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1.
Eur Rev Med Pharmacol Sci ; 26(19): 7204-7211, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36263530

RESUMO

OBJECTIVE: The current post-market study aimed at analyzing women's menstrual bleeding intensity, vaginal infections, and quality of life parameters using the contraceptive vaginal ring Ornibel®. PATIENTS AND METHODS: In Germany and Spain, a multicenter study of healthy female adults (n=211) aged 18 to 45 used the vaginal ring Ornibel® for at least six months. Data collection was conducted using a patient questionnaire. The menstrual bleeding intensity was analyzed using visual analog scales (VAS). A Chi-square linear trend test assessed associations between quality-of-life parameters and continuation and recommendation of vaginal ring use. RESULTS: Three out of four women experienced six menstrual bleedings during the first six months of using the vaginal ring, with a median duration of four days during the study. The use of the vaginal ring led to a significant reduction in menstrual flow intensity (from 60 VAS points to 40 VAS points, p<0.001). In the German cohort, it was shown that dysmenorrhea and unscheduled bleeding and spotting were reduced with the use of Ornibel® as well. Most women (93.7%) agreed or strongly agreed that the vaginal ring was easy to insert, and its use was rated as comfortable or very comfortable by 97.5%. Both parameters were significantly associated with the continuation of the ring (easy to insert p=0.01, feeling comfortable: p=0.002) or its recommendation (easy to insert p=0.002, feeling comfortable: p=0.002). CONCLUSIONS: The observational data demonstrate that the contraceptive vaginal ring provides high acceptability and comfort. It is a well-accepted contraceptive method characterized by high efficacy and positive effects on cycle control.


Assuntos
Anticoncepcionais Femininos , Dispositivos Anticoncepcionais Femininos , Adulto , Feminino , Humanos , Etinilestradiol/efeitos adversos , Qualidade de Vida
2.
Hum Reprod ; 26(8): 2015-27, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632697

RESUMO

BACKGROUND: Gynaecological laparoscopic surgery outcomes can be compromised by the formation of de novo adhesions. This randomized, double-blind study was designed to assess the efficacy and safety of 4% icodextrin solution (Adept(®)) in the reduction of de novo adhesion incidence compared to lactated Ringer's solution (LRS). METHODS: Patients undergoing laparoscopic surgery for removal of myomas or endometriotic cysts were treated with randomized solution as an intra-operative irrigant and 1l post-operative instillate. De novo adhesion incidence (number of sites with adhesions), severity and extent were independently scored at a second-look procedure and the efficacy of the two solutions compared. The effect of surgical covariates on adhesion formation was also investigated. Initial exploratory analysis of individual anatomical sites of clinical importance was progressed. RESULTS Of 498 patients randomized, 330 were evaluable (160 LRS--75% myomectomy/25% endometriotic cysts; 170 Adept--79% myomectomy/21% endometriotic cysts). At study completion, 76.2% LRS and 77.6% Adept had ≥ 1 de novo adhesion. The mean (SD) number of de novo adhesions was 2.58 (2.11) for Adept and 2.58 (2.38) for LRS. The treatment effect difference was not significant (P = 0.909). Assessment of surgical covariates identified significant influences on the mean number of de novo adhesions regardless of treatment, including surgery duration (P = 0.048), blood loss in myomectomy patients (P = 0.019), length of uterine incision in myomectomy patients (P < 0.001) and number of suture knots (P < 0.001). There were 15 adverse events considered treatment-related in the LRS patients (7.2%) and 18 in the Adept group (8.3%). Of 17 reported serious adverse events (9 LRS; 8 Adept) none were considered treatment-related. CONCLUSIONS: The study confirmed the safety of Adept in laparoscopic surgery. The proportion of patients with de novo adhesion formation was considerably higher than previous literature suggested. Overall there was no evidence of a clinical effect but various surgical covariates including surgery duration, blood loss, number and size of incisions, suturing and number of knots were found to influence de novo adhesion formation. The study provides direction for future research into adhesion reduction strategies in site specific surgery.


Assuntos
Glucanos/uso terapêutico , Glucose/uso terapêutico , Procedimentos Cirúrgicos em Ginecologia/efeitos adversos , Laparoscopia/efeitos adversos , Irrigação Terapêutica/métodos , Aderências Teciduais/prevenção & controle , Adolescente , Adulto , Feminino , Humanos , Icodextrina , Mioma/cirurgia , Cirurgia de Second-Look , Gravação em Vídeo
3.
Anaesthesia ; 64(6): 632-7, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19453317

RESUMO

We investigated the influence of resident training on anaesthesia workflow of three standard procedures--laparoscopic cholecystectomy, diagnostic gynaecological laparoscopy and transurethral prostate gland resection (TURP)--comparing 259 non-emergency resident vs 341 consultant cases from 20 German hospitals. Each hospital provided 10 random cases for each procedure, yielding 600 cases for analysis. Standard time intervals as documented in the hospital information system were: 'Case Time' (the time from the start of anaesthesia induction to discharge of the patient to the recovery area) and 'Anaesthesia Control Time' (which was the Case Time minus the time from the start of surgery to the end of surgical closure). Case Time was significantly shorter for consultants in all three procedures (p < 0.05, analysis of variance) and Anaesthesia Control Time shorter for consultants only in gynaecological laparoscopy and TURP. Patient comorbidity, patient age and geographical location of the hospital were not influential factors in the analysis of variance. We conclude that resident training significantly increases duration of elective operative times.


Assuntos
Anestesiologia/educação , Educação de Pós-Graduação em Medicina/organização & administração , Corpo Clínico Hospitalar/educação , Salas Cirúrgicas/organização & administração , Colecistectomia Laparoscópica/estatística & dados numéricos , Consultores/estatística & dados numéricos , Alemanha , Pesquisa sobre Serviços de Saúde/métodos , Humanos , Período Intraoperatório/estatística & dados numéricos , Laparoscopia/estatística & dados numéricos , Masculino , Prostatectomia/estatística & dados numéricos , Estudos Retrospectivos
4.
Curr Pharm Des ; 8(13): 1155-72, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12052225

RESUMO

In large part, antimicrobial drug discovery is driven by the breadth and quality of both potential drug targets and available chemical libraries to screen. Traditionally, targets have been few in number and have been limited to those with known function, from which biochemical assays could be implemented into drug screens. Iterations of this same basic approach, applied to a few biochemically-defined targets have identified a limited set of novel antibiotics and even fewer antifungal agents. Indeed, in the last 50 years less than 30 antimicrobial targets have been exploited commercially. Within infectious disease, the industry was driven largely by chemistry-based approaches, simply making new analogs to existing drugs to overcome the growing problem of drug resistance. Elitra Pharmaceutical s approach has been to enable true functional genomics on a genome-wide scale. Elitra s vision has been to identify all of the essential genes directly in the key pathogenic organisms. Having moved rapidly towards the completion of this goal, we are now faced with the enviable challenge of prioritizing enormous target sets and developing novel sensitive screens for those best suited as definitive drug targets. These highly sensitive, cell-based screening paradigms enable re-screening of even well screened chemical libraries to reveal new chemical entities displaying novel modes of action against new targets. In parallel, we have also begun to shift the paradigm from screening targets singly, towards genome-wide approaches to drug screening.


Assuntos
Anti-Infecciosos/farmacologia , Desenho de Fármacos , Genoma Bacteriano , Genoma Fúngico , Indústria Farmacêutica/métodos , Resistência Microbiana a Medicamentos/genética , Genes Essenciais/efeitos dos fármacos , Genes Essenciais/genética , Saccharomyces cerevisiae/genética
5.
Nature ; 402(6760): 413-8, 1999 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-10586881

RESUMO

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.


Assuntos
Elementos de DNA Transponíveis , Técnicas Genéticas , Genoma Fúngico , Saccharomyces cerevisiae/genética , Algoritmos , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Fenótipo , Reação em Cadeia da Polimerase , Transformação Genética
6.
J Cell Sci ; 111 ( Pt 4): 479-94, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9443897

RESUMO

The Saccharomyces cerevisiae protein Sph1p is both structurally and functionally related to the polarity protein, Spa2p. Sph1p and Spa2p are predicted to share three 100-amino acid domains each exceeding 30% sequence identity, and the amino-terminal domain of each protein contains a direct repeat common to Homo sapiens and Caenorhabditis elegans protein sequences. sph1- and spa2-deleted cells possess defects in mating projection morphology and pseudohyphal growth. sph1(Delta) spa2(Delta) double mutants also exhibit a strong haploid invasive growth defect and an exacerbated mating projection defect relative to either sph1(Delta) or spa2(Delta) single mutants. Consistent with a role in polarized growth, Sph1p localizes to growth sites in a cell cycle-dependent manner: Sph1p concentrates as a cortical patch at the presumptive bud site in unbudded cells, at the tip of small, medium and large buds, and at the bud neck prior to cytokinesis. In pheromone-treated cells, Sph1p localizes to the tip of the mating projection. Proper localization of Sph1p to sites of active growth during budding and mating requires Spa2p. Sph1p interacts in the two-hybrid system with three mitogen-activated protein (MAP) kinase kinases (MAPKKs): Mkk1p and Mkk2p, which function in the cell wall integrity/cell polarization MAP kinase pathway, and Ste7p, which operates in the pheromone and pseudohyphal signaling response pathways. Sph1p also interacts weakly with STE11, the MAPKKK known to activate STE7. Moreover, two-hybrid interactions between SPH1 and STE7 and STE11 occur independently of STE5, a proposed scaffolding protein which interacts with several members of this MAP kinase module. We speculate that Spa2p and Sph1p may function during pseudohyphal and haploid invasive growth to help tether this MAP kinase module to sites of polarized growth. Our results indicate that Spa2p and Sph1p comprise two related proteins important for the control of cell morphogenesis in yeast.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Proteínas Fúngicas/fisiologia , Proteínas dos Microfilamentos , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sequência de Aminoácidos , Polaridade Celular , Proteínas do Citoesqueleto , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais
7.
Genetics ; 147(2): 435-50, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9335584

RESUMO

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.


Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/genética , Membrana Celular/metabolismo , Elementos de DNA Transponíveis , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Fenótipo
8.
Trends Cell Biol ; 6(11): 434-41, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15157515

RESUMO

The budding yeast Saccharomyces cerevisiae responds to intracellular and extracellular cues to direct cell growth. Genetic analysis has revealed many components that participate in this process and has provided insight into the mechanisms by which these proteins function. Several of these components, such as the septins, pheromone receptors and GTPase proteins, have homologues in multicellular eukaryotes, suggesting that many aspects of polarized cell growth may be conserved throughout evolution. This review discusses our current understanding of the molecular mechanisms of growth-site selection during the different stages of the yeast life cycle.

9.
Genes Dev ; 10(7): 777-93, 1996 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-8846915

RESUMO

Spa2p and Cdc10p both participate in bud site selection and cell morphogenesis in yeast, and spa2delta cdc10-10 cells are inviable. To identify additional components important for these processes in yeast, a colony-sectoring assay was used to isolate high-copy suppressors of the spa2delda cdc10-10 lethality. One such gene, AXL2, has been characterized in detail. axl2 cells are defective in bud site selection in haploid cells and bud in a bipolar fashion. Genetic analysis indicates that AXL2 falls into the same epistasis group as BUD3. Axl2p is predicted to be a type I transmembrane protein. Tunicamycin treatment experiments, biochemical fractionation and extraction experiments, and proteinase K protection experiments collectively indicate that Axl2p is an integral membrane glycoprotein at the plasma membrane. Indirect immunofluorescence experiments using either Axl2p tagged with three copies of a hemagglutinin epitope or high-copy AXL2 and anti-Axl2p antibodies reveal a unique localization pattern for Axl2p. The protein is present as a patch at the incipient bud site and in emerging buds, and at the bud periphery in small-budded cells. In cells containing medium-sized or large buds, Axl2p is located as a ring at the neck. Thus, Axl2p is a novel membrane protein critical for selecting proper growth sites in yeast. We suggest that Axl2p acts as an anchor in the plasma membrane that helps direct new growth components and/or polarity establishment components to the cortical axial budding site.


Assuntos
Membrana Celular/fisiologia , Polaridade Celular/genética , Proteínas Fúngicas/genética , Proteínas Ativadoras de GTPase , Genes Fúngicos , Genes Supressores , Glicoproteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sequência de Aminoácidos , Sequência de Bases , Compartimento Celular , Divisão Celular/genética , Fracionamento Celular , Membrana Celular/genética , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Deleção de Genes , Haploidia , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas , Fator de Acasalamento , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Peptídeos , Feromônios , Conformação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA
10.
Mol Gen Genet ; 249(2): 209-16, 1995 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-7500943

RESUMO

The Saccharomyces cerevisiae KRE1 gene encodes a secretory protein required for the production of the cell wall polymer (1-->6)-beta-glucan. Here we report further characterization of the KRE1 gene product, Kre1p. A functional, epitope-tagged Kre1p is shown to be highly modified in a SEC53-dependent manner. Kre1p is O-glycosylated, but the basis for the majority of its post-translational modification is unknown. Fractionation of Kre1p reveals a cell wall-associated form and a less abundant membrane-associated species. Indirect immunoflurorescence demonstrates that Kre1p localizes to the cell surface, where it becomes concentrated at the surface of mother cells. Such a localization of Kre1p seems to parallel the CAL1/CSD2-dependent cell wall deposition of chitin found in S. cerevisiae, and is consistent with evidence from Schizophyllum commune that (1-->6)-beta-glucan accumulates during maturation of the subapical region of the wall distal to the hyphal tip.


Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Glicoproteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Parede Celular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Fúngicas/análise , Proteínas Fúngicas/biossíntese , Glucanos/biossíntese , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/biossíntese , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Mapeamento por Restrição , Saccharomyces cerevisiae/metabolismo , Sitios de Sequências Rotuladas
11.
Yeast ; 10(11): 1527-30, 1994 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7871892

RESUMO

Determination of DNA sequence in the KRE6 region of the Saccharomyces cerevisiae genome completes a 10.4 kbp section on the extreme right arm of chromosome XVI. This segment contains two additional genes, GHP1 and SGV1 (Hwang et al., 1989; Irei et al., 1991) previously assigned physically to chromosome XVI, as well as a new tRNA(Gly) gene, and a novel tRNA(Ala) gene which is flanked by a sigma element.


Assuntos
Genoma Fúngico , RNA de Transferência de Alanina/genética , RNA de Transferência de Glicina/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Cromossomos Fúngicos , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/química , Análise de Sequência de DNA
12.
J Cell Biol ; 127(2): 567-79, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7929594

RESUMO

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.


Assuntos
Matriz Extracelular/metabolismo , Proteínas Fúngicas/genética , Glucanos/biossíntese , Proteínas de Membrana/genética , Proteína Quinase C/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , beta-Glucanas , Sequência de Aminoácidos , Sequência de Bases , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Complexo de Golgi/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteína Quinase 1 Ativada por Mitógeno , Dados de Sequência Molecular , Fenótipo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Homologia de Sequência de Aminoácidos
13.
Mol Cell Biol ; 13(7): 4039-48, 1993 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8321211

RESUMO

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.


Assuntos
Proteínas Fúngicas/genética , Glucanos/biossíntese , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fenótipo , Mapeamento por Restrição , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Supressão Genética
14.
Proc Natl Acad Sci U S A ; 88(24): 11295-9, 1991 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-1837148

RESUMO

The KRE6 gene product is required for synthesis of the major beta-glucans of the yeast cell wall, as mutations in this gene confer reduced levels of both the (1----6)- and (1----3)-beta-D-glucan polymers. Cloning and sequencing of KRE6 reveals a gene encoding a predicted 80-kDa protein with a central transmembrane domain and the topology of a type II membrane protein. Null mutants of KRE6 grow slowly, have larger cells, and show a reduction in alkali-insoluble wall glucans. The mutants show good viability and are not osmotically sensitive, but they are more susceptible to beta-glucanase digestion and mechanical stress than wild-type cells. The specific activity of the GTP-dependent, membrane-associated, in vitro (1----3)-beta-glucan synthase is reduced 50% in kre6 null mutants, and this reduction correlates with the mutation in meiotic tetrads. Transformants of kre6 null mutants with a KRE6 gene expressed from a centomere-based vector show a 4- to 5-fold increase in in vitro (1----3)-beta-glucan synthase activity over transformants with the vector alone. The phenotype and structure of the KRE6 product, Kre6p, suggest that Kre6p may be a beta-glucan synthase, and if so, it implies that beta-glucan synthases are functionally redundant in yeast. Alternatively, Kre6p may be part of a single multiprotein glucan synthase or modulate its activity. Use of KRE6 should permit a genetic analysis of eukaryotic (1----3)-beta-glucan synthesis.


Assuntos
Genes Fúngicos , Glucanos/biossíntese , Glucosiltransferases/genética , Proteínas de Membrana , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Alelos , Sequência de Aminoácidos , Membrana Celular/enzimologia , Deleção Cromossômica , Clonagem Molecular , Biblioteca Genômica , Glucosiltransferases/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia
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