Search

A 1761 bp ORF, which encodes a protein containing 587 residues, is found in the prtVp gene encoding the extracellular protease in Vibrio parahaemolyticus. A 25 amino acid signal peptide and a 562 amino acid extracellular polypeptide form the deduced polypeptide. The molecular mass of the polypeptide is 63156 Da. The protease is probably a metalloenzyme containing Zn as its activity is suppressed by inhibitors of Zn and metals. The structure of the protease is 32% similar to that of Vibrio alginolyticus. Details of cloning and characterization of the gene are given.

An isoleucyl-tRNA synthetase gene (ileS) obtained from a Campylobacter jejuni is 2751 bp in size and similar to that obtained from E. coli. The gene is present in the 1360-1812 kb part of the genome. Isoleucyl-tRNA synthetase is an enzyme encoded by ileS gene, containing 917 amino acids with a molecular mass of 105399 Da. A comparison of the ileS gene product with that of Archea and Eukarya bacteria shows that many sequences have been conserved in these enzymes. Details of the study conducted on the gene are given.

The mxcQ and mxcE loci of Methylobacterium organophilum XX contain two open reading frames and their amino acid sequences are similar to the MxaY and MxaX proteins of the methylotrophic bacteria Paracoccus dentrificans. Cells of Methylobacterium organophilum XX grown on methanol or succinate produce proteins which bind to mxaF, the gene encoding the large subunit of methanol dehyrogenase. However, cells containing mutations in mxcQ or mxcE loci do not form proteins that bind to mxaF. The amino acid sequence of mxcQ and mxcE loci is given.

The obligate pathogenic bacteria Chlamydia psittaci and Chlamydia pneumoniae contain the 9 kDa cysteine-rich protein (CrP) gene upstream from the 60 kDa CrP gene. A monocistronic stimulus induces the transcription of the 9 kDa CrP gene. A bicistronic stimulus induces the transcription of both the genes. Transcription by these stimuli is controlled by one promoter but the termination points are different. The transcription of these genes increases as the bacteria change from the replicative form to the infective form. The structure of the 9 kDa CrP gene is similar to that of C. trachomatis.

The pJV1 circular plasmid of the bacteria Streptomyces phaeochromogenes contains transfer genes needed for chromosome mobilization (Cma) during replication of the plasmid by rolling circle mechanism. These transfer genes are controlled by a repressor gene which is similar to genes of the GntR family of regulators. Uncontrolled expression of the transfer gene causes lethality. The plasmid also contains three genes which are not needed for Cma but increase spreading of the plasmid. The amino acid sequence for the plasmid is given.

The transcription of transferrin receptor genes tbpA and tbpB of Actinobacillus pleuropneumoniae, present in an operon, is controlled by one promoter. The structure of Tbp1 protein, encoded by tbpA, is similar to that of Lbp and Tbp1s proteins of Neisseria and the outer membrane (OM) receptors of Escherichia coil. Most of the recombinant Tbp1 formed by tbpA overexpression is present in the inclusion bodies while most of the recombinant Tbp2, encoded by tbpB, is present in the OM region. Both Tbp1 and Tbp2 bind to the C region of porcine transferrin.

The glucosaminidase, produced by Bacillus subtilis AC327, is a 90 kDa protein containing direct repeats which are the cell wall binding regions. Loss of these repeats decreases the lytic ability of the protein. The lytD gene, encoding the protein, consists of a monocistronic operon containing a signal peptide. The lytD gene is expressed during the exponential growth phase and dissolves the bacterial cell wall of the cell expressing it. The expression of lytD decreases in the presence of a null mutation in sigD.

The pilC1 gene, present in the MS11 strain of Neisseria gonorrhoeae, contains amino acid sequences similar to those present in the opa and pil gene locus. These amino acid sequences are repetitive in nature and are probably involved in recombination during the formation of pili, an antigen. The amino acid structure of pilC1 is 90% similar to that of pilC2 gene. The pilC1 gene is a part of the chromosome that encodes for pili and is formed by DNA duplication. Pili controls attachment of the bacterial cell to the host cell. The amino acid sequence of pilC1 is given.

The mCAT-2 gene encodes a [Na.sup.+]-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system [y.sup.+]. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5' UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.

The extracellular Serratia marcescens endonuclease is used to cleave dsDNA and ssDNA with partly identical sequence favor, but the d(A)- and d(T)-runs in ssDNA are more easily cleaved than in dsDNA. The GC-rich locations in dsDNA are favored by the enzyme. The inability to break d(A)*d(T)-tracts by the enzyme is because of the thin minor groove, the smaller helical repeat and/or the rigidity of dsDNA at such sequences.

The signal transducer and activator of transcription 5a (STAT5a) and its homologue STAT5b show 96% similarity in their amino acid structure but the 5' and 3' untranslated region is not conserved in these proteins. Both these proteins recognize the gamma-interferon-activating sequence site and control transcription in COS cells which is induced by prolactin (PRL). The activation of the whey acidic protein (WAP) gene and the increase in the expression of STAT5 occur simultaneously. The signal for the transcriptional activation of WAP is transducted by STAT5.

The complete sequence of the tRNA-guanine transglycosylase (Tgt) gene of Zymomonas mobilis was determined, revealing four start codons. The gene was overexpressed in Escherichia coli and its Tgt product was purified. The protein's kinetic parameters were almost identical to those of its counterpart in E. coli. The Z. mobilis Tgt, however, is a monomer whereas the E. coli Tgt is a homotrimer whose formation was found to depend on protein concentration.

A gene that is homologous with the ropA gene has been identified in Rhizobium leguminosarum bv. viciae strain 248 and R. leguminosarum bv. trifolii strain LPR5020. The ropA gene encodes the lower-Mr part of antigen group III, which make up part of the membrane proteins of Rhizobium leguminosarum bv. viciae strain 248. RopA2 is a 1,074 bp sequence that encodes a 38.096 kDa protein and is 91.8% homologous to ropA.

The 20kDa subunit of Acetobacter pasteurianus alcohol dehydrogenase is necessary for enzyme function. The product of the gene restores ADH activity in mutant strains that lack the third enzyme subunit and a disruption of the gene abolishes ADH activity in Acetobacter aceti. The small subunit appears to link the 78-kDa subunit to the 48-kDa subunit.

The pagD gene located near the pagC gene of the Salmonella typhimurium PhoP hoQ system is a virulence factor. It is transcribed in the opposite direction from pagC and is regulated by the PhoP hoQ proteins. Bacterial virulence in macrophages is reduced by transposon insertions in pagD but the LD50 in mice is not affected. The gene appears to code for a membrane protein.

The YFW1, YFW12, and YFW16 genes of Saccharomyces cerevisiae can restore resistance to the alkylating agent methyl methanesulfonate (MMS) in E. coli alkB mutants. The protein product of the E. coli AlkB gene confers resistance to alkylating agents. The products of the YFW genes have no sequence homology with the product of the alkB gene and the genes cannot restore resistance to alkylating agents in other alkylation- sensitive E coli strains.

The structure of the murein layer from the cell envelope of Thermus thermophilus has been determined. The monomer has the structure N-acetylglucosamine- N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala and has a Gly-Gly dipeptide at the delta-NH2 group of Orn. Eighty-five percent of total murein consisted of 29 muropeptides and 23% contain a phenylacetic acid residue on the N-terminal Gly.

Using the phoA gene fusion methodology adapted to mycobacteria, several Mycobacterium tuberculosis DNA fragments encoding exported proteins were recently identified. in this paper, the molecular cloning, genomic positioning, nucleotide sequence determination and transcriptional start site mapping of a new M. tuberculosis gene, identified by this methodology, are reported. This gene was called erp (for exported repetitive protein) and has a sequence similar to that of the Mycobacterium leprae 28 kDa antigen irg gene M. tuberculosis erp gene contains a putative iron box close to the mapped transcriptional start site. The predicted Erp protein displays a typical N-terminal signal sequence, a hydrophobic domain at the C-terminus and harbours repeated amino acid motifs. These structural features are reminiscent of cell-wall-associated surface proteins from Gram-positive bacteria. We found that these repeats are conserved among M. tuberculosis isolates, and are absent from the published M. leprae irg gene sequence. in addition to being present in M. leprae, erp sequences were found in other members of the M. tuberculosis complex, but not in other mycobacteria tested. These results suggest that erp might encode a cell surface component shared by major pathogenic mycobacteria.

The gene encoding a 51 kDa polypeptide of Porphyromonas gingivalis 381 was isolated by immunoblotting using an antiserum raised against P. gingivalis alkaline phosphatase. DNA sequence analysis of a 2-5 kb DNA fragment containing a gene encoding the 51 kDa protein revealed one complete and two incomplete ORFs. Database searches using the FASTA program revealed significant homology between the P. gingivalis 51 kDa protein and the MurC protein of Escherichia coli, which functions in peptidoglycan synthesis. The cloned 51 kDa protein encoded a functional product that complemented an E. coli murC mutant. Moreover, the ORF just upstream of murC coded for a protein that was 31% homologous with the E. coli MurG protein. The ORF just downstream of murC coded for a protein that was 17% homologous with the Streptococcus pneumoniae penicillin-binding protein 2B (PBP2B), which functions in peptidoglycan synthesis and is responsible for antibiotic resistance. These results suggest that P. gingivalis contains a homologue of the E. coli peptidoglycan synthesis gene murC and indicate the possibility of a cluster of genes responsible for cell division and cell growth, as in the E. coli mra region. Keywords: Porplhyromonas gingivalis, murC, peptidoglycan synthesis, alkaline phosphatase

Dichelobacter nodosus is the primary pathogen implicated in ovine footrot. In this paper we have delineated a 27 kb locus, termed the virulence-related locus (vrl), that was essentially specific for virulent D. nodosus isolates. The precise ends of this locus were mapped and the sequences of the junction regions from the virulent strain A198 were compared to corresponding sequences from the benign isolate C305. The left end of the vrl locus was located in a sequence similar to that of the small stable 10Sa RNA molecule of Escherichia coli, next to a phage-'attachment-site-like sequence, which indicated that the vrl locus might have arisen by the integration of a phage. However, no attachment-like sequence could be found at the right end of the vrl locus. In the chromosome of the benign strain the sequences bordering vrl were not contiguous but were separated by about 3 kb. it was concluded that the divergence of the benign and virulent strains at this locus was, a multi-step process. Several potential ORFs were identified at the junction regions but only one ORF, encoding a 126 kDa protein, was expressed in a T7 expression system in E. coli.

A Cowdria ruminantium genomic DNA library was constructed in the expression vector [lambda]ZAPII, and an immunoreactive clone, designated [lambda] Acr9.4, was isolated by screening with serum from a C. ruminantium-infected goat. Sequencing of the insert from this clone revealed two open reading frames, encoding peptides of 10462 and 58697 kDa respectively. Database searching indicated that the two genes were homologues of groES and groEL, genes encoding a group of heat shock proteins involved in protein processing, export and assembly. Western blotting experiments showed that the recombinant GroEL protein was recognized by sera raised against four isolates of C. ruminantium which originate from South Africa, West Africa and the Caribbean, but not by antisera to the closely related Ehrlichia species E. ovina, E. [Cytoecetes] ondiri, E. bovis, E. phagocytophila) of African and European ruminants which can be expected to occur in similar geographical areas to C ruminantium. This suggests that this protein may be useful in development of serodiagnostic tests for C. ruminantium infection which are not subject to cross-reactions with antibodies to Ehrlichia species. The cloning and expression of the GroE operon will also facilitate further study of the roles of the Groe proteins in the immune response to C. ruminantium.

Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region. Keywords: clonality, evolutionary lines, flagellin gene, haemolysin gene, invasive associated protein gene

There is a region of close similarity in the class II Escherichia coli synthetase and human alanyl-tRNA synthetase, an acceptor helix G3-U70 that is required for aminoacylation. The similarity is observed only in the N-terminal region in 498 amino acids out of a total of 968 in the polypeptide, while the C-terminal exhibits a lot of difference, indicating that the conservation of the microhelix as an RNA signal for alanine is responsible for the similarity.

A 1747-bp insertion within a lignin peroxidase allele of Phanerochaete chrysosporium BKM-F-1767 is described. Pce1, the element, lies immediately adjacent to the fourth intron of lipI2. Southern blots reveal the presence of Pce1-homologous sequences in other P. chrysosporium strains. Transposon-like features include inverted terminal repeats and a dinucleotide (TA) target duplication. Atypical of transposons, Pce1 is present at very low copy numbers (one to rive copies), and conserved transposase motifs are lacking. The mutation transcriptionally inactivates lipI2 and is inherited in a 1:1 Mendelian fashion among haploid progeny. Thus, Pce1 is a transposon-like element that may play a significant role in generating ligninolytic variation in certain P. chrysosporium strains.

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in >70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.

The substitution of threonine in position 208 with methionine in lysyl-tRNA synthetase results in a mutant that shows affinity to bind lysine, indicating that dimerization of the enzyme is required for active site structuration. Aminoacyl-tRNA synthetases are a group of enzymes that are required for maintaining proper protein synthesis and exhibit vast structural differences in amino acid sequence, quaternary structure and subunit size. Site-directed mutagenesis revealed the role of various amino acids in binding either ATP, or tRNA acceptor stem or in the formation of active site.

A second mouse alpha subunit isoform of prolyl 4-jydroxylase was cloned an named alpha(II). The amino acid sequences of mouse alpha(I) and alpha(II) are 63% identical. Expression of human protein disulphide-isomerase/beta subunit with alpha(II) in insect cells by baculovirus vectors results in the formation of prolyl 4-hydroxylase. This enzyme was inhibited by poly(L-proline) at high concentrations while its activities and catalytic properties were similar to the human alpha(I)2 beta2 tetramer.

The resemblance in amino acid sequence and gene structure of dystrophin and utrophin muscle proteins suggest a common source of gene evolution. G-utrophin, a transcript from utrophin locus, is found in the adult mouse brain and during embryogenesis, it is located in sensory ganglia. Normally, the adult skeletal muscle utrophin is seen at the neuromuscular junction whereas dystrophin is found at the sarcolemma. In spite of evolutionary similarities, there are differences in the functions of both the proteins.

Early mesoderm development and subsequent specification of a differentiated phenotype in subsets of cells of the mesonephrons and sensory neurons of the central nervous system involve the action of Lim-1, the mouse homolog of Xenopus Xlim-1. Nearly 85% identity over the entire protein and a high degree of identity with Xlim-1 in the LIM and homeodomains are evident for nucleotide and deduced amino acid sequences.

Biologically vital noncovalent complexes featuring hydrogen bonding, charge-transfer complexes, ion-dipole effects and electrostatic interactions were determined earlier. The importance of changes in the noncovalent interactions when translocated from an aqueous to a gaseous medium was analyzed. Introduction of leucine zipper peptides into dimers is attributed to hydrophobic interactions. This process disappears during the transfer from an aqueous to a gaseous phase.

An exopolyphosphatase activity has been recently been purified to the homogeneity from a mutant strain of Escherichia coli which lacks the principal exopolypase. The sources used in the experimental study were an ATP, ADP, nonirradiated nucleotides, bovine serum and the ovalbumin from sigma. The study enables the identification of the second enzyme as guanosine pentaphosphate phosphohydrolase. This is used in the responses to stresses and deprivation.

The metalloprotease SepP1 encoded by the gene SepA, is one of the three types of proteolytic enzymes synthesized in Staphylococcus aureus strains and is characterized by a molecular weight of 28,000, insensitivity to sulfhydryl reagents and DFP and inactivation by EDTA. The amino acid sequence of SepP1 reveals the homology between Sep1 and the thermolabile, the thermostable neutral proteases of various bacilli. The amino acid sequences of SepP1 is also homologous to that of Pseudomonas aeruginosa elastase.

A Bacillus pumilus 3,690-bp HindIII fragment containing the gene encoding ferulic acid decarboxylase (fdc) stimulates ferulic acid decarboxylation when expressed in Escherichia coli. The DNA fragment contains a Shine-Dalgarno sequence and a promoter sequence. The fdc has an apparent molecular mass of 42 kDa and is a homodimer. The B. pumilus fdc is structurally similar to the E. coli enzyme and 66.7% identical to yeast phenylacrylic decarboxylases.

The region of DNA next to the dapE gene in Corynebacterium glutamicum contains the aroP gene, which codes for a transporter of aromatic amino acids. After cloning and sequencing the aroP gene, researchers analyzed amino acid transport in wild-types and mutants that either overexpressed or underexpressed aroP. While transport of these amino acids was affected by aroP mutations, other systems appear to be involved in such transport.

An octaketide-producing novel plant-specific type III polyketide synthase (PKS) from aloe (Aloe arborescens) sharing 50-60% amino acid sequence identity with other plant chalcone synthase (CHS)-superfamily enzymes is described. The CHS superfamily of type III PKSs produces a variety of plant secondary metabolites with remarkable structural diversity and biological activities.

The mutations in the Salmonella typhimurium glutamate-inserting ochre suppressor mutants are present in the tRNA(super Glu)(sub 2) gene gltU at 82 min. The mutations in the Escherichia coli mutants are located in the tRNA(super Glu)(sub 2) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min. Transduction studies show that in S. typhimurium, ochre suppressors are linked to ilvE1005::Tn10, and mean cotransduction frequency is 53%. Ochre suppressors of E. coli are linked to pheA18::Tn10, and mean cotransduction frequency is 60%.

13C side-change sequential assignment for 2H-HCA II can be most accurately made using the C(CC)(CO)NH experiment, starting the magnetization transfer steps sequence on 13C6. The increase in sensitivity of this modification over the HC(CC)(CO)NH experiment can be estimated because perdeuterated proteins can be analyzed while possible residue types can be further restricted through correlation peaks and their 13C chemical shifts.

Several unnatural amino acid analogues such as ornithine (aminoethyl), homocysteine (hydroxyethyl), cysteine and (aminoethyl) cysteine were used to substitute Lysine 16 to further comprehend its function in Ras p21 protein. In the Guanosine triphosphate activating protein (GAP), Lys 16 is not concerned with transition-state stabilization or guanosine triphosphate (GTP) binding. GAP-activated guanosine triphosphatase (GTPase) action was totally lost when Lys 16 was substituted by the isoteric unchanged Lys analogue (hydroxyethyl) cysteine, indicating the significance of the changed ammonium side chain.