Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage phi6 is required for single-stranded RNA packaging and transcription

Bacteriophage phi6 has a segmented double-stranded RNA genome. The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7. Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates. Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics. In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4. Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus- or plus-strand synthesis) activity. Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging. Moreover, such particles displayed minus-strand synthesis activity. However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.     

Single nucleotide polymorphism haplotypes in the cholesteryl-ester transfer protein (CETP) gene and lipid phenotypes

We studied the association between high (HDL) and low-density (LDL) cholesterol concentrations and family-derived haplotypes based on six common SNPs in the cholesteryl-ester transfer protein (CETP) gene. We based our analysis on 201 founders from families recruited throughout Germany. The analysis revealed one subhaplotype block with complete, pairwise, linkage disequilibrium between 5 SNPs located in the promoter and intron 1. The sixth SNP was the well known 1405V polymorphism in exon 14, close to the 3' end of the gene. Four haplotypes accounted for 86% of the entire sample. We found that haplotype associations with HDL, LDL, and the LDL/HDL ratio were more robust than associations with individual SNPs. Moreover, the associations were robust for men, but not for women. Our data suggest an interaction between gender and genetic variation within the CETP gene.     

Evidence that hydroxyl radicals mediate auxin-induced extension growth

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.     

Rotatin is a novel gene required for axial rotation and left-right specification in mouse embryos

The genetic cascade that governs left-right (L-R) specification is starting to be elucidated. In the mouse, the lateral asymmetry of the body axis is revealed first by the asymmetric expression of nodal, lefty2 and pitx2 in the left lateral plate mesoderm of the neurulating embryo. Here we describe a novel gene, rotatin, essential for the correct expression of the key L-R specification genes nodal, lefty and Pitx2. Embryos deficient in rotatin show also randomized heart looping and delayed neural tube closure, and fail to undergo the critical morphogenetic step of axial rotation. The amino acid sequence deduced from the cDNA is predicted to contain at least three transmembrane domains. Our results show a novel key player in the genetic cascade that determines L-R specification, and suggest a causal link between this process and axial rotation.     

Green fluorescent protein as a convenient and versatile marker for studies on functional genomics in Drosophila

Gene function can be deduced from lack or gain of activity. For the manipulation of gene doses or activity in Drosophila, a set of P-based vectors was constructed containing green fluorescent protein as marker. pBLUEi, pGREENi and pYELLi were designed for large insert transformation. Mosaicism was generated in vivo with pFlipG which is also ideal for targeted gene disruption. Tissue-specific gene silencing in vivo was performed with the vector set pHIBS and pUdsGFP. pHIBS allows easy cloning and shuttling of double-headed constructs. With pUdsGFP, double stranded RNA can be produced in defined patterns and the area of interference simultaneously visualized by green fluorescence. We demonstrate nearly complete silencing of a ubiquitously expressed gene in a tissue-specific manner.     

parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube

N-cadherin (Ncad) is a classical cadherin that is implicated in several aspects of vertebrate embryonic development, including somitogenesis, heart morphogenesis, neural tube formation and establishment of left-right asymmetry. However, genetic in vivo analyses of its role during neural development have been rather limited. We report the isolation and characterization of the zebrafish parachute (pac) mutations. By mapping and candidate gene analysis, we demonstrate that pac corresponds to a zebrafish n-cadherin (ncad) homolog. Three mutant alleles were sequenced and each is likely to encode a non-functional Ncad protein. All result in a similar neural tube phenotype that is most prominent in the midbrain, hindbrain and the posterior spinal cord. Neuroectodermal cell adhesion is altered, and convergent cell movements during neurulation are severely compromised. In addition, many neurons become progressively displaced along the dorsoventral and the anteroposterior axes. At the cellular level, loss of Ncad affects beta-catenin stabilization/localization and causes mispositioned and increased mitoses in the dorsal midbrain and hindbrain, a phenotype later correlated with enhanced apoptosis and the appearance of ectopic neurons in these areas. Our results thus highlight novel and crucial in vivo roles for Ncad in the control of cell convergence, maintenance of neuronal positioning and dorsal cell proliferation during vertebrate neural tube development.     

The RHO1-GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase-activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7,but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1-controlled PKC1-MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1-mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.     

DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters

The spatial and temporal organization of DNA replication was investigated in living cells with a green fluorescent protein fusion to the DNA polymerase clamp PCNA. In situ extractions and photobleaching experiments revealed that PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery through multiple rounds of Okazaki fragment synthesis. Photobleaching analyses further showed that the transition from earlier to later replicons occurs by disassembly into a nucleoplasmic pool of rapidly diffusing subcomponents and reassembly at newly activated sites. The fact that these replication sites were de novo assembled in close proximity to earlier ones suggests that activation of neighboring origins may occur by a domino effect possibly involving local changes in chromatin structure and accessibility.