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Biomedical subjects

J R Shuster

Publications and source records attributed to J R Shuster.

At least 19 recordsLinked to original sources
PubMed2001

Gene discovery and gene function assignment in filamentous fungi.

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Alleles↗
PubMed2000

Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains.

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH(2)-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong alpha-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 microM at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

Amino Acid Sequence↗
PubMed1999

pH regulation of recombinant glucoamylase production in Fusarium venenatum JeRS 325, a transformant with a Fusarium oxysporum alkaline (trypsin-like) protease promoter.

Fusarium venenatum (formerly Fusarium graminearum) JeRS 325 produces heterologous glucoamylase (GAM) under the regulation of a Fusarium oxysporum alkaline (trypsin-like) protease promoter. The glucoamylase gene was used as a reporter gene to study the effects of ammonium and pH on GAM production under the control of the alkaline protease promoter. Between pH 4.0 and 5.8, GAM production in glucose-limited chemostat cultures of JeRS 325 grown at a dilution rate of 0.10 h-1 (doubling time, 6.9 h) on (NH4)2SO4 medium increased in a linear manner with increase in pH. However, at pH 4.0 and below GAM production was almost completely repressed in glucose-limited chemostat cultures grown on (NH4)2SO4 or NaNO3 medium. Thus GAM production in JeRS 325 is regulated by culture pH, not by the nature of the nitrogen source in the medium. The difficulty of using unbuffered medium when investigating putative ammonium repression is also shown. The study demonstrates the potential for use of the alkaline protease promoter in F. graminearum for the production of recombinant proteins in a pH dependent man ner.

Cells, Cultured↗
PubMed1999

Promoter-tagged restriction enzyme-mediated insertion (PT-REMI) mutagenesis in Aspergillus niger.

Promoter-tagged restriction enzyme-mediated insertion (PT-REMI) mutagenesis was performed in the fungus Aspergillus niger, using a plasmid containing a strong transcriptional promoter. Two DNA-tagged mutants were analyzed in detail. A white-spored mutant was shown to contain a plasmid insertion that disrupted a gene that shows a high degree of homology to the polyketide synthase gene wA of A. nidulans. A morphological mutant was shown to contain a plasmid insertion in the 5' upstream region of a gene that strongly resembles COX5, which encodes the cytochrome c oxidase subunit V. Insertion of the plasmid resulted in enhanced expression of the COX5 RNA, demonstrating that the combination of REMI with a promoter-containing insert can be used to activate gene transcription.

Amino Acid Sequence↗
PubMed1998

Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile.

A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.

Amino Acid Sequence↗
PubMed1994

Genetic stability of protein expression systems in yeast.

For the expression of recombinant proteins in yeast, genetic stability is monitored using a combination of standard microbiology and nucleic acid testing procedures. Process consistency during the cell amplification and product expression phases of fermentation are also reliable indicators of stability. The potential for instability arising from point mutation, gene conversion or recombination has been shown to occur at a low frequency and does not generally affect protein product quality.

Biotechnology↗
PubMed1993

Fructose-1,6-bisphosphatase of the yeast Kluyveromyces lactis.

The fructose-1,6-bisphosphatase [Fru(1,6)P2ase] gene of the budding yeast, Kluyveromyces lactis, was cloned and sequenced. The gene encodes one open reading frame predicting a 354-amino-acid polypeptide. The polypeptide is different from other Fru(1,6)P2ases in that it contains a short amino-acid-insert region close to a basic residue located at the binding site for the allosteric inhibitor AMP. Comparison of the biochemical properties of the K. lactis enzyme with its closest homolog, the Saccharomyces cerevisiae Fru(1,6)P2ase (74% amino acid identity), reveals that the K. lactis enzyme is significantly less sensitive to AMP (Ki = 540 microM) than the S. cerevisiae enzyme (Ki = 190 microM). However, studies with a K. lactis Fru(1,6)P2ase mutant, in which the insert region (amino acids 152-160) was deleted by site-directed mutagenesis [(des-152-160)Fru(1,6)P2ase], showed that the mutant enzyme had higher sensitivity to AMP inhibition (Ki = 280 microM) than the control K. lactis enzyme. Thus, the nine-amino-acid insert region appears to be responsible for the decreased AMP inhibition shown by the K. lactis wild-type enzyme. Catabolite-repression and catabolite-inactivation studies show that, unlike the complete repression of FBP1 mRNA and inactivation of enzyme activity by glucose seen in S. cerevisiae, mRNA levels and enzyme activity of K. lactis Fru(1,6)P2ase decreased only about 2-4-fold due to the presence of glucose in the cell-culture medium.

Amino Acid Sequence↗
PubMed1993

T-lymphocyte response to hepatitis C virus in different clinical courses of infection.

BACKGROUND: To assess the role played by the immune response in the outcome of hepatitis C virus infection, the CD4+ T-lymphocyte response to viral antigens was studied in infected individuals with different clinical courses. METHODS: Using six recombinant proteins of hepatitis C virus, the study assessed the proliferative responses of peripheral blood mononuclear cells from 41 patients with chronic hepatitis C, 11 patients whose chronic hepatitis was successfully treated with interferon alfa and 11 healthy HCV seropositive individuals. RESULTS: (1) Sixty-five percent of hepatitis C virus-seropositive individuals had CD4+ T-cell responses to viral proteins. (2) All viral proteins were immunogenic for T cells, although NS4 was the most immunogenic. (3) There was a significant correlation between the presence of CD4+ T cell responses to Core and a benign course of infection in healthy seropositives, most of whom were viremic. CONCLUSIONS: CD4+ T-cell responses to Core, although they do not coincide with virus clearance, are associated with a benign course of infection and may be required to maintain humoral and cellular responses protective against the disease.

Adolescent↗
PubMed1992

Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease.

Structural and nonstructural regions of the HCV-encoded polyprotein have been expressed in recombinant yeast, bacteria, or insect cells and used to capture and measure reactive antibodies circulating in different individuals. The putative nucleocapsid protein (C) and nonstructural proteins 3-5 (NS3-NS5) were found to contain the most immunodominant epitopes. The NS3, NS4, and C regions were expressed in yeast in the form of a fused, chimeric polyprotein (C25) and a capture assay for reactive antibody was developed. This anti-C25 assay detects all previously identified HCV-seropositive cases and provides a substantially more sensitive diagnostic for both acute and chronic HCV infections than the current anti-C100-3 (NS4) assay. Anti-C25 was detected more frequently than anti-C100-3 in chronic, transfusion-associated non-A, non-B hepatitis patients from the United States (95% vs. 71%) and Japan (98% vs. 82%), in cryptogenic cirrhosis patients from the United States (62% vs. 28%), and in hepatitis B surface antigen-negative cases of hepatocellular carcinoma from Japan (83% vs. 63%). These data indicate that HCV has a greater role in these liver diseases than was previously thought. In volunteer United States blood donors sampled following the introduction of anti-C100-3 screening, the prevalence of anti-C25 and anti-C100-3 was 0.5% and 0.08%, respectively.

Antigens, Viral↗
PubMed1991

Gene expression in yeast: protein secretion.

Maximizing efficiency for the secretion of proteins from yeast requires an understanding of the rate limiting stages in secretion that can result from high levels of gene expression. Recent progress in this area has produced a number of improvements in yeast expression systems for protein secretion.

Animals↗
PubMed1991

Functional and immunological characterization of SIV envelope glycoprotein produced in genetically engineered mammalian cells.

Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immune responses in infected hosts. Macaque simian immunodeficiency virus (SIVmac) is a T-lymphocytopathic lentivirus which causes an AIDS-like disease in rhesus macaques. The envelope gene of SIVmac encodes a precursor glycoprotein (gp160) which is cleaved into an external domain (gp130) and a transmembrane domain (gp32). To investigate the functional and immunological properties of the SIV external envelope glycoprotein, we have used genetically engineered mammalian cells to produce recombinant gp130 (rgp130). The rgp130 has the appropriate molecular weight, is glycosylated, and has native conformation as determined by binding to the cell receptor for SIV, the CD4 antigen. Rhesus macaques immunized with purified rgp130 formulated in muramyl dipeptide adjuvant generated high titers of antienvelope antibodies. Antibodies from these macaques were tested for in vitro virus neutralization; very low or undetectable levels of neutralization were observed. In contrast, neutralizing antibodies were readily detected in sera from goats immunized with rgp130. With respect to cell-mediated immunity, proliferative responses to rgp130 were demonstrated in peripheral blood monocyte cells (PBMC) from macaques immunized with the recombinant glycoprotein as well as in PBMC from SIV-infected animals. These results show that rgp130 is functional and immunogenic; the potential of rgp130 for protective immunization remains to be determined.

Animals↗
PubMed1990

Priming of CD4+ T cells specific for conserved regions of human immunodeficiency virus glycoprotein gp120 in humans immunized with a recombinant envelope protein.

A nonglycosylated denatured form of human immunodeficiency virus (HIV) 1 glycoprotein gp120 (Env 2-3), which does not bind to CD4, was used with muramyl tripeptide as adjuvant to immunize HIV-seronegative healthy volunteers. In all the volunteers, three 50-micrograms injections of Env 2-3 induced priming of CD4+ T cells specific for conserved regions of the native glycosylated gp120. Moreover, we found that several major histocompatibility complex class II (DR) alleles can function as restriction molecules for presentation of conserved epitopes of gp120 to T cells, implying that a T-cell response to these epitopes can be obtained in a large fraction of the population. The possibility to prime CD4+ T cells specific for conserved epitopes of a HIV protein is particularly important in view of the lack of such cells in HIV-infected individuals and of a possible role that CD4+ T cells may play in the development of protective immunity against AIDS.

Alleles↗
PubMed1990

Importance of hypervariable regions of HIV-1 gp120 in the generation of virus neutralizing antibodies.

Variants of the envelope gene of the HIV-SF2 isolate of HIV-1 with deletions of one or more of the hypervariable domains of gp120 were produced in genetically engineered yeast as nonglycosylated denatured polypeptide analogs of gp120. Purified antigens were used to immunize experimental animals to determine whether the removal of hypervariable regions from this type of gp120 immunogen had any effect on (1) the ability of the antigen to elicit virus neutralizing antibodies; and (2) the isolate specificity of the neutralizing antibodies that were elicited. The results of these studies demonstrate that, in addition to the previously identified V3 domain, at least two other hypervariable regions in gp120 are capable of eliciting neutralizing antibodies in experimental animals. However, when all five of the hypervariable regions were deleted, the resulting antigen was no longer capable of eliciting neutralizing antibodies. Finally, the neutralizing antibodies elicited by all of these nonglycosylated antigens were effective against HIV-SF2, the isolate from which the antigens were derived, but were not able to neutralize two divergent isolates, HIV-BRU or HIV-Zr6.

Animals↗
PubMed1989

Yeast mutants conferring resistance to toxic effects of cloned human insulin-like growth factor I.

The production of extracellular human insulin-like growth factor I (IGF-I) in yeast is deleterious to the growth of the host organism. Mutants resistant to the toxic effects of IGF-I production were isolated. A subset of these mutants produced levels of IGF-I greater than the parent strain and were due to chromosomal recessive mutations at a single locus, hpx1. The overproduction of IGF-I was independent of the original promoter and vector expression system. The mutant strains also displayed enhanced extracellular production of other heterologous proteins.

Carboxypeptidases↗
PubMed1989

Cyclic AMP-dependent protein kinase phosphorylates and inactivates the yeast transcriptional activator ADR1.

It has been proposed in several eukaryotic systems that the regulation of gene transcription involves phosphorylation of specific transcription factors. We report here that the yeast transcriptional activator ADR1 is phosphorylated in vitro by cyclic AMP-dependent protein kinase and that mutations which enhance the ability of ADR1 to activate ADH2 expression decrease ADR1 phosphorylation. We also show that increased kinase activity in vivo inhibits ADH2 expression in an ADR1 allele-specific manner. Our data suggest that glucose repression of ADH2 is in part mediated through a cAMP-dependent phosphorylation-inactivation of the ADR1 regulatory protein.

Aldehyde Dehydrogenase↗