Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-06-09
1997-06-24
Hendricks, Keith D.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
4352542, 4353201, 435325, 435419, 530371, 536 2374, C12N 1531, C12N 1581, C12N 1582
Patent
active
056416741
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The invention relates to nucleotide sequences capable of conferring resistance to cydoheximide, cycloheximide resistance proteins and their use as selection markers, for example, to monitor nucleic acid transfer.
DESCRIPTION OF THE RELATED ART
The inventors have, in fact, investigated selectionmarkers suitable for monitoring nudeic acid transfer in eukaryotes, markers which might be more efficient than the markers usually used which are derived from prokaryotic organisms. It is know for example that the markers usually used (geneticin G-418, hygromycin and bleomycin) are derived from resistance genes isolated from prokaryotes and are usually rather inefficient, particularly in fungi. These problems oblige experimenters to use very high concentrations of antibiotics which cause toxidty problems and increase production costs Consequently, other selection agents have been used, for example selection by methotrexate, selection by auxotrophy, etc. However, these selection agents are not generally applicable. There is thus a need for the creation of systems suited to the use of a more effident and/or potent marker than the markers known hitherto and at lower production costs.
Cycloheximide might constitute a potentially useful marker for example for detecting the transfer of heterologous nudeic acid in eukaryotes. For this purpose, the eukaryotes which it is desired to modify in a controllable manner by means of a detection test for resistance to cycloheximide, must be made resistant to this antibiotic under satisfactory conditions in order to ensure reliable monitoring of the transfer of a heterologous nucleic acid made either for research purposes or for the purpose of industrial exploitation.
Cycloheximide is an antibiotic which inhibits protein synthesis by binding to the 60S ribosomal subunit as described by STOCKLEIN et al. (1980, Curt. Genetics 1, 177-183).
Eukaryotic organisms naturally resistant to cycloheximide are rare, up to now observations relating to the mechanisms of resistance have bean described in a mutant of an organism naturally sensitive to cycloheximide, the cyh2 mutant of the yeast Saccharomyces cerevisiae. In this context, the phenomenon of resistance is created by a modification of the ribosomal protein L29 (STOCKLEIN et al., 1980).
These mutant cellular organisms are usually resistant to low concentrations of cycloheximide (of the order of 5-10 .mu.g/ml).
Other authors (TAKAGI et al. in the U.S. Pat. No. 4,857,460) have reported results relating to the study of resistance to cycloheximide in another yeast Candida maltosa. They present a DNA sequence conferring resistance to cycloheximide on C. maltosa. However, neither gene nor open reading frame was identified in this sequence. In other words, the U.S. Pat. No. 4,857,460 does not give the elements for the characterization of a gene elements which could have led to a definition of the conditions for its use, for example, to transform eukaryotes different from Candida maltosa into strains resistant to cycloheximide in a reproducible manner.
SUMMARY OF THE INVENTION
The inventors have concerned themselves with the yeast Kluyveromyces lactis (K. lactis) and demonstrated the fact that cycloheximide resistance in K. lactis depends on the expression of a specific gene. They have also identified the fact that the level of resistance of this yeast to this antibiotic involves other elements linked to the presence of a specific DNA which has the role of cofactor (or cofactor sequence).
In an advantageous embodiment of the invention, the resistance obtained is specific for cycloheximide, the transformed hosts resistant to this antibiotic (cyc.sup.R) being sensitive to several classical inhibitors of ribosomal functions (cryptopleurin, blasticidin, trichodermin, anisomycin, hygromycin).
Thus the inventors obtained in K. lactis a nucleic acid sequence of about 5.2 kb, capable of conferring a level of resistance to a cell host transformed by this sequence sufficiently high for the creation of a selection system as
Davies Julian
Dehoux Pierre
Hendricks Keith D.
Institut Pasteur
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