Cloning and sequencing of the PHO80 gene and CEN15 of Saccharomyces cerevisiae

The PHO80 gene, which is one of the regulatory genes exerting negative control in the pho system of Saccharomyces cerevisiae, was cloned. The 1.8 kb DNA fragment carrying the PHO80 gene was sequenced and one open reading frame large enough to encode 293 amino acids was found in the sequence. Northern blot analysis of poly(A)+-RNA isolated from cells grown under repressed and derepressed conditions revealed that (i) the size of the PHO80 message was around 1.4 kb, (ii) the expression of the PHO80 gene was not affected by the presence or absence of inorganic phosphate in the medium, and (iii) the expression of the PHO80 gene was not affected by pho2, pho4, pho81, or by pho80 itself. A centromere sequence was found downstream of the PHO80 coding region.     

鞣花酸对马兜铃酸I诱导小鼠急性肾损伤的保护作用

目的:研究鞣花酸(ellagic acid,EA)对马兜铃酸I(aristolochic acids I,AAI)诱导的急性肾损伤的保护作用.方法:40只昆明小鼠(雌雄各半)随机分为健康组、模型组(10?mg/kg?mb?AAI)、EA低剂量组(10?mg/kg mb?AAI+10?mg/kg?mb?EA)和EA高剂量...     

Yeast Pho85 kinase is required for proper gene expression during the diauxic shift

The budding yeast Saccharomyces cerevisiae changes its gene expression profile when environmental nutritional conditions are changed. Protein kinases including cyclic AMP-dependent kinase, Snf1 and Tor kinases play important roles in this process. Pho85 kinase, a member of the yeast cyclin-dependent kinase family, is involved in the regulation of phosphate metabolism and reserve carbohydrates, and thus is implicated to function as a nutrient-sensing kinase. Upon depletion of glucose in the medium, yeast cells undergo a diauxic shift, accompanied by a carbon metabolic pathway shift, stimulation of mitochondrial function and downregulation of ribosome biogenesis and protein synthesis. We analysed the effect of a pho85Delta mutation on the expression profiles of the genes in this process to investigate whether Pho85 kinase participates in the yeast diauxy. We found that, in the absence of PHO85, a majority of mitochondrial genes were not properly induced, that proteasome-related and chaperonin genes were more repressed, and that, when glucose was still present in the medium, a certain class of genes involved in ribosome biogenesis (ribosomal protein and rRNA processing genes) was repressed, whereas those involved in gluconeogenesis and the glyoxylate cycle were induced. We also found that PHO85 is required for proper expression of several metal sensor genes and their regulatory genes. These results suggest that Pho85 is required for proper onset of changes in expression profiles of genes responsible for the diauxic shift.     

Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae

The expression of acid phosphatase (APase) from PHO5 and MF alpha-PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MAT alpha 1 gene products respectively. When PHO5 and MF alpha-PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively. The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression. By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, from nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase. A comparison of these three pBR322 fragments revealed 5' ATCGCGCGAG 3' and 5' CGGTGATGNCGG 3' to be the common sequences likely to act as UASs in Saccharomyces cerevisiae. By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.     

Genome-wide survey of non-essential genes required for slowed DNA synthesis-induced filamentous growth in yeast

We recently discovered that slowed DNA synthesis induces filamentous differentiation in S. cerevisiae. We screened the BY yeast deletion strains and identified four classes of non-essential genes that are required for both slowed DNA-induced filamentous growth and classic forms of filamentous growth: (a) genes encoding regulators of the actin cytoskeleton and cell polarity, ABP1, CAP2 and HUF1 (=YOR300W), in addition to the previously known BNI1, BUD2, PEA2, SPA2 and TPM1; (b) genes that are likely involved in cell wall biosynthesis, ECM25, GAS1 and PRS3; (c) genes encoding possible regulators of protein secretion, SEC66, RPL21A and RPL34B; (d) genes encoding factors for normal mitochondrial function, IML1 and UGO1. These results showed that pseudohyphal formation involves not the only previously known regulation of the actin cytoskeleton/cell polarity but also regulation of cell wall synthesis, protein secretion and mitochondrial function. Identification of multiple classes of genes that are required for both slowed DNA synthesis-induced and classic forms of filamentous growth confirms that slowed DNA synthesis-induced filamentous growth is bone fide filamentous differentiation.     

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances, due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses both NADH and NADPH, is hypothesized to reduce the cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that metabolism is still imbalanced, even under aerobic conditions. In this study, we investigated the possible reasons for this imbalance by deleting genes required for NADPH production and gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR-XDH, but not a xylose isomerase, pathway required the oxidative branch of the pentose phosphate pathway (PPP) and gluconeogenic production of glucose-6-P for xylose assimilation. The requirement for generating glucose-6-P from xylose was also shown for Kluyveromyces lactis. When grown in xylose medium, both K. lactis and S. stipitis showed increases in enzyme activity required for producing glucose-6-P. Thus, natural xylose-assimilating yeast respond to xylose, in part, by upregulating enzymes required for recycling xylose back to glucose-6-P for the production of NADPH via the oxidative branch of the PPP. Finally, we show that induction of these enzymes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; S. cerevisiae was not able to increase enzyme activity for glucose-6-P production when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose.     

In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition

The efficacy of ellagic acid (EA), one of the naturally occurring polyphenols, in inhibiting melanogenesis was examined in vitro and in vivo. When mushroom-derived tyrosinase, a metaloprotein containing copper, was incubated with EA, enzymatic activity tended to decrease with decreasing copper concentration. Enzyme activity partially recovered when copper was added to the inactivated enzyme. Tyrosinase activity in the B16 melanoma cells was observed to recover in a dose-dependent manner when copper ions were added to the medium containing EA. Based on these results, EA is thought to react specifically with the copper located at the active centre of the tyrosinase molecule. Furthermore, when EA was applied for 6 weeks to brownish guinea-pigs, which have melanocytes in their skin, at the same time as irradiating for 2 weeks with ultra-violet light, skin pigmentation was clearly suppressed and the skin to which EA had been applied showed features similar to that of non-irradiated skin. These areas were irradiated again when the application of EA had been completed, and skin pigmentation occurred at the former site of EA application. In similar studies with hydroquinone, re-pigmentation did not occur on the sites at which hydroquinone (1%) had been applied. Based on the results reported here, EA is thought to suppress melanogenesis by reacting with activated melanocytes and without injuring cells.