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PubMed1993

Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein.

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described.

Amino Acid Sequence↗
PubMed1981

Integration of the bacteriophage phi 3T-coded thymidylate synthetase gene into the Bacillus subtilis chromosome.

Transformation of Bacillus subtilis 168 Thy- auxotrophs with phi 3T deoxyribonucleic acid (DNA) to thymine independence was found to involve site-specific recombination of phi 3T DNA sequences with their homologous counterparts in the bacterial chromosome. During the transformation, the phage phi 3T-encoded thymidylate synthetase gene, thyP3, was shown to integrate at two genetically distinct sites in the B. Subtilis 168 chromosome. The first site was identified to be in the bacterial thymidylate synthetase gene, thyA. The second site was in a prophage (SPB) known to be carried in the host genome. The frequency of the integration of the thyP3 gene at each of the two loci and some of the parameters affecting this frequency were studied. The common origin of the thyP3 and thyA genes and their molecular evolution are also reported.

Bacillus subtilis↗
PubMed2002

Cloning, characterisation and phylogenetic analysis of the fur gene in Vibrio salmonicida and Vibrio logei.

The gene encoding the ferric uptake regulator protein (fur gene) of Vibrio salmonicida 87/09/1193 was located following hybridisation of an EcoRI digest of chromosomal V. salmonicida DNA with a 316 base pairs (bp) probe internal to the fur gene of Vibrio anguillarum. A 2088 bp fragment including an open reading frame of 441 bp, encoding a protein of 147 amino acids, and homologous with fur, was identified, cloned and sequenced. A plasmid bound V. salmonicida fur gene was found capable of complementing the fur mutation of Escherichia coli H1681. Although no 'iron-box' was identified upstream of the start-codon, beta-galactosidase activity in E. coli H1681 was regulated by iron availability in the media, indicating that in V. salmonicida fur, as in other fur genes, iron functions as a co-repressor. Southern blot hybridizations demonstrated that fur is conserved amongst V. salmonicida strains and several other closely related Vibrio strains in which fur remains as yet, uncharacterized. The fur gene of Vibrio logei NCIMB 2252 was subsequently amplified using polymerase chain reaction primers external to the V. salmonicida fur gene. Comparison of phylogenetic analyses using fur and 16S DNA coding for rRNA sequences, confirmed the usefulness of fur as an evolutionary marker within the genus Vibrio.

Amino Acid Sequence↗
PubMed1984

A functional map of the nopaline synthase promoter.

This paper describes the first functional map of a promoter expressed from the plant chromosome. We have constructed a series of overlapping deletion mutants within the region upstream of the Ti-plasmid encoded nopaline synthase (nos) gene. By monitoring nos expression in tumour tissue we have inferred a functional map of the nos promoter. The maximum length of sequence upstream of the transcription initiation point required to express wild type levels of nopaline synthase is 88 bp. Within this region, the "CAAT" box is essential for maximal activity; deletion of this sequence reduced apparent nos expression by over 80%. Presence of an intact or partial "TATA" box in the absence of the "CAAT" box supports a barely detectable level of nopaline synthase. Removal of all sequences upstream of the nos coding sequence results in no detectable activity.

Amino Acid Oxidoreductases↗
PubMed1990

Characterization of the pilin gene of Moraxella bovis Dalton 2d and expression of pili from M. bovis in Pseudomonas aeruginosa.

The pilin gene of Moraxella bovis Dalton 2d was isolated by cloning in Pseudomonas aeruginosa. The nucleotide sequence of this gene encodes a prepilin of 156 amino acid residues. When high levels of pilin were expressed from the gene in P. aeruginosa, by using the pL promoter of bacteriophage lambda inserted upstream of the coding sequence, pili which were indistinguishable from pili of M. bovis were produced.

Amino Acid Sequence↗
PubMed1994

Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers.

To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.

Amino Acid Sequence↗
PubMed2005

Biochemical and molecular characterisation of the 2,3-dichloro-1-propanol dehalogenase and stereospecific haloalkanoic dehalogenases from a versatile Agrobacterium sp.

We previously reported the presence of both haloalcohol and haloalkanoate dehalogenase activity in the Agrobacterium sp. strain NHG3. The versatile nature of the organism led us to further characterise the genetic basis of these dehalogenation activities. Cloning and sequencing of the haloalcohol dehalogenase and subsequent analysis suggested that it was part of a highly conserved catabolic gene cluster. Characterisation of the haloalkanoate dehalogenase enzyme revealed the presence of two stereospecific enzymes with a narrow substrate range which acted on D-2-chloropropionic and L-2-chloropropionoic acid, respectively. Cloning and sequencing indicated that the two genes were separated by 87 bp of non-coding DNA and were preceded by a putative transporter gene 66 bp upstream of the D-specific enzyme.

Cloning, Molecular↗
PubMedDate not supplied

Induction of antibiotic resistance in Paramecium tetraurelia by the bacterial gene APH-3'-II.

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neor) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neor ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10-20 picoliters of linearized PXV-NEO at > or = 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neor-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neor-specific DNA and neor-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.

Animals↗
PubMed1997

The Gene-Finder computer tools for analysis of human and model organisms genome sequences.

We present a complex of new programs for promoter, 3'-processing, splice sites, coding exons and gene structure identification in genomic DNA of several model species. The human gene structure prediction program FGENEH, exon prediction-FEXH and splice site prediction-HSPL have been modified for sequence analysis of Drosophila (FGENED, FEXD and DSPL), C.elegance (FGENEN, FEXN and NSPL), Yeast (FEXY and YSPL) and Plant (FGENEA, FEXA and ASPL) genomic sequences. We recomputed all frequency and discriminant function parameters for these organisms and adjusted organism specific minimal intron lengths. An accuracy of coding region prediction for these programs is similar with the observed accuracy of FEXH and FGENEH. We have developed FEXHB and FGENEHB programs combining pattern recognition features and information about similarity of predicted exons with known sequences in protein databases. These programs have approximately 10% higher average accuracy of coding region recognition. Two new programs for human promoter site prediction (TSSG and TSSW) have been developed which use Gosh (1993) and Wingender (1994) data bases of functional motifs, respectively. POLYAH program was designed for prediction of 3'-processing regions in human genes and CDSB program was developed for bacterial gene prediction. We have developed a new approach to predict multiple genes based on double dynamic programming, that is very important for analysis of long genomic DNA fragments generated by genome sequencing projects. Analysis of uncharacterized sequences based on our methods is available through the University of Houston, Weizmann Institute of Science email servers and several Web pages at Baylor College of Medicine.

Animals↗
PubMed2004

Diverse members of the AvrBs3/PthA family of type III effectors are major virulence determinants in bacterial blight disease of rice.

AvrXa7 is a member of the avBs3/pthA gene family and the only known type III secretion system effector gene from Xanthomonas oryzae pv. oryzae with a major contribution to bacterial growth and lesion formation in bacterial blight disease of rice. We examined the general requirement for effectors of the AvrBs3/PthA family in bacterial blight of rice by identifying effectors from diverse strains of the pathogen. Inactivation of single effector genes in representative strains from Japan, Korea, and the Philippines resulted in severely limited growth in plants. Five strains harbored one gene of the avrBs3/pthA family, while one strain had two genes with the equivalent virulence activity of avrXa7. Sequence analysis revealed three genes with unique repeat arrangements in comparison to avrXa7. Comparison of the repetitive regions revealed a potential motif for the group that was also present in the repetitive region of avrBs3. However, the repetitive region of avrBs3 could not support virulence activity but, in combination with the C-terminal coding region of avrXa7, triggered a Xa7-dependent avirulence reaction. The results revealed diverse members of the avrBs3/pthA gene family with virulence activity in X. oryzae pv. oryzae and supported the hypothesis that bacterial blight disease of rice is highly dependent on a single class of type III effectors. The results also indicated that avrXa7 avirulence specificity is separable from virulence activity.

Bacterial Proteins↗
PubMed1997

Oligonucleotides identify conserved and variable regions of pspA and pspA-like sequences of Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is an immunogenic surface protein of Streptococcus pneumoniae. PspA of S. pneumoniae strain Rx1 is a 65-kDa protein composed of an alpha-helical N-terminus of 288 amino acids followed by an 82-amino-acid proline-rich region, 10 repeats of 20 amino acids each, and a 17-amino-acid C-terminus. It has been demonstrated that the 3'-half of pspA is relatively conserved among unrelated pneumococcal isolates and the 5'-half of the gene is highly variable. Additionally, nearly all pneumococcal strains contain at least one other locus with sequence homology to pspA. In this study oligonucleotides derived from the DNA sequence of pspA of Rx1 were used both as hybridization probes and as primers in the polymerase chain reaction (PCR) to investigate genetic variation within domains of pspA and in the pspA-like sequences from 18 strains representing 12 capsule and 9 PspA serotypes. Sequences encoding the leader peptide, the proline-rich region, and the repeat region are highly conserved among pspA and pspA-like sequences. The alpha-helical coding domain is highly diverse among pspA and pspA-like sequences of different strains.

Bacterial Proteins↗
PubMed2001

A highly specific one-step PCR - assay for the rapid discrimination of enteropathogenic Yersinia enterocolitica from pathogenic Yersinia pseudotuberculosis and Yersinia pestis.

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y pseudotuberculosis and Y pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y enterocolitica was developed. By this method pathogenic isolates of Y enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.

Animals↗
PubMed1990

Processes of genome evolution reflected by base frequency differences among Serratia marcescens genes.

The G + C content of silent sites in codons varies greatly among Serratia marcescens genes; the value in any one gene seems to reflect a balance between mutation pressure towards high G + C content and natural selection constraining choice among synonymous codons. Interestingly, non-coding sequences have substantially lower G + C content than silent sites thought to be under little selective constraint.

Base Composition↗
PubMed1984

Complete nucleotide sequence of mini-Rts1 and its copy mutant.

The nucleotide sequence of the whole mini-Rts1 genome consisting of 1,855 base pairs was determined. In addition to the cluster of five 24-base-pair direct repeats previously detected (Y. Kamio and Y. Terawaki, J. Bacteriol. 155:1185-1191, 1983), another cluster of three 21-base-pair direct repeats was found. All eight repeats were located in one direction and contained a consensus sequence TTCCCCPyPyPuPuCACACACC. Between the two clusters, a large open reading frame that could encode a 32,980-dalton polypeptide consisting of 288 amino acids was assigned. The molecular size predicted from the amino acid composition was close to the value of a unique mini-Rts1 product, the RepA protein, newly determined in minicells. A copy mutant of mini-Rts1 obtained in vitro by hydroxylamine treatment contained two-base-pair substitutions, one of which was located in the RepA protein coding region, and the other was close to the region where the oriC homologous sequences exist.

Ampicillin↗
PubMed1987

[Detailed mapping of the Hly-region of the plasmid pHly241. Homology of the hemolysis determinants of plasmids pHly241 and pHly195].

Data on the hemolysin synthesis determined by the plasmid pHly241, belonging to the incompatibility group I2, and its derivatives are presented in this paper. The restriction analysis of the pHly241 plasmid has resulted in the detailed mapping of the hly determinant region in the plasmid. The substantial homology has been demonstrated between the regions of the plasmids pHly241 and pHly195 coding for hemolysin synthesis and excretion from the bacterial cell.

Base Sequence↗
PubMed1990

Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili.

The polar pili of Pseudomonas aeruginosa are composed of monomers of the pilin structural subunits. The biogenesis of pili involves the synthesis of pilin precursor, cleavage of a six-amino-acid leader peptide, membrane translocation, and assembly of monomers into a filamentous structure extending from the bacterial surface. This report describes three novel genes necessary for the formation of pili. DNA sequences adjacent to pilA, the pilin structural gene, were cloned and mutagenized with transposon Tn5. Each of the insertions were introduced into the chromosome of P. aeruginosa PAK by gene replacement. The effect of the Tn5 insertions in the bacterial chromosome on pilus assembly was assessed by electron microscopy and sensitivity of mutants to a pilus-specific bacteriophage. The resultant mutants were also tested for synthesis and membrane localization of the pilin antigen in order to define the genes required for maturation, export, and assembly of pilin. A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. This region was sequenced and found to contain three open reading frames coding for 62-, 38- to 45-, and 28- to 32-kilodalton proteins (pilB, pilC, and pilD, respectively). Three proteins of similar molecular weight were expressed in Escherichia coli from the 4.0-kilobase-pair fragment flanking pilA with use of a T7 promoter-polymerase expression system. The results of the analyses of the three genes and the implications for pilin assembly and maturation are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1993

Selection of a 'minimal' glutaminyl-tRNA synthetase and the evolution of class I synthetases.

The evolution of the aminoacyl-tRNA synthetases is intriguing in light of their elaborate relationship with tRNAs and their significance in the decoding process. Based on sequence motifs and structure determination, these enzymes have been assigned to two classes. The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS), a class I enzyme, complexed to tRNA(Gln) and ATP has been described. It is shown here that a 'minimal' GlnRS, i.e. a GlnRS from which domains interacting with the acceptor-end and the anticodon of the tRNA have been deleted, has enzymatic activity and can charge a tRNA(Tyr)-derived amber suppressor (supF) with glutamine. The catalytic core of GlnRS, which is structurally conserved in other class I synthetases, is therefore sufficient for the aminoacylation of tRNA substrates. Some of these truncated enzymes have lost their ability to discriminate against non-cognate tRNAs, implying a more specific role of the acceptor-end-binding domain in the recognition of tRNAs. Our results indicate that the catalytic and substrate recognition properties are carried by distinct domains of GlnRS, and support the notion that class I aminoacyl-tRNA synthetases evolved from a common ancestor, jointly with tRNAs and the genetic code, by the addition of non-catalytic domains conferring new recognition specificities.

Amino Acyl-tRNA Synthetases↗