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H O Halvorson

Publications and source records attributed to H O Halvorson.

At least 37 records · Page 2Linked to original sources
PubMed1984

Cloning the Bacillus subtilis 168 aroC gene encoding dehydroquinase.

An approx. 14-kb Sau3A fragment of Bacillus subtilis DNA containing the aroC and ser-22 genes has been isolated. Gene aroC is expressed in both B. subtilis and Escherichia coli and appears to contain its own promoter, allowing complementation in B. subtilis. However, expression in E. coli is dependent on insert orientation, so the direction of transcription can be deduced. The level of dehydroquinase-specific activity, encoded by the cloned aroC gene, is raised 30- to 40-fold in both E. coli and B. subtilis. The clones are stable in both E. coli and B. subtilis but appear to have undergone several large deletions during their construction.

Bacillus subtilis↗
PubMed1984

Cloning of an unstable spoIIA-tyrA fragment from Bacillus subtilis.

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.

Bacillus subtilis↗
PubMed1984

Cytoplasmic and secreted Saccharomyces cerevisiae invertase mRNAs encoded by one gene can be differentially or coordinately regulated.

A single structural gene, SUC2, encodes both secreted and cytoplasmic invertase in Saccharomyces cerevisiae. It is known that the unprocessed polypeptides which differ by a secretion signal sequence are encoded by separate mRNAs. This unusual transcriptional organization raises the question as to the degree to which the transcripts can be independently regulated. To define a system for studying this problem, we examined invertase transcription after various physiological perturbations of cells: rapid catabolite derepression, heat shock, and cell cycle arrest. With each treatment, fluctuations in mRNA levels for both cytoplasmic and secreted invertase were observed. We concluded that (i) catabolite-derepressed synthesis of the mRNAs occurs rapidly after a drop in glucose, is a sustained response, and does not require de novo protein synthesis; (ii) heat shock transcription of both invertase mRNAs is, in contrast, a brief and transient response requiring de novo protein synthesis; and (iii) alpha-mating hormone treatment (G1 phase arrest and release) results in regular and coordinated synthesis of both mRNAs midway between rounds of histone mRNA synthesis. We propose that invertase mRNA regulation involves constitutively synthesized transcriptional factors (observed during catabolite derepression) and transient factors (observed during heat shock and possibly during synchronous growth). Moreover, the mRNA levels for secreted and cytoplasmic invertase can be independently regulated.

Cell Cycle↗
PubMed1983

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides.

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.

Amino Acid Sequence↗
PubMed1983

Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.

Antipain↗
PubMed1983

Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.

We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.

Acid Phosphatase↗
PubMed1982

Presecretory and cytoplasmic invertase polypeptides encoded by distinct mRNAs derived from the same structural gene differ by a signal sequence.

Saccharomyces cerevisiae strain FH4C carries a single invertase structural gene, SUC2, but produces distinct invertase mRNAs and polypeptides for the secreted and cytoplasmic forms of the enzyme. The two major invertase cell-free translation products are polypeptides of 60,000 daltons (p60) and 62,000 daltons (p62) and correspond to the nonglycosylated cytoplasmic form of invertase and the precursor of glycosylated secreted invertase, respectively. This paper describes amino acid sequence and peptide map analyses of invertase polypeptides. The peptide maps demonstrate that p62, p60, and the in vivo secreted polypeptide have significant structural homology. Sequence analysis, however, revealed differences between p62 and p60 at their amino termini. p62 contains an amino-terminal signal sequence of 19 amino acid residues that is specifically cleaved during secretion in a cell-free system to generate the secreted 87,000-dalton invertase glycopeptide gp87. This signal sequence is not present in p60. p60 synthesis begins with a methionine which can be aligned with a methionine at residue 21 in p 62. During translation, the p60 initiator methionine is removed and the newly generated amino terminus is acetylated. Based on peptide map similarities, partial amino-terminal sequence data, and common genetic origin, it is suggested that p60 and p62 have identical amino acid sequences carboxy-terminal to the p60 initiator methionine (residue 21 of p62). The reciprocal correlations of signal sequence with secretion and absence of signal sequence with cytoplasmic localization provide proof of the signal hypothesis for secreted proteins. Two mechanisms are proposed for the derivation of p60 and p62 from a single structural gene: alternative promoter sites, and differential processing of a single primary transcript.

Amino Acid Sequence↗
PubMed1982

Synthesis of repressible acid phosphatase in Saccharomyces cerevisiae under conditions of enzyme instability.

The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme.

Acid Phosphatase↗
PubMed1981

Distinct repressible mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene.

We have studied regulation of invertase putative structural genes (SUC) in S. cerevisiae and the synthetic relationship between secreted, glycosylated invertase (E.C.3.2.1.26) and the cytoplasmic, nonglycosylated form of the enzyme. Using immunoprecipitation and gel electrophoresis, we have analyzed invertase polypeptides and glycopeptides synthesized in vitro and in vivo. Analysis of size-fractionated mRNA from a SUC2 strain has shown that three mature, catabolite-repressible mRNA species direct the in vitro synthesis of three invertase polypeptides that have differing molecular weights. Two of these polypeptides, P63 and P62 (63 and 62 kd), are larger than the polypeptides of the secreted enzyme and are cotranslationally processed by microsomal membranes in vitro to yield secreted invertase glycopeptides (GP90 and GP87). The smallest polypeptide, P60 (60 kd), which comigrates electrophoretically with cytoplasmic invertase, is not processed. Posttranslationally, a microsomal-membrane detergent extract removes approximately 20 aminoacids from P62 but not from P60. In vitro translations of mRNAs from a genetically confirmed suc3 mutant strain, from the parental SUC3 strain and from derivative meiotic segregants have shown that the three polypeptides (and therefore three mRNA species) are encoded by one gene. Analysis of in vivo radiolabeled invertase from the same SUC3 and suc3 strains has verified that the SUC3 locus contains the structural gene for secreted and cytoplasmic invertase. Through the derepressed synthesis of multiple primary or processed transcripts, the SUC2 and SUC3 genes are regulated to produce multiple invertase polypeptides. The larger two polypeptides appear to be processed and secreted to yield glycosylated invertase, while the smallest remains in the cytoplasm.

Cytoplasm↗
PubMed1980

Post-transcriptional modification of the poly(A) length of galactose-1-phosphate uridyl transferase mRNA in Saccharomyces cerevisiae.

Thermal elution poly(U)-Sepharose chromatography was utilized to fractionate yeast mRNA based on poly(A) size. Analysis of the in vitro translation products of the fractionated RNAs in a wheat-embryo cell-free protein synthesis system shows a heterogeneous but equal distribution of these abundant translatable mRNAs in the different poly(A) size classes. By comparing the translational activity of inducible galactose-1-phosphate uridyl transferase mRNA, which can be monitored as a function of age, to contitutive mRNAs, we demonstrate that initially galactose-1-phosphate uridyl transferase mRNA has a uniformly large poly(A) tail which becomes heterogeneous and shorter with age in the cytoplasm. These observations are consistent with the previously observed cytoplasmic poly(A) catabolism in yeast and with cytoplasmic post-transcriptional modification of the poly(A) length of galactose-1-phosphate uridyl transferase mRNA.

Enzyme Induction↗
PubMed1980

Cloning of randomly sheared DNA fragments from a phi 105 lysogen of Bacillus subtilis identification of prophage-containing clones.

A gene bank for Bacillus subtilis has been developed by cloning randomly sheared DNA fragments of a B. subtilis (phi 105) lysogen DNA in Escherichia coli employing the pMB9 plasmid vector. The DNA was inserted by the oligo(dA)-oligo(dT) method, and the average insert size of the cloned DNA was 7 kilobase pairs (kb). Three clones have been identified which carry DNA from the phi 105 prophage. None of these clones contain the phage-chromosome junction.

Bacillus subtilis↗
PubMed1980

In vitro synthesis of repressible yeast acid phosphatase: identification of multiple mRNAs and products.

Antibodies to repressible nonspecific acid phosphatase [APase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] purified from Saccharomyces cerevisiae were used to detect the in vitro products of APase mRNA. Immunoprecipitation of cell-free synthesized protein and of in vivo enzyme from cell extracts has shown that derepression of enzyme synthesis in situ is the result of de novo appearance of functional mRNA followed by de novo protein synthesis. At least three unique APase polypeptides are synthesized in vitro from separate mRNAs and appear to be glycosylated in vivo to form secreted enzyme.

Acid Phosphatase↗
PubMed1979

The Viking Mission: implications for life on Mars.

The results of the Viking Biology experiments are best explained by non-biological phenomena: The interaction of the reagents with the materials comprising the regolith. Conditions of water activity, temperature, availability of carbon sources and others in most regions of the planet are too extreme for survival and growth of any known Earth microorganisms. Although the possibility persists that some very unusual form of life is somewhere on that planet the evidence is best interpreted as negative. Even though there is no evidence for current life on Mars, whether or not life ever originated there is not known.

Carbon↗
PubMed1979

Specific inhibition of outgrowth of Bacillus subtilis spores by novobiocin.

Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive temperature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while displaying a marginal effect on logarithmic growth during one generation time. Inhibition of outgrowth by novobiocin occurred in the absence of DNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se.

Bacillus subtilis↗