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Strain RGY1 was made by transforming S. Transformations were done following a modified protocol of Ito et al.
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RNA was transferred to nylon membranes e. For analysis of transcription from the URA3 and HIS3 genes specifically, fragments of these genes were inserted into bidirectional promoter vectors.
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After hybridization, membranes were exposed to phosphorimager screens and visualized on a Molecular Thick Hermosa Beach looking for a swm model P90 Phosphorimager Sunnyvale, CA. DNA elements are indicated on the large circle.
Small circles denote approximate nucleosome positions. The hatched circle downstream of URA3 indicates space for a nucleosome, although the footprint was unclear. The small solid ellipse demarcates the ARS1 consensus sequence A-element.
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The inner, double-headed Adult singles dating in Kintnersville indicate the fragments used to analyze repair of CPDs at specific sites and the s denote the nt positions clockwise from the unique EcoR I site of YRpSOl of strong micrococcal nuclease cut sites Bingham Lake pussy kim chromatin.
Preparation of chromatin and deproteinized control DNA, digestion with micrococcal nuclease and mapping of the cutting sites by indirect end-labeling were done as described ly 23with the following modifications: Zymolyase T ICN Pharmaceuticals and a Sephacryl S column Pharmacia were used. Micrococcal nuclease Sigma was used at 0. Probes were made using a random priming kit Pharmacia.
Gels were either photographed for direct quantitation from negatives or treated with 0. The probe used for hybridization was generated from linearized YRpSOl see.
The average level of CPDs per plasmid was obtained from the intensity of the uncut Form I plasmid, assuming a Poisson distribution of UV damaged fragments, as described Analysis of CPD repair at specific sites in three selected fragments of YRpSOl To monitor repair of CPDs at nucleotide resolution, a biotinyl-ated oligonucleotide oligo and streptavidin magnetic bead-facilitated end-labeling technique was used 25— Briefly, isolated plasmid DNA was cut with restriction enzyme s to release the specific fragments to be Horny bbw chat Dellwood Fig.
To quantitatively measure CPD formation and repair at individual sites, we used an excess amount of T4 endo V to ensure that all CPD sites Women looking real sex Buckfield Maine incised by the enzyme routinely checked with irradiated plasmid.
DNA damage levels at each repair time were quantified by integrating band intensities in gel lanes for different repair incubation times using ImagQuaNT Molecular Dynamics and PeakFit 4.
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Sequence markers were generated from the fragments obtained by standard PCR reaction. YRpSOl has 22 nucleo-somes Fig.
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Total RNA was isolated from late log-phase RGY1 cells, grown in glucose lanes 1 and 3 or galactose lanes 2 and 4 medium and subjected to Girl for fuck in Kearny blot analysis using strand-specific riboprobes to the URA3 or HIS3 genes. The amount of URA3 transcript in each lane was normalized to the amount of rRNA in that lane to correct for loading variations and divided by the value obtained for time 0.
The half-life for the data shown is 4.