Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1995-12-05
1999-02-02
Railey, II, Johnny F.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4352521, 4352523, 4352529, 4353201, 426 39, 426 42, 426 52, 426 56, C12N 121, C12N 1574, A23C 9123, A23B 710
Patent
active
058663851
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention provides useful mutants of lactic acid bacteria or plasmids capable of replicating in lactic acid bacteria, comprising nonsense mutation suppressor-encoding genes, the use of such suppressor genes for confining a replicon to a specific lactic acid bacterium or to a lactic acid bacterium growing in a particular environment and for controlling the number of lactic acid bacterial cells in a particular environment.
TECHNICAL BACKGROUND AND PRIOR ART
In the in vivo synthesis of proteins occurring in the ribosomes, mRNA is translated into polypeptide chains. However, the MRNA codons do not directly recognize the amino acids that they specify in the way that an enzyme recognizes a substrate. Translation uses "adaptor" molecules that recognize both an amino acid and a triplet group of nucleotide bases (a codon). These adaptors consist of a set of small RNA molecules known as transfer RNAs (or tRNAs), each of which is only 70 to 90 nucleotides in length. Such tRNA molecules contain unpaired nucleotide residues comprising a CCA triplet at one end of the molecule and, in a central loop, a triplet of varying sequence forming the so-called anticodon that can base-pair to a complementary triplet in the MRNA molecule, while the CCA triplet at the free 3' end of the molecule is attached covalently to a specific amino acid.
The three nucleotide triplets UAG (amber codon), UGA (opal codon) and UAA (ochre codon) do not code for an amino acid. These signals termed stop codons or "nonsense" codons, are involved in polypeptide chain termination. During translation, two protein factors (R1 and R2) recognize these triplets and effect release of the polypeptide chain from the ribosome-mRNA-tRNA complex.
Occasionally a mutation occurs in a cell resulting in a nonsense codon appearing in the middle of a gene, causing premature chain termination and the production of a protein fragment. Such fragments rarely have enzymatic activity.
The effect of such a nonsense mutation can be reversed or suppressed by a second mutation in a gene coding for a tRNA which results in the synthesis of an altered tRNA molecule. Such an altered tRNA recognizes a nonsense codon and inserts an amino acid at that point in the polypeptide chain. The mutated tRNA-encoding gene is termed a suppressor gene and the altered nonsense mutation-suppressing tRNA which it encodes is generally referred to as a nonsense or termination suppressor. Such termination suppressors may be derived by single, double or triple base substitutions in the anticodon region of the tRNA.
Termination suppressors were first detected in E. coli about 25 years ago and have since been extensively studied in this species. It is considered that all termination suppressors in E. coli have been identified. Recently, new suppressor tRNA genes have been synthesized in vitro and subsequently introduced into E. coli. Termination suppressors have also been identified in the E. coli bacteriophage T4 and in Salmonella typhimurium (Eggertson et al., 1988, Microbiological Reviews, 52, 354-374). Furthermore, termination suppressors have been identified in eucaryotic fungi including Neurospora spp., Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Hitherto, nonsense or termination suppressors have not been identified in bacterial species belonging to the industrially important group of lactic acid bacteria which i.a. are commonly utilized as starter cultures in the production of a variety of food products including dairy products, meat products, vegetable products, bakery products and wine, during which production these starter cultures produce lactic acid and other organic acids and in many instances also desirable flavour-enhancing metabolites.
Furthermore, attempts by the present inventors to construct amber-suppressing strains of lactic acid bacteria by introducing cloned known suppressor genes from E. coli proved unsuccessful. Thus, it was attempted to introduce the E. coli supB gene (Thorbjarnadottir et al., 1985), the E. coli supE gene (Nakajima et
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Dickely Fran.cedilla.oise
Hansen Egon Bech
Johansen Eric
Nilsson Dan
Chr. Hansen A/S
Railey II Johnny F.
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