Biomedical subjects

V L Seligy

Publications and source records attributed to V L Seligy.

At least 37 records · Page 2Linked to original sources

PubMed1988

A temporally expressed gene from Schwanniomyces alluvius and detection of homologous sequences in other yeasts.

The boundaries of a temporally expressed gene (EG1) located on a 6.5-kb HindIII chromosomal DNA fragment of the yeast Schwanniomyces alluvius have been defined by cDNA hybridization and S1 nuclease mapping. The sequence of this gene reveals a 474-bp open reading frame (158 aa) and a TATA-like sequence 100 bp upstream from the first start codon. The encoded protein bears little resemblance with other known yeast structural proteins, including histones. A portion of the 3' untranslated region shows 50% identity with a consensus repetitive sequence (B2) found in the mouse genome. Repetitive sequences are also located in both 5' and 3' untranslated regions. Genomic DNA analysis indicated that related sequences were detected at two loci in Schw. alluvius and one locus in Saccharomyces cerevisiae. In contrast to the continuous transcription pattern of the ADE1 genes of Schw. alluvius and S. cerevisiae, the 0.8-kb transcripts of EG1 in these yeasts were most abundant in cells in early-log phase of growth and absent in cells of late-log phase, suggesting discontinuous synthesis.

Amino Acid Sequence↗

PubMed1988

Isolation and expression of cDNA clones coding for two sequence variants of Xenopus laevis histone H5.

We have cloned and characterized cDNAs coding for two variants of Xenopus laevis H5 histone protein (previously called H1s). cDNA was synthesized from RNA of immature erythrocytes in a single reaction using a modification of the method of Gubler and Hoffman [Gene 25 (1983) 263-269], and blunt-end ligated into the HincII site of the phage vector M13mp9. Immunological screening with a polyclonal antibody yielded two clones expressing H5 peptide. Sequence characterization revealed that both clones contained partial cDNA inserts and that the smaller 340-bp clone initiated reverse transcription within the coding region, at a site rich in adenine. Rescreening of the cDNA bank by nucleic acid hybridization produced eleven additional H5 clones, one of which coded for a second variant of H5. These two variants, called XLH5A and XLH5B, are very similar in sequence and code for proteins of 195 and 193 amino acids, respectively, which may be the H1D and H1E variants observed previously. XLH5, avian H5 and human H1O share identity at both nucleotide and amino-acid sequence levels. Further, the XLH5-coding mRNA is likely polyadenylated and lacks the highly conserved, 23-nucleotide dyad symmetry element found within the 3' untranslated regions of most histone-coding mRNAs.

Amino Acid Sequence↗

PubMed1988

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors.

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.

Autoradiography↗

PubMed1988

Molecular Cloning and Expression of a Xylanase Gene from Bacillus polymyxa in Escherichia coli.

Genomic fragments of Bacillus polymyxa derived from separate and complete digestion by EcoRI, HindIII, and BamHI were ligated into the corresponding sites of pBR322, and the resulting chimeric plasmids were transformed into Escherichia coli. Of 6,000 transformants screened, 1 (pBPX-277) produced a clear halo on Remazol brilliant blue xylan plates. The insert in the pBPX-277 recombinant, identified as an 8.0-kilobase BamHI fragment of B. polymyxa, was subsequently subjected to extensive mapping and a series of subclonings into pUC19. A 2.9-kilobase BamHI-EcoRI subfragment was found to code for xylanase activity. Xylanase activity expressed by E. coli harboring the cloned gene was located primarily in the periplasm and corresponded to one of two distinct xylanases produced by B. polymyxa. Xylanase expression by the cloned gene occurred in the absence of xylan and was reduced by glucose and xylose. Southern blot hybridization with the cloned fragment as a probe against complete genomic digests of the bacilli B. polymyxa, B. circulans, and B. subtilis revealed that the cloned xylanase gene was unique to B. polymyxa. The xylanase expressed by the cloned gene had a molecular weight of approximately 48,000 and an isoelectric point of 4.9.

Journal Article↗

PubMed1988

Bacillus subtilis beta-1,4-endoglucanase products from intact and truncated genes are secreted into the extracellular medium by Escherichia coli.

We compared the secretion of a Bacillus subtilis endo-beta-1,4-glucanase (EC 3.2.1.4) in B. subtilis and of the product from the cloned gene (pC6.3) expressed in Escherichia coli. The cloned enzyme has been isolated previously as the 52.2-kilodalton (kDa) species predicted from the gene sequence (R. M. MacKay, A. Lo, G. Willick, M. Zuker, S. Baird, M. Dove, F. Moranelli, and V. Seligy, Nucleic Acids Res., 14:9159-9170, 1986); this 52.2-kDa species is then converted to an active 35.8-kDa species. The 35.8-kDa species has a segment removed from the COOH terminus. Endoglucanase products were identified by use of an antibody directed to the 35.8-kDa enzyme. Time course studies of the secretion in B. subtilis showed that the enzyme was first secreted as a 52.2-kDa proenzyme. This was cleaved progressively to a product of about 32 kDa. Time course analysis of the expression of the cloned product from pC6.3 in E. coli showed that about 70% of the endoglucanase activity was found extracellularly. Analysis of active products from three deletion clones showed that the expression pattern of the endoglucanase was not affected by removal of the transcription termination signal and that neither expression nor secretion was substantially altered by removal of a region coding for up to 163 residues of the carboxyl terminus.

Bacillus subtilis↗

PubMed1987

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin.

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.

Amino Acid Sequence↗

PubMed1986

Complete nucleotide sequence and mRNA-mapping of the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas moewusii.

Nucleotide (nt) sequence of the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase from the green alga, Chlamydomonas moewusii, and mapping of transcription ends was achieved by two new strategies. The deduced LS sequence of 475 amino acid residues was compared with similar genes from six other species; cyanobacteria, land plants and a related alga (C. reinhardtii). The most conserved regions are the three ribulose bisphosphate binding sites and the CO2 activator site. The nt sequence conservation outside the coding region is limited to only three segments within the 5'-flanking region: a region of tandem repeats, TATAA box and ribosome-binding site. Termination point of transcription is an 'A' residue 3' to the first of two 18-nt inverted repeats, which has the potential to form a stem-loop hairpin structure. The possible role of these potential regulatory features for transcription and translation, and similar structures in other LS genes is presented.

Amino Acid Sequence↗

PubMed1986

A clone coding for Schizophyllum commune beta-glucosidase: homology with a yeast beta-glucosidase.

Three identical clones coding for a partial sequence of the Schizophyllum commune beta-glucosidase were isolated from a cDNA library in lambda gt11, using polyclonal antibody to the enzyme. The identity was confirmed by comparison of the amino-terminus of a peptide from a protease lys-C digest with the sequence inferred from the cDNA sequence. A comparison of the sequence with that reported for a beta-glucosidase from Candida pelliculosa revealed a region in the latter with 43% identity in amino acid sequence. There was also a similarity in the S. commune beta-glucosidase to an active site sequence proposed for a S. commune endoglucanase, suggesting the possibility of a common catalytic mechanism for these two glucolytic enzymes.

Amino Acid Sequence↗

PubMed1985

Oncogene related sequences in fungi: linkage of some to actin.

We used v-rasKi, v-bas, Ras-l, v-mos and v-abl DNA fragments as probes to detect homologous sequences in genomic DNA from a variety of fungi. Cellular homologs were identified in most of them and the number of related fragments detected varied with the probe used. In addition, we found that some onc gene homologs were linked to actin-related sequences.

Actins↗

PubMed1985

Multiplicity in cellulases of Schizophyllum commune. Derivation partly from heterogeneity in transcription and glycosylation.

The white-rot fungus, Schizophyllum commune, secretes a member of each of three classes of cellulases: a beta-glucosidase, an exoglucanase, and an endoglucanase. Antibodies were developed to members of each of these three enzyme classes. Secretion of these cellulases is induced when a mycelium is transferred from a glucose to cellulose medium. The maximum level of cellulase transcripts, as indicated by the ability to direct biosynthesis of these cellulases in the rabbit reticulocyte cell-free translation system, occurred when the rate of secretion was maximum. This implied that initial regulation, at least, of cellulase biosynthesis occurs at the transcriptional level. There were two distinct mRNA-directed products for each of the cellulases, with sizes estimated to be, for the beta-glucosidase, 95700 and 93800, for the exoglucanase, 59300 and 58200, and for the carboxymethylcellulase, 40600 and 39400. The secreted cellulases are largely glycosylated, as indicated by their binding to concanavalin A and their incorporation of D-[3H]mannose. The labelled protein was fractionated on concanavalin-A-agarose; about 70% of the label was bound. A small amount of each of the cellulases appeared in the unbound fraction; the remainder appeared in fractions eluted with 10 mM methyl glucoside or with 100 mM methyl glucoside plus 500 mM methyl mannoside. These results indicated each of the cellulases had an additional heterogeneity in glycosylation, with the most heavily glycosylated and highest molecular weight form eluting last from the concanavalin-A-agarose. Although tunicamycin (5 micrograms/ml) blocked glycosylation, there was still some secretion but at a reduced rate which was more pronounced for the beta-glucosidase than for the carboxymethylcellulase activity. The size of the tunicamycin-secreted product in each case was, within experimental error, equivalent to that of the mRNA-directed one.

Agaricales↗

PubMed1985

Glucanase gene diversity in prokaryotic and eukaryotic organisms.

A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.

Amino Acid Sequence↗

PubMed1984

Uncoupled synthesis of H1o-like histone H1s during late erythropoiesis in Xenopus laevis.

This study investigated the synthesis of Xenopus histones during erythropoiesis. Although cessation of DNA replication in the mid-stages of erythroid maturation is accompanied by arrested synthesis of histone H1 and core histones, synthesis of H1o (an H1o-like histone) was found to continue into late stages of erythropoiesis, as has been reported for avian erythrocyte histone H5. This was accompanied by a threefold increase in the relative amount of Xenopus H1s, similar to the accumulation reported for H5 during avian erythropoiesis and for H1o in some differentiated mammalian cells. The structural and metabolic homologies of avian H5, mammalian H1o, and Xenopus H1s imply that these lysine-rich histones have closely related functions distinct from those of H1, and thus represent a subclass of lysine-rich histones.

Anemia↗

PubMed1984

Molecular cloning of the ADE1 gene of Saccharomyces cerevisiae and stability of the transformants.

Plasmid YEp ( ADE1 )1a, containing a 2.7-kb Sau3A fragment of Saccharomyces cerevisiae DNA inserted at the BamHI site of the yeast shuttle vector pBTI -1 (Morris et al., 1981), results in high frequency, unstable transformation of ade 1 yeast strains. A second plasmid, YRp ( ADE1 )2, containing adjacent 0.5-kb and 3.0-kb BamHI fragments in pBR322 gave three types of yeast transformants: (1) transformants carrying extrachromosomal copies of the plasmid which indicate the presence of a functional ars sequence, (2) transformants indistinguishable from ade 1 strains by hybridization analysis, and (3) a transformant carrying a multimeric form of YRp ( ADE1 )2. Cells transformed with either of the plasmids are free of the red pigment characteristic of ade 1 mutants and indicate potential for direct colour-based selection of yeast transformants using ADE1 plasmids.

Cloning, Molecular↗

PubMed1984

Applications of recombinant DNA technology to the pulp and paper industry.

New gene selection techniques (Recombinant DNA) are currently available to exploit useful properties of various biological systems hitherto regarded as interesting but of little or no immediate commercial value. The application of genetic engineering techniques to problems in the Pulp and Paper Industry are many. As a first step these techniques are being used to provide much needed fundamental information on the cellular and molecular mechanisms involved in the expression of extra-cellular enzymes that degrade lignocellulosic pulping wastes. The information gleaned from the studies on cellulolytic fungi and bacteria can be used to genetically engineer a yeast or bacterium capable of converting pulping wastes into ethanol and other useful by-products.

Journal Article↗

PubMed1984

Chloroplast DNA recombination in interspecific hybrids of Chlamydomonas: Linkage between a nonmendelian locus for streptomycin resistance and restriction fragments coding for 16S rRNA.

Differences in the distribution of Ava I and BstEII restriction sites in the chloroplast DNA (cpDNA) of Chlamydomonas eugametos and C. moewusii have been used to detect extensive cpDNA recombination in the hybrid progeny of these interfertile algae. In the present study, the inheritance of these restriction-site differences was tested for recombination with nonmendelian genetic markers for resistance to streptomycin and erythromycin in interspecific crosses and in hybrid backcrosses to C. moewusii. Most of the restriction-pattern markers appear linked to the antibiotic-resistance markers, thus supporting the chloroplast localization of the resistance markers. The streptomycin marker, in particular, shows perfect coordinate inheritance with an Ava I band containing one cpDNA fragment and a BstEII band containing two comigrating cpDNA fragments. Molecular hybridization experiments using DNA from the Ava I band as a probe show sequence homology between this DNA, the two comigrating BstEII fragments, and cpDNA fragments from C. reinhardtii containing the genes for 16S rRNA. The results show the feasibility of using C. eugametos-C. moewusii hybrids to identify cpDNA sequences that either contain or are closely linked to nonmendelian genetic markers.

Journal Article↗

PubMed1984

Extracellular proteins secreted by the basidiomycete Schizophyllum commune in response to carbon source.

The secretion of 1,4-beta-D-glucanases by the basidiomycete Schizophyllum commune in response to cellulose or cellobiose has been studied. The proteins were labeled with 35S, and the secretion of enzymes was measured by beta-glucosidase and carboxymethyl cellulase activities and by immunoprecipitation with specific antibodies. The antigen proteins used were a beta-glucosidase (Mr, 93,000), an avicelase (avicelase II; Mr, 64,000), and a carboxymethyl cellulose (carboxymethyl cellulase I; Mr 41,000). The beta-glucosidase was initially secreted as an Mr 110,000 form, which was followed later by lower-molecular-weight (88,000 to 93,000) forms. The avicelase II, which accounted for about 50% of the secreted labeled protein, had an Mr of 64,000. Secretion of the related avicelase I (Mr 61,000) followed later. The carboxymethyl cellulose I was secreted in two molecular weight forms, Mr 44,000 and 41,000. The evidence is consistent with the idea that three genes account for the secreted glucanase activities. Other species result from different glycosylation or proteolytic cleavage processing, which may occur during or after secretion. The beta-glucosidase secretion appears to be regulated differently than that of avicelase II or carboxymethyl cellulase I; the latter two were regulated coordinately under the conditions used in this work. No common immune determinants between the three antigens were observed.

Agaricales↗

PubMed1984

The induction of alpha-amylase by starch in Aspergillus oryzae: evidence for controlled mRNA expression.

The induction of alpha-amylase by starch has been studied in the filamentous fungus Aspergillus oryzae. Low levels of alpha-amylase activity were found in both intracellular and extracellular samples from glucose-grown cultures. However, alpha-amylase activity increased when starch was the sole carbon source. The intracellular enzyme activity was induced by a factor of approximately 6.5, while the extracellular activity increased 20-fold over that found in the glucose-grown cultures. Regardless of the carbon source or cellular location, the molecular weight of the active protein was 52 500 +/- 1800 and only this protein reacted with antibodies specific for alpha-amylase. A parallel study of the in vitro translated proteins directed by poly(A)+ RNA fractions indicated a presumptive alpha-amylase with a similar isoelectric point but with a molecular weight of approximately 54 000. This protein was most prevalent using RNA from early, exponentially growing cultures in starch medium. Immunoprecipitation data indicate that the abundance of alpha-amylase transcripts decreases dramatically after the first 12 h, reflecting an initial transcription control for the expression of this enzyme.

Animals↗

PubMed1982

Possible mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae.

A model is proposed for the mechanism of flocculation interactions in yeasts in which flocculent cells have a recognition factor which attaches to alpha-mannan sites on other cells. This factor may be governed by the expression of the single, dominant gene FLO1. Isogenic strains of Saccharomyces cerevisiae, differing only at FLO1 and the marker genes ade1 and trp1, were developed to examine the components involved in flocculene. Electron microscopy and concanavalin Aferritin labeling of aggregated cells showed that extensive and intense interactions between cell wall mannan layers mediated cell aggregation. The components of the mannan layer essential for flocculence were Ca2+ ions, alpha-mannan carbohydrates, and proteins. By studying the divalent cation dependence at various pH values and in the presence of competing monovalent cations, flocculation was found to be Ca2+ dependent; however, Mg2+ and Mn2+ ions substituted for Ca2+ under certain conditions. Reversible inhibition of flocculation by concanavalin A and succinylated concanavalin A implicated alpha-branched mannan carbohydrates as one essential component which alone did not determine the strain specificity of flocculence, since nonflocculent strains interacted with and competed for binding sites on flocculent cells. FLO1 may govern the expression of a proteinaceous, lectin-like activity, firmly associated with the cell walls of flocculent cells, which bind to the alpha-mannan carbohydrates of adjoining cells. It was selectively and irreversibly inhibited by proteolysis and reduction of disulfide bonds. The potential of this system as a model for the genetic and biochemical control of cell-cell interactions is discussed.

Agglutination↗