Antisigma Factors
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.
Selection Vectors
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:
U.S. Pat. No. 5,300,431; PA0 Burns and Beacham, Gene 27 (1984) 323-325; PA0 Kuhn et al., Gene 42 (1986) 253-263; PA0 Heinrich and Plapp, Gene 42 (1986) 345-349; PA0 Gay et al., J. Bacteriol. (1985) 918-921; PA0 Cheng and Modrich, J. Bacteriol. (1983) 1005-1008; PA0 Dean, Gene 15 (1981) 99-102; PA0 Hagan and Warren, Gene 19 (1982) 147-151; PA0 Hennecke et al., Gene 19 (1982) 231-234; PA0 Honigman & Oppenheim, Gene 13 (1981) 289-298; PA0 Ozaki et al., Gene 8 (1980) 801-314; PA0 Roberts et al., Gene 12 (1980) 123-127; PA0 Schumann, Molec. gen. Genet 174 (1979) 221-224
However, the use of an antisigma gene in a positive selection vector has not previously been described.