Mitochondrial Molecular Adaptations and Life History Strategies Coevolve in Plants

Messenger RNA secondary structure prevents mutations at functionally important sites. Mutations at exposed sites would cause micro-adaptations, niche-specialization, and therefore, can be thought to promote K-strategists. Exposing, rather than protecting, conserved sites, is also potentially adaptive because they probably promote macro-adaptive changes. This presumably fits r-strategists: their population dynamics tolerate decreased survival. We found that helix-forming tendencies are greater at evolutionary conserved sites of plant mitochondrial mRNAs than at evolutionary variable sites in a majority (73%) of species–gene combinations. K-strategists preferentially protect conserved sites in short genes, r-strategists protect them most in larger genes. This adaptive scenario resembles our earlier findings in chloroplast genes. Protection levels at various codon positions also display disparity with respect to life history strategies of the plants. Conserved site protection increases overall mRNA folding stabilities for some genes, while decreases it for some others. This contrast exists between homologous genes of r- and K- strategists. Such compensating interactions between variability, mRNA size, codon position, and secondary structure factors within r- and K-strategists are most likely, molecular adaptations of plants belonging to the two extreme life history strategies. Our results suggest coevolution between molecular and ecological adaptive strategies

Phylogenetic influence of complex, evolutionary models: a Bayesian approach

Molecular evolution recovers the history of living species by comparing genetic information, exploring genome structure and function from an evolutionary perspective. Here we infer substitution rates and ancestral reconstructions, to better understand mutation responses to some known biochemical phenomena. Mutation processes are commonly inferred using parsimony, maximum likelihood and Bayesian. Parsimony is not explicitly model-based, and is statistically biased due to unrealistic assumptions. The model-based maximum likelihood approaches become computationally inefficient while analyzing large or high-dimensional datasets, leaving little opportunities to incorporate complex evolutionary models. We implemented a posterior probability (Bayesian) approach that evaluates evolutionary models, applying it to primate mitochondrial genomes. The species nucleotide sequence data were augmented with ancestral states at the internal nodes of the phylogeny. We simplified probability calculations for substitution events along the branches by assuming that only up to one or two substitution events occurred per branch per site. These conditional pathway calculations introduce very little bias into the inferred reconstructions, while increasing the feasibility of incorporating complex evolutionary models with higher dimensions. Compositional bias tests, including functional predictions of ancestral tRNAs, show that ancestral sequences from the Bayesian approach are more biologically realistic than those reconstructed by maximum likelihood. To explore other model complexity, we allowed substitution rates to vary among sites by having a different model at each site. With a strand-symmetric model as the base model, asymmetric substitution probabilities for specific substitution types were varied among sites. This model would not be feasible with standard matrix exponentiation methods, particularly maximum likelihood. We observed for A--\u3eG and C--\u3eT substitutions almost linear, respectively, almost asymptotic responses (with some regional deviations). Note that the HMM models had no a priori response built in them. Observed responses fitted predictions from earlier gene by gene likelihood analyses. For A--\u3eG substitutions, deviations from the expected linear response correlated positively with the loop-forming propensity of the corresponding site in the mRNA secondary structure. In the COI region, C--\u3eT substitutions have a prominent dip, suggesting protection against mutations. The C--\u3eT substitution responses differed significantly between primate sub-groups defined based on their single genome A--\u3eG responses

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications

<p>Abstract</p> <p>Background</p> <p>Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems.</p> <p>Results</p> <p>We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in <it>Psilotum nudum </it>and <it>Nephroselmis olivacea </it>mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: <it>r</it>-strategists minimize mutation costs in large genes; <it>K</it>-strategists do the opposite.</p> <p>Conclusion</p> <p>Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure <it>a priori </it>biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for <it>r</it>- and <it>K-</it>strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of <it>K</it>-strategists, perhaps because domestication increases reproductive output.</p

Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

<p>Abstract</p> <p>Background</p> <p>Human Rad51 (RAD51), analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA (dsDNA) in the presence of certain nucleotide cofactors. ATP hydrolysis is not required for this process, because even ATP non hydrolysable analogs like AMP-PNP and ATPγS, support DNA unwinding. Even ADP, the product of ATP hydrolysis, feebly supports DNA unwinding.</p> <p>Results</p> <p>We find that human Rad52 (RAD52) stimulates RAD51 mediated DNA unwinding in the presence of all Adenine nucleotide cofactors, (except in AMP and no nucleotide conditions that intrinsically fail to support unwinding reaction) while enhancing aggregation of RAD51-dsDNA complexes in parallel. Interestingly, salt at low concentration can substitute the role of RAD52, in facilitating aggregation of RAD51-dsDNA complexes, that concomitantly also leads to better unwinding.</p> <p>Conclusion</p> <p>RAD52 itself being a highly aggregated protein perhaps acts as scaffold to bring together RAD51 and DNA molecules into large co-aggregates of RAD52-RAD51-DNA complexes to promote RAD51 mediated DNA unwinding reaction, when appropriate nucleotide cofactors are available, presumably through macromolecular crowding effects. Our work highlights the functional link between aggregation of protein-DNA complexes and DNA unwinding in RAD51 system.</p

Functional genomics screen with pooled shRNA library and gene expression profiling with extracts of Azadirachta indica identify potential pathways for therapeutic targets in head and neck squamous cell carcinoma

Tumor suppression by the extracts of Azadirachta indica (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that neem extracts and their associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell lines treated with crude neem leaf extracts, known for their anti-tumorigenic activity. We analyzed differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing. We found 225 genes affected the cancer cell growth in the shRNA-infected cells treated with neem extract. Pathway enrichment analyses of whole-genome gene expression data from cells temporally treated with neem extract revealed important roles played by the TGF-β pathway and HSF-1-related gene network. Our results indicate that neem extract affects various important molecular signaling pathways in head and neck cancer cells, some of which may be therapeutic targets for this devastating tumor

A minimal set of internal control genes for gene expression studies in head and neck squamous cell carcinoma

Selection of the right reference gene(s) is crucial in the analysis and interpretation of gene expression data. The aim of the present study was to discover and validate a minimal set of internal control genes in head and neck tumor studies. We analyzed data from multiple sources (in house whole-genome gene expression microarrays, previously published quantitative real-time PCR (qPCR) data and RNA-seq data from TCGA) to come up with a list of 18 genes (discovery set) that had the lowest variance, a high level of expression across tumors, and their matched normal samples. The genes in the discovery set were ranked using four different algorithms (BestKeeper, geNorm, NormFinder, and comparative delta Ct) and a web-based comparative tool, RefFinder, for their stability and variance in expression across tissues. Finally, we validated their expression using qPCR in an additional set of tumor:matched normal samples that resulted in five genes (RPL30, RPL27, PSMC5, MTCH1, and OAZ1), out of which RPL30 and RPL27 were most stable and were abundantly expressed across the tissues. Our data suggest that RPL30 or RPL27 in combination with either PSMC5 or MTCH1 or OAZ1 can be used as a minimal set of control genes in head and neck tumor gene expression studies

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell’s viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell’s viper studied previously, we found 45–100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers

A comparative approach to elucidate chloroplast genome replication

Abstract Background Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts. Results We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Small Single Copy (SSC) and the Large Single Copy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication. Conclusion Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population</p

Augmenting transcriptome assembly by combining de novo and genome-guided tools

Researchers interested in studying and constructing transcriptomes, especially for non-model species, face the conundrum of choosing from a number of available de novo and genome-guided assemblers. None of the popular assembly tools in use today achieve requisite sensitivity, specificity or recovery of full-length transcripts on their own. Here, we present a comprehensive comparative study of the performance of various assemblers. Additionally, we present an approach to combinatorially augment transciptome assembly by using both de novo and genome-guided tools. In our study, we obtained the best recovery and most full-length transcripts with Trinity and TopHat1-Cufflinks, respectively. The sensitivity of the assembly and isoform recovery was superior, without compromising much on the specificity, when transcripts from Trinity were augmented with those from TopHat1-Cufflinks