Effect of heme and vacuole deficiency on FRE1 gene expression and ferrireductase activity in Saccharomyces cerevisiae

Doppler sonography of the fetal descending aorta, renal artery, middle cerebral artery and umbilical artery in a population of 74 fetuses with a abdominal circumference below the fifth percentile of the reference limits were done. All fetuses were free from structural and chromosomal abnormalities. The pulsatility- and the resistance indices as well as the ratios between these indices from peripheral and cerebral vessels were calculated and correlated to the fetal distress. The measurement of the pulsatility index of the middle cerebral artery provided the best results in predicting the development of fetal distress. Better results were achieved by the use of ratios of pulsatility-indices of various vessels than by the examination of the vessels alone. Our results suggest the usefulness of the examination of the middle cerebral artery and their ratios compared to the renal artery.     

Genome-wide expression monitoring in Saccharomyces cerevisiae

The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.     

Control of glycolytic gene expression in the budding yeast (Saccharomyces cerevisiae)

Fascin is an actin-bundling protein that was first isolated from cytoplasmic extracts of sea urchin eggs [Kane, 1975: J. Cell Biol. 66:305-315] and was the first bundling protein to be characterized in vitro. Subsequent work has shown that fascin bundles actin filaments in fertilized egg microvilli and filopodia of phagocytic coelomocytes [Otto et al., 1980: Cell Motil. 1:31-40; Otto and Bryan, 1981: Cell Motil. 1:179-192]. Fifteen years later, the molecular cloning of sea urchin fascin [Bryan et al., 1993: Proc. Natl. Acad. Sci. U.S.A. 90:9115-9119] has led to the identification and characterization of homologous proteins in Drosophila [Cant et al., 1994: J. Cell Biol. 125:369-380], Xenopus [Holthuis et al., 1994: Biochim. Biophys. Acta. 1219:184-188], rodents [Edwards et al,. 1995: J. Biol. Chem. 270:10764-10770], and humans [Duh et al., 1994: DNA Cell Biol. 13:821-827; Mosialos et al., 1994: J. Virol. 68:7320-7328] that bundle actin filaments into structures which stabilize cellular processes ranging from mechanosensory bristles to the filopodia of nerve growth cones. Fascin has emerged from relative obscurity as an exotic invertebrate egg protein to being recognized as a widely expressed protein found in a broad spectrum of tissues and organisms. The purpose of this review is to relate the early studies done on the sea urchin and HeLa cell fascins to the recent molecular biology that defines a family of bundling proteins, and discuss the current state of knowledge regarding fascin structure and function.     

Involvement of the Saccharomyces cerevisiae UTH1 gene in the oxidative-stress response

Despite lack of empirical support, facilitated communication was rapidly adopted and used with individuals who have severe communication disorders. An overview of the psychological literature was provided here to support theoretical explanations for this rapid adoption. The literature suggests that cognitive biases, ambiguous stimuli, and biases in data may be associated with a tendency to adopt interventions such as facilitated communication. Psychosocial influences associated with autism, the helping relationship, and the professional career cycle may enhance a readiness to adopt alternative treatments. Social influences may create an environment in which fads arise. Suggestions were provided for students and professionals in the broad fields of rehabilitation and education on how to improve their participation in developing and monitoring innovative treatment methods.     

Characterization of a novel alpha-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae

A highly active alpha-amylase (76,250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae alpha-amylase acted by endo-hydrolysis on glucose polymers containing alpha-1,4 and alpha-1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH2-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro- Glu-Ser-Val-Thr-Gly. The L. kononenkoae alpha-amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.     

Suppression of sdh1 mutations by the SDH1b gene of Saccharomyces cerevisiae

Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the beta-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100-500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b.     

Application of mRNA differential display to investigate gene expression in thermotolerant cells of Saccharomyces cerevisiae

In nephrogenic diabetes insipidus, the kidney is unable to concentrate urine despite normal or elevated concentrations of the antidiuretic hormone arginine vasopressin (AVP). In congenital nephrogenic diabetes insipidus (NDI), the obvious clinical manifestations of the disease, that is polyuria and polydipsia, are present at birth and need to be immediately recognized to avoid severe episodes of dehydration. Most (>90%) congenital NDI patients have mutations in the AVPR2 gene, the Xq28 gene coding for the vasopressin V2 (antidiuretic) receptor. In <10% of the families studied, congenital NDI has an autosomal recessive inheritance and mutations of the aquaporin-2 gene (AQP2), ie, the vasopressin-sensitive water channel, have been identified. When studied in vitro, most AVPR2 mutations lead to receptors that are trapped intracellularly and are unable to reach the plasma membrane. A minority of the mutant receptors reach the cell surface but are unable to bind AVP or to trigger an intracellular cyclic adenosine-monophosphate (cAMP) signal. Similarly AQP2 mutant proteins are trapped intracellularly and cannot be expressed at the luminal membrane. The acquired form of NDI is much more common than the congenital form, is almost always less severe, and is associated with downregulation of AQP2. The advances described here are examples of "bedside physiology" and provide diagnostic tools for physicians caring for these patients.     

Cross regulation of four GATA factors that control nitrogen catabolic gene expression in Saccharomyces cerevisiae

Nitrogen catabolic gene expression in Saccharomyces cerevisiae has been reported to be regulated by three GATA family proteins, the positive regulators Gln3p and Gat1p/Nil1p and the negative regulator Dal80p/Uga43p. We show here that a fourth member of the yeast GATA family, the Dal80p homolog Deh1p, also negatively regulates expression of some, but not all, nitrogen catabolic genes, i.e., GAP1, DAL80, and UGA4 expression increases in a deh1 delta mutant. Consistent with Deh1p regulation of these genes is the observation that Deh1p forms specific DNA-protein complexes with GATAA-containing UGA4 and GAP1 promoter fragments in electrophoretic mobility shift assays. Deh1p function is demonstrable, however, only when a repressive nitrogen source such as glutamine is present; deh1 delta mutants exhibit no detectable phenotype with a poor nitrogen source such as proline. Our experiments also demonstrate that GATA factor gene expression is highly regulated by the GATA factors themselves in an interdependent manner. DAL80 expression is Gln3p and Gat1p dependent and Dal80p regulated. Moreover, Gln3p and Dal80p bind to DAL80 promoter fragments. In turn, GAT1 expression is Gln3p dependent and Dal80p regulated but is not autogenously regulated like DAL80. DEH1 expression is largely Gln3p independent, modestly Gat1p dependent, and most highly regulated by Dal80p. Paradoxically, the high-level DEH1 expression observed in a dal80::hisG disruption mutant is highly sensitive to nitrogen catabolite repression.     

Integration of glucoamylase gene from Aspergillus niger into Saccharomyces cerevisiae genome and its stable expression

The proteolitic enzyme pepsin (EC 3.4.23.1) was purified from chicken stomach by a modification of the method of Bohak (1970): after homogenazing the raw material, the zymogen was extracted with NaCl and NaHCO3, activated with 3N HCl and precipitated with NaCl (28% final concentration). The precipitate was lyophilised; fractions of it were suspended in 0.02N HCl. The solution was filtered through a column (2.6 x 80 cm) of Sephadex G-100 at an elution rate of 8-10 ml x cm-2 x h-1. Two protein peaks were obtained, the first one corresponding to the pepsin (2.0 mg/ml in pooled fractions). The milk clotting activity of the enzyme was determined on skimmed milk as a substrate (Berridge, 1955). Its proteolitic activity on the artificial substrate N-acetyl-L-phenylalanyl-L-3, 5-diiodo tyrosin (APD) also was determined (Rick-Fritsch, 1974). Mean clotting activity value was 5.52 UC, higher (P < 0.01) than that of the reference chymosin (0.64 UC). The activity with APD was unsatisfactory, due to very high absorbance values of the blanks. It is concluded, that the purification steps followed in this trial are simple and rapid, conferring a strong stimulus to using chicken pepsin as a clotting agent for the industrial production of pasteurized white cheese.     

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.     

Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Delta mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae.     

Effect of aeration and unsaturated fatty acids on expression of the Saccharomyces cerevisiae alcohol acetyltransferase gene

The reduction of acetate ester synthesis by aeration and the addition of unsaturated fatty acids to the medium has been reported to be the result of the reduction in alcohol acetyltransferase (AATase) activity induced by inhibition of this enzyme. However, regulation of the AATase gene ATF1 has not been reported. In this study, ATF1 gene expression was studied by Northern analysis, and the results showed that the ATF1 gene was repressed both by aeration and by unsaturated fatty acids. The results also showed that the reduction of AATase activity is closely related to the degree of repression of ATF1 mRNA, which suggested that the gene repression is the primary means of reducing AATase activity in vivo. Using the Escherichia coli lacZ gene as a reporter gene, it was shown that a 150-bp fragment of the 5' flanking sequence played a major role in the repression by aeration and unsaturated fatty acid addition.