M13 endopeptidases: New conserved motifs correlated with structure,and simultaneous phylogenetic occurrence of PHEX and the bony fish

M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures. Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis. This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome). New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated. Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures. Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue. Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX. Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates.     

Limitations on the recombinant plasmid selection by Lac(+)/Lac(-) colony phenotype detection

Lac(+)/Lac(-) selection of recombinant plasmids based on the insertional inactivation of LacZalpha gene cannot differentiate recombinant clones in some cases. Several fragments of exon 11 of human brca1 gene were cloned in LacZalpha-containing plasmids so that frameshift appeared at the 5(')-end of the fragments tested but these fragments were in frame with the part of LacZalpha situated downstream of the polylinker. All plasmids except one caused blue colonies formation after being transformed in Escherichia coli LacZDeltaM15 cells in spite of the frameshift. The fact may be explained by reinitiation of translation within the mRNA transcribed from the inserted DNA fragments at in-frame AUG, GUG, and UUG. The data demonstrated limitations on the Lac(+)/Lac(-) selection of LacZalpha-based recombinant plasmids.     

Differentiation of Arcobacter species by numerical analysis of AFLP profiles and description of a novel Arcobacter from pig abortions and turkey faeces

AIMS: To evaluate the efficacy of amplified fragment length polymorphism (AFLP)-based genetic profiling for taxonomic and epidemiological analyses of diverse Arcobacter species. METHODS AND RESULTS: Seventy-two isolates of A. butzleri, A. cryaerophilus, A. skirrowii and A. nitrofigilis, and a previously unclassified porcine abortion strain were studied. AFLP profiling was performed using a BglII-Csp6I-based protocol previously used to characterize Campylobacter species. Duplicate profiles of 20 isolates were 93.25% similar, indicating high reproducibility. Numerical analysis of all 72 strains revealed five phenons at the 29% similarity level, four of which represented each of the known species studied. The remaining phenon was further characterized by phenotypic and 16S rDNA sequence analyses, the results of which indicated it to be a novel Arcobacter species. The genetically distinct subgroups of A. cryaerophilus were differentiated at the 39.5% similarity level. For strain typing, 62 distinct types were defined, with evidence of clonal lineages within A. butzleri, A. cryaerophilus and A. skirrowii. CONCLUSIONS: AFLP profiling is an effective means of determining taxonomic and strain relationships for arcobacters. SIGNIFICANCE AND IMPACT OF THE STUDY: First use of AFLP profiling for diverse Arcobacter species; indication of clonality in A. butzleri, A. cryaerophilus and A. skirrowii; potentially novel Arcobacter taxon identified.     

Cross-contamination with Salmonella on a broiler slaughterhouse line demonstrated by use of epidemiological markers

AIMS: To investigate contamination of surfaces on a poultry slaughter line from infected poultry and subsequent cross-contamination of non-infected poultry. METHODS AND RESULTS: A broiler slaughterhouse was investigated for the presence of Salmonella on 17 defined points over two 1-week periods. Flocks supplied to slaughter and neck skin samples from processed chicken were likewise investigated. Salmonella was detected in 10 out of 18 flocks at ante-mortem inspection, while seven flocks tested positive in the finished products. Equipment at all but one control point at the slaughter line tested positive at least once during the study. The chicken receiving area was the most contaminated. By comparison of typing results from serotyping, plasmid profile typing and phage typing, direct evidence for cross-contamination with Salm. serotype Typhimurium, Salm. Serotype 4.12:b:- and Salm. serotype Virchow on the slaughter line was obtained for four of the flocks. The cleaning procedure in place did not remove all Salmonella from the contaminated areas. CONCLUSIONS: Evidence for contamination of equipment on a slaughter line and subsequent cross-contamination to non-infected chicken was provided by typing methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided detailed information on cross-contamination on a slaughter line by the use of phage typing and plasmid profiling. The study stresses the importance of controlling Salmonella in the primary production, as contamination of the equipment on the slaughter line will act as a vehicle to contaminate finished products. Cleaning procedures on slaughter lines cannot be expected to control this problem with the current equipment.     

Subunit E of mitochondrial ATP synthase: a bioinformatic analysis reveals a phosphopeptide binding motif supporting a multifunctional regulatory role and identifies a related human brain protein with the same motif

The mitochondrial adenosine triphosphate (ATP) synthase is located in the inner membrane and consists of at least 16 subunit types in animals, one of which is subunit e, the function of which is not clearly defined. A highly homologous protein is located in the nucleus and named progesterone receptor binding protein (RBF), to designate its role in this organelle. In addition, the expression level of subunit e in mammalian cells fluctuates greatly and is induced by certain carcinogens and elevated in liver cancers. Because these previous observations suggested to us that subunit e may play multifunctional regulatory roles, we employed a bioinformatic approach to test this view. First, from sequence alignment studies, secondary structure analyses, and basic local alignment search tool (BLAST) searches, we concluded that mitochondrial subunit e and the homologous nuclear protein RBF are most likely the same protein. Second, we examined the known sequence and structure of one of the most common multifunctional cell regulatory proteins, the 14-3-3 protein, involved in phosphopeptide binding, and deduced that it has an apparent binding motif (-KX(6)R---RY-). Third, from careful examination of the conserved residues within all subunit e sequences in the database, we discovered that this protein has a comparable binding motif (-RY---KX(6)R-). Finally, in a BLAST search for additional homologs of subunit e, we found a human brain protein, KIAA1578, the C-terminal 30 amino acids of which are identical to those of human subunit e. This protein also contains a potential phosphopeptide binding motif. In summary, these studies provide support for the view that subunit e is a multifunctional cell regulator involved in cell signaling, and implicate the involvement of the KIAA1578 protein in cell signaling as well. These studies suggest also that, while functioning as a subunit of mitochondrial ATP synthases, subunit e may help regulate these complexes by binding to phosphopeptides within one or more of the other subunit types.     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers     

Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676

The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed >99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1. Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.     

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki

A 4 kb BamHI-HindIII fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

VIRTUAL2D: A web-accessible predictive database for proteomics analysis

The available archive of sequence databases compiled from whole genome projects and budding proteomics efforts have enabled us to develop VIRTUAL2D, an interactive system for the assembly of virtual protein expression maps computed on the basis of theoretical isoelectric focusing point, molecular weight, tissue specificity and relative abundance for any set of proteins currently catalogued. This tool will assist in the preliminary, albeit putative, prediction of the identity and location of unknown and/or low abundance proteins in experimentally derived two-dimensional polyacrylamide gel electrophoresis maps.