In yeast, a new mutation called suc was recently discovered. The suc mutant cann
ID: 96515 • Letter: I
Question
In yeast, a new mutation called suc was recently discovered. The suc mutant cannot use sucrose as a source of carbon, and will not grow if the culture medium contains only sucrose as a carbon source. A plasmid library made with cDNA prepared from wild type yeast was used to transform a strain of yeast with two mutations: suc and ura (Suc- ura- strain). In the Suc-Ura- strain the URA gene, which allows yeast to grow in medium depleted in uracil, has been deleted. After transformation the yeast cells were plated on culture medium without uracil and containing sucrose as the only carbon source. a) Why must the plasmid chosen to construct the library contain a functional URA gene? b) Although 80% of the yeast cells were transformed, only a fraction of the transformed yeast formed colonies on the medium. Explain this observation. c) The recombinant plasmid extracted from a surviving colony contains a gene called SUC2. SUC 2 codes for convertase, an enzyme needed to metabolize sucrose. Mutations in another gene called SNF prevent the SNF mutant to use sucrose as a source of carbon. However, SNF mutants can still grow on glucose. Scientists have shown that in SNF mutants the SUC2 gene does not contain any mutations but these mutants have no convertase activity. We know that the SNF gene is not involved in translation. Is the SNF gene involved in DNA replication or transcription? Briefly justify your answer. d) The SNF mutation is rescued by another mutation in the gene for histone H4. What is the likely function of the SNF gene?
Explanation / Answer
Since the Suc-Ura strain does not have a functional URA gene that allows the yeast to grow in a medium depleted in uracil, the plasmid chosen to construct the library should have a functional URA gene. Mutant Suc-Ura yeast strain transformed with plasmid having a functional URA gene can synthesize uracil, a nitrogenous base found in RNA, when grown in a medium without uracil. Although 80% of the yeast cells were transformed, only a fraction of the transformed yeast formed colonies on the medium. This is because yeast after transformation is grown in a medium without uracil and containing sucrose as the only carbon source. Only those yeast cells that are transformed with plasmid construct having cDNA gene construct for metabolizing sucrose can only grow in medium with sucrose as the only carbon source. Hence not all transformed cells can form colonies in the medium with sucrose. The SNF gene is involved in transcription and hence can be considered as a transcriptional regulator. SNF gene must be required for the transcription of SUC2 gene that codes for invertase enzyme required for metabolizing sucrose. However mutations in SNF gene prevent the SNF protein from transcribing the SUC2 gene. As a result SUC2 gene is not transcribed and invertase enzyme is not produced. Due to this SUC2 mutant yeast strains cannot grow in a medium with only sucrose as carbon source. However, they can grow in a medium containing glucose as carbon source. For transcription to occur chromatin remodeling should take place so that DNA gets relaxed and becomes transcriptionally active. SNF gene must be promoting chromatin remodeling of SUC2 gene so that the gene is transcribed in presence of sucrose. However, mutations in SNF gene cannot cause chromatin remodeling and thus SUC2 gene is not expressed in SNF mutant yeast strains. Mutation in the gene for H4 rescued SNF mutation. It is because H4 is a histone protein that is involved in chromatin structure in eukaryotic cells. Acetylation and methylation modifications of these histone proteins alter gene expression. Acetylation of histone proteins promotes chromatin decondensation resulting in gene expression. Mutation in H4 gene must have resulted in a non-functional H4 protein due to which histone complex cannot assemble on DNA. This results in the DNA to remain in a decondensed or relaxed state thus promoting the transcription of SUC2 gene.