The cloning and characterization of a second alpha-amylase of A-hydrophila JMP636

Aims: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA. Methods and Results: The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases. Conclusions: AmyB has been isolated, characterized and then compared to AmyA. Significance and Impact of Study: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of. which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA, variant, may be functional. (C) 2002 Elsevier Science B.V. All rights reserved.

Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.

The Cys282Tyr polymorphism in the HFE gene in Australian Parkinson's disease patients

Iron homeostasis is altered in Parkinson's disease (PD). The HFE protein is an important regulator of cellular iron homeostasis and variations within this gene can result in iron overload and the disorder known as hereditary haemochromatosis. We studied the Cys282Tyr single nucleotide polymorphism as a genetic risk factor for PD in two distinct and separately collected cohorts of Australian PD patients and controls. In the combined cohort comprising 438 PD patients and 485 control subjects, we revealed an odds ratio for possession of the 282Tyr allele of 0.61 (95% confidence interval, Cl = 0.42-0.90, P = 0.011) from univariate chi-squared and 0.59 (95% Cl = 0.39-0.90, P = 0.014) after logistic regression analyses (correcting for potential confounding factors). These results suggest that possession of the 282Tyr allele may offer some protection against the development of PD. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

The mitochondrial 12S gene is a suitable marker of populations of Sarcoptes scabiei from wombats, dogs and humans in Australia

We sequenced part of the mitochondrial 12S ribosomal RNA gene of 23 specimens of Sarcoptes scabiei from eight wombats, one dog and three humans. Twelve of the 326 nucleotide positions varied among these mites and there were nine haplotypes (sequences) that differed by 1-8 nucleotides. Phylogenetic analyses indicated that these mites were from two lineages: (1) mites from wombats from Victoria, Australia, and mites from the humans and dog from the Northern Territory, Australia (haplotypes 1-4, 9); and (2) mites from the humans and dog from the Northern Territory (haplotypes 5-8). Mites from the three different hosts (wombats, a dog and humans) had not diverged phylogenetically; rather, these mites had similar 12S sequences. Thus, we conclude that these mites from wombats, humans and a dog are closely related, and that they diverged from a common ancestor relatively recently. This conclusion is consistent with the argument that people and/or their dogs introduced to Australia the S. scabiei mites that infect wombats Australia. So, S. scabiei, which has been blamed for the extinction of populations of wombats in Australia, may be a parasitic mite that was introduced to Australia with people and/or their dogs. These data show that the mitochondrial 12S rRNA gene may be a suitable population marker of S. scabiei from wombats, dogs and humans in Australia.

Genetic variability in cultivated common bean beyond the two major gene pools

It is generally accepted that two major gene pools exist in cultivated common bean (Phaseolus vulgaris L.), a Middle American and an Andean one. Some evidence, based on unique phaseolin morphotypes and AFLP analysis, suggests that at least one more gene pool exists in cultivated common bean. To investigate this hypothesis, 1072 accessions from a common bean core collection from the primary centres of origin, held at CIAT, were investigated. Various agronomic and morphological attributes (14 categorical and 11 quantitative) were measured. Multivariate analyses, consisting of homogeneity analysis and clustering for categorical data, clustering and ordination techniques for quantitative data and nonlinear principal component analysis for mixed data, were undertaken. The results of most analyses supported the existence of the two major gene pools. However, the analysis of categorical data of protein types showed an additional minor gene pool. The minor gene pool is designated North Andean and includes phaseolin types CH, S and T; lectin types 312, Pr, B and K; and mostly A5, A6 and A4 types alpha-amylase inhibitor. Analysis of the combined categorical data of protein types and some plant categorical data also suggested that some other germplasm with C type phaseolin are distinguished from the major gene pools.

Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a

Voltage-gated sodium channels drive the initial depolarization phase of the cardiac action potential and therefore critically determine conduction of excitation through the heart. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene, SCN5A, have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing), atrioventricular conduction delay, and ventricular fibrillation. The pathophysiological basis of these clinical conditions is unresolved. Here we show that disruption of the mouse cardiac sodium channel gene, Scn5a, causes intrauterine lethality in homozygotes with severe defects in ventricular morphogenesis whereas heterozygotes show normal survival. Whole-cell patch clamp analyses of isolated ventricular myocytes from adult Scn5a(+/-) mice demonstrate a approximate to50% reduction in sodium conductance. Scn5a(+/-) hearts have several defects including impaired atrioventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. These findings reconcile reduced activity of the cardiac sodium channel leading to slowed conduction with several apparently diverse clinical phenotypes, providing a model for the detailed analysis of the pathophysiology of arrhythmias.

Testing the applicability of molecular genetic markers to population analyses of scleratinian corals

The abundance of coral reefs worldwide is in decline, and despite the ecological importance of reefs, only a limited number of DNA markers have been identified for scleractinian coral genetic studies. This paper addresses the search for new coral molecular markers and investigates the applicability of the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITS1), and the pocilloporin gene to the question of intraspecific variation in the scleractinian coral Pocillopora verrucosa along the southeast African coastline. The COI fragment was 710 bp long and was identical for P. verrucosa (n = 10) and P. damicornis (n = 3). Only two different ITS1 sequences were found (differing by 13 bp insertion), but more importantly, 24% of the sequences were heterogenous indicating that different multiple copies of the sequence exist. Pocilloporin is an intronless gene that was absolutely conserved throughout all P. verrucosa populations (n = 50). Thus, the three DNA regions studied appear unsuitable for the population genetic analyses of P. verrucosa.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

A case of myoepithelial carcinoma displaying biallelic inactivation of the tumour suppressor gene APC in a patient with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by mutation of the APC gene. It is characterised by the appearance of hundreds to thousands of colorectal adenomas in adolescence and the subsequent development of colorectal cancer. Various extracolonic malignancies are associated with FAP, including desmoids and neoplasms of the stomach, duodenum, pancreas, liver, and brain. We present a family affected by FAP with an exon 14 APC mutation displaying two rare extracolonic lesions, a hepatoblastoma and a myoepithelial carcinoma. The hepatoblastoma was found in a male patient aged 2 years. The second lesion, a myoepithelial carcinoma of the right cheek, was found in a female patient aged 14 years. Inactivation of the normal APC allele was demonstrated in this lesion by loss of heterozygosity analysis, thus implicating APC in the initiation or progression of this neoplasm. This is the first reported case of this lesion in a family affected by FAP.

D2 dopamine receptor gene polymorphism discriminates two kinds of novelty seeking

Cloninger's psychobiological model of personality as applied to substance misuse has received mixed support. Contrary to the model, recent data suggest that a combination of high novelty seeking (NS) and high harm avoidance (HA) represents a significant risk for the development of severe substance misuse. A genetic polymorphism previously implicated in severe substance dependence, the A1 allele of the D2 dopamine receptor (DRD2) gene, was examined in relation to NS and HA amongst 203 adolescent boys. Specifically, we hypothesized that subjects with the A1 + allele (A1/A1 and A1/A2 genotypes) would report stronger NS and would exhibit a more positive relationship between NS and HA than those with the A1-allele (A2/A2 genotypes). These predictions were supported. The correlation between NS and HA in 81 A1 + allelic boys (r = 0.27, P = 0.02), and that in the 122 A1- allelic boys (r = -0.15, P = 0.09), indicated that this relationship differed according to allelic status (F = 8.52, P < 0:004). Among those with the A1-allele, the present results are consistent with the traditional view that novelty seeking provides positive reinforcement, or the fulfillment of appetitive drives. In contrast, novelty seeking in those with the A1 + allele appears to include a negative reinforcement or self-medicating function. (C) 2002 Elsevier Science Ltd. All rights reserved.