Search PubMed⌕ Search

Biomedical subjects

S A Henry

Publications and source records attributed to S A Henry.

At least 37 records · Page 2Linked to original sources
PubMed1995

Functional characterization of an inositol-sensitive upstream activation sequence in yeast. A cis-regulatory element responsible for inositol-choline mediated regulation of phospholipid biosynthesis.

A repeated element, the inositol-sensitive upstream activation sequence (UASINO), having the consensus sequence, 5'-CATGTGAAAT-3', is present in the promoters of genes encoding enzymes of phospholipid biosynthesis that are regulated in response to the phospholipid precursors, inositol and choline. None of the naturally occurring variants of the UASINO element exactly recapitulates the consensus (for review, see Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf, F., Kolwhein, S., and Henry, S. A. (1992) in Molecular Biology of the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., and Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The first six bases of the UASINO element are homologous with canonical binding motif for proteins of the basic helix-loop-helix (bHLH) family. Two bHLH regulatory proteins, Ino2p and Ino4p from yeast, were previously shown to bind to promoter fragments containing this element. In the present study, an extensive analysis of UASINO function has been conducted. We report that any base substitution within the putative bHLH binding site resulted either in a dramatic reduction or in a complete obliteration of UASINO function as tested in an expression assay in vivo. Base substitutions in the 5' region that flanks the 10-base pair repeat, as well as sequences within the repeat itself at its 3' end outside the bHLH core, were also assessed. The two bases immediately flanking the 5' end of the element proved to be very important to its function as a UAS element as did the two bases immediately 3' of the bHLH core motif. Substitutions of the final two bases of the original ten base pair consensus (i.e. 5'-CATGTGAAAT-3') had less dramatic effects. We also tested a subset of the altered elements for their ability to serve as competitors in an assay of Ino2p x Ino4p binding. The strength of any given sequence as a UASINO element, as assayed in vivo, was strongly correlated with its strength as a competitor for Ino2p x Ino4p binding. We also tested a subset of the modified UASINO elements for their effects on expression in vivo in a strain carrying an opi1 mutation. The opi1 mutation renders the coregulated enzymes of phospholipid synthesis constitutive in the presence of phospholipid precursors. All elements that retained some residual UASINO activity when tested in the wild-type strain were constitutively expressed at a level comparable with the wild-type derepressed level when tested in the opi1 mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1995

SIN3 works through two different promoter elements to regulate INO1 gene expression in yeast.

The SIN3 global regulatory factor affects expression of many yeast genes, including the phospholipid biosynthetic gene, INO1. Mutations in the SIN3 gene result in elevated levels of INO1 expression under conditions that normally confer full repression of INO1 transcription, indicating that SIN3 is a negative regulator of INO1. In this study, the INO1 promoter was analyzed for sequences that play a role in responding to SIN3-mediated repression. Two distinct promoter elements, the upstream repression sequence (URS1) and the INO1 upstream activation sequence (UASINO) both were found to be involved in enabling SIN3 to repress INO1 expression.

Base Sequence↗
PubMed1995

Physical map locations of the phospholipid biosynthetic structural and regulatory genes of Saccharomyces cerevisiae.

Here we report the physical map locations of five genes required for phospholipid biosynthesis in Saccharomyces cerevisiae. These include four structural genes (INO1, CHO2, OPI3 and PIS1) and one global negative regulatory gene (UME6). Collectively, this information completes the mapping of all phospholipid biosynthetic structural and regulatory genes identified to date.

Chromosome Mapping↗
PubMed1995

Production and reutilization of an extracellular phosphatidylinositol catabolite, glycerophosphoinositol, by Saccharomyces cerevisiae.

Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns). We now report that S. cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability. A wild-type strain uniformly prelabeled with [3H] inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol. This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization. In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered. An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho [3H]inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2. Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporter-independent mechanism. The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent. Labeling studies employing [14C]glycerophospho[3H] inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.

Biological Transport, Active↗
PubMed1994

The Saccharomyces cerevisiae PLB1 gene encodes a protein required for lysophospholipase and phospholipase B activity.

Several enzymes with lysophospholipase/phospholipase B activity have been described from the budding yeast Saccharomyces cerevisiae. In vitro, these enzymes are capable of hydrolyzing all phospholipids that can be extracted from yeast cells. Two forms of the enzyme have been isolated from plasma membranes and a third from culture supernatants and the periplasmic space, but their biological roles have not been determined. These highly glycosylated enzymes were reported to have very similar catalytic properties but differed with respect to apparent molecular weight. We isolated a gene from S. cerevisiae, encoding a protein predicted to share 45% amino acid sequence identity with phospholipase B from Penicillium notatum. This yeast gene, designated PLB1, was mapped to the left arm of chromosome VIII. No residual lysophospholipase/phospholipase B activity was detected upon assay of extracts or culture supernatants of a plb1 delta mutant. Thus, either the PLB1 gene encodes all of the previously detected isoforms of phospholipase B or its gene product is required for their expression or activation. Deletion of PLB1 did not result in any apparent phenotypic defect, suggesting either that we failed to identify the growth conditions that would betray such a defect or that Plb1p is functionally redundant with another protein, whose activity has gone undetected. A plb1 delta mutant released wild-type levels of the soluble phosphatidylinositol metabolite glycerophosphoinositol into the growth medium but released greatly reduced levels of the corresponding phosphatidylcholine and phosphatidylethanolamine metabolites. These results indicate that PLB1 is principally responsible for the production of the deacylation products of phosphatidylcholine and phosphatidylethanolamine but not phosphatidylinositol.

Amino Acid Sequence↗
PubMed1994

INO2 and INO4 gene products, positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INO1 promoter.

The INO4 gene encodes a protein required for derepression of a number of structural genes encoding enzymes involved in phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. Ino4p shows structural similarity to the basic helix-loop-helix (bHLH) family of regulatory proteins (Hoshizaki, D. K., Hill, J. E., and Henry, S. A. (1990) J. Biol Chem. 265, 4736-4745). In this report we demonstrate that Ino4p translated in vitro forms a complex with Ino2p, another positive regulator of phospholipid biosynthesis that contains a bHLH domain. The Ino2p.Ino4p complex binds to a fragment of the INO1 promoter containing two copies of the consensus binding site for the bHLH family of proteins. The complex formed when this DNA fragment is incubated with in vitro translated Ino2p and Ino4p is identical in mobility to the complex formed when this DNA fragment is incubated with whole cell extracts. The binding of DNA by the Ino2p.Ino4p complex is competed by an oligonucleotide containing the consensus binding sequence for bHLH proteins. Neither Ino2p nor Ino4p translated alone is capable of forming a complex with the INO1 promoter fragment. The two products, translated separately and mixed, show only reduced capability to form a complex compared with cotranslated proteins. Immunoprecipitation experiments demonstrate that Ino2p and Ino4p interact in the absence of DNA. Ino2p and Ino4p are, thus, both necessary and sufficient for formation of a complex with the INO1 promoter.

Base Sequence↗
PubMed1994

Functional characterization of the INO2 gene of Saccharomyces cerevisiae. A positive regulator of phospholipid biosynthesis.

The INO2 locus encodes a novel product showing structural similarity to the basic helix-loop-helix (b-HLH) family of regulatory proteins (Nikoloff, D.M., McGraw, P., and Henry, S.A. (1992) Nucleic Acids Res. 20, 3253). The ino2 mutants exhibit pleiotropic defects in phospholipid metabolism including inability to derepress the biosynthetic enzyme inositol-1-phosphate synthase. Localization of mutations in ino2 strains has demonstrated that the b-HLH domain is required for biological activity and is sensitive to perturbation, thereby establishing a correlation between the structure and function of Ino2p. Defects in the b-HLH domain of Ino2p resulted in reduced DNA binding activity. In addition, the absence of a specific DNA-protein complex correlated with a reduction or loss of INO1 transcription. Studies using Ino2p-specific antibody revealed that Ino2p participates in the formation of specific DNA-protein complexes. Ino2p-dependent binding activity overlapped with a region of the INO1 promoter that contains two potential HLH consensus binding sites. Furthermore, Ino2p showed single base pair discrimination in a putative binding site, establishing a relationship between Ino2p and its target binding site.

Amino Acid Sequence↗
PubMed1994

A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes.

SEC14p is required for protein transport from the yeast Golgi complex. We describe a quantitative analysis of yeast bulk membrane and Golgi membrane phospholipid composition under conditions where Golgi secretory function has been uncoupled from its usual SEC14p requirement. The data demonstrate that SEC14p specifically functions to maintain a reduced phosphatidylcholine content in Golgi membranes and indicate that overproduction of SEC14p markedly reduces the apparent rate of phosphatidylcholine biosynthesis via the CDP-choline pathway in vivo. We suggest that SEC14p serves as a sensor of Golgi membrane phospholipid composition through which the activity of the CDP-choline pathway in Golgi membranes is regulated such that a phosphatidylcholine content that is compatible with the essential secretory function of these membranes is maintained.

Carrier Proteins↗
PubMed1994

A pleiotropic phospholipid biosynthetic regulatory mutation in Saccharomyces cerevisiae is allelic to sin3 (sdi1, ume4, rpd1).

Three mutants were identified in a genetic screen using an INO1-lacZ fusion to detect altered INO1 regulation in Saccharomyces cerevisiae. These strains harbor mutations that render the cell unable to fully repress expression of INO1, the structural gene for inositol-1-phosphate synthase. The Cpe-(constitutive phospholipid gene expression) phenotype associated with these mutations segregated 2:2, indicating that it was the result of a single gene mutation. The mutations were shown to be recessive and allelic. A strain carrying the tightest of the three alleles was examined in detail and was found to express the set of co-regulated phospholipid structural genes (INO1, CHO1, CHO2 and OP13) constitutively. The Cpe- mutants also exhibited a pleiotropic defect in sporulation. The mutations were mapped to the right arm of chromosome XV, close to the centromere, where it was discovered that they were allelic to the previously identified regulatory mutation sin3 (sdi1, ume4, rpd1, gam2). A sin3 null mutation failed to complement the mutation conferring the Cpe- phenotype. A mutant harboring a sin3 null allele exhibited the same altered INO1 expression pattern observed in strains carrying the Cpe- mutations isolated in this study.

Alleles↗
PubMed1994

Isolation and characterization of a mutant of Saccharomyces cerevisiae with pleiotropic deficiencies in transcriptional activation and repression.

The isolation of the dep1 mutant of Saccharomyces cerevisiae is reported. The mutant was identified by its disability to regulate expression of structural genes involved in phospholipid biosynthesis, INO1, CHO1 and OPI3, in response to supplementation with soluble lipid precursors. Expression of the INO1, CHO1 and OPI3 genes was not fully derepressed in the absence of soluble lipid precursors, inositol and choline in the dep1 mutant, as compared to wild type. The mutant also exhibited incomplete repression of these same genes in the presence of inositol and choline. Repression by phosphate of the PHO5 gene was reduced in the mutant, as was derepression of this gene in the absence of phosphate. In addition, we show that expression of INO1 and OPI3 structural genes is strongly dependent on the growth phase both in wild-type and dep1 mutant strains. However, in the mutant, elevated basal steady-state mRNA levels were reached in the late stationary growth phase, independent of supplementation conditions. The dep1 mutation represents a new complementation group with respect to phospholipid synthesis and was mapped to a position of about 12 cM distal from the centromere on the left arm of chromosome I. Deficiencies in transcription activation and repression of metabolically unrelated genes, as well as reduced mating efficiency and lack of sporulation of homozygous diploid dep1/dep1 mutants indicate a pleiotropic regulatory function of the DEP1 gene product. Thus, Dep1p appears to be a new member of a class of transcriptional modulators, including Rpd1p/Sin3p/Ume4p/Sdi1p/Gam3p, Rpd3p, Spt10p and Spt21p.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
PubMed1993

Molecular cloning of the yeast OPI3 gene as a high copy number suppressor of the cho2 mutation.

By functional complementation of the auxotrophic requirements for choline of a cdg1, cho2 double-mutant, by transformation with a genomic DNA library in a high copy number plasmid, two different types of complementing DNA inserts were identified. One type of insert was earlier shown to represent the CHO2 structural gene. In this report we describe the molecular and biochemical chemical characterization of the second type of complementing activity. The transcript encoded by the cloned gene was about 1000-nt in length and was regulated in response to the soluble phospholipid precursors, inositol and choline. A gene disruption resulted in no obvious growth phenotype at 23 degrees C or 30 degrees C, but in a lack of growth at 37 degrees C in the presence of monomethylethanolamine. Null-mutants exhibited an inositol-secretion phenotype, indicative of mutations in the lipid biosynthetic pathway. Complementation analysis, biochemical analysis of the phospholipid methylation pathway in vivo, and comparison of the restriction pattern of the cloned gene to published sequences, unequivocally identified the cloned gene as the OPI3 gene, encoding phospholipid-N-methyltransferase in yeast. When present in multiple copies the OPI3 gene efficiently suppresses the phospholipid methylation defect of a cho2 mutation. As a result of impaired synthesis of phosphatidylcholine, the INO1-deregulation phenotype is abolished in cho2 mutants transformed with the OPI3 gene on a high copy number plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗
PubMed1992

Cis and trans regulatory elements required for regulation of the CHO1 gene of Saccharomyces cerevisiae.

A 34 base-pair (bp) fragment spanning sequences -154 to -120 of the promoter of the CHO1 gene (structural gene for phosphatidylserine synthase) from the yeast Saccharomyces cerevisiae has been shown to place transcription of a promoter-less Escherichia coli lacZ gene under control of the phospholipid precursors inositol and choline. Furthermore, in deletion experiments the CHO1 UASINO was localized to sequences between -151 and -123 of the CHO1 promoter. A nine bp sequence was identified in the promoter region of the CHO1 gene that shares an eight out of nine bp match with a sequence (consensus 5' ATGTGAAAT 3') that is repeated a total of 23 times upstream from several coregulated phospholipid biosynthetic genes. This sequence is contained within the -151 to -123 region to which the CHO1 UAS has been localized. The nine bp repeated element is believed to be involved in the control of phospholipid biosynthetic gene transcription in response to changing levels of inositol and choline in the growth medium. This control has been shown to require activities encoded by the products of the three regulatory genes: INO2, INO4, and OPI1. A mutation in any of these regulatory genes results in aberrant CHO1-lacZ gene regulation, and affects regulation of the construct containing the 34 bp (-154 to -120) CHO1 fragment demonstrating that the regulatory signal generated by these genes interacts with the 5' end of the CHO1 gene.

Amino Acid Sequence↗
PubMed1991

Regulation of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase by phospholipid precursors in Saccharomyces cerevisiae.

Phosphatidylethanolamine methyltransferase (PEMT) and phospholipid methyltransferase (PLMT), which are encoded by the CHO2 and OPI3 genes, respectively, catalyze the three-step methylation of phosphatidylethanolamine to phosphatidylcholine in Saccharomyces cerevisiae. Regulation of PEMT and PLMT as well as CHO2 mRNA and OPI3 mRNA abundance was examined in S. cerevisiae cells supplemented with phospholipid precursors. The addition of choline to inositol-containing growth medium repressed the levels of CHO2 mRNA and OPI3 mRNA abundance in wild-type cells. The major effect on the levels of the CHO2 mRNA and OPI3 mRNA occurred in response to inositol. Regulation was also examined in cho2 and opi3 mutants, which are defective in PEMT and PLMT activities, respectively. These mutants can synthesize phosphatidylcholine when they are supplemented with choline by the CDP-choline-based pathway but they are not auxotrophic for choline. CHO2 mRNA and OPI3 mRNA were regulated by inositol plus choline in opi3 and cho2 mutants, respectively. However, there was no regulation in response to inositol when the mutants were not supplemented with choline. This analysis showed that the regulation of CHO2 mRNA and OPI3 mRNA abundance by inositol required phosphatidylcholine synthesis by the CDP-choline-based pathway. The regulation of CHO2 mRNA and OPI3 mRNA abundance generally correlated with the activities of PEMT and PLMT, respectively. CDP-diacylglycerol synthase and phosphatidylserine synthase, which are regulated by inositol in wild-type cells, were examined in the cho2 and opi3 mutants. Phosphatidylcholine synthesis was not required for the regulation of CDP-diacylglycerol synthase and phosphatidylserine synthase by inositol.

Cloning, Molecular↗
PubMed1991

Interaction of trans and cis regulatory elements in the INO1 promoter of Saccharomyces cerevisiae.

Electrophoretic mobility shift assays (EMSA) were used to define the regions of the INO1 promoter that interact with factors present in extracts prepared from the yeast, Saccharomyces cerevisae. These experiments identified three different types of protein:DNA complexes that assemble with the INO1 promoter. Formation of one type of complex depended on functional alleles of previously described regulatory genes, INO2 and INO4, that encode positive regulatory factors. Formation of the INO2/INO4-dependent complexes was increased when extracts prepared from cells grown under derepressing conditions (i.e. absence of inositol and choline). A second type of complex was dependent on the OPI1 gene, that encodes a negative regulator. Computer-aided sequence analysis of the promoters of several genes encoding phospholipid biosynthetic enzymes revealed a highly conserved nine basepair element (5'-ATGTGAAAT-3'), designated 'nonamer' motif, that is similar to the octamer motif identified in the promoters of mammalian immunoglobulin genes. The nonamer motif was shown to form a specific complex with a factor, designated nonamer binding factor (NBF). Extracts prepared from Schizosaccharomyces pombe formed a nonamer-specific complex.

Base Sequence↗
PubMed1991

Analysis of sequences in the INO1 promoter that are involved in its regulation by phospholipid precursors.

The promoter region of the highly regulated INO1 structural gene of yeast has been investigated. The major transcription initiation start site (+1) was mapped to a position located five nucleotides upstream of the previously identified initiation codon. The INO1 TATA is located at -116 to -111. The INO1 promoter region was used to construct fusions to the Escherichia coli lacZ gene. All INO1 fusion constructs that retained regulation in response to the phospholipid precursors inositol and choline, contained at least one copy of a nine bp repeated element (consensus, 5'-ATGTG-AAAT-3'). The smallest fragment of the INO1 promoter found to activate and regulate transcription of the fusion gene from a heterologous TATA element was 40 nucleotides in length. This fragment contained one copy of the nine bp repeat and spanned the INO1 promoter region from -259 to -219. However, when an oligonucleotide containing the nine bp repeated sequence was inserted 5' to the CYC1 TATA element, it failed to activate transcription.

Base Sequence↗