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S Silver

Publications and source records attributed to S Silver.

At least 109 records · Page 6Linked to original sources
PubMed1988

Promoters and transcription of the plasmid-mediated citrate-utilization system in Escherichia coli.

The nucleotide sequence of an 878-bp BamHI-BglII restriction endonuclease fragment from citrate utilization transposon Tn3411 was determined, and was compared with that from plasmid pMS185 [Sasatsu et al., J. Bacteriol. 164 (1985) 983-993]. A long open reading frame for a 379-amino acid (aa) polypeptide (citB) was found 5' to the citA gene (431-aa membrane protein) in Tn3411 as well as in pMS185. Promoter regions were identified by RNA polymerase filter-binding assays, S1 nuclease mapping and cit-lac fusion experiments. The results indicated that two genes (citA and citB) have separate promoters, and the location of the promoter for the citB gene in the Tn3411 nucleotide sequence was different from that in pMS185. The regulation of transcription of the two genes (citA and citB) was characterized by the use of cit-lacZ fusions. The level of the citB promoter activity was about five-fold higher than that of the citA gene promoter, and transcription from both was not induced by citrate. Synthesis of the mRNA for the citB gene (especially with the wild-type Cit+ determinant) was suppressed by citrate, accompanying growth suppression of Escherichia coli. The citB gene expressed in E. coli minicells produced a membrane-associated 37.5-kDa polypeptide.

Amino Acid Sequence↗
PubMed1988

The role of operator position in SV40 T-antigen-mediated repression.

We have manipulated positional relationships between the SV40 early operator and promoter to study the repression of transcription by T antigen. Single or multiple insertions of T-antigen-binding region I resulted in only weak repression even when the operator was placed immediately adjacent to known promoter elements. The low levels of repression appear to reflect the intrinsic strength of the T-antigen-operator interaction.

Antigens, Polyomavirus Transforming↗
PubMed1988

Addition of growth hormone secretion signal to basic fibroblast growth factor results in cell transformation and secretion of aberrant forms of the protein.

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types. Unlike most growth factors, the primary translation product for bFGF appears to lack a secretory signal peptide. To explore the normal mode of bFGF release, as well as to investigate the growth factor's oncogenic potential, expression vectors were created for a bFGF cDNA and for a chimeric molecule in which the bFGF coding sequence was linked to the human growth hormone signal peptide sequence. Transfection of NIH3T3 cells with the bFGF cDNA vectors caused the synthesis of high levels of biologically active, cell-associated bFGF, but no evidence of transformation was detected. In contrast, the chimeric bFGF-signal peptide expression vector induced foci of transformation at a very high frequency. The transformed cells grew in soft agar and were tumorigenic in nude mice. The majority of the immunoreactive bFGF species made by the transformed cells was found in the conditioned medium and appeared to be posttranslationally modified, indicating that the chimeric bFGF-signal peptide molecule was processed through the secretory pathway. The secreted bFGF exhibited little mitogenic activity, suggesting that interaction of bFGF with its receptor likely occurs while the fusion protein is being processed along the secretory pathway.

Animals↗
PubMed1987

Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358.

The broad-spectrum mercurial-resistance plasmid pDU1358 was analyzed by cloning the resistance determinants and preparing a physical and genetic map of a 45-kilobase (kb) region of the plasmid that contains two separate mercurial-resistance operons that mapped about 20 kb apart. One encoded narrow-spectrum mercurial resistance to Hg2+ and a few organomercurials; the other specified broad-spectrum resistance to phenylmercury and additional organomercurials. Each determinant governed mercurial transport functions. Southern DNA X DNA hybridization experiments using gene-specific probes from the plasmid R100 mer operon indicated close homology with the R100 determinant. The 2153 base pairs of the promoter-distal part of the broad-spectrum Hg2+-resistance operon of pDU1358 were sequenced. This region included the 3'-terminal part of the merA gene, merD, unidentified reading frame URF1, and a part of URF2 homologous to previously sequenced determinants of plasmid R100. Between the merA and merD genes, an open reading frame encoding a 212 amino acid polypeptide was identified as the merB gene that determines the enzyme organomercurial lyase that cleaves the C--Hg bond of phenylmercury.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258.

The mercurial-resistance determinant from Staphylococcus aureus plasmid pI258 is located on a 6.4-kilobase-pair Bgl II fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA sequence of the Bgl II fragment was determined. The mer DNA sequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and merB (organomercurial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage identity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mer sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons.

Bacillus subtilis↗
PubMed1987

Decreased chromate uptake in Pseudomonas fluorescens carrying a chromate resistance plasmid.

CrO4(2-) resistance in Pseudomonas fluorescens LB300(pLHB1) was related to reduced uptake of CrO4(2-) relative to the plasmidless strain LB303. 51CrO4(2-) was transported mainly via the SO4(2-) active transport system; thus, cells grown with 0.15 mM cysteine, a repressor of the SO4(2-) transport system, were much more resistant to CrO4(2-) than those grown with 0.15 mM djenkolic acid, which derepressed the 35SrO4(2-) uptake system. Kinetics of 51CrO4(2-) uptake by P. fluorescens with and without the plasmid showed that the Vmax for 51CrO4(2-) uptake with the resistant strain was 2.2 times less than the Vmax for the sensitive strain, whereas the Km remained constant.

Biological Transport, Active↗
PubMed1986

Nucleotide sequence of the structural genes for an anion pump. The plasmid-encoded arsenical resistance operon.

The structural genes for the arsenical pump of the conjugative R-factor R773 contained on a HindIII fragment of 4.3 kilobase pairs were cloned into bacteriophage M13. A series of ordered deletions was created using Bal31 digestion, and the nucleotide sequence of the operon determined. Three open reading frames for genes arsA, arsB, and arsC were found. The arsA gene encodes a hydrophilic protein of 63,169 Da with two potential adenylate-binding sites. The arsB gene encodes a potentially membrane protein of 45,577 Da. The arsC gene encodes a 15,811-Da hydrophilic protein. The arsA and arsC gene products correspond to cytosolic proteins previously identified from minicell experiments. Isolated ArsA protein was shown to bind to dye-agarose columns which act as affinity resins for nucleotide-binding proteins. A model is proposed in which these gene products form an anion translocating ATPase for extrusion of arsenite and arsenate from resistant cells.

Adenosine Triphosphatases↗
PubMed1986

An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen.

Seventeen base pairs of DNA from SV40 origin region I encode a tripartite binding site for a dimeric mass of SV40 large T antigen. Two binding components are the directly repeated pentanucleotide sequences 5'-GAGGC-3'/5'-GCCTC-3'. The third component is the asymmetric sequence 5'-TTTTTTG-3'/5'-CAAAAAA-3' that separates the pentanucleotides. Nucleotide-specific features of this spacer element stabilize binding to the adjacent pentanucleotides. We report here that the spacer sequence determines a DNA conformation that correlates with high affinity binding of T antigen. The nature of the spacer sequence suggests that the DNA is bent. We propose that binding of T antigen to region I proceeds through monomer-pentanucleotide interactions and either protein-protein or protein-spacer interactions directed by the spacer-encoded structure.

Antigens, Viral, Tumor↗
PubMed1986

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system.

The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence, and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.

Amino Acid Sequence↗
PubMed1986

Regulation of herpes simplex virus 1 genes: alpha gene sequence requirements for transient induction of indicator genes regulated by beta or late (gamma 2) promoters.

This laboratory reported earlier that chimeric genes consisting of the structural sequences of the thymidine kinase (TK) gene fused to the promoter-regulatory domains of late (gamma 2) genes were regulated as bonafide gamma 2 genes when resident in the herpes simplex virus 1 genome but could not be differentiated from beta genes when introduced by transfection and stably integrated into the environment of the host genome (S. Silver and B. Roizman, Mol. Cell. Biol. 5, 518-528, 1985). We report here that beta-TK and the chimeric gamma 2-TK gene transfected into TK- baby hamster kidney (BHKtk-) were induced by alpha 4 and alpha 0 but not by the other alpha genes. Specifically: Both TK genes were induced by cotransfection with DNA fragments carrying an intact alpha 4 or an intact alpha 0 gene, but not by fragments carrying only the promoter-regulatory domain or the structural sequences of the alpha 4 gene or intact alpha 22, alpha 27, and alpha 47 genes. An alpha 4 gene carrying a 2700-bp deletion in its 3' coding sequence also induced both genes, although less efficiently. RNA homologous to the alpha 4 gene recovered from the cytoplasm of cells transfected with either the intact or truncated alpha 4 gene mapped to the bonafide site of transcription initiation of the alpha 4 gene. RNA homologous to the chimeric TK gene extracted from the cytoplasm of cells transfected with both gamma 2-TK and the alpha 4 gene was transcribed from the bonafide gamma 2 gene capping site fused to the TK gene. These results indicate that the alpha 4 gene and the alpha 0 gene are each capable of inducing the expression of both beta and gamma 2 genes resident in the environment of the cellular genome, that the active site responsible for induction is located near the N terminus of the alpha 4 protein, and reinforce the conclusion that gamma 2 genes resident in the environment of the host cell cannot be used to identify the authentic determinants of gamma 2 gene regulation by currently available tests.

Animals↗
PubMed1986

Resistance to mercury and to cadmium in chromosomally resistant Staphylococcus aureus.

Apparently chromosomally located mercury resistance determinants in five methicillin-resistant Staphylococcus aureus strains of different geographical origin were structurally homologous to plasmid-located mercury resistance determinants in S. aureus. These were all located on a 6.3-kilobase (kb) Bg/II fragment, as evident from Southern hybridization experiments with the 6.3-kb Bg/II fragment of plasmid pI258 as the probe. These methicillin-resistant S. aureus strains exhibited similar phage susceptibility patterns and biochemical reactions. They differed, however, in the DNA location of the mercury resistance determinants, as evidenced by neighboring cleavage sites for restriction endonucleases EcoRI, HindIII, and PstI. In an environmental (nonhospital) strain in which mercury resistance was also apparently chromosomally conferred, these determinants were also homologous to pI258 DNA, but they were located on a 6.6-kb Bg/II fragment. Cadmium resistance determinants in the five methicillin-resistant S. aureus strains and the environmental S. aureus strain were not similar to the known plasmid-located determinants cadA and cadB. Cd2+ resistance was based on an efflux mechanism for Cd2+. However, no parallel resistance to zinc was conferred. The 3.2-kb XbaI-Bg/II fragment obtained from plasmid pI258 and used as a cadA-specific probe did not hybridize to total DNA digests of the strains with apparently chromosomally determined cadmium resistance.

Cadmium↗
PubMed1985

Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme.

The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn501. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn501) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecyl-nucleotide sequence in the R100 sequence without counterpart in Tn501. Thus the predicted merA polypeptide of Tn501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259-267], for which there is a 2 A resolution electron density map [Thieme et al.: J. Mol. Biol. 152 (1981) 763-782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.

Amino Acid Sequence↗
PubMed1985

Mercuric reductase enzymes from Streptomyces species and group B Streptococcus.

Mercury volatilization (Hg2+ reductase) activity has been found with Hg2+-resistant isolates of three Streptomyces species and with three Hg2+-resistant strains of group B Streptococcus from clinical sources in Japan. Hg2+ reductase activities in crude cell extracts showed the temperature sensitivity, the requirement for an added thiol compound and the characteristic dependence on NAD(P)H cofactors of similar enzymes isolated from other bacteria.

Drug Resistance, Microbial↗
PubMed1985

Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.

Biological Transport↗