTyH3-neo also designated pJEF1105) ( Figure 3) was constructed by inserting a 1 Kb BamHI frag ⁇ ment encoding neomycin phosphotransferase from plasmid pGH54 (Joyce et al. , Journal of Bacteri ⁇ ology, 158:636, 1984) into a plasmid pGTyH3 which had been partially digested with Bglll.
  • Plasmid pGTyH3 was constructed as described by Boeke et al. , in Cell, 4_ :491-500 (1985) , the teachings of which are incorporated herein by reference.
  • pJEFH05 carries the neo fragment in the same transcriptional orientation as TyH3 in the 3' Bglll site.
  • pJEFll05 has been deposited with the American Type Culture Collection (Rockville, MD) under deposit number 67247. It had previously been shown that it is possible to insert genetic information into this site without disruption of transposition. Boeke, J.D. et. al., Cell, 0:491-500 (1985). The pJEFllOS plasmid was transformed into various yeast strains, including BWGl-7a, by selecting for Ura+ colonies.
  • Yeast transformants containing pJEFH05 and some of the related plasmids described above were examined for transposition competency.
  • the structures of the related plasmids differed from that of pJEFll05 in both the site of insertion of the neo gene into TyH3 (site A, B or C) and the orientation of the neo gene (+, indicating the same transciptional orientation as TyH3; -, indicating transcriptional orientation opposite to that of TyH3) .
  • Sites A and B are within the open reading frame tyb; Site C is outside the open reading frames.
  • the cells were plated to form single colonies on SC "" ura plates containing 2% galactose as the carbon source for 5 days at 22°C (shown to be the optimal regimen for obtaining transposition of lacO-marked Ty elements into chromosomal DNA) . Randomly selected colonies were streaked to SC-ura glucose plates (to select for plasmid-containing cells) and allowed to grow into colonies at 30°C. The plasmids were then segregated from the cells by growth on YPD (rich) medium. Plasmid-free cells were identified by their Ura "" phenotype. Results of this work are shown in Table 1.
  • a , B and C in the table refer to the sites at which the neo gene was inserted in the Ty element . See Figure 4 . 80-100% of the pJEF1105-transformed cells which went through this procedure were found to be resistant to G418. In contrast, the untransformed strain, and pJEFH05 transformants which were not 5 exposed to galactose failed to show any G418 resistance following loss of the plasmid. Moreover, insertion of the neo fragment at either of the other two Bglll sites within the pGTyH3 plasmid (which would lead to frameshift mutations in the Ty-encoded
  • Chromosomal DNA was prepared from the plasmid-free segregants and digested with the restriction enzyme EcoRI. The resulting DNA fragments were separated by electrophoresis on a 0.6% agarose gel and transferred to nitrocellulose
  • Example 4 Amplification of TRPl gene
  • the Saccharomyces cerevisiae TRPl gene was inserted into pGTyH3, forming plasmid pGTy-TRPl, using techniques similar to those described above.
  • This construct appears to work as well as the pGTyH3—neo construct in that it produces multiple copies of a Ty-T Pl cassette in the yeast cell genome.
  • the sequence of TRPl was removed prior to insertion of TRPl into pGTyH3, the sequence of TRPl was removed. It is possible that the presence of a strong transcriptional terminator within the "passenger gene" might destroy the ability of the Ty-passenger gene cassette to trans ⁇ pose because Ty transposes through an RNA intermedi ⁇ ate in a retrovirus-like mode.
  • Truncation of the Ty-passenger gene transcript might render it incom ⁇ petent for transposition, because the repeated "R" region normally found in both ends of the Ty trans- cript would be lacking. This region is essential for proper reverse transcription.
  • Varmus, H.E. and Sawnstram, R. In: RNA Tumor Viruses, (2d ed.) (1984); Gilboa, E. et. a . , Cell, 1 ⁇ :93-100 (1979).
  • Proper neo transcription is likely to be essential for proper transposition.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Procédé d'amplification d'un gène intéressant dans des cellules eukaryotiques, et cellules dans lesquelles l'amplification peut être effectuée. Selon la présente invention, un gène intéressant est introduit dans un rétrotransposon fonctionnel, dont la transposition ou le mouvement dans l'ADN génomique des cellules hôtes peut être contrôlé par un promoteur inductible, pour former une cassette d'amplification. Celle-ci est introduite dans des cellules hôtes appropriées dans des conditions de non-induction. L'induction du promoteur se traduit par une abondante transcription de la cassette d'amplification, ce qui s'accompagne de la transposition fréquente de la cassette dans des sites multiples de l'ADN génomique hôte. Dans une forme préférée d'exécution, un gène intéressant est introduit dans un transposon de levure, ou élément Ty. La cassette résultante gène-Ty est introduite dans un plasmide contenant également le promoteur inductible GAL1, un fragment d'origine de réplication d'une longueur de 2 microns et un gène URA3. Les cellules de levure sont transformées avec le plasmide dans des conditions de non-induction (c'est-à-dire dans des milieux contenant du glucose). Les cellules contenant le plasmide sont cultivées dans des milieux contenant du galactose, ce qui provoque l'induction du promoteur GAL1, une abondante transcription de la cassette gène-Ty et sa fréquente transposition dans l'ADN génomique des cellules de levure. Il en résulte une amplification du gène intéressant. L'expression du nombre accru de copies se traduit par un accroissement de production de la protéine codée.
PCT/US1987/002788 1986-10-27 1987-10-26 Amplification de genes utilisant de retrotransposons WO1988003169A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DK348488A DK348488D0 (da) 1986-10-27 1988-06-24 Genopformering under anvendelse af retrotransposoner

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US92384086A 1986-10-27 1986-10-27
US923,840 1992-08-03

Publications (1)

Publication Number Publication Date
WO1988003169A1 true WO1988003169A1 (fr) 1988-05-05

Family

ID=25449337

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1987/002788 WO1988003169A1 (fr) 1986-10-27 1987-10-26 Amplification de genes utilisant de retrotransposons

Country Status (2)

Country Link
AU (1) AU8171087A (fr)
WO (1) WO1988003169A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001800A1 (fr) * 1990-07-20 1992-02-06 Chiron Corporation Procede de transformation integrante de levure au moyen d'elements repetitifs disperses
FR2670502A1 (fr) * 1990-12-13 1992-06-19 Eurolysine Cassette d'integration "multisite" dans le genome d'une levure, levure transformee et procede de preparation d'un produit d'interet par une levure ainsi transformee.
EP0506945A1 (fr) * 1990-10-25 1992-10-07 HODGSON, Clague Pitman Procede de transfert de genes au moyen de retrotransposons
US5395763A (en) * 1992-06-24 1995-03-07 Monsanto Company Chromosomal expression vector
US5482853A (en) * 1988-11-23 1996-01-09 The Regents Of The University Of California Position-specific insertion vectors and method of using same
US5756313A (en) * 1989-05-22 1998-05-26 The Green Cross Corporation Albumin gene-containing plasmid, transformant carrying same, production of such transformant and production of albumin
WO1999061651A2 (fr) * 1998-05-27 1999-12-02 Novo Nordisk Biotech, Inc. Procede de production d'un polypeptide en modifiant le nombre de copies d'un gene
EP1124978A1 (fr) * 1998-10-30 2001-08-22 Janssen Pharmaceutica N.V. Retrotransposon inhabituel provenant de $i(candida albicans)
WO2001081598A2 (fr) * 2000-04-26 2001-11-01 Janssen Pharmaceutica N.V. Familles d'un retrotransposon multiple du champignon asexue candida albicans
EP1700914A1 (fr) * 2003-11-21 2006-09-13 Osaka Industrial Promotion Organization Mise au point d'une technique de modification du genome d'un mammifere a l'aide d'un retrotransposon
US7135337B2 (en) 1997-03-27 2006-11-14 Grigliatti Tom A Insect expression vectors
EP2055784A1 (fr) * 2007-10-31 2009-05-06 Bundesrepublik Deutschland, letztvertreten durch den Präsidenten des Paul-Ehrlich-Instituts Prof. Dr. Johannes Löwer Activation contrôlée des rétrotransposons non LTR chez les mammifères

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8615701D0 (en) * 1986-06-27 1986-08-06 Delta Biotechnology Ltd Stable gene integration vector
ES2052712T3 (es) * 1987-04-09 1994-07-16 Delta Biotechnology Ltd Vector de levadura.

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NATURE, Volume 313, 17 January 1985, J. MELLOR et al., "A Retrovirus-Like Strategy for Expression of a Fusion Protein Encoded by Yeast Transposon TY1", pages 243-246. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 82, May 1985, J. CLARE et al., "Nucleotide Sequence of a Yeast TY Element: Evidence for an Unusual Mechanism of Gene Expression", pages 2829-2833. *
TRENDS IN GENETICS, Volume 2, No. 5, May 1986, ELSEVIER SCIENCE PUBLISHERS B.V., (Amsterdam, NL), G.R. FINK et al., "The Mechanism and Consequences of Retrotransposition", pages 118-123. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5482853A (en) * 1988-11-23 1996-01-09 The Regents Of The University Of California Position-specific insertion vectors and method of using same
US5756313A (en) * 1989-05-22 1998-05-26 The Green Cross Corporation Albumin gene-containing plasmid, transformant carrying same, production of such transformant and production of albumin
WO1992001800A1 (fr) * 1990-07-20 1992-02-06 Chiron Corporation Procede de transformation integrante de levure au moyen d'elements repetitifs disperses
US5629203A (en) * 1990-07-20 1997-05-13 Chiron Corporation Method for integrative transformation of yeast using dispersed repetitive elements
EP0506945A1 (fr) * 1990-10-25 1992-10-07 HODGSON, Clague Pitman Procede de transfert de genes au moyen de retrotransposons
EP0506945A4 (en) * 1990-10-25 1993-04-28 Clague Pitman Hodgson Method of gene transfer using retrotransposons
FR2670502A1 (fr) * 1990-12-13 1992-06-19 Eurolysine Cassette d'integration "multisite" dans le genome d'une levure, levure transformee et procede de preparation d'un produit d'interet par une levure ainsi transformee.
WO1992010577A1 (fr) * 1990-12-13 1992-06-25 Eurolysine Cassette d'integration 'multisite' pour le genome d'une levure
US5395763A (en) * 1992-06-24 1995-03-07 Monsanto Company Chromosomal expression vector
US7135337B2 (en) 1997-03-27 2006-11-14 Grigliatti Tom A Insect expression vectors
WO1999061651A3 (fr) * 1998-05-27 2000-03-02 Novo Nordisk Biotech Inc Procede de production d'un polypeptide en modifiant le nombre de copies d'un gene
WO1999061651A2 (fr) * 1998-05-27 1999-12-02 Novo Nordisk Biotech, Inc. Procede de production d'un polypeptide en modifiant le nombre de copies d'un gene
EP1124978A1 (fr) * 1998-10-30 2001-08-22 Janssen Pharmaceutica N.V. Retrotransposon inhabituel provenant de $i(candida albicans)
EP1124978A4 (fr) * 1998-10-30 2003-11-12 Janssen Pharmaceutica Nv Retrotransposon inhabituel provenant de candida albicans
WO2001081598A2 (fr) * 2000-04-26 2001-11-01 Janssen Pharmaceutica N.V. Familles d'un retrotransposon multiple du champignon asexue candida albicans
WO2001081598A3 (fr) * 2000-04-26 2002-08-08 Janssen Pharmaceutica Nv Familles d'un retrotransposon multiple du champignon asexue candida albicans
EP1700914A1 (fr) * 2003-11-21 2006-09-13 Osaka Industrial Promotion Organization Mise au point d'une technique de modification du genome d'un mammifere a l'aide d'un retrotransposon
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
EP2055784A1 (fr) * 2007-10-31 2009-05-06 Bundesrepublik Deutschland, letztvertreten durch den Präsidenten des Paul-Ehrlich-Instituts Prof. Dr. Johannes Löwer Activation contrôlée des rétrotransposons non LTR chez les mammifères
WO2009056321A1 (fr) * 2007-10-31 2009-05-07 BUNDESREPUBLIK DEUTSCHLAND,letztvertreten durch den Präsidenten des Paul-Ehrlich-Instituts Activation contrôlée de rétrotransposons sans ltr chez les mammifères
US9481892B2 (en) 2007-10-31 2016-11-01 Liliana Layer Controlled activation of non-LTR retrotransposons in mammals

Also Published As

Publication number Publication date
AU8171087A (en) 1988-05-25

Similar Documents

Publication Publication Date Title
Clark-Adams et al. The SPT6 gene is essential for growth and is required for δ-mediated transcription in Saccharomyces cerevisiae
Volkert et al. Site-specific recombination promotes plasmid amplification in yeast
JP6646020B2 (ja) タンパク質の製造のための酵母株
Boeke et al. Ty elements transpose through an RNA intermediate
US4959317A (en) Site-specific recombination of DNA in eukaryotic cells
Herskowitz et al. [8] Putting the HO gene to work: practical uses for mating-type switching
US5776745A (en) Recombinationally targeted cloning in yeast artificial chromosomes
Sikorski et al. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Clarke et al. The structure and function of yeast centromeres
Sherman An introduction to the genetics and molecular biology of the yeast Saccharomyces cerevisiae
JP2614215B2 (ja) ピチア属酵母の部位選択的ゲノム修飾
Watson et al. Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe
DK2250270T3 (en) Method for Targeted Integration of Multiple Copies of an Interesting Gene into a Yarrowia Strain
Spencer et al. Targeted recombination-based cloning and manipulation of large DNA segments in yeast
WO1988003169A1 (fr) Amplification de genes utilisant de retrotransposons
EP0539490B1 (fr) Procede de triage de banques
AU2011202192A1 (en) Generation of recombinant genes in Saccharomyces cerevisiae
Tsalik et al. Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted DNA damage
EP0045573A2 (fr) Segment eucaryotique à replication autonome
Flagg et al. Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration
Barton et al. Site-directed, recombination-mediated mutagenesis of a complex gene locus
EP0399455B1 (fr) Cellules de levure stablement transformées et leur utilisation pour la production d'albumine
Kumar et al. Insertional mutagenesis: transposon-insertion libraries as mutagens in yeast
Roy et al. The mcm17 mutation of yeast shows a size-dependent segregational defect of a mini-chromosome
US4464472A (en) Construction and use of functional minichromosomes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU DK JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE