Dihydrofolate Reductases

Decent Essays
1. The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases by Ron Ortenberg, Orit Rozenblatt-Rosen and Moshe Mevarech.
2. When working with Haloferax volcanii the scientists were trying to figure out what genes control the dihydrofolate reductases and removed the hdrA gene from the H.volcanii and challenged the cells with trimethylprim. When a trimethoprim resistant mutant occurred it was analyzed and there was DNA sequence similar to the amino acid sequence of dihydrofolate reductase. When the scientists used the amplification unit as a probe the chromosomal locus containing hdrB gene was detected. Wanting to determine which hdr gene would have a greater effect on the organism ability to have
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In order to achieve this goal they created variants of each gene. These genes were all removed by a similar process by which the pieces of the gene were cut with different restriction enzymes and purified and cloned in order to give us different plasmids. They were amplified using PCR and these were then integrated into the regions and then grown on media and observed. When there was excision of the gene they were looking to remove these strains were cultivated and given designations. Strain WR 441(hDHFR-2) has the hdrA was deleted, WR445 has a deletion of both genes and WR446 (hDHFR-1) has a deletion of hdrB, WR 447 had hts deleted from it. These strains in turn were grown on three different Medias HY which was the rich media, M which was the minimal media and agar plates. In these experiments they found that WR441 needs nothing else and grew on each media with no other enrichment. However WR446 and WR 445 could grow on minimal medium containing both trimethoprim and thymidine, but only when that medium was supplemented with glycine, methionine, pantothenic acid and hypoxanthine. Another strain WR447 was grown and found that it was auxotrophic for thymidine. However when plasmid pHE4 was added to it could grow on minimal medium containing trimethoprim and thymidine. When the hts was removed the hdrB gene did not have a strong promotor which appeared when the hDHFR-2 didn’t express itself enough to give the organism the trimethoprim resistance it need to get a high copy number. Because of this we can use the hdr gene as an expression point for Hf.volcanii. At the lowest level of expression, only complementation of purine, glycine, pantothenate and methionine autotrophy will occur When the gene has a higher level of expression we can see that there will be prototrophic growth in minimal medium, and when the gene is working at its highest level it will give a resistance to
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