Identification of DNA polymorphisms associated with the V type alpha1-antitrypsin gene.
alpha1-Antitrypsin (alpha1-AT) is a highly polymorphic protein. The V allele of alpha1-AT has been shown to be associated with focal glomerulosclerosis (FGS) in Negroid and mixed race South African patients. To identify mutations and polymorphisms in the gene for the V allele of alpha1-AT in five South African patients with FGS nephrotic syndrome DNA sequence analysis and restriction fragment length polymorphisms of the coding exons were carried out. Four of the patients were heterozygous for the BstEII RFLP in exon III [M1(Val213)(Ala213)] and one patient was a M1(Ala213) homozygote. The mutation for the V allele was identified in exon II as Gly-148 (GGG)-->Arg (AGG) and in all patients was associated with a silent mutation at position 158 (AAC-->AAT). The patient who was homozygous for (Ala213) also had a silent mutation at position 256 in exon III (GAT-->GAC) which was not present in any of the other four patients. Although the V allele of alpha1-AT is not associated with severe plasma deficiency, it may be in linkage disequilibrium with other genes on chromosome 14 that predispose to FGS. Furthermore, the associated silent mutation at position 158 and the Ala213 polymorphism are of interest, as these could represent an evolutionary intermediate between the M1(Ala213) and M1(Val213) subtypes. (+info)
Identification of a cytolethal distending toxin gene locus and features of a virulence-associated region in Actinobacillus actinomycetemcomitans.
A genetic locus for a cytolethal distending toxin (CDT) was identified in a polymorphic region of the chromosome of Actinobacillus actinomycetemcomitans, a predominant oral pathogen. The locus was comprised of three open reading frames (ORFs) that had significant amino acid sequence similarity and more than 90% sequence identity to the cdtABC genes of some pathogenic Escherichia coli strains and Haemophilus ducreyi, respectively. Sonic extracts from recombinant E. coli, containing the A. actinomycetemcomitans ORFs, caused the distension and killing of Chinese hamster ovary cells characteristic of a CDT. Monoclonal antibodies made reactive with the CdtA, CdtB, and CdtC proteins of H. ducreyi recognized the corresponding gene products from the recombinant strain. CDT-like activities were no longer expressed by the recombinant strain when an OmegaKan-2 interposon was inserted into the cdtA and cdtB genes. Expression of the CDT-like activities in A. actinomycetemcomitans was strain specific. Naturally occurring expression-negative strains had large deletions within the region of the cdt locus. The cdtABC genes were flanked by an ORF (virulence plasmid protein), a partial ORF (integrase), and DNA sequences (bacteriophage integration site) characteristic of virulence-associated regions. These results provide evidence for a functional CDT in a human oral pathogen. (+info)
Role of the angiotensin type 2 receptor gene in congenital anomalies of the kidney and urinary tract, CAKUT, of mice and men.
Angiotensin type 2 receptor gene null mutant mice display congenital anomalies of the kidney and urinary tract (CAKUT). Various features of mouse CAKUT impressively mimic human CAKUT. Studies of the human type 2 receptor (AGTR2) gene in two independent cohorts found that a significant association exists between CAKUT and a nucleotide transition within the lariat branchpoint motif of intron 1, which perturbs AGTR2 mRNA splicing efficiency. AGTR2, therefore, has a significant ontogenic role for the kidney and urinary tract system. Studies revealed that the establishment of CAKUT is preceded by delayed apoptosis of undifferentiated mesenchymal cells surrounding the urinary tract during key ontogenic events, from the ureteral budding to the expansive growth of the kidney and ureter. (+info)
Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and description of Mesorhizobium amorphae sp. nov.
Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-fixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplified 16S rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 16S rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16S rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 16S rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 16S rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov. (+info)
RFLP of rRNA genes and sequencing of the 16S-23S rDNA intergenic spacer region of ammonia-oxidizing bacteria: a phylogenetic approach.
It has been established that 16S rRNA gene-based phylogeny gives a low resolution between members of the chemoautotrophic ammonia-oxidizing bacteria (AOB) belonging to the beta-subclass of the Proteobacteria. In this study, 12 isolates of AOB were ribotyped, and the sequences of the 16S-23S rDNA intergenic spacer region (ISR) were determined and used in a phylogenetic study. 16S and 23S rDNA ribotyping revealed that the AOB studied contain only one rrn operon per genome, in contrast to most bacteria, which have 5-10 copies of the rRNA genes per genome. It is likely that the presence of only one set of rRNA genes is related to the slow growth of the AOB. The 16S and 23S rRNA genes of the AOB were shown to be arranged in the classical way: a 16S rRNA gene, an ISR and a 23S rRNA gene. Despite the close phylogenetic relationship among the AOB, the relative location of the rRNA genes in the genome appears to vary considerably. The size of the ISR was approximately 400 bp in the Nitrosomonas isolates and 645-694 bp in the Nitrosospira isolates, suggesting a species-specific size difference in the ISR. The ISR contained two potential tRNA genes in the 5' end in all isolates studied. The similarity values between the ISR sequences of the AOB are low (42.9-96.2%) compared with the 16S rDNA sequence similarity values, and therefore the ISR sequences are valuable as a complementary phylogenetic tool in combination with 16S rRNA gene sequences. The phylogenetic analysis of the AOB based on ISR sequences confirms the 16S rRNA gene-based phylogeny but has the benefit of giving a higher resolution. (+info)
Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers.
The identification and classification of yeasts have traditionally been based on morphological, physiological and biochemical traits. Various kits have been developed as rapid systems for yeast identification, but mostly for clinical diagnosis. In recent years, different molecular biology techniques have been developed for yeast identification, but there is no available database to identify a large number of species. In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts. In many cases, the size of the PCR products and the restriction patterns obtained with endonucleases CfoI, HaeIII and HinfI yielded a unique profile for each species. Accordingly, the use of this molecular approach is proposed as a new rapid and easy method of routine yeast identification. (+info)
Epidemiological characterization of methicillin-resistant Staphylococcus aureus isolated in the North West of England by protein A (spa) and coagulase (coa) gene polymorphisms.
In a comparative study, isolates of methicillin-resistant Staphylococcus aureus (MRSA) with known pulsed-field gel electrophoresis (PFGE) and bacteriophage type were analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) for additional discriminatory subtyping information. PFGE was previously performed using standardized, commercially available kits and pre-programmed software. Isolates were examined for coagulase (coa) and protein A (spa) gene polymorphisms following PCR amplification of the coa hypervariable and spa repeat regions. Coa gene RFLPs produced a total of 38 distinct combined patterns after digestion with HaeIII and AluI and identified the predominant epidemic (EMRSA) types 15 and 16. A unique HaeIII restriction site was identified by RFLP and sequence analysis in the coa gene for EMRSA 15 but not EMRSA 16. The spa gene PCR yielded a total of 14 different profiles ranging from 3-18 repeats with the 2 predominant EMRSA types falling into 2 distinct groups. PCR detection of coa and spa polymorphisms offer a rapid preliminary strain identification and discriminatory subtyping information for surveillance of MRSA. (+info)
Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.
We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones. (+info)