Nuclear DNA content of Pongamia pinnata L. and genome size stability of in vitro-regenerated plantlets

Pongamia pinnata L. is a multipurpose versatile legume that is well known as a prospective feedstock biodiesel species. However, to date, there has been little genomic research aimed at the exploitation of the biotechnological potential of this species. Genetic characterization of any plant is a challenging task when there is no information about the genome size and organization of the species. Therefore, the genome size of P. pinnata was estimated by flow cytometry with respect to two standards (Zea mays and Pisum sativum), and compared with that of in vitro-raised plants (nodal segment, in vitro-rooted plantlets and acclimatized in vitro plants) to study the potential effect of somaclonal variation on genome size. This method can be used to support the establishment of true-to-type plants to encourage afforestation programs. Modified propidium iodide/hypotonic citrate buffer was used for isolation of the intact nuclei. The 2C DNA value of this species was estimated to be 2.51?±?0.01 pg. Statistically, there was no significant difference in the DNA content of the in vitro-grown plants and mother plant at α?=?0.05. As a result of the low genome size of P. pinnata, a species that has adapted itself to a wide range of edaphic and ecological condition, we can now proceed for its next generation sequencing and genomic diversity studies.     

Stress induced MAPK genes show distinct pattern of codon usage in Arabidopsis thaliana,Glycine max and Oryza sativa

Mitogen activated protein kinase (MAPK) genes provide resistance to various biotic and abiotic stresses. Codon usage profiling of the genes reveals the characteristic features of the genes like nucleotide composition, gene expressivity, optimal codons etc. The present study is a comparative analysis of codon usage patterns for different MAPK genes in three organisms, viz. Arabidopsis thaliana, Glycine max (soybean) and Oryza sativa (rice). The study has revealed a high AT content in MAPK genes of Arabidopsis and soybean whereas in rice a balanced AT-GC content at the third synonymous position of codon. The genes show a low bias in codon usage profile as reflected in the higher values (50.83 to 56.55) of effective number of codons (N_c). The prediction of gene expression profile in the MAPK genes revealed that these genes might be under the selective pressure of translational optimization as reflected in the low codon adaptation index (CAI) values ranging from 0.147 to 0.208.     

Lipid rafts alter the stability and activity of the cholera toxin A1 subunit

Cholera toxin (CT) travels from the cell surface to the endoplasmic reticulum (ER) as an AB holotoxin. ER-specific conditions then promote the dissociation of the catalytic CTA1 subunit from the rest of the toxin. CTA1 is held in a stable conformation by its assembly in the CT holotoxin, but the dissociated CTA1 subunit is an unstable protein that spontaneously assumes a disordered state at physiological temperature. This unfolding event triggers the ER-to-cytosol translocation of CTA1 through the quality control mechanism of ER-associated degradation. The translocated pool of CTA1 must regain a folded, active structure to modify its G protein target which is located in lipid rafts at the cytoplasmic face of the plasma membrane. Here, we report that lipid rafts place disordered CTA1 in a functional conformation. The hydrophobic C-terminal domain of CTA1 is essential for binding to the plasma membrane and lipid rafts. These interactions inhibit the temperature-induced unfolding of CTA1. Moreover, lipid rafts could promote a gain of structure in the disordered, 37 °C conformation of CTA1. This gain of structure corresponded to a gain of function: whereas CTA1 by itself exhibited minimal in vitro activity at 37 °C, exposure to lipid rafts resulted in substantial toxin activity at 37 °C. In vivo, the disruption of lipid rafts with filipin substantially reduced the activity of cytosolic CTA1. Lipid rafts thus exhibit a chaperone-like function that returns disordered CTA1 to an active state and is required for the optimal in vivo activity of CTA1.     

Gene expression,nucleotide composition and codon usage bias of genes associated with human Y chromosome

Analysis of codon usage pattern is important to understand the genetic and evolutionary characteristics of genomes. We have used bioinformatic approaches to analyze the codon usage bias (CUB) of the genes located in human Y chromosome. Codon bias index (CBI) indicated that the overall extent of codon usage bias was low. The relative synonymous codon usage (RSCU) analysis suggested that approximately half of the codons out of 59 synonymous codons were most frequently used, and possessed a T or G at the third codon position. The codon usage pattern was different in different genes as revealed from correspondence analysis (COA). A significant correlation between effective number of codons (ENC) and various GC contents suggests that both mutation pressure and natural selection affect the codon usage pattern of genes located in human Y chromosome. In addition, Y-linked genes have significant difference in GC contents at the second and third codon positions, expression level, and codon usage pattern of some codons like the SPANX genes in X chromosome.     

Context dependent function of APPb enhancer identified using enhancer trap-containing BACs as transgenes in zebrafish

An enhancer within intron 1 of the amyloid precursor protein gene (APPb) of zebrafish is identified functionally using a novel approach. Bacterial artificial chromosomes (BACs) were retrofitted with enhancer traps, and expressed as transgenes in zebrafish. Expression from both transient assays and stable lines were used for analysis. Although the enhancer was active in specific nonneural cells of the notochord when placed with APPb gene promoter proximal elements its function was restricted to, and absolutely required for, specific expression in neurons when juxtaposed with additional far-upstream promoter elements of the gene. We demonstrate that expression of green fluorescent protein fluorescence resembling the tissue distribution of APPb mRNA requires both the intron 1 enhancer and ~28 kb of DNA upstream of the gene. The results indicate that tissue-specificity of an isolated enhancer may be quite different from that in the context of its own gene. Using this enhancer and upstream sequence, polymorphic variants of APPb can now more closely recapitulate the endogenous pattern and regulation of APPb expression in animal models for Alzheimer's disease. The methodology should help functionally map multiple noncontiguous regulatory elements in BACs with or without gene-coding sequences.     

Allele frequency for Cystic fibrosis in Indians vis-a/-vis global populations

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This gene encodes a protein involved in epithelial anion channel. Cystic fibrosis is the most common life-limiting genetic disorder in Caucasians; it also affects other ethnic groups like the Blacks and the Native Americans. Cystic fibrosis is considered to be rare among individuals from the Indian subcontinent. We analyzed a total of 29 world׳s populations for cystic fibrosis on the basis of gene frequency and heterozygosity. Among 29 countries Switzerland revealed the highest gene frequency and heterozygosity for CF (0.022, 0.043) whereas Japan recorded the lowest values (0.002, 0.004) followed by India (0.004, 0.008). Our analysis suggests that the prevalence of cystic fibrosis is very low in India.     

Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in the liver and secreted as biliary glycoprotein 1 (BGP1) via bile canaliculi (BCs). CEACAM1-LF is a 72 amino acid cytoplasmic domain mRNA splice isoform with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ceacam1^−/− or Ser503Ala transgenic mice have been shown to develop insulin resistance and nonalcoholic fatty liver disease; however, the role of the human equivalent residue, Ser508, in lipid dysregulation is unknown. Human HepG2 hepatocytes that express CEACAM1 and form BC in vitro were compared with CEACAM1^−/− cells and CEACAM1^−/− cells expressing Ser508Ala null or Ser508Asp phosphorylation mimic mutations or to phosphorylation null mutations in the tyrosine ITIMs known to be phosphorylated by the tyrosine kinase Src. CEACAM1^−/− cells and the Ser508Asp and Tyr520Phe mutants strongly retained lipids, while Ser508Ala and Tyr493Phe mutants had low lipid levels compared with wild-type cells, indicating that the ITIM mutants phenocopied the Ser508 mutants. We found that the fatty acid transporter CD36 was upregulated in the S508A mutant, coexpressed in BCs with CEACAM1, co-IPed with CEACAM1 and Src, and when downregulated via RNAi, an increase in lipid droplet content was observed. Nuclear translocation of CD36 associated kinase LKB1 was increased sevenfold in the S508A mutant versus CEACAM1^−/− cells and correlated with increased activation of CD36-associated kinase AMPK in CEACAM1^−/− cells. Thus, while CEACAM1^−/− HepG2 cells upregulate lipid storage similar to Ceacam1^−/− in murine liver, the null mutation Ser508Ala led to decreased lipid storage, emphasizing evolutionary changes between the CEACAM1 genes in mouse and humans.     

Chronic obstructive pulmonary disease: A crosstalk on nucleotide compositional dynamics and codon usage patterns of the genes involved in disease

Chronic obstructive pulmonary disease (COPD), a lung disease, affects a large number of people worldwide, leading to death. Here, we analyzed the compositional features and trends of codon usage of the genes influencing COPD to understand molecular biology, genetics, and evolutionary relationships of these genes as no work was reported yet. Coding sequences of COPD genes were found to be rich in guanine-cytosine (GC) content. A high value (34-60) of the effective number of codons of the genes indicated low codon usage bias (CUB). Correspondence analysis suggested that the COPD genes were distinct in their codon usage patterns. Relative synonymous codon usage values of codons differed between the more preferred codons and the less-preferred ones. Correlation analysis between overall nucleotides and those at third codon position revealed that mutation pressure might influence the CUB of the genes. The high correlation between GC12 and GC3 signified that directional mutation pressure might have operated at all the three codon positions in COPD genes.