Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-07-18
2000-02-29
Guzo, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 71, 435 29, 536 231, 536 237, C12Q 168
Patent
active
060307773
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to recombinant vectors which are useful for direct selection of colonies harboring recombinant plasmids, on basis of the loss of expression of a known gene, the expression of which would normally have resulted in toxicity to the bacterial host. The invention also relates to host cells harboring the said vectors, as well as to methods utilizing the said vectors, for the selection of nucleic acid clones.
BACKGROUND ART
In eubacteria, the core RNA polymerase is composed of .alpha., .beta., and .beta.' subunits in the ratio 2:1:1. To direct RNA polymerase to promoters of specific genes to be transcribed, bacteria produce a variety of proteins, known as sigma (.sigma.) factors, which interact with RNA polymerase to form an active holoenzyme. The resulting complexes are able to recognize and attach to selected nucleotide sequences in promoters.
Antisigma (Asi) proteins, i.e. proteins which inhibit the sigma subunit of RNA polymerase, are known in the art. A gene called asiA, coding for the 10 kDa anti-sigma.sup.70 factor of bacteriophage T4 (hereinafter referred to as AsiA), has been identified by Orsini et al. (1993) J. Bacteriol. 175, 85-93. The open reading frame encoded a 90 amino acid protein having the deduced sequence shown as SEQ ID NO: 1.
The asiA-encoded protein was overproduced in a phage T7 expression system and partially purified. It showed a strong inhibitory activity towards sigma.sup.70 -directed transcription by RNA polymerase holoenzyme. The nucleotide sequence of gene asiA has been deposited in the GenBank data base under accession no. M99441.
Examples of proteins regulating the sigma subunit of RNA polymerase are known from other systems such as Salmonella typhimurium (Ohnishi et al. (1992) Mol. Microbiol. 6, 3149-3157) and Bacillus subtilis (Duncan & Losick (1993) Proc. Natl. Acad. Sd. U.S.A. 90, 2325-2329; Benson & Haldenwang (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2330-2334). The nucleotide sequences of these antisigma factors do not show any gross similarity with the asiA sequence disclosed by Orsini et al. Therefore, although the different antisigma factors are functionally similar, it is not possible to anticipate that an antisigma factor from E. coli will neutralize a RNA polymerase sigma subunit from another bacterial species.
Recombinant DNA technology has led to the development of a variety of vectors that enable cloning and expression of heterologous genes. Generally, the heterologous genes are engineered in such a way that a known marker gene is either interrupted or replaced by the gene. Correct recombinants are selected by screening transformants for the loss of the said marker. This requires screening several hundreds of colonies for the loss of the marker gene. In addition, several of the selected clones generally turn out to be false positives for a variety of reasons.
In order to overcome these disadvantages, researchers have developed vectors that enable a direct positive selection of correct recombinants. Generally, such vectors harbor a gene wherein the encoded product on expression is toxic to the host. This toxicity could be lethal to the host thereby killing the organism or render the host cells to be sick. When a heterologous gene interrupts or replaces the toxic gene, the resulting recombinant grows normally in solid media.
Positive selection vectors, useful for direct selection of colonies harboring recombinant plasmids, are thus known in the art, e.g. from:
However, the use of an antisigma gene in a positive selection vector has not previously been described.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Plasmid map of vector pARC 8173
FIG. 2: Plasmid map of vector pARC 8177
FIG. 3: Plasmid map of vector pARC 8235
FIG. 4: Plasmid map of vector pARC 8233
DISCLOSURE OF THE INVENTION
The present invention provides a vector comprising a positive selection cassette, the said positive selection cassette comprising a DNA sequence coding for an antisigma polypeptide, said DNA sequence including a multiple cloning site fo
REFERENCES:
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Astra Aktiebolag
Guzo David
Shuman Jon
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