Molecular Cloning and Characterization of a Calmodulin Gene from Arabidopsis thaliana

The calcium binding protein, calmodulin Is involved in regulating various cellular and biochemical processes. A gene tor calmodulin (CaM) has been Isolated from a genomic library of Arabidopsis thaliana constructed in ; EMBL-4 using a heterologous cDNA probe from electric eel. One of the positive clones was characterized and the region containing the calmodulin gene sequences was Identified, excised using appropriate restriction enzymes and subcloned Into a plasmid vector. The genomic clone contains a complete copy of the calmodulin gene. A comparison of the nucleotide sequence of the part of the clone with those of the other plant and animal systems confirms that the clone In fact contains the calmodulin gene sequences. Southern hybridization ulling the calmodulin gene sequences as a probe reveals the presence of more than one copy of the calmodulin gene. The results of this investigation taken together with those Iff the other. indicate that the calmodulin gene belongs to a small mutigene family consisting of atieast four member. In the Arabidopsis genome.     

Aerotolerant growth inAzospirillum brasilense induced by dihydroxyphenyl iron-binding compound

High concentrations of phosphate drastically inhibited the aerobic growth ofAzospirillum brasilense in liquid medium having a limiting concentration of combined nitrogen. No growth inhibition occurred if a high concentration of combined nitrogen was present. Aerobic growth occurred only in the presence of a very low amount of phosphate and this could be enhanced by adding norepinephrine. Addition of norepinephrine enhanced the O₂ uptake by cell suspensions by almost a factor of 3. All of the intermediates of the tricarboxylic acid metabolic cycle were actively oxidized in cell-free extracts and their rate of oxidation increased in the presence of norepinephrine.     

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.     

Development and characterization of microsatellite markers from tropical forage Stylosanthes species and analysis of genetic variability and cross-species transferability

A limited number of functional molecular markers has slowed the desired genetic improvement of Stylosanthes species. Hence, in an attempt to develop simple sequence repeat (SSR) markers, genomic libraries from Stylosanthes seabrana B.L. Maass & 't Mannetje (2n=2x=20) using 5' anchored degenerate microsatellite primers were constructed. Of the 76 new microsatellites, 21 functional primer pairs were designed. Because of the small number of primer pairs designed, 428 expressed sequence tag (EST) sequences from seven Stylosanthes species were also examined for SSR detection. Approximately 10% of sequences delivered functional primer pairs, and after redundancy elimination, 57 microsatellite repeats were selected. Tetranucleotides followed by trinucleotides were the major repeated sequences in Stylosanthes ESTs. In total, a robust set of 21 genomic-SSR (gSSR) and 20 EST-SSR (eSSR) markers were developed. These markers were analyzed for intraspecific diversity within 20 S. seabrana accessions and for their cross-species transferability. Mean expected (He) and observed (Ho) heterozygosity values with gSSR markers were 0.64 and 0.372, respectively, whereas with eSSR markers these were 0.297 and 0.214, respectively. Dendrograms having moderate bootstrap value (23%-94%) were able to distinguish all accessions of S. seabrana with gSSR markers, whereas eSSR markers showed 100% similarities between few accessions. The set of 21 gSSRs, from S. seabrana, and 20 eSSRs, from selected Stylosanthes species, with their high cross-species transferability (45% with gSSRs, 86% with eSSRs) will facilitate genetic improvement of Stylosanthes species globally.     

Novel splice variant of mouse insulin2 mRNA: Implications for insulin expression

Insulin is a secreted peptide that controls glucose homeostasis in mammals, and insulin biosynthesis is regulated by glucose at many levels. Rodent insulin is encoded by two non-allelic genes. We have identified a novel splice variant of the insulin2 gene in mice that constitutes about 75% of total insulin2 mRNA. The alternate splicing does not alter the ORF but reduces the 5′UTR by 12 bases. A reporter gene containing the novel short 5′UTR, is more efficiently expressed in cells, suggesting that alternative splicing of insulin mRNA in mice could result in an additional level of regulation in insulin biosynthesis.     

Evaluation of genus <Emphasis Type="Italic">Cenchrus</Emphasis> based on malondialdehyde,proline content,specific leaf area and carbon isotope discrimination for drought tolerance and divergence of species at DNA level

Cenchrus (family Poaceae) is an important component of major grass covers of the world. Largely it is apomictic and both annual and perennial species exist in nature. Variations in contents of malondialdehyde, proline, specific leaf area and carbon isotope discrimination for drought tolerance were estimated among eight prominent species of Cenchrus. Simultaneously, genetic variations were also estimated by employing 187 RAPD primers. Of these, 23 primers did not react, 2 performed poorly and 7 produced many non-scorable bands and one primer yielded a single monomorphic band. Rest of the 154 primers generated one or more unambiguously scorable fragments. Twelve hundred and four of the 1,296 putative loci were polymorphic (93%) between at least one pair-wise comparisons among eight species. Dice coefficient and neighbor-joining algorithm analyses showed clustering patterns that fit with the known habitat of the species except perennial, C. myosuroides which formed a node between two annuals species. When these species were subjected to water stress tolerance test, a correlation (r = 0.612) between specific leaf area (SLA) and carbon isotope discrimination (CID) and difference in levels of drought tolerance based parameters among eight species were observed. Of the eight species investigated two annuals viz., C. biflorus and C. echinatus showed highest level of genetic similarity which was also evident from the similar levels of SLA, MDA, proline contents and carbon isotope discrimination values observed in these two species.     

Novel screening protocol for precise phenotyping of salt-tolerance at reproductive stage in rice

The present study reports an unequivocal and improved protocol for efficient screening of salt tolerance at flowering stage in rice, which can aid phenotyping of population for subsequent identification of QTLs associated with salinity stress, particularly at reproductive stage. To validate the new method, the selection criteria, level and time of imposition of stress; plant growth medium were standardized using three rice genotypes. The setup was established with a piezometer placed in a perforated pot for continuous monitoring of soil EC and pH throughout the period of study. Further, fertilizer enriched soil was partially substituted by gravels for stabilization and maintaining the uniformity of soil EC in pots without hindering its buffering capacity. The protocol including modified medium (Soil:Stone, 4:1) at 8 dS m^?1 salinity level was validated using seven different genotypes possessing differential salt sensitivity. Based on the important selection traits such as high stability index for plant yield, harvest index and number of grains/panicle and also high K⁺ concentration and low Na⁺– K⁺ ratio in flag leaf at grain filling stage were validated and employed in the evaluation of a mapping population in the modified screening medium. The method was found significantly efficient for easy maintenance of desired level of soil salinity and identification of genotypes tolerant to salinity at reproductive stage.     

Identification of Diploid <Emphasis Type="Italic">Stylosanthes seabrana</Emphasis> Accessions from Existing Germplasm of <Emphasis Type="Italic">S. scabra</Emphasis> Utilizing Genome-Specific STS Markers and Flow Cytometry,and Their Molecular Characterization

Stylosanthes seabrana (Maass and ‘t Mannetje) (2n = 2x = 20), commonly known as Caatinga stylo, is an important tropical perennial forage legume. In nature, it largely co-exist with S. scabra, an allotetraploid (2n = 4x = 40) species, sharing a very high similarity for morphological traits like growth habit, perenniality, fruit shape and presence of small appendage at the base of the pod or loment. This makes the two species difficult to distinguish morphologically, leading to chances of contamination in respective germplasm collections. In present study, 10 S. seabrana accessions were discovered from the existing global germplasm stock of S. scabra represented by 48 diverse collections, utilizing sequence-tagged-sites (STS) genome-specific markers. All the newly identified S. seabrana accessions displayed STS phenotypes of typical diploid species. Earlier reports have conclusively indicated S. seabrana and S. viscosa as two diploid progenitors of allotetraploid S. scabra. With primer pairs SHST3F3/R3, all putative S. seabrana yielded single band of ~550 bp and S. viscosa of ~870 bp whereas both of these bands were observed in allotetraploid S. scabra. Since SHST3F3/R3 primer pairs are known to amplify single or no band with diploid and two bands with tetraploid species, the amplification patterns corroborated that all newly identified S. seabrana lines were diploid in nature. Flow cytometric measurement of DNA content of the species, along with distinguishing morphological traits such as flowering time and seedling vigour, which significantly differ from S. scabra, confirmed all identified lines as S. seabrana. These newly identified lines exhibited high level of similarity among themselves as revealed by RAPD and STS markers (>92% and 80% respectively). Along with the enrichment in genetic resources of Stylosanthes, these newly identified and characterized accessions of S. seabrana can be better exploited in breeding programs targeted to quality.     

Glucose-stimulated translation regulation of insulin by the 5' UTR-binding proteins

Insulin is the key regulator of glucose homeostasis in mammals, and glucose-stimulated insulin biosynthesis is essential for maintaining glucose levels in a narrow range in mammals. Glucose specifically promotes the translation of insulin in pancreatic β-islet, and the untranslated regions of insulin mRNA play a role in such regulation. Specific factors in the β-islets bind to the insulin 5' UTR and regulate its translation. In the present study we identify protein-disulfide isomerase (PDI) as a key regulator of glucose-stimulated insulin biosynthesis. We show that both in vitro and in vivo PDI can specifically associate with the 5' UTR of insulin mRNA. Immunodepletion of PDI from the islet extract results in loss of glucose-stimulated translation indicating a critical role for PDI in insulin biosynthesis. Similarly, transient overexpression of PDI resulted in specific translation activation by glucose. We show that the RNA binding activity of PDI is mediated through PABP. PDI catalyzes the reduction of the PABP disulfide bond resulting in specific binding of PABP to the insulin 5' UTR. We also show that glucose stimulation of the islets results in activation of a specific kinase that can phosphorylate PDI. These findings identify PDI and PABP as important players in glucose homeostasis.