Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

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Molecular biologists are increasingly faced with the problem of assigning a function to genes that have been cloned. A new approach to this problem involves the manipulation of the cloned gene to create what are known as 'dominant negative' mutations. These encode mutant polypeptides that when overexpressed disrupt the activity of the wild-type gene. There are many precedents for this kind of behaviour in the literature--some oncogenes might be examples of naturally occurring dominant negative mutations.

Cisplatin is a chemotherapeutic drug used to treat a variety of cancers. Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness. Molecular mechanisms that underlie cisplatin resistance are poorly understood. Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin. Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug. Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells. Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage. We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals. The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug. C isplatin [cis-diamminedichloroplatinum(II)] chemotherapy is curative for the majority of patients with advanced testicular cancer, which was almost uniformly fatal before its use, and is also effective for ovarian, bladder, cervical, head and neck, and small-cell lung cancers (1). Many patients with these cancers, however, eventually relapse and become refractory to this drug (2). In contrast to the cancers treatable with cisplatin, common cancers such as colorectal are intrinsically resistant to this drug. Increasing dosage to overcome resistance can cause serious nephrotoxicity and ototoxicity. Understanding the mechanism of intrinsic and acquired resistance to cisplatin is thus critical for developing more effective treatment for cancer.The mechanism by which cisplatin enters cells is unknown. The drug becomes activated once it enters the cytoplasm, where the chloride atoms on cisplatin are displaced by water molecules. This aquated product is a potent electrophile that can react with any nucleophile, including the sulfhydryl groups on proteins and nitrogen donor atoms on nucleic acids. Work from many laboratories has implicated DNA as a critical target for cisplatin cytotoxicity, the most revealing evidence being the hypersensitivity to cisplatin of both prokaryotic and eukaryotic cells deficient in DNA repair (3-5). The most prevalent cisplatininduced DNA adduct is an intrastrand crosslink in which the platinum is covalently bound to the N 7 positions of the imidazole ring of two adjacent guanines (6). The intrastrand crosslinks are repaired by the nucleotide excision repair pathway (7).Studies in mammalian cells have identified several genes that affect sensitivity of cells to cisplatin (8). Overexpression of a multidrug resistance protein MRP2 or a copper-transporting P-type ATPase ATP7B res...

Meiosis is thought to require the protein kinase Ime2 early for DNA replication and the cyclin-dependent kinase Cdc28 late for chromosome segregation. To elucidate the roles of these kinases, we inhibited their activities early and late using conditional mutants that are sensitive to chemical inhibitors. Our studies reveal that both Cdc28 and Ime2 have critical roles in meiotic S phase and M phase. Early inhibition of analog-sensitive cdc28-as1 blocked DNA replication, revealing a previously undetected role for Cdc28. Yet Cdc28 was dispensable for one of its functions in the mitotic cell cycle, degradation of Sic1. Late addition of inhibitor to ime2-as1 revealed unexpected roles of Ime2 in the initiation and execution of chromosome segregation. The requirement of Ime2 for M phase is partially explained by its stimulation of the key meiotic transcription factor Ndt80, which is needed in turn for high Cdc28 activity. In accordance with a late role for Ime2, we observed an increase in its activity during M phase that depended on Cdc28 and Ndt80. We speculate that several unique features of the meiotic cell division reflect a division of labor and regulatory coordination between Ime2 and Cdc28. Meiosis is a specialized cell division that produces haploid cells needed for sexual reproduction. Successful completion of meiosis requires several sequential landmark events: DNA replication, recombination and synapsis of homologs, and chromosome segregation. Chromosome segregation occurs in two successive meiotic divisions to yield four haploid meiotic products that are packaged into gametes. Progression through meiosis requires three major decisions. The first is whether to enter the meiotic differentiation program or continue with mitotic proliferation. The second is whether to embark on chromosome preparation, including meiotic S phase, and then recombination and synapsis of homologs. The third decision, whether to proceed into M phase, depends on the successful completion of replication and recombination. Transcription factors and protein kinases are central components of the pathways that control these key decisions. In particular, the transcription factor Ime1 and the kinase Ime2 are thought to participate in the G1-S transition, whereas the transcription factor Ndt80 and the kinase Cdc28 take part in the G2-M transition.Intertwined with the landmark events of meiosis is a well-characterized transcriptional cascade Primig et al. 2000). Ime1 stimulates transcription of the early class of genes and thus is required for entry into the meiotic program and for the meiotic G1-S transition (Vershon and Pierce 2000). An important target of Ime1 is IME2, which encodes a serine-threonine protein kinase (Mitchell et al. 1990). Ime2 is required for meiotic DNA replication (Foiani et al. 1996) and appears to control the G1-S transition by decreasing the level of Sic1 (Dirick et al. 1998), an inhibitor of the cyclin-dependent kinase (CDK) Cdc28 in the mitotic cell cycle (Mendenhall and Hodge 1998). It has been proposed that Ime2-d...

The MAPKKK Ste11p functions in three Saccharomyces cerevisiae MAPK cascades [the high osmolarity glycerol (HOG), pheromone response, and pseudohyphal/invasive growth pathways], but its activation in response to high osmolarity stimulates only the HOG pathway. To determine what restricts cross-activation of MAPK cascades (cross talk), we have studied mutants in which the pheromone response pathway is activated by high osmolarity (1 M sorbitol). We found that mutations in the HOG1 gene, encoding the p38-type MAPK of the HOG pathway, and in the PBS2 gene, encoding the activating kinase for Hog1p, allowed osmolarity-induced activation of the pheromone response pathway. This cross talk required the osmosensor Sho1p, as well as Ste20p, Ste50p, the pheromone response MAPK cascade (Ste11p, Ste7p, and Fus3p or Kss1p), and Ste12p but not Ste4p or the MAPK scaffold protein, Ste5p. The cross talk in hog1 mutants induced multiple responses of the pheromone response pathway: induction of a FUS1::lacZ reporter, morphological changes, and mating in ste4 and ste5 mutants. We suggest that Hog1p may prevent osmolarity-induced cross talk by inhibiting Sho1p, perhaps as part of a feedback control on the HOG pathway. We have also shown that Ste20p and Ste50p function in the Sho1p branch of the HOG pathway and that a second osmosensor in addition to Sho1p may activate Ste11p. Finally, we have found that pseudohyphal growth exhibited by wild-type (HOG1) strains depends on SHO1, suggesting that Sho1p may be a receptor that feeds into the pseudohyphal growth pathway.

Gametogenesis requires the successful coordination of two key processes, meiotic nuclear division and gamete morphogenesis. A central regulatory step in progression through gametogenesis occurs at the pachytene stage of meiotic prophase. We find that Ndt80 functions at pachytene of yeast gametogenesis (sporulation) to activate transcription of a set of genes required for both meiotic division (e.g., B-type cyclins) and gamete formation (e.g., SPS1). Ectopic synthesis of Ndt80 in vegetative cells induces transcription of these genes, and recombinant Ndt80 protein binds to a conserved sequence in their upstream region. Transcription of NDT80 itself is dependent on Ime1, which activates expression of early sporulation genes. Transcription of the Ndt80-regulated gene CLB1 is mediated by the checkpoint gene RAD17. Thus Ndt80 is a pivotal component of a transcriptional cascade programming yeast gametogenesis and may also be a target of meiotic checkpoint control.

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