Phylogenetic and recombination analysis of Begomoviruses associated with Cotton leaf curl disease and in silico analysis of viral-host protein interactions.

Cotton leaf curl disease (CLCuD), caused by numerous begomoviruses (BGVs), is a highly disastrous disease in cotton crops worldwide. To date, several efforts have shown limited success in controlling this disease. CLCuD-associated BGVs (CABs) are known for their high rate of intra and interspecific recombinations, which raises an urgent need to find an efficient and conserved target region to combat disease. In the present study, phylogenetic analysis of selected 11 CABs, along with associated alphasatellites, and betasatellites revealed a close evolutionary relationship among them. Recombination analysis of 1374 isolates of CABs revealed 54 recombination events for the major players of CLCuD in cotton and the Cotton leaf curl Multan virus (CLCuMuV) as the most recombinant CAB. Recombination breakpoints were frequent in all regions except C2 and C3. C3-encoded protein, known as viral replication enhancer (REn), promotes viral replication by enhancing the activity of replicase (Rep) protein. Both proteins were found to contain significantly conserved domains and motifs. The identified motifs were found crucial for their interaction with host protein PCNA (Proliferating cell nuclear antigen), facilitating viral replication. Interruption at the REn-PCNA and Rep-PCNA interactions by targeting the identified conserved motifs is proposed as a prospect to halt viral replication, after suitable experimental validation.

The rising threat of geminiviruses: molecular insights into the disease mechanism and mitigation strategies.

BACKGROUND: Geminiviruses are among the most threatening emerging plant viruses, accountable for a huge loss to agricultural production worldwide. These viruses have been responsible for some serious outbreaks during the last few decades across different parts of the world. Sincere efforts have been made to regulate the disease incidence by incorporating a multi-dimensional approach, and this process has been facilitated greatly by the advent of molecular techniques. But, the mixed infection due to the polyphagous nature of vectors results in viral recombination followed by the emergence of novel viral strains which thus renders the existing mitigation strategies ineffective. Hence, a multifaceted insight into the molecular mechanism of the disease is really needed to understand the regulatory points; much has been done in this direction during the last few years. The present review aims to explore all the latest developments made so far and to organize the information in a comprehensive manner so that some novel hypotheses for controlling the disease may be generated. METHODS AND RESULTS: Starting with the background information, diverse genera of geminiviruses are listed along with their pathological and economic impacts. A comprehensive and detailed mechanism of infection is elaborated to study the interactions between vector, host, and virus at different stages in the life cycle of geminiviruses. Finally, an effort isalso made to analyze the progress made at the molecular level for the development of various mitigation strategies and suggest more effective and better approaches for controlling the disease. CONCLUSION: The study has provided a thorough understanding of molecular mechanism of geminivirus infection.

Molecular dynamics simulation-based trinucleotide and tetranucleotide level structural and energy characterization of the functional units of genomic DNA.

Genomes of most organisms on earth are written in a universal language of life, made up of four units - adenine (A), thymine (T), guanine (G), and cytosine (C), and understanding the way they are put together has been a great challenge to date. Multiple efforts have been made to annotate this wonderfully engineered string of DNA using different methods but they lack a universal character. In this article, we have investigated the structural and energetic profiles of both prokaryotes and eukaryotes by considering two essential genomic sites, viz., the transcription start sites (TSS) and exon-intron boundaries. We have characterized these sites by mapping the structural and energy features of DNA obtained from molecular dynamics simulations, which considers all possible trinucleotide and tetranucleotide steps. For DNA, these physicochemical properties show distinct signatures at the TSS and intron-exon boundaries. Our results firmly convey the idea that DNA uses the same dialect for prokaryotes and eukaryotes and that it is worth going beyond sequence-level analyses to physicochemical space to determine the functional destiny of DNA sequences.

Intron exon boundary junctions in human genome have in-built unique structural and energetic signals.

Precise identification of correct exon-intron boundaries is a prerequisite to analyze the location and structure of genes. The existing framework for genomic signals, delineating exon and introns in a genomic segment, seems insufficient, predominantly due to poor sequence consensus as well as limitations of training on available experimental data sets. We present here a novel concept for characterizing exon-intron boundaries in genomic segments on the basis of structural and energetic properties. We analyzed boundary junctions on both sides of all the exons (3 28 368) of protein coding genes from human genome (GENCODE database) using 28 structural and three energy parameters. Study of sequence conservation at these sites shows very poor consensus. It is observed that DNA adopts a unique structural and energy state at the boundary junctions. Also, signals are somewhat different for housekeeping and tissue specific genes. Clustering of 31 parameters into four derived vectors gives some additional insights into the physical mechanisms involved in this biological process. Sites of structural and energy signals correlate well to the positions playing important roles in pre-mRNA splicing.

A novel method SEProm for prokaryotic promoter prediction based on DNA structure and energetics.

MOTIVATION: Despite conservation in general architecture of promoters and protein-DNA interaction interface of RNA polymerases among various prokaryotes, identification of promoter regions in the whole genome sequences remains a daunting challenge. The available tools for promoter prediction do not seem to address the problem satisfactorily, apparently because the biochemical nature of promoter signals is yet to be understood fully. Using 28 structural and 3 energetic parameters, we found that prokaryotic promoter regions have a unique structural and energy state, quite distinct from that of coding regions and the information for this signature state is in-built in their sequences. We developed a novel promoter prediction tool from these 31 parameters using various statistical techniques. RESULTS: Here, we introduce SEProm, a novel tool that is developed by studying and utilizing the in-built structural and energy information of DNA sequences, which is applicable to all prokaryotes including archaea. Compared to five most recent, diverged and current best available tools, SEProm performs much better, predicting promoters with an 'F-value' of 82.04 and 'Precision' of 81.08. The next best 'F-value' was obtained with PromPredict (72.14) followed by BProm (68.37). On the basis of 'Precision' value, the next best 'Precision' was observed for Pepper (75.39) followed by PromPredict (72.01). SEProm maintained the lead even when comparison was done on two test organisms (not involved in training for SEProm). AVAILABILITY AND IMPLEMENTATION: The software is freely available with easy to follow instructions (www.scfbio-iitd.res.in/software/TSS_Predict.jsp). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Toward a Universal Structural and Energetic Model for Prokaryotic Promoters.

With almost no consensus promoter sequence in prokaryotes, recruitment of RNA polymerase (RNAP) to precise transcriptional start sites (TSSs) has remained an unsolved puzzle. Uncovering the underlying mechanism is critical for understanding the principle of gene regulation. We attempted to search the hidden code in ∼16,500 promoters of 12 prokaryotes representing two kingdoms in their structure and energetics. Twenty-eight fundamental parameters of DNA structure including backbone angles, basepair axis, and interbasepair and intrabasepair parameters were used, and information was extracted from x-ray crystallography data. Three parameters (solvation energy, hydrogen-bond energy, and stacking energy) were selected for creating energetics profiles using in-house programs. DNA of promoter regions was found to be inherently designed to undergo a change in every parameter undertaken for the study, in all prokaryotes. The change starts from some distance upstream of TSSs and continues past some distance from TSS, hence giving a signature state to promoter regions. These signature states might be the universal hidden codes recognized by RNAP. This observation was reiterated when randomly selected promoter sequences (with little sequence conservation) were subjected to structure generation; all developed into very similar three-dimensional structures quite distinct from those of conventional B-DNA and coding sequences. Fine structural details at important motifs (viz. -11, -35, and -75 positions relative to TSS) of promoters reveal novel to our knowledge and pointed insights for RNAP interaction at these locations; it could be correlated with how some particular structural changes at the -11 region may allow insertion of RNAP amino acids in interbasepair space as well as facilitate the flipping out of bases from the DNA duplex.

Assessing genetic diversity among six populations of Gossypium arboreum L. using microsatellites markers.

Among the four cultivated cotton species, G. hirsutum (allotetraploid) presently holds a primary place in cultivation. Efforts to further improve this primary cotton face the constraints of its narrow genetic base due to repeated selective breeding and hence demands enrichment of diversity in the gene pool. G. arboreum (diploid species) is an invaluable genetic resource with great potential in this direction. Based on the dispersal and domestication in different directions from Indus valley, different races of G. arboreum have evolved, each having certain traits like drought and disease resistance, which the tetraploid cotton lack. Due to lack of systematic, race wise characterization of G. arboreum germplasm, it  has not been explored fully. During the present study, 100 polymorphic SSR loci were  used to genotype 95 accessions belonging to 6 races of G. arboreum producing 246 polymorphic alleles; mean number of effective alleles was 1.505. AMOVA showed 14 % of molecular variance among population groups, 34 % among individuals and remaining 52 % within individuals. UPGMA dendrogram, based on Nei's genetic distance, distributed the six populations in two major clusters of 3 populations each; race 'bengalense' was found more close to 'cernuum' than the others. The clustering of 95 genotypes by UPGMA tree generation as well as PCoA analysis clustered 'bengalense' genotypes into one group along with some genotypes of 'cernuum', while rest of the genotypes made separate clusters. Outcomes of this research should be helpful in identifying the genotypes for their further utilization in hybridization program to obtain high level of germplasm diversity.

Physiological traits associated with heat tolerance in bread wheat (Triticum aestivum L.).

Field experiments for evaluating heat tolerance-related physiological traits were conducted for two consecutive years using a mapping population of recombinant inbred lines (RILs) from the cross RAJ4014/WH730. Chlorophyll content (Chl) and chlorophyll fluorescence (CFL) were recorded under timely sown (TS) and late sown (LS) conditions. Late sowing exposes the terminal stage of plants to high temperature stress. Pooled analysis showed that CFL and Chl differed significantly under TS and LS conditions. The mean value of CFL (Fv/Fm) and Chl under both timely and late sown conditions were used as physiological traits for association with markers. Regression analysis revealed significant association of microsatellite markers viz., Xpsp3094 and Xgwm131 with coefficients of determination (R (2)) values for CFL (Fv/Fm) and Chl as 12 and 8 %, respectively. The correlation between thousand grain weight (TGW) with Chl and CFL were 14 and 7 % and correlation between grain wt./spike with Chl and CFL were 15 and 8 %, respectively. The genotypes showing tolerance to terminal heat stress as manifested by low heat susceptibility index (HSI = 0.43) for thousand grain weight, were also found having very low Chl, HSI (-0.52). These results suggest that these physiological traits may be used as a secondary character for screening heat-tolerant genotypes.

Studying fluctuating trajectories of optically confined passive tracers inside cells provides familiar active forces.

In recent years, there has been a growing interest in studying the trajectories of microparticles inside living cells. Among other things, such studies are useful in understanding the spatio-temporal properties of a cell. In this work, we study the stochastic trajectories of a passive microparticle inside a cell using experiments and theory. Our theory is based on modeling the microparticle inside a cell as an active particle in a viscoelastic medium. The activity is included in our model from an additional stochastic term with non-zero persistence in the Langevin equation describing the dynamics of the microparticle. Using this model, we are able to predict the power spectral density (PSD) measured in the experiment and compute active forces. This caters to the situation where a tracer particle is optically confined and then yields a PSD for positional fluctuations. The low frequency part of the PSD yields information about the active forces that the particle feels. The fit to the model extracts such active force. Thus, we can conclude that trapping the particle does not affect the values of the forces extracted from the active fits if accounted for appropriately by proper theoretical models. In addition, the fit also provides system properties and optical tweezers trap stiffness.

Enhanced shoot multiplication in Ficus religiosa L. in the presence of adenine sulphate, glutamine and phloroglucinol.

Ficus religiosa (Pipal) is a long-lived valuable multipurpose forest tree. The tree is exploited because of its religious, ornamental and medicinal value and the regeneration rate in natural habitat is low. An in vitro propagation protocol has been developed from nodal segments obtained from a 45-50-year old tree. The highest bud break frequency (100 %) followed by maximum number of multiple shoots (13.9) as well as length (2.47 cm) were obtained on Woody Plant medium (WPM) supplemented with 1.0 mg/l BAP along with 0.5 mg/l IAA. Two modifications in this medium resulted in enhanced shoot regeneration-one with 200 mg/l glutamine + 150 mg/l ADS (called as MM-1) giving 32.5 shoots per nodal explant while another modification-with 200 mg/l glutamine + 150 mg/l ADS + 100 mg/l phloroglucinol (called as MM-2) giving 35.65 shoots per explant. These two media were used for sub-culturing of shoots for 4 months. The rate of shoot multiplication was same during the first three sub-cultures on MM-1 and the shoots regenerated were healthy, afterwards shoot multiplication declined. While on MM-2, shoot multiplication declined after first sub-culture and shoots underwent the problem of early leaf fall. Rooting was best induced in micro-shoots excised from proliferated shoot cultures on semi-solid as well as liquid WPM modified with 2.0 mg/l IBA and 0.5 mg/l IAA. The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 25-30 days before transfer to soil conditions, where the established plants showed more than 90 % survival.

ChemGenome2.1: An Ab Initio Gene Prediction Software.

Gene prediction, also known as gene identification, gene finding, gene recognition, or gene discovery, is among one of the important problems of molecular biology and is receiving increasing attention due to the advent of large-scale genome sequencing projects. We designed an ab initio model (called ChemGenome) for gene prediction in prokaryotic genomes based on physicochemical characteristics of codons. In this chapter, we present the methodology of the latest version of this model ChemGenome2.1 (CG2.1). The first module of the protocol builds a three-dimensional vector from three calculated quantities for each codon-the double-helical trinucleotide base pairing energy, the base pair stacking energy, and an index of the propensity of a codon for protein-nucleic acid interactions. As this three-dimensional vector moves along any genome, the net orientation of the resultant vector should differ significantly for gene and non-genic regions to make a distinction feasible. The predicted putative protein-coding genes from above parameters are passed through a second module of the protocol which reduces the number of false positives by utilizing a filter based on stereochemical properties of protein sequences. The chemical properties of amino acid side chains taken into consideration are the presence of sp3 hybridized γ carbon atom, hydrogen bond donor ability, short/absence of δ carbon and linearity of the side chains/non-occurrence of bi-dentate forks with terminal hydrogen atoms in the side chain. The final prediction of the potential protein-coding genes is based on the frequency of occurrence of amino acids in the predicted protein sequences and their deviation from the frequency values of Swissprot protein sequences, both at monomer and tripeptide levels. The final screening is based on Z-score. Though CG2.1 is a gene finding tool for prokaryotes, considering the underlying similarity in the chemical and physical properties of DNA among prokaryotes and eukaryotes, we attempted to evaluate its applicability for gene finding in the lower eukaryotes. The results give a hope that the concept of gene finding based on physicochemical model of codons is a viable idea for eukaryotes as well, though, undoubtedly, improvements are needed.

Recombinant horse interleukin-4 and interleukin-10 induced a mixed inflammatory cytokine response in horse peripheral blood mononuclear cells.

BACKGROUND AND AIM: Interleukin (IL)-4 and IL-10 activate plethora of immune cells and induce the humoral immune response. However, recombinant version of horse IL-4 and IL-10 has not been investigated to understand their immunomodulating activities. This study aimed to produce recombinant horse mature IL-4 and IL-10 in Escherichia coli. Immune-modulating activities of recombinant horse IL-4 and IL-10 were investigated in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Equine PBMCs were stimulated with recombinant IL-4 and IL-10. A proliferation of PBMCs was measured by XTT assay and cytokines induction was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis displayed a molecular weight of 15 kDa for IL-4 and 19 kDa for IL-10. Recombinant IL-4 and IL-10 significantly induced cell proliferation at 250 ng/ml. The results demonstrated that IL-4 enhanced expression of interferon-gamma (IFN-γ), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10, while recombinant horse IL-10 induced expression of IL-6, IFN-γ, and TNF-α. CONCLUSION: The present study demonstrated that biologically active horse IL-4 and IL-10 could be produced in E. coli.

Promoter Trapping and Deletion Analysis Show Arabidopsis thaliana APETALA2 Gene Promoter Is Bidirectional and Functions as a Pollen- and Ovule-Specific Promoter in the Reverse Orientation.

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at -309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5' UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300 bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.

Micropropagation of Ficus religiosa L. via leaf explants and comparative evaluation of acetylcholinesterase inhibitory activity in the micropropagated and conventionally grown plants.

A high-frequency, season-independent, in vitro regeneration of Ficusreligiosa was developed, followed by comparative acetylcholinesterase inhibitory (AChEI) activity assay of the in vitro raised and conventionally grown plants. The use of AChEI activity is the most accepted strategy for the treatment of Alzheimer disease. Fully expanded, mature leaves were cut into different segments to initiate the cultures. The middle section of the leaf in vertical orientation with cut portion inserted inside the medium was found most suitable for direct shoot regeneration. Leaf explants responded with nearly consistent frequency (60-66.67 %) throughout the year. To obtain high frequency response with enhanced shoot multiplication rate, 32 plant growth regulator regimes were screened amongst which benzylaminopurine at 5.0 mg/l was found most suitable, yielding 100 % response and maximum number of shoots per explant (7.93); same concentration was also most supportive for repeated multiplication (6.53 shoots). The quality of the shoots and multiplication rate could be significantly enhanced (24.35 shoots) when adenine sulphate, glutamine and phloroglucinol, in an optimised concentration, were additionally supplemented. The clonal nature of the micropropagated plants was confirmed by random amplified polymorphic DNA analysis. A comparative analysis of AChEI activity was carried out amongst the methanolic extracts of stem segments of the mother plant, randomly selected seedlings of different age (4 and 6 months old) of the same mother plant and randomly selected micropropagated plants of different age (3 and 6 months age). The mother plant sample showed effective AChEI activity, with IC50 of 66.46 µg/ml while seedlings, of different age groups, performed poorly (6-month-old seedlings, Se-16M, yielded IC50 of 20,538.46 µg/ml, while two randomly selected 4 months' aged seedlings, Se-24M and Se-34M exhibited IC50 of 19,341.03 and 24,281.70 µg/ml). On the other hand, various micropropagated plants, 2 of 3 months (MiP-13M, MiP-23M) and 2 of 6 months (MiP-36M and MiP-46M) age behaved like the mother plant, exhibiting IC50 values of 71.87, 72.91, 67.65 and 69.65 µg/ml, respectively.