Stuart M. Arfin

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.

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 ∼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 ∼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 ∼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 nta1Δ 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.

The Sequence of Porcine Protein NH-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.

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.

Dipeptide inhibitors of uniquitin-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.

Stereochemistry of valine and isoleucine biosynthesis: IV. Synthesis, configuration, and enzymatic specificity of α-acetolactate and α-aceto-α-hydroxybutyrate☆

Abstract A new synthetic route, involving acetylenic intermediates, has been developed for the preparation of the valine and isoleucine biosynthetic intermediates α-acetolactic acid ( III ) and α-aceto-α-hydroxybutyric acid ( IV ) including the optically active form of these labile acids. The absolute configuration of acetolactate methyl ester XV was confirmed as (R)-(−), and the configuration of XVI was also established as (R)-(−). Two trideuterio analogs of acetolactate were prepared by this route. The acetolactate anion was found to undergo a rapid room-temperature degenerate rearrangement resulting in racemization and methyl interchange. The isomeroreductase of Salmonella typhimurium was found to be specific for the (S) enantiomers of III and IV , allowing conclusions about the conformation of IV during the ethyl migration step in isoleucine biosynthesis. Acetolactate decarboxylase of Acidobacterium aerogenes was found to decarboxylate specifically the (S) enantiomers of III and IV , forming (−)-acetoin from III with inversion of configuration.

Assignment of Structural Gene for Asparagine Synthetase to Human Chromosome 7

Somatic cell hybrids obtained from the fusion of human B lymphocytes and an asparagine synthetase-deficient Chinese hamster ovary cell line were isolated after growth in asparagine-free medium. The human and hamster forms of asparagine synthetase differ significantly in their rate of inactivation at 47.5° C. The asparagine synthetase activity expressed in the hybrids was inactivated at 47.5° C at the same rate as the human form of the enzyme. Karyotypic analysis and analysis for chromosome-specific enzyme markers showed that the structural gene for asparagine synthetase is located on chromosome 7 in humans. The heat-inactivation profile for asparagine synthetase in extracts of hybrids formed between human peripheral leukocytes and a hamster cell line expressing asparagine synthetase activity was intermediate between the two parental types when human chromosome 7 was present, but was identical to the hamster parent when chromosome 7 was absent.

Acetolactate synthase of Pseudomomas aeruginos. 1. Purification and allosteric properties

Abstract Acetolactate synthase (acetolactate pyruvate-lyase (carboxylating), EC 4.1.3.18, formely also known as acetohydroxy acid sythetase) from Pseudomonas aeruginosa was purified approx. 380-fold to homogeneity. Feedback inhibition by all three branched-chain amino acids was observed and was potentiated at low temperatures. Plots of percent inhibition vs inhibitor concentration were sigmoid for all three ligands. At subsaturating levels, inhibition by combinations of branched-chain amino acids appears to be cumulative.

Amino acid analogs as tools for the study of amino acid metabolism in mammalian cells

Abstract Amino acid analogs can inhibit several steps in the metabolism and/or utilization of the natural amino acids in mammalian cells. Mutants can be selected on the basis of their resistance to these analogs. Such mutants may be altered in their transport of amino acids, in the regulation of amino acid biosynthesis or in the utilization of amino acids for protein synthesis. Analysis of these mutants will help clarify the mechanisms which regulate these various processes.

Comparison of the properties of histidine ammonia-lyase in normal and histidinemic mutant mice.

The histidinemic (his/his) mutant mouse shows greatly reduced skin and liver histidine:ammonia-lyase (HAL; EC 4.3.1.3) activity compared with normal mice. Liver HAL activity in the mutant is heat and salt labile and is inhibited at high substrate concentrations. Two HAL components have been identified in the normal mouse liver, a minor component with properties similar to those of HAL of the mutant mouse and a major component which is heat and salt stable and insensitive to substrate inhibition. Immunotitration with anti-HAL antibody shows that the livers of mutant mice contain no detectable antigenically cross-reacting HAL protein. It is concluded, therefore, that the his allele is a null allele at a structural or regulatory locus for the major HAL enzyme and maps close to the HAL-regulatory locus Hsd and that the low residual HAL activity in the mutant is due to another enzyme.