Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products al and a2, which determine the a/a cell type. These signals lead to increased expression of the IMEI (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IMEI expression from the GAL] promoter. The deduced IMEI product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression Sporulation of the yeast Saccharomyces cerevisiae is a cellular differentiation pathway (reviewed in references 12 and 26). It is normally restricted to one type of cell, the a/a cell, and is induced by nitrogen starvation. These circumstances lead to arrest of the mitotic cell cycle, to expression of sporulation-specific genes, and to initiation of the sporulation program. Cells engage in meiotic DNA synthesis, recombination, and two meiotic divisions. Each of the four meiotic products is packaged into a spore, and the four spores of the cell are encased in a sac, the ascus. Sporulation thus includes meiosis and spore formation.One of the earliest unique events in starved a/a cells is elevated accumulation of IME1 RNA (22,28,38; reviewed in reference 27). The IME1 product is thought to play a pivotal role in activating meiosis, because multicopy IME1 plasmids permit sporulation in cells that lack the determinants of a/a cell type, the MATal and MATa2 gene products, and also permit meiotic recombination in the absence of nitrogen starvation (22,38 Sporulation is accompanied by express'ion of a unique set of genes, the sporulation-specific genes. Some of these genes are essential for particular meiotic events, and others have no essential role in sporulation under laboratory conditions (1,6,11,14,17,21,25,26,33,34,42,45,47). These genes fall into early, middle, and late expression classes (1,23,25,26 The imel-12, ime2-2, gal80, ho::LYS2, his4-G, and his4-N mutations have previously been described, as have the auxotrophic markers in these strains (28,38). We note that the ho::LYS2 insertion confers a weak Lys' phenotype.The a/a diploid (strain 545) was derived from an a/a diploid (strain 537) after mild UV irradiation by screening colonies for mating-factor production through a halo assay (46). Engebrecht and Roeder observed that a/a and a/a diploids in the SK1 background were able to sporulate at a low level (strains J254 and J256 [11]). In side-by-side comparisons, we confirmed that J254 and J256 were able to sporulate and that our strain 545 was unable to sporulate. Fourteen four-spored tetrads were analyzed from an a/a/a/a tetraploid derived from crossing strains 545 and J256. Nine segregants were able to mate and able to sporulate weakly; 25 segregants were able to mate but unable to sporulate. These observations indicate that the difference in sporulation abilities 6103 on May 10, 2018 by guest

In the yeast Saccharomyces cerevisiae, genetic studies suggest that the RIM1 gene encodes a positive regulator of meiosis. rim1 mutations cause reduced expression of IME1, which is required for expression of many meiotic genes, and thus lead to a partial defect in meiosis and spore formation. We report the sequence of RIM1 and functional analysis of its coding region. The RIM1 gene product (RIM1) contains three regions similar to C2H2 zinc fingers. Serine substitutions for cysteine in each of the putative zinc fingers abolish RIM1 function. The carboxyl-terminus of RIM1 is enriched in acidic amino acids and is required for full RIM1 activity. RIM1 also contains two putative cAMP-dependent protein kinase (cAPK) phosphorylation sites. At one site, substitution of alanine for serine does not affect RIM1 activity; at the other site, this substitution impairs activity. This analysis of RIM1 suggests that the protein may function as a transcriptional activator. We have used the cloned RIM1 gene to create a complete rim1 deletion. This null allele, like previously isolated rim1 mutations, causes a partial meiotic defect. In addition to RIM1, maximum IME1 expression requires the MCK1 and IME4 gene products. Defects associated with rim1, mck1, and ime4 mutations in expression of a meiotic reporter gene (ime2-lacZ) and in sporulation are additive. These findings suggest that RIM1 acts independently of MCK1 and IME4 to stimulate IME1 expression.

Fission yeast cells either remain in the mitotic cell cycle or exit to meiotic sporulation from an uncommitted G1 state dependent on the presence or absence of nitrogen source in the medium (Nurse and Bissett, 1981). We examined how heterothallic haploid cells, which cannot sporulate, behave under nitrogen-starvation for longer than 25 days at 26 degrees C. These cells were shown to enter a stable state (designated the dormant G0) with nearly full viability. Maintaining the dormant cells required glucose, suggesting that the cells remained metabolically active although cell division had ceased. They differed dramatically from mitotic and uncommitted G1 cells in heat resistance, and also in cytoplasmic and nuclear morphologies. After nitrogen replenishment, the initial responses of dormant G0 cells were investigated. The kinetics for reentry into the proliferative state were delayed considerably, and the changes in cell shape were enhanced particularly for those recovering from extended nitrogen starvation. A part of the delay could be accounted for by the duration of nuclear decondensation and cell elongation for the first cell division.

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It has been reported that apoptosis is a significant contributor to myocardial cell death as a result of reperfusion injury. However, whether the extent of cardiomyocyte apoptosis following ischemia and reperfusion varies in different pathophysiological backgrounds is still uncertain. In this study, we examined whether hypercholesterolemia increases the extent of myocardial reperfusion injury by aggravating cardiomyocyte apoptosis and the effects of hypercholesterolemia on the expression of Bcl-2 and Bax proteins and the activation of caspase-3. Twenty-eight male New Zealand white rabbits were fed standard chow (control, n = 14) or chow supplemented with 10% cholesterol (hypercholesterolemic, n = 14) for 8 wk. Anesthetized rabbits were then subjected to 30 min of left circumflex artery occlusion followed by 4 h of reperfusion. Apoptosis was identified as "DNA ladders" by gel electrophoresis and confirmed histologically using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The infarct size (% of risk region) was significantly greater in hypercholesterolemic rabbits than in controls (39 +/- 6 vs. 23 +/- 2%, P = 0.02). Very few TUNEL-positive cardiomyocytes could be identified in the nonischemic regions in both groups, consistent with an absence of DNA laddering. In contrast, TUNEL-positive cardiomyocytes were significantly displayed in the ischemic, nonnecrotic myocardium, and DNA ladder occurred in all animals. The percentage of TUNEL-positive cardiomyocytes in the ischemic nonnecrotic myocardium was significantly higher in hypercholesterolemic rabbits compared with controls (40 +/- 5 vs. 17 +/- 11%, P < 0.001). Western blot analysis showed that, in the nonischemic myocardium, hypercholesterolemic rabbits exhibited an approximately 50% increase in the expression of Bcl-2 (P < 0.05), but not Bax, than control rabbits. However, compared with controls, hypercholesterolemic rabbits exhibited a more pronounced decrease in the expression of Bcl-2 (42 +/- 4 vs. 26 +/- 2%, P < 0.01) and a similar extent of increase in the expression of Bax in the ischemic myocardium. Furthermore, hypercholesterolemic rabbits were associated with a markedly increased activation of caspase-3 within the ischemic myocardium compared to control rabbits. This study demonstrates that although hypercholesterolemia is associated with an increased myocardial Bcl-2/Bax ratio at baseline, it still significantly exacerbates cardiac reperfusion injury, not only by increasing the infarct size but also by increasing the extent of cardiomyocyte apoptosis.