Complete nucleotide sequence of polyomavirus SA12

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.     

Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis

Callose (beta-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.     

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

Aim: The aim of this study was to develop a multiplex real‐time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens. Methods and Results: A primer set and three species‐specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia‐associated non‐Erysipelothrix spp. bacterial isolates. Cross‐reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection. Conclusion: The multiplex real‐time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive. Significance and Impact of the Study: The developed real‐time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.     

Role of a novel coiled-coil domain-containing protein CCDC69 in regulating central spindle assembly

The formation of the central spindle (or the spindle midzone) is essential for cytokinesis in animal cells. In this study, we report that coiled-coil domain-containing protein 69 (CCDC69) is implicated in controlling the assembly of central spindles and the recruitment of midzone components. Exogenous expression of CCDC69 in HeLa cells interfered with microtubule polymerization and disrupted the formation of bipolar mitotic spindles. Endogenous CCDC69 proteins were localized to the central spindle during anaphase. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and disrupted the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and polo-like kinase 1 (Plk1) at the central spindle. Aurora B kinase was found to bind to CCDC69 and this binding depended on the coiled-coil domains at the C-terminus of CCDC69. Further, disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Our results indicate that CCDC69 acts as a scaffold to regulate the recruitment of midzone components and the assembly of central spindles.Key words: CCDC69, aurora B, Plk1, central spindles, midzone components, cytokinesis     

Metabolic Syndrome Is Associated with Increased Oxo-Nitrative Stress and Asthma-Like Changes in Lungs

Epidemiological studies have shown an increased obesity-related risk of asthma. In support, obese mice develop airway hyperresponsiveness (AHR). However, it remains unclear whether the increased risk is a consequence of obesity, adipogenic diet, or the metabolic syndrome (MetS). Altered L-arginine and nitric oxide (NO) metabolism is a common feature between asthma and metabolic syndrome that appears independent of body mass. Increased asthma risk resulting from such metabolic changes would have important consequences in global health. Since high-sugar diets can induce MetS, without necessarily causing obesity, studies of their effect on arginine/NO metabolism and airway function could clarify this aspect. We investigated whether normal-weight mice with MetS, due to high-fructose diet, had dysfunctional arginine/NO metabolism and features of asthma. Mice were fed chow-diet, high-fat-diet, or high-fructose-diet for 18 weeks. Only the high-fat-diet group developed obesity or adiposity. Hyperinsulinemia, hyperglycaemia, and hyperlipidaemia were common to both high-fat-diet and high-fructose-diet groups and the high-fructose-diet group additionally developed hypertension. At 18 weeks, airway hyperresponsiveness (AHR) could be seen in obese high-fat-diet mice as well as non-obese high-fructose-diet mice, when compared to standard chow-diet mice. No inflammatory cell infiltrate or goblet cell metaplasia was seen in either high-fat-diet or high-fructose-diet mice. Exhaled NO was reduced in both these groups. This reduction in exhaled NO correlated with reduced arginine bioavailability in lungs. In summary, mice with normal weight but metabolic obesity show reduced arginine bioavailability, reduced NO production, and asthma-like features. Reduced NO related bronchodilation and increased oxo-nitrosative stress may contribute to the pathogenesis.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

RAS Transformation Requires CUX1-Dependent Repair of Oxidative DNA Damage

The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1^+/− MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1''s enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras^G12V and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.     

Callus Cultures from Zygotic Embryos of Costus speciosus and Their Morphogenetic Responses

Soft, nodular and hard types of calli were initiated on mature zygotic embryo explants of two tetraploid clones of Costus speciosus, of which, only the hard calli were amenable to morphogenetic responses. The two clones differed in their growth regulator requirements both for the initiation of calli and for shoot regeneration. De novo formation of both shoot bud meristems and somatic embryoids were observed. Latter were encased partially or fUlly by coleoptilar sheath. Embryoids could be isolated as discrete units. On maturity, a stock like appendage developed from the base and finally embryoids got detached from the subtending tissue. Both shoot-bud meristems and somatic embryoids developed into complete plantlets, the former upon sequential transfer of calli on Schenk and Hildebrandt’s (SH) basal medium containing lower levels of growth hormones, while the latter only on basal medium. These culture regenerants were subsequently transferred to the field. The morphogenetic behaviour of these two tetraploid clones reflects their marked genotypic difference inspite of their same ploidy status.     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

Molecular Assortment of Lens Species with Different Adaptations to Drought Conditions Using SSR Markers

The success of drought tolerance breeding programs can be enhanced through molecular assortment of germplasm. This study was designed to characterize molecular diversity within and between Lens species with different adaptations to drought stress conditions using SSR markers. Drought stress was applied at seedling stage to study the effects on morpho-physiological traits under controlled condition, where tolerant cultivars and wilds showed 12.8–27.6% and 9.5–23.2% reduction in seed yield per plant respectively. When juxtaposed to field conditions, the tolerant cultivars (PDL-1 and PDL-2) and wild (ILWL-314 and ILWL-436) accessions showed 10.5–26.5% and 7.5%–15.6% reduction in seed yield per plant, respectively under rain-fed conditions. The reductions in seed yield in the two tolerant cultivars and wilds under severe drought condition were 48–49% and 30.5–45.3% respectively. A set of 258 alleles were identified among 278 genotypes using 35 SSR markers. Genetic diversity and polymorphism information contents varied between 0.321–0.854 and 0.299–0.836, with mean value of 0.682 and 0.643, respectively. All the genotypes were clustered into 11 groups based on SSR markers. Tolerant genotypes were grouped in cluster 6 while sensitive ones were mainly grouped into cluster 7. Wild accessions were separated from cultivars on the basis of both population structure and cluster analysis. Cluster analysis has further grouped the wild accessions on the basis of species and sub-species into 5 clusters. Physiological and morphological characters under drought stress were significantly (P = 0.05) different among microsatellite clusters. These findings suggest that drought adaptation is variable among wild and cultivated genotypes. Also, genotypes from contrasting clusters can be selected for hybridization which could help in evolution of better segregants for improving drought tolerance in lentil.