E. coli translation initiation factor IF2--an extremely conserved protein. Comparative sequence analysis of the infB gene in clinical isolates of E. coli

The functionally uncharacterised N-terminal of translation initiation factor IF2 has been found to be extremely variable when comparing different bacterial species. In order to study the intraspecies variability of IF2 the 2670 basepairs nucleotide sequence of the infB gene (encoding IF2) was determined in 10 clinical isolates of E. coli. The N-terminal domains (I, II and III) were completely conserved indicating a specific function of this region of IF2. Only one polymorphic position was found in the deduced 890 amino acid sequence. This Gln/Gly490 is located within the central GTP/GDP-binding domain IV of IF2. The results are further evidence that IF2 from E. coli has reached a highly defined level of structural and functional development.     

Growth factor-independent expression of the gene encoding eukaryotic translation initiation factor 4E in transformed cell lines

Activation of protein synthesis is necessary for the transition of cells from quiescence to proliferation, while withdrawal of growth factors leads to decrease in protein synthesis and transition of normal cells into the resting period. It is shown in this paper that serum growth factors are required for activation of expression of gene encoding translation initiation factor 4E (eIF-4E) in non-transformed NIH 3T3 and Rat-1 fibroblasts but this requirement is lost in C6 glioblastoma, A431 carcinoma and N-myc transformed Rat-1 cells. These data raise the possibility that neoplastic transformation leads to growth factor-independent expression of eIF-4E, thus facilitating continuous growth and replication of transformed cells.     

The sequence of the ribosomal 16S RNA from Proteus vulgaris. Sequence comparison with E. coli 16S RNA and its use in secondary model building

The complete nucleotide sequence of the 16S RNA from Proteus vulgaris has been determined. The molecule (1544 nucleotides) shows 93% homology with the sequence of E. coli 16S RNA. Six methylated nucleotides have been localized in positions homologous to those observed in the E. coli RNA molecule. Both E. coli and P. vulgaris 16S RNA chains can be folded up into a common secondary structure scheme. Comparative sequence analysis of the two molecules has provided a valuable contribution to 16S RNA secondary structure model building.     

Periodic selection, infectious gene exchange and the genetic structure of E. coli populations

As a consequence of sequential replacements by clones of higher fitness (periodic selection), bacterial populations would be continually purged of genetic variability, and the fate of selectively neutral alleles in very large populations of bacteria would be similar to that in demes of sexually reproducing organisms with small genetically effective population sizes. The significance of periodic selection in reducing genetic variability in these clonally reproducing species is dependent on the amount of genetic exchange between clones (recombination). In an effort to determine the relationship between the rates of periodic selection, recombination and the genetically effective sizes of bacterial populations, a model for periodic selection and infectious gene exchange has been developed and its properties analyzed. It shows that, for a given periodic selection regime, genetically effective population size increases exponentially with the rate of recombination.--With the parameters of this model in the range anticipated for natural populations of E. coli, the purging effects of periodic selection on genetic variability are significant; individual populations or lineages of this bacterial species would have very small genetically effective population sizes.--Based on this result, some other a priori considerations and a review of the results of epidemiological and genetic variability studies, it is postulated that E. coli is composed of a relatively limited number of geographically widespread and genetically nearly isolated and monomorphic lineages. The implications of these considerations of the genetic structure of E. coli populations on the interpretation of protein variation and the neutral gene hypothesis are discussed.     

Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10(7) per g of dry soil for the H2-CO2-utilizing methanogens and of up to 10(6) for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H2-CO2, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 microM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.     

Oligomeric interaction of the PapB transcriptional regulator with the upstream activating region of pili adhesin gene promoters in Escherichia coli

1. Prostanoid receptor-mediated sensitization, or excitation, of sensory nerve fibres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F-11 (embryonic rat DRG x neuroblastoma hybrid) cell line. 2. In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI2) stimulated cyclic AMP accumulation. Prostaglandin E2 (PGE2) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A2 (cTxA2), PGD2 and PGF2alpha had negligible effects. The rank order of agonist potency was cicaprost>PGE2=BMY45778=cPGI2=PGI2. In the F-11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost>iloprost=cPGI2=PGI2=BMY45778>PGE2=cTXA2++ +. In DRG cultures, cicaprost induced significantly more accumulation of inositol phosphates than PGE2. 3. To examine the effects of prostanoids on C-fibre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the 'grease-gap' technique. PGI2 (0.1 nM-10 microM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI2=cPGI2=PGE1>cTXA2>PGE2=PGD2=TXB2>PGF2alpha. 4. Prior depolarization of nerves with either forskolin (10 microM) or phorbol dibutyrate (1 microM) alone significantly reduced the response to PGI2 (10 microM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI2 responses almost completely. 5. Putative EP1 and/or TP receptor-selective antagonists had no effect on the responses to PGI2, cPGI2 or PGE2 in the three preparations studied. 6. Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These findings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.     

The use of a chimera HIV-1/HIV-2 envelope protein for immunodiagnosis of HIV infection: its expression and purification in E. coli by use of a translation initiation site within HIV-1 env gene

A chimera HIV-1/HIV-2 envelope sequence composed of multiple conserved immunodominant epitopes of HIV-1 envelope protein (HIV-1 IIIB: env482-518 + env548-675) and the HIV-2 gp36 immunodominant epitope (env592-603), was constructed and directly over-expressed in E. coli by using a prokaryotic translation initiation sequence contained within the gene of HIV-1 envelope. The recombinant product was purified and applied in antibody-screening assay. The purified chimera antigen reacted with all the thirty-eight HIV-1 positive serum samples, the two HIV-2 serum samples, and had no cross-reaction with all the eighty-eight normal healthy serum sample. The results indicated that this recombinant chimera HIV-1/HIV-2 envelope protein could be useful for diagnostic purposes of HIV infection.     

The gene for 16S rRNA methyltransferase (ksgA) functions as a multicopy suppressor for a cold-sensitive mutant of era, an essential RAS-like GTP-binding protein in Escherichia coli

Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an E. coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the E. coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.     

Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli

BACKGROUND AND AIMS OF THE STUDY: This study investigated the efficacy of postoperative ticlopidine as antiplatelet therapy in patients shortly after heart valve repair or replacement. METHODS: Between 1990 and 1995, 235 consecutive patients underwent either valve repair (n = 67) or replacement with a bioprosthesis (n = 168). The bioprostheses used were Carpentier-Edwards porcine or pericardial (n = 158) valves, Prima stentless valves (n = 3) and cryopreserved homografts (n = 7). Types of repair were aortic (one), mitral annuloplasty with Carpentier ring (65) and tricuspid repair (one). Mean patient age was 67 (range: 16 to 83) years for valve replacement and 57 (range: 32 to 74) years for repair (p < 0.01). Atrial fibrillation occurred in 34% of patients. The hospital mortality rate was 11% (26 patients). Of the 209 survivors, 137 were assigned to antiplatelet treatment with ticlopidine for the first three months of follow up. The other 72 received either oral anticoagulation (coumadin; n = 40), aspirin (n = 14) or no medication (n = 18). In 15 patients, ticlopidine treatment was interrupted due to diarrhea (13 cases), mild allergic reaction (one) or anemia (one). The mean follow up was 3.2 years (range: 1 month to 6 years); cumulative follow up was 684 patient-years (pt-yr) and was complete in 96% of cases. RESULTS: There were two episodes of thromboembolism in the ticlopidine group at 1 month and 6 months respectively, with a linearized incidence of 0.5% pt-yr. In the coumadin group there were four episodes of thromboembolism, three within the first three months of follow up. The linearized incidence was 3% pt-yr (p < 0.01). There were three episodes of hemorrhage in the ticlopidine group in the first three months of follow up and one in the coumadin group. The linearized incidence was 0.75% pt-yr. CONCLUSIONS: Following heart valve repair or replacement with a bioprosthesis, the first three months is a high-risk period for thromboembolism. Ticlopidine seems to prevent this complication better than conventional therapy with oral anticoagulants. Nevertheless, hemorrhage continues to be a problem with ticlopidine therapy.     

16S rRNA gene sequence of Rubrobacter radiotolerans and its phylogenetic alignment with members of the genus Arthrobacter, gram-positive bacteria, and members of the family Deinococcaceae

The nearly complete sequence of the 16S rRNA gene of an extremely highly radiotolerant bacterium, Rubrobacter radiotolerans (reclassified from Arthrobacter radiotolerans based on chemical characteristics), was determined by PCR amplification of the genomic DNA followed by cloning of the amplified gene and sequencing by the dideoxynucleotide method. The sequence was aligned with the sequences of members of the genus Arthrobacter and also with the sequences of representatives of the gram-positive bacteria having high G + C contents and the family Deinococcaceae (radioresistant micrococci and their relatives). The results of our phylogenetic analysis confirmed that R. radiotolerans is not a member of the Arthrobacter group and thus supported the previous reclassification. Moreover, although it is radioresistant and has a high G+C content, R. radiotolerans is more closely related to the gram-positive bacteria with high G+C contents than to the radioresistant members of the Deinococcaceae.     

Physicochemical interaction and structure development during the formation of metal gas-transfer coatings on diamond (review). 1. Kinetics

Work devoted to studying the phase composition and thickness of chromium, titanium, molybdenum, vanadium, and tungsten coatings which form on diamond powder during vacuum annealing of this powder mixed with chromium powder or oxidized powders of Ti, Mo, V, and W is analyzed. Coatings consist of metal (Cr, Ti, Mo, V, W) and carbide (Cr₇C₃, Cr₃C₂, TiC, -Mo₂C, V₂C, VC, W₂C, WC) phases. Diffusion of carbon during coating growth with increased metallizing time and temperature causes carbidization of chromium, titanium, and vanadium (it causes a reduction in the content of metal phase and an increase in carbide phases is coatings), growth of higher carbides (Cr₃C₂, VC, WC) at the expense of lower carbides (Cr₇C₃, V₂C, W₂C), and filling of carbon vacancies in the lattices of TiC and VC. Saturation of coatings with carbides correlates with the temperature-time range in which a further increase in coating weight slows down.Translated from Poroshkovaya Metallurgiya, No. 7(355), pp. 34–40, July, 1992.     

A 6.1 kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.     

Selective structure of the gene pool. III. Technology of determination from F(ST)-statistics using numerical resampling

The selection pressure on the ith gene was determined on the basis of the heterogeneity of interpopulation diversity FST(i), assumpting that the significant deviation of FST(i) (observed differentiation of the ith gene) from Fe (selectively neutral differentiation) indicates selection. A selectively neutral variation Fe was estimated from the mean FST, averaged over the total set of genes. Since the validity of the equation FST approximately Fe = (4NeMe + 1)-1 depends on the fulfillment of conditions of the theoretical model as well as on organization of sampling of genes and subpopulations rather than on statistical parameters of the gene sample, an additional correction by boot-strapping is suggested. Simulation of sampling of the gene pool using numerical resampling (10(6) samples from each of the 50 markers) allowed us to characterize the selective structure of the gene pool. This structure consists of the following: one class of selectively neutral genes, NEUTRAL (FST(i) approximately Fe); and two classes of selective genes, LOWER DIFF (FST(i) < Fe) and SUPER DIFF (FST(i) > Fe). Interclass regions N-LOWER and N-SUPER between the classes of neutral and selective genes were defined with the use of bootstrapping. The hypothesis on the presence or absence of selection cannot be formulated with regard to the genes included in the interclass regions. A general trend for all gene pools of the world was revealed: the ratio between the boundary values of selective classes and Fe is constant. On the basis of this, a rapid and simple technique for estimating selective values of genes was developed. The positions of more than 80 immunobiochemical gene markers in the selective structure of six gene pools of the world (Europe, Asia, Africa, Australia, America, and northeastern Eurasia) are presented. The selective structure of five subregions of northeastern Eurasia (Caucasus, Europe, Ural, Siberia, and Central Asia) are characterized as well.     

Refined localization of the Batten disease gene (CLN3) by haplotype and linkage disequilibrium mapping to D16S288-D16S383 and exclusion from this region of a variant form of Batten disease with granular osmiophilic deposits

Haplotype analysis in a collaborative collection of 143 families with juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten (Spielmeyer-Vogt-Sj?gren) disease has permitted refined localization of the disease gene, CLN3, which was assigned to chromosome 16 in 1989. Recombination events in four maternal meioses delimit new flanking genetic markers for CLN3 which localize the gene to the chromosome interval 16p12.1-11.2 between microsatellite markers D16S288 and D16S383. This narrows the position of CLN3 to a region of 2.1 cM, a significant reduction from the previous best interval. Using haplotypes, analysis of the strong linkage disequilibrium that exists between genetic markers within the D16S288-D16S383 interval and CLN3 shows that CLN3 is in closest proximity to loci D16S299 and D16S298. Analysis of markers across the D16S288-D16S383 region in four families with a variant form of JNCL characterized histologically by cytosomal granular osmiophilic deposits (GROD) has excluded linkage of the gene locus to the CLN3 region of chromosome 16, suggesting that JNCL with GROD is not an allelic form of JNCL.     

Genetic diversity of the biofilm covering Montacuta ferruginosa (Mollusca, bivalvia) as evaluated by denaturing gradient gel electrophoresis analysis and cloning of PCR-amplified gene fragments coding for 16S rRNA

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the gamma-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.