The SP013 gene, required for meiosis I segregation in Saccharomyces cerevisiae, produces two developmentally regulated transcripts (1.0 and 1.4 kilobases) that differ in length at their 5' ends. The shorter transcript is sufficient to complement the spol3-1 mutation and contains a major open reading frame encoding a highly basic protein of 33.4 kilodaltons. A fragment upstream (-170 to -8) of the open reading frame confers meiosis-specific transcription on a spol3-HIS3 fusion. Deletions at the 5' end of spo3-lacZ fusions define a region between -140 and -80 that is essential for meiosis-specific expression. This region acts in an orientation-independent manner and is responsive to the MAT-RME regulatory cascade. It contains a 10-base-pair sequence, TAGCCGCCGA, found in a number of meiosis-specific genes, that appears to be required for SP013 expression. This sequence is identical to URS1, a ubiquitous mitotic repressor element.Initiation of meiosis and spore formation in Saccharomyces cerevisiae is controlled by cell type and nutritional status (1, 2). Cell-type control occurs through the products of the MATa and MATa loci, which combine to negatively regulate transcription of RMEI (3, 4). RMEI negatively regulates IMEI, whose expression appears to be sufficient to induce meiosis (5). IMEI is also under negative control by the system monitoring nutritional status (5, 6).SP013 is required for the proper separation of homologs at meiosis I (7). SP013 has been cloned and encodes two overlapping transcripts with the same 3' end (8 MATERIALS AND METHODS Strains. RE944 (spol3-1/spol3-1 ura3/ura3) is from our strain collection. LP112 (MATa/MATa ade2/ade2 canl-100/ canl-100 his3-1 1, 15/his3-1 1,15 leu2-3,1 12/leu2-3, 112 trpl-1/ trpl-l ura3-1/ura3-1), obtained from S. Lindquist (University of Chicago), is a cross of W303-1A and W303-1B from R. Rothstein (Columbia University). Isogenic diploids SFY67 (MATa/MATa) and SFY68 (MATa/MATa) were obtained from J. Segall (University of Toronto). S104 (MATa/MATa leul/LEUI ura3/ura3 trpl/trpl cani/cani his4-519/HIS4 RMEI/rmel::LEU2), S150 (MATa/MATa leu2/leu2 ura3/ ura3 trpl/trpl his4-519/his4-712 canl/CANl lysliL YSI RMEl/rmel::LEU2), S151 (MATa/MATa leu2/leu2 ura3/ ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/L YSI rmel ::LEU2/rmel ::LEU2), S152 (matal-50/MATa leu2/ leu2 ura3/ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/ LYSI RMEJ/rmel ::LEU2), and S153 (matal-50/MATa leu2/leu2 ura3/ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/LYSI rmel::LEU2/rmel::LEU2) were acquired from A. Mitchell (Columbia University).Growth and Sporulation. General methods for growth and sporulation have been described (7,8,14). Strains were grown to midlogarithmic phase in synthetic acetate medium [1% potassium acetate plus 0.68% Difco yeast nitrogen base without amino acids, buffered to pH 6.5 with 0.05 M potassium phthalate (pH 5.5) and supplemented with auxotrophic requirements] prior to sporulation.DNA Sequence Analysis. Dideoxy sequencing of the cloned SP013 gene was carried out as ...

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The primary objective of this study is to identify prognostic site-specific epigenetic changes in surgically treated Stage I and II nonsmall cell lung cancer (NSCLC) patients by quantifying methylation levels at multiple CpG sites within each gene promoter. Paraffin-embedded tumors from stage Ib, IIa and IIb in training and validation groups of 75 and 57 surgically treated NSCLC patients, respectively, were analyzed for p16, MGMT, RASSF1, RASSF5, CDH1, LET7, DAPK and PTEN promoter hypermethylation. Hypermethylation status was quantified individually at multiple CpG sites within each promoter by pyrosequencing. Molecular and clinical characteristics with time to recurrence (TTR) and overall survival (OS) were evaluated. Overall average promoter methylation levels of MGMT and RASSF1 were significantly higher in smokers than in nonsmokers (p 5 0.006 and p 5 0.029, respectively). Methylation levels of the p16 promoter were significantly higher in squamous cell carcinoma than in adenocarcinoma (p 5 0.020). In univariate analysis, hypermethylation of RASSF1 at CpG sites 253 and 248 and PTEN at CpG site 21310 were the significantly associated with shorter TTR (p 5 0.002 and p < 0.000, respectively). Hypermethylation of PTEN at 21310 and DAPK at 21482 were most significantly associated with outcome in multivariate analysis. These results show that methylation of specific promoter CpG sites in PTEN, RASSF1 and DAPK is associated with outcome in early stage surgically treated NSCLC.