Integration of genomic tools to assist breeding in the <Emphasis Type="Italic">japonica</Emphasis> subspecies of rice

The cultivated rice (Oryza sativa L.) has two subspecies, indica and japonica. The japonica rice germplasm has a narrower genetic diversity compared to the indica subspecies. Rice breeders aim to develop new varieties with a higher yield potential, with enhanced resistances to biotic and abiotic stresses, and improved adaptation to environmental changes. In order to face some of these challenges, japonica rice germplasm will have to be diversified and new breeding strategies developed. Indica rice improvement could also profit from more “genepool mingling” for which japonica rice could play an important role. Interesting traits such as low-temperature tolerance, and wider climate adaptation could be introgressed into the indica subspecies. In the past decade, huge developments in rice genomics have expanded our available knowledge on this crop and it is now time to use these technologies for improving and accelerating rice breeding research. With the full sequence of the rice genome, breeders may take advantage of new genes. Also new genes may be discovered from the genepool of wild relatives, or landraces of the genus Oryza, and incorporated into elite japonica cultivars in a kind of “gene revolution” program. Expectedly, new technologies that are currently being optimized, aiming for novel gene discovery or for tracking the regions under selection, will be suggested as new breeding approaches. This paper revisits breeding strategies successfully employed in indica rice, and discusses their application in japonica rice improvement (e.g. ideotype breeding, wide hybridization and hybrid performance).     

Age-related responses of hepatic ATPases to starvation and cold stress in male garden lizards

Hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of male green lizards declined during the maturation phase (juvenile to 1-year-old) and stabilized thereafter. On the other hand, the Ca²⁺-ATPase activity of the liver declined during the later half of the life span (1-year-old to 2–4-year-old). Starvation stress induced a decline in hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of juvenile lizards and caused an increase in 1-year-old and 2–4-year-old counterparts. The Ca²⁺-ATPase activity declined only in starved 1-year-old lizards. Following cold stress, the hepatic Na⁺-K⁺-ATPase activity of juvenile lizards showed a higher degree of decline than 2–4-year-old counterparts. The Mg²⁺-ATPase activity declined in cold-stressed juvenile lizards, but the parameter was elevated in similarly treated 1-year-old lizards. On the other hand, the increase in Ca²⁺-ATPase activity in response to cold stress was confined only to 2–4-year-old lizards.     

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.     

Turtle isochore structure is intermediate between amphibians and other amniotes

Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.     

SNAREs in opposing bilayers interact in a circular array to form conducting pores

The process of fusion at the nerve terminal is mediated via a specialized set of proteins in the synaptic vesicles and the presynaptic membrane. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. The structure and arrangement of these SNAREs associated with lipid bilayers were examined using atomic force microscopy. A bilayer electrophysiological setup allowed for measurements of membrane conductance and capacitance. Here we demonstrate that the interaction of these proteins to form a fusion pore is dependent on the presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane. However when t-SNARE vesicles were added to a v-SNARE membrane, SNAREs assembles in a ring pattern and a stepwise increase in capacitance, and increase in conductance were observed. Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion.     

Antagonistic Activity of Cellular Components of Potential Probiotic Bacteria, Isolated from the Gut of Labeo rohita, Against Aeromonas hydrophila

The objective of this study was to characterise the antagonistic activity of cellular components of potential probiotic bacteria isolated from the gut of healthy rohu (Labeo rohita), a tropical freshwater fish, against the fish pathogen, Aeromonas hydrophila. Three potential probiotic strains (referred to as R1, R2, and R5) were screened using a well diffusion, and their antagonistic activity against A. hydrophila was determined. Biochemical tests and 16S rRNA gene analysis confirmed that R1, R2, and R5 were Lactobacillus plantarum VSG3, Pseudomonas aeruginosa VSG2, and Bacillus subtilis VSG1, respectively. Four different fractions of cellular components (i.e. the whole-cell product, heat-killed whole-cell product [HKWCP], intracellular product [ICP], and extracellular product) of these selected strains were effective in an in vitro sensitivity test against 6 A. hydrophila strains. Among the cellular components, the ICP of R1, HKWCP of R2, and ICP of R5 exhibited the strongest antagonistic activities, as evidenced by their inhibition zones. The antimicrobial compounds from these selected cellular components were partially purified by thin-layer and high-performance liquid chromatography, and their properties were analysed. The ranges of pH stability of the purified compounds were wide (3.0–10.0), and compounds were thermally stable up to 90 °C. Considering these results, isolated probiotic strains may find potential applications in the prevention and treatment of aquatic aeromonosis.     

Effect of age on lipid peroxides, lipofuscin and ascorbic acid contents of the lungs of male garden lizard

Oxidative damage was assessed through the estimation of lipid peroxides (LP) in the lungs of an ageing short-lived species of reptile, Calotes versicolor, commonly known as the garden lizard. Attempts were also made to trace its relationship with the age pigment, lipofuscin and the antioxidant ascorbic acid. While LP increased with advancing age the contents of both lipofuscin and ascorbic acid did not show appreciable change during maturation ( < 1-1 year old) but declined during senescence phase (1 to 2-4 year old). While the pattern of age associated changes in LP and ascorbic acid indicate similarity with the pattern observed in most of the mammals, the reduction of lipofuscin in older lizards is a significant departure from the common trend.     

Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages

Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.     

Mitochondrial ATPase 6/8 genes reveal genetic divergence in the Coilia dussumieri (Valenciennes, 1848) populations of north east and northwest coasts of India

The golden anchovy, Coilia dussumieri, though possessing discontinuous distribution along northeast and northwest coasts of India, it is being managed as unit stock for fishery assessment purposes. By considering the need for stock specific management of the species, mitochondrial ATP synthase 6 and 8 (ATPase 6/8) genes were analyzed for delineating genetic stock structure of the species. Sequence analysis revealed a total of 34 haplotypes across four populations from both the east and west coasts of India. Haplotype diversity (h) was found in the range of 0.7421–0.9368. Similarly, nucleotide diversity (π) varied from 0.0012 to 0.0025. AMOVA results indicated a high total variance of 72.66 % between east and west coast populations and less (1.34 %) among populations within the respective coast. Phylogenetic tree constructed using pair wise F_ST also indicated the genetic divergence of populations of east and west coasts of India. The findings of the present study will be helpful in developing stock specific management measures for conservation and sustainable utilization of the species.     

Direct interaction between SNAP-23 and L-type Ca2+ channel

During secretion, membrane-bound secretory vesicles dock and fuse at the base of porosomes in the cell plasma membrane. Among other proteins, the porosome is composed of SNAREs and Ca2+-channels. Ca2+-channels and SNAREs have been implicated in cell secretion. Several immunoprecipitation and binding studies suggest the physical interaction of the t-SNARE proteins, Syntaxin-1 and SNAP-25 with various Ca2+-channels. In this study, using yeast two-hybrid and immunoanalysis, we demonstrate for the first time, direct interaction of SNAP-23 and a L-type Ca2+-channel at the plasma membrane in pancreas.