Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1986-04-30
1991-06-04
Teskin, Robin L.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 34, 435 691, 435 701, 435 711, 435 91, 4351721, 4352402, 4352403, 435254, 4353201, 435839, 435849, 435878, 435942, 935 9, 935 22, 935 55, 935 59, 935 66, 935 69, 935 70, 935 72, 935 73, 935 74, C12N 1570, C12N 1564, C12N 1574, C12N 1579
Patent
active
050213440
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the application of phleomycins as a selection agent in the field of genetic engineering.
For bacteria there are available at the present time reliable and sensitive selection markers which are constituted by characters of resistance to certain antibiotics such as ampicillin, on the other hand, for eucaryotic cells the selection labelers are, generally, insensitive or require the use of a complicated technology to be usable.
It is, in addition, interesting to have available a selection labeler which can be expressed both in prokaryotic cells and in eukaryotic cells since often the vector constructions in the eukaryotic cells are partly effected in prokaryotic cells.
The present invention rests on the demonstration of new selection labelers which confer on the cells a resistance to antibiotics of the phleomycin type.
The present invention relates to the application of antibiotics of the phleomycin family as selection agent of cells which have been artificially modified by incorporation of a phleo.sup.r gene of resistance to an antibiotic of the family of phleomycins.
By "antibiotic of the family of phleomycins" is meant to denote, besides the phleomycins themselves, related antibiotics such as bleomycins, zorbamycins, victomycin, platomycins, tallysomycins or the antibiotics SF 1771, SF 1961 and YA 56, as well as mixtures of these antibiotics.
These antibiotics are characterized by a basic structure which differentiates them distinctly from the other antibiotics and the tests carried out within the scope of the present invention have demonstrated that the exact nature of the antibiotic used for the selection does not notably modify the result obtained provided that the antibiotic is indeed of the phleomycin type.
These antibiotics obtained commercially (bleomycine) or prepared from productive strains ATCC 21890 and 21892 (phleomycins), ATCC 21807 (victomycine), ATCC 21893 (platomycines), ATCC 31158 (tallysomycines), ATCC 31248 (SF 1771) are relatively toxic for bacteria, the lower eukaryotics like the yeasts and the fungi and the higher eukaryotics both animal and plant cells in cultivation.
Recently isolated strains of Actinomycetes produce antibiotics identical with those mentioned above or different. Particularly, the strain V9, Streptoverticillium in the course of taxonomic identification, synthesizes substances belonging to the family of phleomycines. The absorption curves in the ultra-violet of the purified products of the V9 strain are significantly different from those of the products mentioned above.
The genes which confer resistance on antibiotics of the phleomycin type are named below generically as phleo.sup.r genes.
It should be pointed out that up to the present numerous distinct phleo.sup.r genes have been established as will be demonstrated below.
The phleo.sup.r genes are borne by natural plasmids isolated from bacterial strains, for example found in a hospital environment, or can be cloned from the DNA of various organisms, particularly the DNA of organisms producing antibiotics of the phleomycin type. But these phleo.sup.r genes also exist in genetic materials widely available since it has been possible to establish the presence of such a gene on the transposon Tn5 of Escherichia coli or on the plasmid pUB110.
A gene of great importance for the present invention is that borne by the transposon Tn5 to E. coli well known to molecular biologists. This gene whose function has not yet been described is situated in the central portion of Tn5, between the gene which results in the resistance among others to kanamycin and that which results in the resistance in certain bacteria to streptomycin.
The exact location of the gene concerned, denoted by Tn5 phleo.sup.r, has been determined exactly by dissection of the Tn5 DNA with various restriction enzyme and by determination of the corresponding nucleotidic sequence.
The phleo.sup.r Tn5 gene results in the resistance to all the antibiotics mentioned above, including here those of the V9 strain, when the latter is rende
REFERENCES:
Jimenez et al, Nature, 287:869-871 (1980).
Maniatis et al, 1982, Molecular Cloning-Cold Spring Harbor Laboratory, pp. 11-13.
Suzuki et al, 1969, J. Antibiotics 22(9):446-448.
Putnoky et al. 1983, Mol. Gen. Genet. 191:288-294.
Gritz et al, 1983, Gene, 25:179.
Takeshi Murakami et al, "Cloning of Anti-biotic Resistance Genes in Streptomyces", Journal of Antibiotics, vol. 36, No. 10, Oct. 1983, pp. 1305-1311.
Yasutoshi Takeichi et al, "Cloning of Bacillus Subtilis Alpha-Amylase Structural Gene in Plasmid pUB110", Agric. Bid. Chem., vol. 47, No. 1, Jan. 1983, pp. 159-161.
P. Attfield et al., "R Plasmids Mediate Protection and Sensitivity to Bleomycin", J. Pharm. Pharmacol. 1980, 32 (Suppl.), p. 31p.
Takado Iijima et al., "Mutability of the Phleomycin Resistant Mutants of Bacillus Subtilis. I. Isolation of Genetically Unstable Mutants," J. Gen. Appl. Microbiol., 1970, vol. 16, No. 5, pp. 419-427.
Armau Elise
Drocourt Daniel
Etienne Gilles
Tiraby Gerard
Centre National de la Recherche Scientifique "CNRS"
Peet Richard C.
Teskin Robin L.
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