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PubMed1985

Physical and genetic analysis of DNA regions encoding the immunoglobulin A proteases of different specificities produced by Haemophilus influenzae.

The structural gene for immunoglobulin A protease (iga) from Haemophilus influenzae serotype d was cloned in pBR322. The gene was used as a probe for Southern hybridization analysis of chromosomal DNA from the five other H. influenzae serotypes (a, b, c, e, and f). In most cases strains from a single serotype exhibited a distinct pattern of restriction fragment(s) homologous to the iga gene probe which was unique for that serotype. Serotype f strains were unique in that they gave two distinct patterns of homologous restriction fragments which correlated well with the production of two different protease types by members of this group. An iga mutant of H. influenzae serotype d was isolated by introducing a 4-base-pair insertion into the cloned iga gene and using the altered DNA for transformation of an H. influenzae recipient. The resulting iga- mutant produced no immunoglobulin A protease but was otherwise indistinguishable from its iga+ parent in growth characteristics. Transformation of mutant cells with chromosomal DNA isolated from either a serotype d or a serotype c strain gave rise to iga+ transformants. Those obtained with serotype d DNA produced a type 1 protease, whereas those obtained with serotype c DNA produced either a type 1 protease (characteristic of serotype d) or a type 2 protease (characteristic of serotype c). Southern analysis of the latter transformants, using the iga gene probe, indicated that the type 1 transformants had a serotype d pattern of restriction fragments whereas the type 2 transformants had either a serotype c or a novel pattern of restriction fragments. These results indicate that there is considerable homology between the iga genes of the various serotypes and that the homologous sequences identified with the serotype d probe are the immunoglobulin A protease-coding sequences in each case.

Bacterial Proteins↗

PubMed1986

Expression of Escherichia coli infC: identification of a promoter in an upstream thrS coding sequence.

infC, the gene which codes for translation initiation factor 3, is situated in a cluster in the genome of Escherichia coli with genes for several other components of the translation apparatus. Only three nucleotides separate the termination codon of thrS from the initiation codon of infC. This implies that infC is either cotranscribed with thrS from a thrS promoter or that the transcriptional signals for infC are embedded within the upstream thrS coding region. In the present work, several plasmids have been constructed which encompass infC and various amounts of the upstream thrS sequence. The ability of the plasmid DNA, or derived restriction fragments, to direct the synthesis of initiation factor 3 was tested in an in vitro DNA-dependent coupled transcription-translation system and in plasmid-transformed maxicells. The results indicate that initiation factor 3 is synthesized in the absence of the thrS promoter. A promoter whose presence is sufficient for the expression of infC has been localized to an 89-base-pair region which lies 178 to 267 base pairs upstream of the infC initiation codon. S1 nuclease mapping of in vivo transcripts confirms that a transcription initiation site is located in this region. These studies demonstrate that infC can be transcribed from a promoter within the upstream thrS coding sequence.

Amino Acyl-tRNA Synthetases↗

PubMed2004

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum.

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.

Amino Acid Sequence↗

PubMed1997

Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene.

The Bacillus subtilis araC locus, mapped at about 294 degrees on the genetic map, was defined by mutations conferring an Ara- phenotype to strains bearing the metabolic araA, araB, and araD wild-type alleles (located at about 256 degrees on the genetic map) and by mutants showing constitutive expression of the three genes. In previous work, it has been postulated that the gene in which these mutations lie exerts its effect on the ara metabolic operon in trans, and this locus was named araC by analogy to the Escherichia coli regulatory gene. Here, we report the cloning and sequencing of the araC locus. This region comprises two open reading frames with divergently arranged promoters, the regulatory gene, araC, encoding a 41-kDa polypeptide, and a partially cloned gene, termed araE, which most probably codes for a permease involved in the transport of L-arabinose. The DNA sequence of araC revealed that its putative product is very similar to a number of bacterial negative regulators (the GalR-LacI family). However, a helix-turn-helix motif was identified in the N-terminal region by its identity to the consensus signature sequence of another group of repressors, the GntR family. The lack of similarity between the predicted primary structure of the product encoded by the B. subtilis regulatory gene and the AraC regulator from E. coli and the apparently different modes of action of these two proteins lead us to propose a new name, araR, for this gene. The araR gene is monocistronic, and the promoter region contains -10 and -35 regions (as determined by primer extension analysis) similar to those recognized by RNA polymerase containing the major vegetative cell sigma factor sigmaA. An insertion-deletion mutation in the araR gene leads to constitutive expression of the L-arabinose metabolic operon. We demonstrate that the araR gene codes for a negative regulator of the ara operon and that the expression of araR is repressed by its own product.

Amino Acid Sequence↗

PubMed1993

Cloning of the dihydroxyacid dehydratase-encoding gene (ILV3) from Saccharomyces cerevisiae.

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or alpha-ketobutyrate are converted into alpha-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes (ILV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (cM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ILV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILV genes have been cloned, thus allowing direct molecular analyses of their regulation.

Amino Acid Sequence↗

PubMed2002

Genetic analysis of jumbled spine and ribs (Jsr) mutation affecting the vertebral development in mice.

The jumbled spine and ribs (Jsr) mouse was derived from a spontaneous mutation. As the phenotype, a shortened trunk and kinky tail are characteristic Jsr traits. In this study, on high resolution mapping it was found that Lunatic fringe (Lfng) mapped at the same position as Jsr. Lfng was identified as the candidate gene for Jsr, but sequence analysis of this gene revealed no substitution in the coding region of cDNA. Therefore, we adopted the strategy of positional cloning for Jsr using a mouse bacterial artificial chromosome (BAC) library. A BAC contig was constructed from three BAC clones showing positive signals of Lfng and 11MMHAP75FRD8.seq near the Jsr locus on chromosome 5. Based on the genetic mapping of both T7 and sp6 ends of a clone of BAC382-O-7 (BAC382), the Jsr gene was considered to exist in BAC382 and to be positioned near the sp6 side.

Animals↗

PubMed1991

New adhesive factor (antigen 8786) on a human enterotoxigenic Escherichia coli O117:H4 strain isolated in Africa.

An enterotoxigenic Escherichia coli strain, E. coli 8786, of serotype O117:H4 produced only heat-stable enterotoxin and gave mannose-resistant hemagglutination with human and bovine erythrocytes. The strain adhered to the brush border of human enterocytes and to enterocytelike cell line Caco-2. Adhesion inhibition assays using Caco-2 cells with different adhesive factor extracts showed that the adhesive factor of E. coli 8786 is different from colonization factor antigen I (CFA/I). CFA/II, CFA/III of Darfeuille et al. (A. Darfeuille, B. Lafeuille, B. Joly, and R. Cluzel, Ann. Microbiol. Inst. Pasteur 134A:53-64, 1983), CS6, and antigen 2230. A bacterial surface protein, designated antigen 8786, with a molecular mass of 16,300 Da was responsible for the adhesion to intestinal cells. It was immunologically different from previously described adhesive factors as determined by immunoblotting. Antigen 8786 was detected on the bacterial cell surface and appeared to be nonfimbrial. NH2-terminal analysis of antigen 8786 showed no homology with the previously described adhesive factors. Nevertheless, antigen 8786 is closely related to the NH2-terminal sequence of Salmonella enteritidis fimbrin. A hybridization experiment using a synthetic oligonucleotide probe based on the NH2-terminal amino acid sequence of antigen 8786 revealed that the coding region was located on a 70-MDa plasmid.

Amino Acid Sequence↗

PubMed1987

Biosynthesis of bacterial glycogen: primary structure of Salmonella typhimurium ADPglucose synthetase as deduced from the nucleotide sequence of the glgC gene.

The nucleotide sequence of a 1.4-kilobase-pair fragment containing the Salmonella typhimurium LT2 glgC gene coding for ADPglucose synthetase was determined. The glgC structural gene contains 1,293 base pairs, having a coding capacity of 431 amino acids. The amino acid sequence deduced from the nucleotide sequence shows that the molecular weight of ADPglucose synthetase is 45,580. Previous results of the total amino acid composition analysis and amino acid sequencing (M. Lehmann and J. Preiss, J. Bacteriol. 143:120-127, 1980) of the first 27 amino acids from the N terminus agree with that deduced from nucleotide sequencing data. Comparison of the Escherichia coli K-12 and S. typhimurium LT2 ADPglucose synthetase shows that there is 80% homology in their nucleotide sequence and 90% homology in their deduced amino acid sequence. Moreover, the amino acid residues of the putative allosteric sites for the physiological activator fructose bisphosphate (amino acid residue 39) and inhibitor AMP (amino acid residue 114) are identical between the two enzymes. There is also extensive homology in the putative ADPglucose binding site. In both E. coli K-12 and S. typhimurium LT2, the first base of the translational start ATG of glgA overlaps with the third base TAA stop codon of the glgC gene.

Amino Acid Sequence↗

PubMed1993

Detecting frame shifts by amino acid sequence comparison.

Various amino acid substitution scoring matrices are used in conjunction with local alignments programs to detect regions of similarity and infer potential common ancestry between proteins. The usual scoring schemes derive from the implicit hypothesis that related proteins evolve from a common ancestor by the accumulation of point mutations and that amino acids tend to be progressively substituted by others with similar properties. However, other frequent single mutation events, like nucleotide insertion or deletion and gene inversion, change the translation reading frame and cause previously encoded amino acid sequences to become unrecognizable at once. Here, I derive five new types of scoring matrix, each capable of detecting a specific frame shift (deletion, insertion and inversion in 3 frames) and use them with a regular local alignments program to detect amino acid sequences that may have derived from alternative reading frames of the same nucleotide sequence. Frame shifts are inferred from the sole comparison of the protein sequences. The five scoring matrices were used with the BLASTP program to compare all the protein sequences in the Swissprot database. Surprisingly, the searches revealed hundreds of highly significant frame shift matches, of which many are likely to represent sequencing errors. Others provide some evidence that frame shift mutations might be used in protein evolution as a way to create new amino acid sequences from pre-existing coding regions.

Adenosine Triphosphatases↗

PubMed1998

Isolation and characterisation of an aryl-beta-D-glucoside uptake and utilisation system (abg) from the gram-positive ruminal Clostridium species C. longisporum.

A phosphotransferase-dependent aryl-beta-glucoside uptake and utilisation system (abg) was isolated from the ruminal Clostridium ("C. Longisporum"). The system is composed of three genes, abgG, abgF and abgA, and a number of regulatory regions, including terminator/antiterminator type stem-loop structures preceding the abgG and abgF genes. Similarity analysis of the proteins encoded by these genes indicated that they were responsible for the regulation of the abg system through antitermination (AbgG), the uptake and phosphorylation of aryl-beta-glucosides (AbgF) and the hydrolysis of the intracellular phosphorylated glycosides (AbgA). Experimental evidence for the functions of AbgF and AbgA was obtained. Although it was not possible to demonstrate any function for AbgG, a promoter 5' to the abgG gene was identified which was responsible for expression of the downstream genes. The abg system is remarkably similar to operons from the gram negative Enterobacteriaceae, both in the coding and non-coding regulatory regions.

Amino Acid Sequence↗

PubMed1989

Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA.

The metY gene coding for a minor form of the initiator tRNA is the first gene of a complex polycistronic operon also encoding the transcription termination factor NusA and the translation initiation factor IF2. The mixed tRNA-mRNA polycistronic transcript is cleaved by RNase III in a hairpin structure downstream from the tRNA. This cleavage separates the tRNA from the mRNA and initiates the rapid degradation of the 5' extremity of the downstream mRNA. Dissociation of the structural (tRNA) and informational (mRNA) RNAs from this operon is also achieved by independent transcription in vivo. The presence of two transcription terminators located downstream from metY produces a small tRNAMetf2 precursor transcript, whereas an internal promoter situated between metY and the first open reading frame directs the transcription of only the protein-coding part of the operon.

Bacterial Proteins↗

PubMed1984

A method for measuring the non-random bias of a codon usage table.

We describe a new statistical method for measuring bias in the codon usage table of a gene. The test is based on the multinomial and Poisson distributions. The method is used to scan DNA sequences and measure the strength of codon preference. For E. Coli we show that the strength of codon preference is related to levels of gene expression. The method can also be used to compare base triplet frequencies with those expected from the base composition. This second type of codon bias test is useful for distinguishing coding from non-coding regions.

Base Sequence↗

PubMed1978

HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli genome, and location of the tonB gene.

The HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli chromosome are presented. Analysis of phage DNAs carrying tonB mutations has allowed identification of a 1,730-base pair HindII fragment containing at least part of the tonB gene. This fragment is 4,020 base pairs from the end of trpA, with the total distance from attphi80 to trpA being 6,550 +/- 800 base pairs. Properties of hybrid plasmids containing insertions of various tonB+ restriction fragments suggest that tonB lies completely within the 1,730-base pair fragment. In addition, apparent fusions of beta-galactoside to proteins within the tonB region suggest that the entire region codes for more than one polypeptide.

Bacterial Proteins↗

PubMed1986

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose:alpha-1,4-glucan, 4-glucosyltransferase as deduced from the nucleotide sequence of the glgA gene.

The nucleotide sequence of the glgA gene, coding for glycogen synthase (EC 2.4.1.21) was elucidated. It consists of 1431 base pairs specifying a protein of 477 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis obtained with the pure protein as well as with the molecular weight as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal acid sequence and amino acid sequence analysis of various peptides obtained from CNBr degradation of purified glycogen synthase.

Amino Acid Sequence↗

PubMed1979

dnaG (primase)-dependent origins of DNA replication. Nucleotide sequences of the negative strand initiation sites of bacteriophages St-1, phi K, and alpha 3.

The simplest known origins of DNA replication occur in the single-stranded bacteriophages. In one set of phages, negative strand synthesis is initiated by a single protein, the product of the Escherichia coli replication gene dnaG. Evidently, in these phages--G4, St-1, phi K, and alpha 3--the origin for negative strand synthesis consists of a nucleic acid element capable of direct recognition by the dnaG priming protein. We have located and sequenced the origins of negative strand synthesis in St-1, phi K, and alpha 3, and compared them with the origin sequence previously determined for G4. In each case, the point at which the negative strand is initiated can be identified at the nucleotide level. The data lead to the following conclusions: 1. In all four phages, the negative strand initiation site occurs within an intercistronic region of approximately 135 bases. While in G4, the origin lies between genes specifying the viral coat proteins F and G, the origin is shifted in St-1, phi K, and alpha 3 to a position between coat protein genes G and H. 2. Extensive nucleotide conservation exists at the negative strand origin, but does not extend into the adjacent coding regions. The conserved origin DNA occurs in two regions, 42 and 45 bases long, which are separated by 13 bases of divergent sequence. 3. Correlated with the two stretches of conserved nucleotide sequence are two regions of potential secondary structure. The start point of negative strand synthesis lies just prior to one of these hairpins. Similarities in both primary sequence and secondary structure can be found between the negative strand origins of G4, St-1, phi K, and alpha 3 and the general origin regions of bacteriophage lambda and of E. coli.

Base Sequence↗

PubMed2004

Construction and characterization of transposon TnphoZ for the identification of genes encoding exported proteins in Streptococcus agalactiae.

Bacterial virulence often depends on exported proteins. To identify genes encoding exported proteins in the neonatal pathogen, group B streptococcus, the transposon TnphoZ was constructed. Here, the coding sequence for the secretion-dependent enzyme alkaline phosphatase from Enterococcus faecalis was fused to the left terminal repeat of Tn917, generating TnphoZ. A collection of TnphoZ mutants was isolated and the DNA flanking the transposon insertion sites was sequenced. Sequence data correlated the expression of high AP activity with transposon insertion into genes encoding predicted exported proteins. It is anticipated that TnphoZ will be suitable for use in other Gram-positive hosts.

Alkaline Phosphatase↗

PubMed2006

Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli.

Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.

Chromosomes, Artificial, Bacterial↗

PubMed1986

The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.

Amino Acid Sequence↗