Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalyt...

Cyclin B is degraded at the onset of anaphase by a ubiquitin-dependent proteolytic system. We have fractionated mitotic Xenopus egg extracts to identify components required for this process. We find that UBC4 and at least one other ubiquitin-conjugating enzyme can support cyclin B ubiquitination. The mitotic specificity of cyclin ubiquitination is determined by a 20S complex that contains homol...

The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory...

The origin recognition complex (ORC) binds to origins of replication in budding yeast. We have cloned a Xenopus homolog of the largest ORC polypeptide (XORC1). Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication. We have purified Xenopus ORC, consisting of a protein complex similar to yeast ORC. In Xenopus egg extracts, ORC associates with chromatin throu...

Mycelial extracts of an Aspergillus wentii strain grown on yeast-extract sucrose medium and initially isolated from country-cured ham were highly toxic when inoculated into chicken embryos or fed to mice. Moldy corn and rice were less toxic when fed to mice. Water extracts of moldy corn or rice or culture filtrates from yeast-extract sucrose medium were not toxic. Purification by thin-layer chr...

A method is described for measuring cytosolic free Ca2+ and its time-dependent changes in the yeast Saccharomyces cerevisiae by using the luminescent protein aequorin as a Ca(2+)-specific indicator. This method with intact yeast cells is labeled "in vivo" to distinguish it from methods with cell extracts, labeled "in vitro." A plasmid in which the apoaequorin cDNA was joined downstream from the...

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-β-D-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated i...

p53 over expression in yeast results in cell death with typical markers of apoptosis such as DNA fragmentation and phosphatidylserine externalization. We aimed to substitute/supplement classical fluorescent techniques (TUNEL, Annexin V, ROS detection) usually used to detect biochemical changes occurring during yeast apoptosis mediated by p53 over expression and the effect of anti-apoptotic puri...

A heat-stable protein has been detected in Saccharomyces cerevisiae which inhibits mitochondrial ATPase activity. The protein inhibitor has been isolated from extracts prepared by brief heat treatment of unbroken cell suspensions. The isolated inhibitor is a small basic protein (molecular weight close to 7000, isoelectric proint 9.05) devoid of tryptophan, tyrosine, and cysteine as well as prol...

Photoreactivating enzyme (DNA photolyase; deoxyribocyclobutadipyrimidine pyrimidine-lyase, EC 4.1.99.3) repairs UV damage to DNA by utilizing the energy of near-UV/visible light to split pyrimidine dimers into monomers. The enzyme is widespread in nature but is absent in certain species in a seemingly unpredictable manner. Its presence in humans has been a source of considerable controversy. To...