Effect of heat stress during flowering and pod formation in pea ( Pisum sativum L.)

Heat stress is a major constraint of yield in grain legumes including peas. Increasing global warming and human population now urge to develop climate resilient varieties. The present experiment was conducted over 2 years to evaluate the heat tolerance of 211 pea genotypes. In the present study, the field pea genotypes showed a wide variation for reproductive stage heat stress (RSHS) quantitative traits. Significant positive correlations were found between no. of seeds per plant and no. of pods per plant; seed diameter (mm) and 25-seed weight (g) in heat tolerant as well as heat susceptible genotypes. Principal component analysis revealed two major principal components contributed approximately 91% of total variations and heat tolerant and susceptible genotypes separately formed two major clusters. Stepwise multiple regression analysis revealed that no. of seeds per plant was the best predictor for no. of pods per plant. On the basis of four RSHS traits, the most prominent heat tolerant pea genotypes identified in the present study JP-625, IARI-2877, PMR-38 II, EC-318760, EC-328758 and IARI-2904 would better combat RSHS and provide yield stability under changing climatic conditions.     

Genomewide scan for nonsyndromic cleft lip and palate in multigenerational Indian families reveals significant evidence of linkage at 13q33.1-34

Nonsyndromic cleft lip with or without cleft palate (CL-P) is a common congenital anomaly with incidence ranging from 1 in 300 to 1 in 2,500 live births. We analyzed two Indian pedigrees (UR017 and UR019) with isolated, nonsyndromic CL-P, in which the anomaly segregates as an autosomal dominant trait. The phenotype was variable, ranging from unilateral to bilateral CL-P. A genomewide linkage scan that used approximately 10,000 SNPs was performed. Nonparametric linkage (NPL) analysis identified 11 genomic regions (NPL>3.5; P<.005) that could potentially harbor CL-P susceptibility variations. Among those, the most significant evidence was for chromosome 13q33.1-34 at marker rs1830756 (NPL=5.57; P=.00024). This was also supported by parametric linkage; MOD score (LOD scores maximized over genetic model parameters) analysis favored an autosomal dominant model. The maximum LOD score was 4.45, and heterogeneity LOD was 4.45 (alpha =100%). Haplotype analysis with informative crossovers enabled the mapping of the CL-P locus to a region of approximately 20.17 cM (7.42 Mb) between SNPs rs951095 and rs726455. Thus, we have identified a novel genomic region on 13q33.1-34 that harbors a high-risk variant for CL-P in these Indian families.     

Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution

ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either half-transporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "inside-out" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5- and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP > CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer.     

Structural and functional evidence for the role of the TLR2 DD loop in TLR1/TLR2 heterodimerization and signaling

The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the alphaD region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM(3)CSK(4) mediated NF-kappaB activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains.     

Cell cycle arrest and apoptosis by expression of a novel TPIP (TPIP-C2) cDNA encoding a C2-domain in HEK-293 cells

The human TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belongs to the PTEN (Phosphatase and TENsin homologue deleted on chromosome 10) family of dual-specific phosphatases and is expressed from the human chromosome 13 as multiple splice-variants, e.g., TPIPα, β, γ mRNAs. PTEN is a well characterized tumor suppressor, which controls survival, adhesion, motility and migration of mammalian cells, its C2-domain plays crucial role in controlling these functions. However, role of isolated C2-domain protein in regulation of cell proliferation and apoptosis is not reported. We report sequence analysis and function of a novel human TPIP (TPIP-C2) cDNA encoding a 193 amino acid C2-domain in cell proliferation and apoptosis regulation. In silico analysis and homology modelling revealed that the C2-domain of TPIP-C2 is similar to that of PTEN but with short disorder sequences overlapping or adjacent to the post-translational modification sites. Overexpression of TPIP-C2 cDNA in human embryonic kidney (HEK-293) cells caused cell cycle arrest, inhibition of cell proliferation and induced apoptosis in an activated caspase 3 and PARP-dependent manner in comparison to overexpression of the full length human PTEN cDNA. TPIP-C2 overexpressed cells also showed S-phase cell cycle arrest. We suggest that C2-domain of TPIP-C2 may act as a dominant negative effector, which may bind to and arrest the cell proliferation signalling complex and isolated TPIP-C2-domain-like proteins expressed in mammalian cells/tissues may play important role in regulation of cell proliferation and apoptosis. The TPIP-C2 cDNA may be exploited for inducing cell cycle-inhibition and apoptosis in human cancer cells and tissues.     

Draft genome sequence of Thermus sp. strain RL, isolated from a hot water spring located atop the Himalayan ranges at Manikaran, India

Thermus sp. strain RL was isolated from a hot water spring (90°C to 98°C) at Manikaran, Himachal Pradesh, India. Here we report the draft genome sequence (20,36,600 bp) of this strain. The draft genome sequence consists of 17 contigs and 1,986 protein-coding sequences and has an average G+C content of 68.77%.