Gene structure in Saccharomyces cerevisiae : DNase I hypersensitive sites in galactose gene cluster and non-detection of 5-methylc[yt]osine Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/gx41mm72z

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  • The positions of the DNase I hypersensitive sites within the galactose gene cluster of the yeast Saccharomyces cerevisiaa have been determined. When yeast nuclei were incubated with DNase I, the purified DNA restriction endonuclease digested and analyzed by Southern hybridization, five hypersensitive regions were observed in this cluster of three closely spaced genes. Areas between the 5' end of GAL 7 and the 3' end of GAL 10 and between the two 5' ends of the divergently transcribed GAL 10 and GAL 1 genes contain DNase hypersensitive regions. DNase I hypersensitive sites were also observed in the areas near the 3' ends of the GAL 7 and GAL 1 genes. An additional site was observed within the GAL 10 structural gene. The region between GAL 10 and GAL 1 contains three tightly grouped cutting sites which map within a localized region of very high GC content. None of the five hypersensitive regions were observed when isolated DNA was digested with DNase I. Hypersensitive sites were present irrespective of the level of transcription of the galactose genes. A deletion which eliminates the 5' end of GAL 10 and all of GAL 1 does not destroy the internal GAL 10 hypersensitive site. The DNA of Saccharomyces cerevisiae has previously been reported to contain the modified base 5-methylcytosine. In order to determine if this methylation might occur in CCGG sequences, restriction endonuclease digestions were performed on DNA of strain B273-10B with the isoschizomer pair HpaII and Mspi. These two enzymes differ in their ability to cut the sequence C(5MeC)GG. Ethidium bromide stained gel tracks of DNA restricted with these two enzymes appeared identical. The patterns of HpaII and Mspl fragments within a 7.5 kb region about the gene for iso-2 cytochrome c and within the 6.3 kb two micron circle plasmid were also identical. Failing to have found any methylation in CCGG sequences, the genomic content of 5-methylcytosine was re-evaluated. HPLC analysis of highly purified yeast DNA showed 5-methylcytosine to be undetectable (less than 0.05% of total cytosine).
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