Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-07-17
1999-03-16
Elliott, George C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 914, 435243, 43525231, 4353201, 4351723, 536 231, 536 242, C12P 2100, C12N 121, C12N 1570, C07H 2104
Patent
active
058828885
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK96/00038 filed Jan. 23, 1996 and claims priority under 35 U.S.C. 119 of Danish applications 0083/95 and 0799/95 filed Jan. 23, 1996 and Jul. 6, 1995, respectively, the contents of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to a novel DNA construct useful for the construction of a bacterial cell having integrated more than one copy of a DNA sequence of interest into its genome, which cell may be free of any selection markers, and a method of constructing such cells.
BACKGROUND OF THE INVENTION
Prokaryotic transposable elements are discrete DNA sequences capable of insertion at single or multiple sites within a prokaryotic genome. Normally, such elements consist of a gene encoding a transposase protein and a transposable cassette comprising a resistance gene flanked by sequences recognized by the transposase protein. Transposition of the transposable cassette into the genome of a host cell (which may, e.g. take place at random or at hot spot sites) occurs via recognition and interaction with the flanking sequences of the transposable cassette by the transposase protein.
Different classes of transposable elements exist. One class comprises i) insertion sequences (IS) which are small (less than 2 kb) DNA fragments encoding transposase proteins or other determinants mediating transposition, and ii) composite transposons, i.e. DNA fragments flanked by two copies of an insertion sequence. The terminal portions of all IS sequences comprises inverted repeat sequences. The transposase protein functions by recognizing these terminal sequences and interacting with the sequences to effect transpositions within the genome.
The second class of transposons is the Tn3 family of tranposons. These transposons encode two products involved in a two-step transposition process: a transposase and a resolvase.
Transposons belonging to this second class have inverted terminal repeats of approximately 35-40 bp.
The third class includes bacteriophage Mu and related phages. Bacteriophage Mu is large relative to other transposons with a genome of 36 kb. Mu encodes two gene products which are involved in the transposition process, a 70 kDa transposase and an accessory protein of approximately 33 kDa. An unusual feature of Mu that distinguishes it from other tranposons is that its ends are not inverted repeat sequences. The Mu transposase has, however, been shown to bind to both ends in an in vitro binding assay.
Transposons have been used extensively for mutagenesis and cloning in gram-positive and gram-negative bacteria: Youngman, P. J., Perkins, J. B., Losick, R. (1983) Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917, Proc. Natl. Acad. Sci. USA, 80, 2305-2309; Youngman, P., Perkins, J. B., Losick, R. (1984), Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene, Plasmid 12, 1-9; Youngman, P. (1985) Plasmid vectors for recovering and expoliting Tn917 transpositions in Bacillus subtilis and other gram positives, p. 79-103 in K. Hardy (ed.), Plasmids: a practical approach, IRL Press, Oxford; Kleckner, N., Roth, J., Botstein, D. (1977) Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics, J. Mol. Biol., 116, 125-159; Wati, M. R., Priest, F. G., Mitchell, W. J. (1990) Mutagenesis using Tn917 in Bacillus licheniformis. FEMS microbiol. Lett., 71, 211-214; Petit, M.-A., Bruand, C., Janniere, L., Ehrlich, S. D. (1990) Tn10-derived transposons active in Bacillus subtilis. J. Bacteriol., 172, 6736-6740.
The latter reference describes pHV1248 and pHV1249, plasmids that are thermosensitive for replication, which carry a transposase gene from Tn10 modified to be expressed in B. subtilis, and sufficient sequences from the
REFERENCES:
patent: 5102797 (1992-04-01), Tucker et al.
patent: 5527695 (1996-06-01), Hodges et al.
Kristensen et al. Site-specific deletion of chromosomally ligated DNA segments with the multimer resolution system of briad-host range plasmid RP4. J. Bacteriol. Vol. 177(1):52-58, Jan. 12, 1995.
Elliott George C.
Gregg, Esq. Valeta
Novo Nordisk A S
Sandals William
Zelson Esq. Steve T.
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