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S M Arfin

Publications and source records attributed to S M Arfin.

At least 19 recordsLinked to original sources
PubMed2001

The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons.

Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling). The effect of this transcription-induced DNA supercoiling on these promoters depends on their intrinsic properties. As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose. Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co-ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions. As DNA supercoiling-dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.

Bacterial Proteins↗
PubMed2000

Global gene expression profiling in Escherichia coli K12. The effects of integration host factor.

We have used nylon membranes spotted in duplicate with full-length polymerase chain reaction-generated products of each of the 4,290 predicted Escherichia coli K12 open reading frames (ORFs) to measure the gene expression profiles in otherwise isogenic integration host factor IHF(+) and IHF(-) strains. Our results demonstrate that random hexamer rather than 3' ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria. This is explained by the fact that the currently available set of 4,290 unique 3' ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing degradation rates (steady-state levels) are observed for the 25-base pair region of each message complementary to each ORF-specific primer. To evaluate the DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design. These statistical methods allowed us to identify and appropriately correct for experimental variables that affect the reproducibility and accuracy of DNA microarray measurements and allowed us to determine the statistical significance of gene expression differences between our IHF(+) and IHF(-) strains. Our results demonstrate that small differences in gene expression levels can be accurately measured and that the significance of differential gene expression measurements cannot be assessed simply by the magnitude of the fold difference. Our statistical criteria, supported by excellent agreement between previously determined effects of IHF on gene expression and the results reported here, have allowed us to identify new genes regulated by IHF with a high degree of confidence.

Bacterial Proteins↗
PubMed1999

Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli.

The twin-domain model [Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024-7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the ilvYC operon of Escherichia coli. Double-reporter constructs with the lacZ gene under transcriptional control of the ilvC promoter and the galK gene under control of the divergent ilvY promoter were used to demonstrate that a down-promoter mutation in the ilvY promoter severely decreases in vivo transcription from the ilvC promoter. However, a down-promoter mutation in the ilvC promoter only slightly affects transcription from the ilvY promoter. In vitro transcription assays with DNA topoisomers showed that transcription from the ilvC promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the ilvY promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in ilvY promoter activity and a decrease in ilvC promoter activity predicted by the in vitro data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the ilvYC operon with the nutritional and environmental conditions of cell growth.

Bacterial Proteins↗
PubMed1996

A mouse amidase specific for N-terminal asparagine. The gene, the enzyme, and their function in the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine. The approximately 17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length. The approximately 1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes. The Ntan1 promoter is located approximately 500 base pairs upstream of the Ntan1 start codon. The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine. The expression of mouse NtN-amidase in S. cerevisiae nta1Delta was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule. Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown.

Amidohydrolases↗
PubMed1995

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

Amino Acid Sequence↗
PubMed1995

The sequence of porcine protein NH2-terminal asparagine amidohydrolase. A new component of the N-end Rule pathway.

Co- and post-translational amino-terminal processing of proteins is one mechanism by which intracellular proteins can be either protected from or targeted to degradation by the N-end Rule pathway (Bachmair, A., Finley, D., and Varshavsky, A. (1986) Science 234, 179-186). A novel enzyme, protein NH2-terminal asparagine amidohydrolase, which can function in this pathway by potentially directing critical regulatory proteins possessing an amino-terminal asparagine residue formed from the removal of N-acetylmethionine, has recently been purified and characterized (Stewart, A.E., Arfin, S. M., and Bradshaw, R. A. (1994) J. Biol. Chem. 269, 23509-23517). Here, we report the isolation and characterization of a cDNA for porcine protein NH2-terminal asparagine amidohydrolase, which indicates that it is a new type of enzyme, not homologous to any previously identified protein. This provides strong evidence for the importance of regulated protein degradation in cellular functioning.

Amino Acid Sequence↗
PubMed1994

Protein NH2-terminal asparagine deamidase. Isolation and characterization of a new enzyme.

An apparently unique enzyme, designated protein NH2-terminal asparagine deamidase (PNAD), that specifically converts NH2-terminal asparagine residues of peptide and protein substrates to aspartic acid, has been isolated to homogeneity from porcine liver by an eight-step procedure. PNAD is a relatively low abundance protein, is readily solubilized, and exists as a monomeric species of approximately 33 kDa. PNAD does not act on internal asparagine residues and requires a free N alpha-amino group. It has reduced or no activity on NH2-terminal asparagine dipeptides and no activity toward free asparagine or asparagine amide. It does not act on any NH2-terminal glutamine substrates. PNAD does not show a strong pH dependence suggesting that the enzyme can act equally well on substrates with ionized or unionized alpha-amino groups. The properties and specificity of PNAD are consistent with those expected for the enzyme required for the ubiquitin-dependent turnover of intracellular proteins that initiate with Met-Asn-. Such proteins should be N alpha-acetylated on the retained initiator methionine and can subsequently be modified by the removal of N-acetyl methionine by acylaminoacid hydrolase. Conversion of the resulting NH2-terminal asparagine to aspartic acid by PNAD would render the protein susceptible to arginylation, polyubiquitinylation and degradation as specified by the N-end rule.

Amidohydrolases↗
PubMed1994

Nucleotide and deduced amino acid sequence of human cysteinyl-tRNA synthetase.

We have determined the nucleotide sequence of a cDNA coding human cysteinyl-tRNA synthetase. The predicted protein sequence of 638 amino acids has substantial sequence similarity to the E. coli cysteinyl-tRNA synthetase in the nucleotide-binding fold region that constitutes the potential active site. The results are consistent with the hypothesis that evolution of the full complement of aminoacyl-tRNA synthetases occurred prior to the divergence of prokaryotes and eukaryotes.

Amino Acid Sequence↗
PubMed1992

Dipeptide inhibitors of ubiquitin-mediated protein turnover prevent growth factor-induced neurite outgrowth in rat pheochromocytoma PC12 cells.

Dipeptide inhibitors of the ubiquitin-dependent proteolysis pathway governed by N-terminal recognition (N-end rule) in reticulocyte lysates significantly suppress NGF- and bFGF-induced neurite outgrowth in rat pheochromocytoma PC12 cells, but do not cause retraction of already formed neurites. Peptides which do not inhibit proteolysis are also without effect on PC12 cell differentiation. Suppression of neurite outgrowth is readily reversible upon removal of the inhibitors. These data demonstrate a requirement for specific protein turnover in the process of neuron-like differentiation in PC12 cells and provide the first demonstration of a physiological role for the N-end rule.

Animals↗
PubMed1991

Cloning and characterization of a Saccharomyces cerevisiae gene encoding a new member of the ubiquitin-conjugating protein family.

Ubiquitin-conjugating enzymes (E2s), which participate in the post-translational conjugation of ubiquitin to proteins, are encoded by a multigene family in the yeast Saccharomyces cerevisiae. E2s function in a variety of cellular activities including intracellular proteolysis, DNA repair, sporulation, and cell cycle traverse. Here, we report the cloning and characterization of a new member of the yeast UBC gene family, UBC8. UBC8 encodes a 206-amino acid protein containing a highly acidic carboxyl terminus. The primary structure of the protein is similar to that of all other known E2s, with the highest homology being to the E2 (23 kDa) of wheat germ. Haploid strains in which the UBC8 gene is disrupted are viable, and the disruption does not produce any obvious phenotype. The UBC8 protein, produced in Escherichia coli, forms thiol ester adducts with ubiquitin and, apparently, diubiquitin, but does not transfer ubiquitin to histones.

Amino Acid Sequence↗
PubMed1991

Nucleotide and deduced amino acid sequence of human threonyl-tRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes.

The nucleotide sequence of a cDNA coding human threonyl-tRNA synthetase has been determined. The predicted protein sequence is highly homologous to that of the yeast cytoplasmic, yeast mitochondria and Escherichia coli threonyl-tRNA synthetases. In particular, the three structural motifs recently shown to be common to class II aminoacyl-tRNA synthetases are present in the threonyl-tRNA synthetases from all sources. Primer extension and S1 nuclease analyses indicate that transcription initiates approximately 220-230 nucleotides upstream of the putative initiator methionine codon. This region contains a 10-nucleotide interrupted inverted repeat flanked by a 13-nucleotide interrupted direct repeat.

Amino Acid Sequence↗
PubMed1989

Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines.

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Histidyl-tRNA synthetase of Chinese hamster ovary cells contains phosphoserine.

The Chinese hamster ovary cell line 2H2-5, which expresses elevated levels of histidyl-tRNA synthetase, was used to demonstrate that histidyl-tRNA synthetase contains phosphoserine. After growth of the cells in medium containing [32P]orthophosphate, histidyl-tRNA synthetase was isolated by partial purification and immunoprecipitation and shown to be a phosphoprotein. Phosphoamino acid analysis showed that histidyl-tRNA synthetase is phosphorylated on serine, as has previously been shown for threonyl-tRNA synthetase of CHO cells.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Threonyl-tRNA synthetase gene maps close to leucyl-tRNA synthetase gene on human chromosome 5.

The structural gene for threonyl-tRNA synthetase was mapped to human chromosome 5 by an analysis of the isoelectric focusing patterns of this enzyme from human X Chinese hamster interspecific somatic cell hybrids. The threonyl-tRNA synthetase gene is the fourth of seven aminoacyl-tRNA synthetase genes mapped in humans to be assigned to this chromosome. Regional mapping studies showed that the threonyl-tRNA synthetase gene is on the short arm of chromosome 5, p13-cen, and is close to, but separable from, the gene for leucyl-tRNA synthetase which maps to 5cen-5q11.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Threonyl-tRNA synthetase from Chinese hamster ovary cells is phosphorylated on serine.

A Chinese hamster ovary cell line, 2000A, in which threonyl-tRNA synthetase accounts for 1.5% of the total soluble protein, was used to demonstrate that this enzyme is a phosphoprotein. Threonyl-tRNA synthetase was isolated by immunoprecipitation from cells labeled with 32Pi for 18 h. Phosphoamino acid analysis of radiolabeled threonyl-tRNA synthetase showed that phosphorylation occurs on serine.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Chinese hamster ovary cells resistant to borrelidin overproduce threonyl-tRNA synthetase.

Chinese hamster ovary cell lines that are 1000-fold more resistant to the threonyl-tRNA synthetase inhibitor borrelidin than the sensitive parental cells were isolated after stepwise selection for growth in increasing concentrations of the drug. These cells show a 10-20-fold increase in threonyl-tRNA synthetase activity. Quantitation of the amount of threonyl-tRNA synthetase protein by immunological techniques indicated a 60-100-fold increase compared to sensitive cells. No significant changes in the Km for substrates, inhibition by borrelidin or thermal stability were found for the threonyl-tRNA synthetase of resistant cells. These data suggest that the resistant cell lines may have amplified the gene encoding threonyl-tRNA synthetase, but no evidence of homogeneously staining regions or double minute chromosomes was found. The resistant cell lines should prove useful for the study of the regulation of threonyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗