Genetic analyses for deciphering the status and role of photoperiodic and maturity genes in major Indian soybean cultivars.

Allelic combinations of major photoperiodic (E1, E3, E4) and maturity (E2) genes have extended the adaptation of quantitative photoperiod sensitive soybean crop from its origin (China ∼35◦N latitude) to both north (up to ∼50◦N) and south (up to 40◦S) latitudes, but their allelic status and role in India (6-35◦N) are unknown. Loss of function and hypoactive alleles of these genes are known to confer photoinsensitivity to long days and early maturity. Early maturity has helped to adapt soybean to short growing season of India. We had earlier found that all the Indian cultivars are sensitive to incandescent long day (ILD) and could identify six insensitive accessions through screening 2071 accessions under ILD. Available models for ILD insensitivity suggested that identified insensitive genotypes should be either e3/e4 or e1 (e1-nl or e1-fs) with either e3 or e4. We found that one of the insensitive accessions (EC 390977) was of e3/e4 genotype and hybridized it with four ILD sensitive cultivars JS 335, JS 95-60, JS 93-05, NRC 37 and an accession EC 538828. Inheritance studies and marker-based cosegregation analyses confirmed the segregation of E3 and E4 genes and identified JS 93-05 and NRC 37 as E3E3E4E4 and EC 538828 as e3e3E4E4. Further, genotyping through sequencing, derived cleaved amplified polymorphic sequences (dCAPS) and cleaved amplified polymorphic sequences (CAPS) markers identified JS 95-60 with hypoactive e1-as and JS 335 with loss of function e3-fs alleles. Presence of photoperiodic recessive alleles in these two most popular Indian cultivars suggested for their role in conferring early flowering and maturity. This observation could be confirmed in F2 population derived from the cross JS 95-60 × EC 390977, where individuals with e1-as e1-as and e4e4 genotypes could flower 7 and 2.4 days earlier, respectively. Possibility of identification of new alleles ormechanism for ILD insensitivity and use of photoinsensitivity in Indian conditions have been discussed.

Identification of Indian sub-continent as hotspot for HCV genotype 3a origin by Bayesian evolutionary reconstruction.

BACKGROUND: Recent emphasis in Hepatitis C virus (HCV) evolutionary biology has focused on analysis using Core, E1/E2 and/or NS5b regions, with limited appreciation of full length genome. While HCV genotypes have been described as endemic in the Indian subcontinent, there has been no confirmation at the molecular evolutionary level of these genotypes. We have attempted here to determine the status of Indian HCV genotype 3a sequences in relation to similar genotypes from other parts of the world. METHODS: Cloning, sequencing and molecular characterization was performed on 9 Indian sequences and comparative analyses were performed with 46 sequences from other countries. Evolutionary-rate and molecular-clock hypothesis testing was addressed by Bayesian MCMC. RESULTS: Genetic analysis of full length genome revealed two hypervariable regions (HVR) in E2 region - HVR496 and HVR576, with a variable 5-8 amino-acid insertion sequence and a putative N-glycosylation site. Phylogenetic analysis revealed a divergence resulting in 2 distinct clades: clade-1 represented by HCV 3a subtype and clade-2 represented by other 3 subtype genomes. Clade-2 shows earlier divergence than clade-1. Analysis revealed that genotype 3a genomes from India roots out first (∼99 years ago) in clade1. Bayesian skyline plot analysis revealed an increase in effective number of infections from 1940s to 1990s, followed by a gradual decrease after 2000. CONCLUSIONS: Genotype 3a sequences appear to have originated in India and later dispersed to United Kingdom around mid 1940s, most likely around the time of Indian independence and World War II.

Cell cycle regulation by the pro-apoptotic gene Scotin.

Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in a p53-dependent manner. In this report, we have used Drosophila as a model system to obtain a preliminary insight into the molecular mechanism of Scotin function, which was further validated using the mammalian system. Targeted expression of Scotin in developing Drosophila induced apoptosis and developmental defects in wings and eyes. Co-expression of Scotin with the anti-apoptotic protein p35, while inhibited the apoptosis in both dividing and non-dividing cells, rescued adult wing or eye phenotypes only when Scotin was expressed in non-dividing cells. This suggests that mechanisms of Scotin-induced apoptosis in dividing and non-dividing cells may vary. Suppressor-enhancer screen using cell cycle regulators suggested that Scotin may mediate cell cycle arrest at both G(1)/S and G(2)/M phases. Overexpression of Scotin in mammalian cells resulted in mitotic arrest and subsequently apoptosis. Furthermore, a larger proportion of cells overexpressing Scotin showed sequestration of Cyclin B1 in the cytoplasm. These results suggest that one of the ways by which Scotin induces apoptosis is by causing cell cycle arrest.

Cullin4B/E3-ubiquitin ligase negatively regulates beta-catenin.

Beta-catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, beta-catenin is targeted to ubiquitin-proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in beta-catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased beta-catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and beta-catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of beta-catenin levels.

Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase.

Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5'extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/modification of nuclear proteins.