Biomedical subjects

A F Neuwald

Publications and source records attributed to A F Neuwald.

26 records · Page 2Linked to original sources

PubMed1993

Detecting subtle sequence signals: a Gibbs sampling strategy for multiple alignment.

A wealth of protein and DNA sequence data is being generated by genome projects and other sequencing efforts. A crucial barrier to deciphering these sequences and understanding the relations among them is the difficulty of detecting subtle local residue patterns common to multiple sequences. Such patterns frequently reflect similar molecular structures and biological properties. A mathematical definition of this "local multiple alignment" problem suitable for full computer automation has been used to develop a new and sensitive algorithm, based on the statistical method of iterative sampling. This algorithm finds an optimized local alignment model for N sequences in N-linear time, requiring only seconds on current workstations, and allows the simultaneous detection and optimization of multiple patterns and pattern repeats. The method is illustrated as applied to helix-turn-helix proteins, lipocalins, and prenyltransferases.

Algorithms↗

PubMed1993

Conditional dihydrofolate reductase deficiency due to transposon Tn5tac1 insertion downstream from the folA gene in Escherichia coli.

Transposon Tn5tac1 can generate conditional mutations by virtue of an outward-facing tac promoter, which is regulated by the lac repressor and isopropyl-beta-D-thiogalactopyranoside (IPTG). We report here on a Tn5tac1 insertion in Escherichia coli that results in a conditional (IPTG-elicited) folA mutant phenotype: During aerobic growth, IPTG caused decreased synthesis of dihydrofolate reductase (DHFR; encoded by the folA gene) and hypersensitivity to trimethoprim (a DHFR inhibitor); during anaerobic growth, IPTG elicited auxotrophy that was satisfied by thymine or glycine or threonine. The Tn5tac1 insertion was downstream from folA, with the tac promoter pointing into the gene (antisense direction). Complementation tests indicated that the conditional folA deficiency was a cis effect of transcription from the tac promoter, perhaps due to head-to-head collision between converging RNA polymerases.

Alleles↗

PubMed1992

Mutational analysis of the Escherichia coli serB promoter region reveals transcriptional linkage to a downstream gene.

Genes encoding proteins with unrelated functions can be cotranscribed, and this may be used by cells to coordinate different metabolic pathways during growth. We describe a gene, designated sms, which is downstream from the serine biosynthetic gene serB in Escherichia coli but does not appear to be involved in amino acid (aa) biosynthesis. The sms gene is 1380 bp long. The Sms product migrates at 55 kDa on sodium dodecyl sulfate(SDS)-polyacrylamide gels and has a M(r) of 49472 (460 aa residues) calculated from the nucleotide sequence. The deduced Sms aa sequence shares regions of similarity with two ATP-dependent proteases, Lon and RecA, and contains two motifs: a C-x(2)-C-x(n)-C-x(2)-C motif, which is found in some nucleic acid binding proteins, and an ATP/GTP binding site motif. Insertional inactivation of sms led to increased sensitivity to the alkylating agent methylmethane sulfonate, but not to a requirement for serine or other metabolites. Several promoter mutations were isolated and characterized, which suggest that serB has a typical promoter recognized by sigma 70. After the serB coding sequence there is a 48-bp region with no obvious promoter sequence preceding the sms translation start codon. Analyses using sms'-lacZ fusions cloned downstream from wild-type and mutant serB promoters showed that sms is cotranscribed with serB.

ATP-Dependent Proteases↗

PubMed1992

cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth.

The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.

Aerobiosis↗

PubMed1991

Diverse proteins homologous to inositol monophosphatase.

Bovine inositol monophosphatase (IMP) and several homologous proteins were found to share two sequence motifs with bovine inositol polyphosphate 1-phosphatase (IPP). These motifs may correspond to binding sites within IMP and IPP for inositol phosphates or for lithium, since both substances are bound by these proteins. This suggests that the proteins homologous to IMP, which have diverse biological roles but whose function is not clear, may act by enhancing the synthesis or degradation of phosphorylated compounds.

Amino Acid Sequence↗

PubMed1990

IS30 activation of an smp'-lacZ gene fusion in Escherichia coli.

The Escherichia coli serB gene is divergently transcribed from the gene. Six independently isolated IS30 insertions within the 5' end of the serB structural gene resulted in increased expression of an smp'-lacZ gene fusion. DNA sequence analysis of two of the insertions suggests that a promoter was created upon IS30 insertion.

Base Sequence↗

PubMed1989

An Escherichia coli membrane protein with a unique signal sequence.

The smp gene, whose promoter divergently overlaps with the Escherichia coli serB promoter, encodes a 24-kDa membrane protein of unknown function. An smp fusion to the lacZ gene was constructed to select for smp promoter up-mutations. The mutations isolated, however, were located within the smp structural gene rather than within the promoter region. A 15-bp deletion and two different point mutations, corresponding to the hydrophobic region of the Smp signal sequence, resulted in a Lac+ phenotype. The point mutations introduced a positively charged amino acid into the hydrophobic region. The Smp signal sequence is unique in that it is encoded by an mRNA which has the potential to form a 9-bp hairpin structure with a 39-nt loop, that conforms to the idealized mirror symmetric sequence GC(RGUGR)(YUGUY)5 (RGUGR)CG. This highly symmetrical region may encode additional intragenic information important for the expression of smp. A computer search revealed that the smp gene product shares homology with elongation factor Ts and ribosomal subunit L4, two components of the E. coli translational apparatus.

Amino Acid Sequence↗

PubMed1985

DNA sequence and characterization of the Escherichia coli serB gene.

We have determined the sequence of a DNA fragment containing the Escherichia coli serB gene. An open reading frame of 966 nucleotides was identified that encodes a polypeptide of 322 amino acids with a molecular weight of 35,002 daltons. The transcription start site was determined by Mung Bean nuclease mapping. The -10 and -35 regions of the serB promoter lack homology to the consensus sequences. In addition, the -35 region of the serB promoter overlaps the -35 region of a second divergent promoter. Frameshift mutations were constructed at three different sites within the serB gene. When plasmids carrying these mutations were used as templates in a minicell system, mutations closer to the proposed transcription and translation start sites resulted in smaller polypeptides than those further away, confirming the proposed direction of transcription and translation. The observed sizes of the truncated and native polypeptides were in agreement with those predicted from the DNA sequence. A very stable stem and loop structure (delta G= -32 kcal/mole) that does not fit the criteria of known transcription terminators was found one nucleotide downstream from the putative UAA translation stop codon.

Amino Acid Sequence↗