RE & Complementation

Because the media contains a methionine supplement

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because the media contains a methionine supplement, which means that transformed YMP 12 strain yeast cells can grow even with a repressed promoter. Growth for all transformed YMP 12 strain yeast would be expected in YC + glu + met – ura, because the media provides methionine that allows the transformed S. cerevisiae cell to grow even with a repressed GAL1 promoter. Significant growth was observed for YMP 12 yeast cells transformed with pBG1805+ MET3 , pYES2.1+ Met3, but not pYES2.1- lacZ on YC +met – ura –gal media (Fig.2 BEH). Yeast cells transformed with pYES2.1- lacZ were not expected to grow because successful complementation depends on the expression of the fusion protein and the ability of the fusion protein to restore enzymatic activity that is missing in the met3 mutant strain. The pYES2.1- lacZ overexpression plasmid does not contain a MET3 homologue, and even if its fusion protein were expressed it would not restore the cells ability to grow, therefore complementation did not occur. YMP 12 yeast cells transformed with pBG1805+ MET3 grew, and were expected to grow because MET3 was expected to restore enzymatic activity lost in the met3 mutant. This growth suggests that the YMP 12 yeast strain is the
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met3 mutant strain because growth YMP 12 yeast only grew when the enzymatic activity of MET3’s gene products, or homologues of MET3’s gene products, restored enzymatic activity lost in the met3 mutant. YMP 12 yeast cells also grew after a transformation with pYES2.1+ Met3 , which suggests that Met3 is a homologue of MET3 because it was able to restore enzymatic activity lost in the met3 mutant, and induce YMP 12 yeast cell growth. Baseline transcription occurred in transformed YMP 12 yeast cells grown on YC + glu – met – ura media (Fig. 2 C,F,I); no growth was expected because glucose represses the GAL1 promoter that would mostly prevent all yeast cells from producing their own methionine. Without an additional supplement of methionine in the media, yeast cells should be unable to grow in these conditions. A leaky GAL1 promoter would be the likely explanation for any baseline transcription under the repressed condition of a glucose-rich media. Further research also suggests that sua1 is a homologue of MET3 because both genes produce proteins with the same enzyme commission number: EC 2.7.7.4 [5]. The enzyme commission number indicates an enzyme-catalyzed reaction, and because both Met3p and Sua1p share the same EC number they must catalyze the same reaction, even though they are not the same protein. A pairwise alignment of Met3p, ATP sulfurylase, and the Sua1p, sulfate adenylyltransferase, has an e-value of 1e-112, suggesting a significant match of a large amino acid alignment with a query coverage of 57% [4]. Although only 57% of the protein sequence aligns overall, there is a sequence of 252 amino acids in which both Sua1p and Met3p match 73% of the time [4]. This area of the protein is most likely associated with essential enzymatic activity, such as a substrate-binding site. According to this BLAST data, the S. pombe
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