Transcriptional status of known and novel genes tagged with consensus of 33.15 repeat loci employing minisatellite-associated sequence amplification (MASA) and real-time PCR in water buffalo, Bubalus bubalis

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.     

Sympathetic control after cardiac resynchronization therapy: responders versus nonresponders

Cardiac resynchronization therapy (CRT) decreases muscle sympathetic nerve activity (MSNA) in patients with severe congestive heart failure (CHF) and cardiac asynchrony. Whether this affects equally patients who clinically respond or not to CRT is unknown. We tested the hypothesis that the favorable effects of CRT on MSNA disappear on CRT interruption only in those who respond to CRT. Twenty-three consecutive CHF patients participated in the study, among whom 16 presented a symptomatic improvement by one or more New York Heart Association (NYHA) functional classes 15 +/- 5 mo after CRT (responders), and seven had not improved after 12 +/- 4 mo of CRT (nonresponders). MSNA and echocardiographic recordings were obtained in random order during atrio-right ventricular pacing (ARV), without stimulation in patients who were not pacemaker dependent (OFF, n = 17), and during atrio-biventricular pacing (BIV). Responders had a longer 6-min walking distance, a lower NYHA class and brain natriuretic peptide levels, and a better quality of life than did nonresponders (all P < 0.05). MSNA increased by 25 +/- 7% in the responders, whereas it remained unchanged in the nonresponders, when shifting from the BIV mode to a nonsynchronous condition (ARV and OFF modes) (P < 0.01). Cardiac output decreased by 0.7 +/- 0.2 l/min in the responders but did not change when shifting from the BIV mode to the nonsynchronous pacing mode in the nonresponders (P < 0.01). In conclusion, reversible sympathoinhibition is a marker of the clinical response to CRT.     

Characterization, mapping, and distribution of the two XMRV parental proviruses

Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1(n) receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.     

Molecular characterization of <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek genotypes based on nuclear ribosomal DNA and RAPD polymorphism

Mungbean germplasm characterization, evaluation and improvement are fundamentally based on morpho-agronomic traits. The lack of break-through in mungbean production has been due to non-availability of genetic variability for high yield potential. Forty-four genotypes of mungbean [Vigna radiata (L.)Wilczek] were subjected to random amplified polymorphic DNA (RAPD) analysis to assess the genetic diversity and relationships among the genotypes. Multilocus genotyping by twelve RAPD primers generated 166 markers and detected an average of intraspecific variation amounting to 82% polymorphism in banding patterns. Dendrogram obtained from cluster analysis delineated all the 44 genotypes into six clusters. Higher values of Nei’s gene diversity (h) and Shannon information index (i) and genetic distance analysis validate existence of wide genetic diversity among mungbean genotypes tested. Besides internal transcribed spacer (ITS) length variations, single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELS) were detected at number of sites in nuclear rDNA region and the sequences of representatives of each sub-cluster and all distinct genotypes have been submitted to NCBI database and assigned Gen accession numbers HQ 148136-148147. Multiple sequence alignment revealed further lineages of distinct genotypes with main RAPD clusters. The measures of relative genetic distances among the genotypes of mungbean did not completely correlate the geographical places of their development. The homogeneous phenotypic markers proved insufficient in exhibiting genetic divergence among mungbean genotypes studied. RMG-62, RMG-976, and NDM-56 have been identified as potential source of parents for crop improvement. RAPD primers, OPA-9 and OPA-2 as polymorphic genetic markers and number of pods/plant and number of seeds/plant as dependable phenotypic markers have been identified for improving yield potentials. This genetic diversity will be of significance in developing intraspecific crosses in mungbean crop improvement programme.     

Antioxidant responses of pea genotypes to zinc deficiency

The effects of Zn deficiency on antioxidant responses of two pea (Pisum sativum L.) genotypes, a Zn-efficient IPFD-99-13 and Zn-inefficient KPMR-500, grown in sand culture were studied. In the pea genotype KPMR-500, Zn deficiency decreased dry matter yield, tissue Zn concentration, and antioxidant enzyme activities istronger than in the genotype IPFD-99-13. Genotype IPFD-99-13 developed more efficient antioxidant system to scavenge ROS than genotype KPMR-500. Zinc deficiency produced oxidative damage to pea genotypes due to enhanced accumulation of TBARS and H₂O₂ and decreased activities of antioxidant enzymes (Cu/Zn superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX)). In the leaves of IPFD-99-13 genotype, the higher activity of ROS-scavenging enzyme, e.g., SOD, CAT, POD, and glutathione reductase, and antioxidants, such as ascorbate and non-protein thiols, led to the lower accumulation of H₂O₂ and lipid peroxides. These results suggest that, by maintaining an efficient antioxidant defense system, the IPFD-99-13 genotype shows a lower sensivity to Zn deficiency than the KPMR-500 genotype.     

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.     

FBIS: A regional DNA barcode archival & analysis system for Indian fishes

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. AVAILABILITY: The database is available for free at http://mail.nbfgr.res.in/fbis/