horylated CTD. Antibodies specific for a-tubulin were used to show that equal amounts of protein were loaded for each sample. Interestingly, Karagiannis and Balasubramanian and others reported that lsk1D mutations completely abolish Ser-2 phosphorylation. In contrast, the above experiment demonstrated only a reduction in the levels of Ser-2 phosphorylation. It was thus of interest to identify the origin of this discrepancy. Since the original protocols did not include phosphatase inhibitors, the experiment was repeated with and without this treatment. Similar to the previously published work, bands were completely absent from lsk1D and lsg1D samples when no phosphatase inhibitors were included in the extraction buffer. We thus conclude that the inclusion of phosphatase inhibitors in the extraction buffer was the source of the discrepancy in results. Importantly, these data suggest the existence of at least one other 14726663” kinase capable of phosphorylating Ser-2 residues in the CTD. Gene expression analysis To this point all data were supportive of Lsg1p functioning in a complex with Lsk1p and Lsc1p to phosphorylate Ser-2 residues within the heptad repeats of the Rpb1p subunit of RNA polymerase II. Furthermore, genetic analysis of lsg1D, lsk1D, and lsc1D mutants clearly demonstrated that all three sub-units were important for the reliable execution of cytokinesis. Since misregulation of Ser-2 phosphorylation might alter the control of transcription, we decided to analyze the global gene expression profiles of mutants impaired in Ser-2 phosphorylation of the RNA polymerase II CTD. We hypothesized that the transcription of genes involved in cytokinesis or the cytoskeleton might be selectively affected by impairment of Ser-2 phosphorylation. Two YM-155 web strains were used in these studies: a mutant strain bearing the rpb1-12XS2ACTD mutation and a control strain of the genotype rpb1-12XWTCTD. The rpb1 gene in the rpb1-12XS2ACTD strain has been mutated so as to encode a total of 12 mutant heptad repeats in which alanine residues have replaced serine residues at the “two”position. Interestingly, this mutant phenocopies lsk1D, lsc1D, and lsg1D strains in terms of the 17062696” strain’s inability to complete cytokinesis upon LatA treatment, and in its capacity to rescue the temperature sensitive cdc16116 mutation. The rpb1 gene in the rpb1-12XWTCTD strain, on the other hand, encodes a molecule bearing 12 wild-type heptad repeats. This strain displays no obvious growth or morphological phenotypes and does not exhibit any cell cycle or cytokinesis defects. These strains can thus be employed as helpful tools in understanding the effects of the impairment of Ser-2 phosphorylation on cellular physiology and transcription. To obtain global gene expression profiles, rpb1-12XCTD and rpb1-12XS2ACTD strains were grown to mid-log phase at 30uC, and then treated with either DMSO or LatA for three hours. Total RNA was then extracted and used in microarray hybridizations using Yeast Genome 2.0 Gene Chips purchased from Affymetrix. Three replicates of each strain Cells of the indicated genotypes were grown to mid-log phase in YES. Total lysates were then resolved by SDS-PAGE and immunoblotted with antibodies specific for the unphosphorylated form of the CTD, the Ser-5 phosphorylated form of the CTD, the Ser-2 phosphorylated form of the CTD, as well as antibodies 
specific for alphatubulin. Cells of the indicated genotypes were grown to mid-log phase in YES. Total lysates were then prep
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