Showing papers by "Public Health Research Institute published in 1988"
TL;DR: The cloning of this gene in Escherichia coli by using an inserted transposon (Tn551) as a cloning probe is described, and the cloned gene showed considerable variation with respect to different exoproteins and different host strains compared with the chromosomal agr determinant.
Abstract: We have previously identified a gene in Staphylococcus aureus, agr, whose activity is required for high-level post-exponential-phase expression of a series of secreted proteins. In this paper, we describe the cloning of this gene in Escherichia coli by using an inserted transposon (Tn551) as a cloning probe. The cloned gene, consisting of a 241-codon open reading frame containing the site of the transposon insertion, was recloned to an S. aureus vector, pSK265, and shown to be functional in S. aureus. Activity was evaluated by determinations of alpha-hemolysin, beta-hemolysin, and toxic shock syndrome toxin-1 production in early-stationary-phase cultures. The cloned gene showed considerable variation with respect to different exoproteins and different host strains compared with the chromosomal agr determinant; this variation could not be attributed to the higher copy number of the cloned gene and probably reflects inapparent subtleties of the regulatory system.
572 citations
332 citations
TL;DR: The synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they serve the dual role of specific probe and amplifiable reporter.
Abstract: We have synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase. Each recombinant consists of a sequence specific for the protozoan parasite, Plasmodium falciparum, embedded within the sequence of MDV-1 RNA, which is a natural template for Qβ replicase. The probe sequence was inserted within a hairpin loop that occurs on the exterior of MDV-1 RNA. The recombinant RNAs hybridize specifically to complementary DNA, despite topological constraints on the probe domain, are replicated at the same rate as MDV-1 RNA, despite their additional length, and are able to serve as templates for the synthesis of a large number of RNA copies. A Qβ replicase reaction initiated with only 0.14 femtograms of recombinant RNA (1,000 molecules) can produce 129 nanograms of recombinant RNA product in 30 minutes. This represents a one-billion fold amplification. Our results demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they would serve the dual role of specific probe and amplifiable reporter.
326 citations
TL;DR: The DNA sequences of the spo0H genes from Bacillus licheniformis and B. subtilis are described, and the predicted open reading frames code for proteins of 26,097 and 25,447 daltons, respectively, typical of sigma factors.
Abstract: The DNA sequences of the spo0H genes from Bacillus licheniformis and B. subtilis are described, and the predicted open reading frames code for proteins of 26,097 and 25,447 daltons, respectively. The two spo0H gene products are 91% identical to one another and about 25% identical to most of the procaryotic sigma factors. The predicted proteins have a conserved 14-amino-acid sequence at their amino terminal end, typical of sigma factors. Antibodies raised against the spo0H gene product of B. licheniformis specifically react with RNA polymerase sigma factor protein, sigma 30, purified from B. subtilis. We conclude that the spo0H genes of B. licheniformis and B. subtilis code for sigma 30, now known as sigma H.
156 citations
Patent•
30 Sep 1988TL;DR: In this article, a probe for the detection of a nucleic acid target sequence containing a molecular switch comprising three essential elements: a probe sequence of 20-60 nucleotides surrounded by switch sequences of 10-40 nucleotide which are complementary to each other, wherein the state of the switch is useful for selectively generating a detectable signal if the probe is hybridized to a target.
Abstract: A probe for the detection of a nucleic acid target sequence containing a molecular switch comprising three essential elements: a probe sequence of 20-60 nucleotides surrounded by switch sequences of 10-40 nucleotides which are complementary to each other, wherein the state of the switch is useful for selectively generating a detectable signal if the probe is hybridized to a target; also, assays and kits utilizing such probes.
135 citations
TL;DR: The results suggest that the electrogenic behavior of proton transport by the H+-ATPase may be altered in pma1 mutants and that resistance to hygromycin B may be mediated via depolarization of the cellular membrane potential.
118 citations
TL;DR: The genomic organization of five small multicopy staphylococcal plasmids comprising the pT181 family has been analyzed and it is suggested that the replication, resistance, and relaxation complex regions of these plasmid can be regarded as conserved segments assembled in various combinations, but always with the same spatial arrangement.
114 citations
TL;DR: Results indicated that 6 ppm or more of dietary NIV for 1 year showed a characteristic toxic effect of trichothecene mycotoxins in mice.
104 citations
TL;DR: This report completes the sequence analysis of phi 6 and determines the nucleotide sequence of cDNA derived from the largest RNA segment (L), which constitutes the first complete sequence of a double-stranded RNA genome virus.
Abstract: The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA. We determined the nucleotide sequence of cDNA derived from the largest RNA segment (L). This segment specifies the procapsid proteins necessary for transcription and replication of the phi 6 genome. The coding sequences of the four proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through analysis of isolated proteins. This report completes the sequence analysis of phi 6. This constitutes the first complete sequence of a double-stranded RNA genome virus.
102 citations
TL;DR: S1 mapping and translational lacZ fusion studies indicated that ORF1 and the sin gene are regulated differently, and three prominent termination sites at the end of the Sin gene were identified.
Abstract: The newly identified sin gene affects late growth processes in Bacillus subtilis when it is overexpressed or inactivated in the chromosome. S1 nuclease mapping of the sin gene transcripts in vivo reveals the existence of three transcripts (RNAI, RNAII, and RNAIII). By correlating 59 ends of sin gene transcripts with DNA sequence, we have identified three different promoterlike sequences (P1, P2, and P3) for these transcripts. 39-End mapping of these transcripts identified three prominent termination sites at the end of the sin gene. These termination sites are localized on two hairpin structures previously identified from the DNA sequence. The most abundant transcript, RNAIII, coded only for the sin gene, while the polycistronic transcripts RNAII and RNAI coded for the sin gene and ORF1 that precedes the sin gene. S1 mapping and translational lacZ fusion studies indicated that ORF1 and the sin gene are regulated differently. ORF1 expression is under developmental control, increasing at the end of vegetative growth, and requires functional spo0A and spo0H gene products. The sin gene is expressed at an almost constant and relatively low level throughout growth and remains largely unaffected by spo0A and spo0H mutations. Images
102 citations
TL;DR: In this paper, N-glycanase F (PNGase F) was used to remove the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-Mr (49K) product.
Abstract: Treatment of [3H]glucosamine-labeled Friend mink cell focus-forming virus (FrMCF) gp70 with excess peptide:N-glycanase F (PNGase F) resulted in removal of the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-Mr (49K) product. For [3H]mannose-labeled gp70, similar treatment led to removal of all the carbohydrate label from the protein. Prior digestion of the PNGase F-treated gp70 with neuraminidase resulted in an additional size shift, and treatment with O-glycanase led to the removal of almost all of the PNGase F-resistant sugars. These results indicate that gp70 possesses sialic acid-containing O-linked oligosaccharides. Analysis of intracellular env precursors demonstrated that O-linked sugars were present in gPr90env, the polyprotein intermediate which contains complex sugars, but not in the primary translation product, gPr80env, and proteolytic digestion studies allowed localization of the O-linked carbohydrates to a 10K region near the center of the gp70 molecule. Similar substituents were detected on the gp70s of ecotropic and xenotropic murine leukemia viruses and two subgroups of feline leukemia virus, indicating that O-linked glycosylation is a conserved feature of retroviral env proteins.
TL;DR: The homologous terminal noncoding regions between the middle and small dsRNA segments are found to be more extensive than RNA sequencing had previously indicated.
TL;DR: A rapid method to quantitate specific bacterial RNA species, which produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis.
TL;DR: Bacteriophage Φ6 has two unique aspects that have resulted in its utility as a model system for processes in more complex systems: the viral nucleocapsid is enveloped by a lipid-containing membrane that is very simple in its protein composition and the assembly inside the infected cells offers a model for the study of membrane differentiation and translocation.
Abstract: Publisher Summary Bacteriophage Φ6 has two unique aspects that have resulted in its utility as a model system for processes in more complex systems. The virus contains a genome composed of three segments of double-stranded (ds) RNA. The replication of the genome appears to be similar to that of the dsRNA viruses that infect eukaryotic organisms. Φ6 is therefore a useful system for studying the basic mechanisms of dsRNA replication and genomic packaging. In addition, the viral nucleocapsid is enveloped by a lipid-containing membrane that is very simple in its protein composition. The assembly inside the infected cells offers a model for the study of membrane differentiation and translocation. The Φ6 system has proved to be amenable to analysis. The entire genome is cloned as cDNA and the nucleotide sequence of the cDNA is determined. In addition, it has been possible to isolate nonsense mutants for 9 of the 12 genes of Φ6 and temperature-sensitive (ts) or missense mutants for two others. The virion contains a very active transcriptase that is capable of directing the synthesis of full-length messages of the three genomic segments.
TL;DR: The dye release assay is more sensitive and more accurate than the previously described turbidimetric assay and measured spectrophotometrically at 595 nm after the insoluble substrate is removed by filtration or centrifugation.
TL;DR: The results indicate that the assembly of the phi procapsid is independent of other phage proteins and of normal phage RNA.
Abstract: A polyhedral particle that resembles in composition and structure the procapsid of bacteriophage phi 6 was produced in Escherichia coli containing cDNA copies of the entire large genomic segment inserted into expression vector plasmids under the control of lac or tac promoters. The particles were composed of proteins P1, P2, P4, and P7 in the same stoichiometry as in the intact virion. In electron micrographs of negatively stained samples, the particles appeared as hexagons, stars, or rings of 10 knobs, which are characteristic of the five-, three-, and twofold axes of symmetry characteristic of phi 6 procapsids. Stable particles were also produced from cDNA deletions that produce only P1 and P4. Other cDNA deletions producing P1 and P7 and P1 alone resulted in unstable particles which could only be visualized in electron micrographs of thin sections of E. coli transformed by the recombinant plasmids. Our results indicate that the assembly of the phi procapsid is independent of other phage proteins and of normal phage RNA.
TL;DR: The present study indicates that CD8 is complexed with HLA heavy chain on the same cells, and the complex may have functional relevance in the T-cell recognition process.
Abstract: Immune recognition by cytotoxic effector T cells requires participation of the CD8 and major histocompatibility complex class I antigens. We found that the CD8 molecule is noncovalently associated with the HLA class I heavy chain on the surface of human T cells activated by Con A. Accordingly, anti-CD8 monoclonal antibodies precipitated a heterodimer containing polypeptides of 32 and 43 kDa from the lysates of activated T cells. The 43-kDa chain of this heterodimer can be adsorbed from cell lysates with anti-HLA-A, -B, and -C antibodies. Endoglycosidase F treatment and chymotryptic peptide mapping identified a structural similarity between this 43-kDa molecule and the HLA class I heavy chain precipitated by the anti-HLA-A, -B, and -C antibody W6/32. Analysis of anti-CD8 precipitates under nonreducing and reducing conditions indicated a lack of interchain disulfide bonding between the CD8 and HLA heavy chain molecules. The CD8-HLA heavy chain complex was also detected in mixed lymphocyte cultures and a cloned cytotoxic T-lymphocyte line but not in purified natural killer cells. The present study indicates that CD8 is complexed with HLA heavy chain on the same cells, and the complex may have functional relevance in the T-cell recognition process.
TL;DR: The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames potentially encoding proteins of greater than 100 amino acids.
Abstract: pS194 is a naturally occurring Staphylococcus aureus plasmid encoding streptomycin resistance. The plasmid has a copy number of about 25 per cell, and belongs to the inc5 incompatibility group. The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames potentially encoding proteins of greater than 100 amino acids. All four of these reading frames are on the same coding strand. The first reading frame, repE, encodes a 38 kd protein specifically required for pS194 replication. The second open reading frame, str, encodes a 34 kd polypeptide required for streptomycin resistance, probably a streptomycin adenylyltransferase. The third potential polypeptide, rlx, would be 37 kd and is probably required for relaxation complex formation and plasmid mobilization by conjugative plasmids. The fourth, orfD, overlapping the rlx reading frame, is potentially 27 kd, and may also be involved in mobilization.
TL;DR: It appears that the tnpC gene product is required for correct orientation of the element upon insertion and that this protein may interact with the carboxy‐terminal portion of the tNPB gene product.
Abstract: Tn554 is a high-frequency, site-specific, transposable element having integrative properties resembling those of lysogenic bacteriophages. Nucleotide sequence analysis indicates that Tn554 has three transposition genes, designated tnpA, tnpB and tnpC. Mutations in each of these were complemented efficiently in trans by clones containing internal fragments of Tn554; thus the products of these genes function in trans. Elements carrying deletions of the Tn554 termini could not be complemented. The product of tnpC is not absolutely required for transposition, since deletion mutations encompassing 80% of tnpC, as well as frameshift mutations located near the amino terminus of tnpC, transposed at frequencies as high as 2% of that observed with wild-type Tn554. However, such mutations affected the orientation of insertion. With wild-type Tn554 insertion occurs in a single orientation regardless of the orientation of the donor. In tnpC mutants insertion orientation was dictated by the orientation of Tn554 in the donor molecule. A mutant lacking the carboxy-terminal 59 residues of tnpB also exhibited altered insertion orientation. Thus it appears that the tnpC gene product is required for correct orientation of the element upon insertion and that this protein may interact with the carboxy-terminal portion of the tnpB gene product.
TL;DR: The availability of many sites of differing strengths may be an important factor in the ability of gyrase to fine-tune the level of supercoiling or provide local swivels in bacterial DNA.
TL;DR: It is shown, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to Vmax rates following addition of 5 microM ATP as a cold chase.
TL;DR: A versatile vector is constructed, pIS112, in which lacZ translational fusions can be made in Escherichia coli and then analyzed in Bacillus subtilis in three contexts, without recloning, to analyze the expression of the early sporulation gene, spoOF, which, during early stationary phase, is induced 10-fold from a basal vegetative level.
TL;DR: This is the first evidence for the natural occurrence of NIV in cereals grown in Canada, though its level was far less than DON.
Abstract: An investigation for the occurrence of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) in cereals (ten wheat, one rye and one corn) harvested in Canada have been carried out using a procedure, which is rapid and sensitive for Fusarium mycotoxins. NIV, DON and ZEN were detected in 4, 9 and 9 out of ten wheat samples, and their average levels in the positives were 23 ng/g, 1257 ng/g and 9 ng/g, respectively. One rye and one corn were also contaminated with a minor amount of NIV. This is the first evidence for the natural occurrence of NIV in cereals grown in Canada, though its level was far less than DON.
TL;DR: Data is presented which indicates that the origin of replication of plasmids pT181 and pC221 can also function as termination signals, indicating the existence of a specific relationship between the initiation and termination of a replication event.
Abstract: We present data which indicate that (i) the origin of replication of plasmids pT181 and pC221 can also function as termination signals; (ii) termination of replication occurs when a round of replication initiated either by RepC at the pT181 origin or by RepD at the pC221 origin reaches either of these origins, proving that the two plasmids cross-react for termination of replication; and (iii) the replication initiated at the origin of another staphylococcal plasmid, pE194, does not terminate at the origin of pT181 or pC221, indicating the existence of a specific relationship between the initiation and termination of a replication event.
TL;DR: Cmp, a nucleotide sequence element in the plasmid pT181 of Staphylococcus aureus, acts as an enhancer of DNA replication and reduces by twofold the frequency ofplasmid replication under derepressed conditions.
Abstract: cmp, a nucleotide sequence element in the plasmid pT181 of Staphylococcus aureus, acts as an enhancer of DNA replication. When cmp is present on an unrelated vector along with the pT181 origin of replication, it increases the ability of the linked pT181 origin to compete with a coresident pT181 plasmid for the initiator protein RepC. cmp is contained within a 156-base-pair segment, and its deletion from pT181 reduces by twofold the frequency of plasmid replication under derepressed conditions. The enhancer sequence contains a locus of DNA bending, and enhancer activity decreases with distance from the replication origin.
TL;DR: Tumorigenicity assays indicate that the 9,10-dihydrodiol derivatives of cholanthrene and its 3- and 6-methyl derivatives are all potent tumor initiators on mouse skin, consistent with the hypothesis that the diol epoxide metabolites of these dihydrodiols are the active carcinogenic forms of the parent hydrocarbons.
Abstract: Syntheses of the trans-dihydrodiol derivatives implicated as the proximate carcinogenic metabolites of the polycyclic hydrocarbons cholanthrene, 6-methylcholanthrene, benz[a]anthracene, and 7- and 12-methylbenz[a]anthracene are described. These compounds are useful models for research to determine the molecular basis of the strong enhancement of carcinogenicity consequent upon methyl substitution in nonbenzo bay molecular sites and meso regions of polycyclic hydrocarbons. Synthesis of the bay region anti-diol epoxide derivative of cholanthrene, its putative ultimate carcinogenic metabolite, is also described. Tumorigenicity assays indicate that the 9,10-dihydrodiol derivatives of cholanthrene and its 3- and 6-methyl derivatives are all potent tumor initiators on mouse skin. The most active member of the series is the dihydrodiol derivative of 6-methylcholanthrene, which contains a bay region methyl group. The ability of the dihydrodiols 3a-c and the trans-3,4-dihydrodiol of 7,12-dimethylbenz[a]anthracene (3d) to induce chromosomal aberrations in rat bone marrow cells was also examined. The observed order of activity was 3d greater than 3c greater than 3b greater than 3a. These findings are consistent with the hypothesis that the diol epoxide metabolites of these dihydrodiols are the active carcinogenic forms of the parent hydrocarbons.
TL;DR: Description d'un protocole de dosage de ces substances (vitamines du groupe B), qui presentent en outre une activite de fixation de la couleur de the viande mais dont l'ingestion a haute dose presente des inconvenients avec parfois des symptomes pathologiques.
TL;DR: The close-packing geometry used helps explain the near constant linear mass density of known filamentous phages and the set of equations obtained yields a calculated x-ray diffraction pattern for Pf1 which compares reasonably with experimental patterns.
TL;DR: This procedure, referred to as BH-RIP, provides a simple assay for characterizing and titering antibodies against HIV which is equivalent in specificity, and more sensitive and efficient than the Western blotting method.