Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor.

The nucleotide sequence of black beetle virion (BBV) RNA2 has been determined. RNA2 is 1399 b long. Its 5' terminus is capped. Its 3' terminus has an unidentified moiety that renders the RNA resistent to polyadenylation and ligation. The first AUG codon at base 23 is followed by an open reading frame for a protein 407 amino acids long, the predicted size of coat protein precursor. A second open reading frame for a putative protein 72 amino acid residues long begins at base 1110. No other large open reading frames exist. The 5' half of the RNA can be folded into a long, imperfect hairpin of high predicted stability. The 3' half of the RNA can fold into a complex set of multiply bifurcated stem and loop regions.     

A novel p53 rescue compound induces p53-dependent growth arrest and sensitises glioma cells to Apo2L/TRAIL-induced apoptosis

Reactivation of mutant p53 in tumours is a promising strategy for cancer therapy. Here we characterise the novel p53 rescue compound P53R3 that restores sequence-specific DNA binding of the endogenously expressed p53(R175H) and p53(R273H) mutants in gel-shift assays. Overexpression of the paradigmatic p53 mutants p53(R175H), p53(R248W) and p53(R273H) in the p53 null glioma cell line LN-308 reveals that P53R3 induces p53-dependent antiproliferative effects with much higher specificity and over a wider range of concentrations than the previously described p53 rescue drug p53 reactivation and induction of massive apoptosis (PRIMA-1). Furthermore, P53R3 enhances recruitment of endogenous p53 to several target promoters in glioma cells bearing mutant (T98G) and wild-type (LNT-229) p53 and induces mRNA expression of numerous p53 target genes in a p53-dependent manner. Interestingly, P53R3 strongly enhances the mRNA, total protein and cell surface expression of the death receptor death receptor 5 (DR5) whereas CD95 and TNF receptor 1 levels are unaffected. Accordingly, P53R3 does not sensitise for CD95 ligand- or tumour necrosis factor alpha-induced cell death, but displays synergy with Apo2L.0 in 9 of 12 glioma cell lines. Both DR5 surface induction and synergy with Apo2L.0 are sensitive to siRNA-mediated downregulation of p53. Thus this new p53 rescue compound may open up novel perspectives for the treatment of cancers currently considered resistant to the therapeutic induction of apoptosis.     

Effect of Age-Stiffening Tissues and Intraocular Pressure on Optic Nerve Damages

Age-stiffening of ocular tissues is statistically linked to glaucoma in the elderly. In this study, the effects of age-stiffening on the lamina cribrosa, the primary site of glaucomatous nerve damages, were modeled using computational finite element analysis. We showed that glaucomatous nerve damages and peripheral vision loss behavior can be phenomenologically modeled by shear-based damage criterion. Using this damage criterion, the potential vision loss for 30 years old with mild hypertension of 25mmHg intraocular pressure (IOP) was estimated to be 4%. When the IOP was elevated to 35mmHg, the potential vision loss rose to 45%; and age-stiffening from 35 to 60 years old increased the potential vision loss to 52%. These results showed that while IOP plays a central role in glaucomatous damages, age-stiffening facilitates glaucomatous damages and may be the principal factor that resulted in a higher rate of glaucoma in the elderly than the general population.     

Inferences from a rapidly moving hybrid zone

Anartia fatima and Anartia amathea (Lepidoptera: Nymphalidae) are sister taxa whose ranges abut in a narrow hybrid zone in eastern Panama. At the center of the zone, hybrids are abundant, although deviations from Hardy-Weinberg and linkage disequilibria are strong, due in part to assortative mating. We measured differences across the zone in four wing color-pattern characters, three allozyme loci, and mitochondrial haplotype. Wing pattern, allozyme, and mitochondrial clines were coincident (i.e., had the same positions) and concordant (i.e.. all markers had similar cline shapes, about 28 km wide). Repeated samples demonstrated that the hybrid zone has been moving eastwards at an average rate of 2.5 km/year over the past 20 years, accompanied by an equivalent movement of the mtDNA cline. No introgression of mtDNA haplotypes were found in the 'wake" of the moving cline, as might be expected for a neutral marker. The concordance of morphological and mtDNA clines between 1994 and 2000, in spite of hybrid zone movement, suggests strong epistasis between the mitochondrial genome and nuclear loci. Cline movement is achieved mainly by pure fatima immigrating into amathea populations; hybrids had little effect, and were presumably outcompeted by fitter pure fatima genotypes. This movement can be explained if random dispersal of 7-19 km x gen(1/2) is coupled with a competitive advantage to A. fatima genomes of 2-5%. Hybrid zone motion is equivalent to Phase III of Wright's shifting balance. Hybrid zone movement has rarely been considered likely in the past, but our results show that it may be more important in biogeography and evolution than generally realized.     

Hepatitis C NS3 protease: restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides

The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide significantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease.     

Valproate-induced teratogenesis in Japanese rice fish (Oryzias latipes) embryogenesis

Fertilized eggs of Japanese rice fish (medaka) at three developmental stages (Iwamatsu stages 4-30) were exposed to waterborne valproic acid (VPA) (0-80 mM) in hatching solution for 48 h. The amount of valproate to cause 50% mortality (IC(50)) is found to be developmental stage-specific. The embryos were more sensitive to valproate at early stages of development (Iwamatsu stages 4-10) than in the embryos in late stages (Iwamatsu stages 17-30). Valproate exposed embryos have microcephaly and disrupted cardiovasculature with delayed vessel circulation, thrombus formation, and slow heart rate. The hatching efficiency is also reduced by valproate exposure due to developmental delay. The mRNA analysis of nine genes belong to oxidative stress (catalase, gsr, gst), neurogenesis (iro3, wnt1, shh, otx2, nlgn3b) and cell cycle regulation (ccna2) have been done. It was observed that the genes belong to oxidative stress remained unaltered after valproate exposure. However, some of the genes belong to neurogenesis (wnt1,shh, otx2 and nlgn3b) and cell cycle (ccna2) showed developmental stage-specific alteration after valproate exposure. This study indicates that valproate is able to induce some of the phenotypic features which are analogous to human fetal valproate syndrome (FVS). Modulation of genes expressed in neural tissues indicates that this fish can be used to analyze the mechanisms of many neurobehavioral disorders like Autism spectrum disorder (ASD) in human.     

Genomic islands of divergence in hybridizing Heliconius butterflies identified by large-scale targeted sequencing

Heliconius butterflies represent a recent radiation of species, in which wing pattern divergence has been implicated in speciation. Several loci that control wing pattern phenotypes have been mapped and two were identified through sequencing. These same gene regions play a role in adaptation across the whole Heliconius radiation. Previous studies of population genetic patterns at these regions have sequenced small amplicons. Here, we use targeted next-generation sequence capture to survey patterns of divergence across these entire regions in divergent geographical races and species of Heliconius. This technique was successful both within and between species for obtaining high coverage of almost all coding regions and sufficient coverage of non-coding regions to perform population genetic analyses. We find major peaks of elevated population differentiation between races across hybrid zones, which indicate regions under strong divergent selection. These 'islands' of divergence appear to be more extensive between closely related species, but there is less clear evidence for such islands between more distantly related species at two further points along the 'speciation continuum'. We also sequence fosmid clones across these regions in different Heliconius melpomene races. We find no major structural rearrangements but many relatively large (greater than 1 kb) insertion/deletion events (including gain/loss of transposable elements) that are variable between races.     

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.     

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.