|dc.contributor.author||Nagel, Michael G.||en_US
|dc.description.abstract||The cloned dihydrofolate reductase gene of Saccharomyces
cerevisiae (DFR 1) is expressed in Escherichia coli. Bacterial strain
JF1754 transformed with plasmids containing DFR 1 is at least 5X more
resistant to inhibition by the folate antagonist trimethoprim.
Expression of yeast DFR 1 in E. coli suggests it is likely that the
gene lacks intervening sequences. The 1.8 kbp DNA fragment encoding
yeast dhfr activity probably has its own promotor, as the gene is
expressed in both orientations in E. coli.
Expression of the yeast dhfr gene cloned into M13 viral vectors
allowed positive selection of DFR 1 - M13 bacterial transfectants in
medium supplemented with trimethoprim. A series of nested deletions
generated by nuclease Bal 31 digestion and by restriction endonuclease
cleavage of plasmids containing DFR 1 physically mapped the gene to a
930 bp region between the Pst 1 and Sal 1 cut sites. This is
consistent with the 21,000 molecular weight attributed to yeast dhfr
in previous reports.
From preliminary DNA sequence analysis of the dhfr DNA fragment
the 3' terminus of DFR 1 was assigned to a position 27 nucleotides
from the Eco Rl cut site on the Bam Hi - Eco Rl DNA segment. Several
putative yeast transcription termination consensus sequences were
identified 3' to the opal stop codon.
DFR 1 is expressed in yeast and it confers resistance to the
antifolate methotrexate when the gene is present in 2 - 10 copies per
cell. Plasmid-dependent resistance to methotrexate is also observed in
a rad 6 background although the effect is somewhat less than that conferred to wild-type or rad 18 cells. Integration of DFR 1 into the
yeast genome showed an intermediate sensitivity to folate antagonists.
This may suggest a gene dosage effect. No change in petite induction
in these yeast strains was observed in transformed cells containing
yeast dhfr plasmids.
The sensitivity of rad 6 , rad 18 and wild-type cell populations
to trimethoprim were unaffected by the presence of DFR 1 in
transformants. Moreover, trimethoprim did not induce petites in any
strain tested, which normally results if dhfr is inhibited by other
antifolates such as methotrexate. This may suggest that the dhfr
enzyme is not the only possible target of trimethoprim in yeast.
rad 6 mutants showed a very low level of spontaneous petite
formation. Methotrexate failed to induce respiratory deficient
mutants in this strain which suggested that rad 6 might be an
obligate grande. However, ethidium bromide induced petites to a level
approximately 50% of that exhibited by wild-type and rad 18 strains.||en_US
|dc.title||Partial characterization of the cloned dihydrofolate reductase gene of Saccharomyces cerevisiae||en_US
|dc.type||Electronic Thesis or Dissertation||en_US
|dc.degree.name||M.Sc. Biological Sciences||en_US
|dc.contributor.department||Department of Biological Sciences||en_US
|dc.degree.discipline||Faculty of Mathematics and Science||en_US