Phosphorylation of Pnut in the Early Stages of Drosophila Embryo Development Affects Association of the Septin Complex with the Membrane and Is Important for Viability.

Septin proteins are polymerizing GTPases that are found in most eukaryotic species. Septins are important for cytokinesis and participate in many processes involving spatial modifications of the cell cortex. In Drosophila, septin proteins Pnut, Sep1, and Sep2 form a hexameric septin complex. Here, we found that septin protein Pnut is phosphorylated during the first 2 hr of Drosophila embryo development. To study the effect of Pnut phosphorylation in a live organism, we created a new Drosophila pnut null mutant that allows for the analysis of Pnut mutations during embryogenesis. To understand the functional significance of Pnut phosphorylation, Drosophila strains carrying nonphosphorylatable and phospho-mimetic mutant pnut transgenes were established. The expression of the nonphosphorylatable Pnut protein resulted in semilethality and abnormal protein localization, whereas the expression of the phospho-mimetic mutant form of Pnut disrupted the assembly of a functional septin complex and septin filament formation in vitro Overall, our findings indicate that the controlled phosphorylation of Pnut plays an important role in regulating septin complex functions during organism development.

Genome-Wide Analyses Reveal Gene Influence on HIV Disease Progression and HIV-1C Acquisition in Southern Africa.

Sub-Saharan Africans infected with HIV-1C make up the largest AIDS patient population in the world and exhibit large heterogeneity in disease progression before initiating antiretroviral therapy. To identify host variants associated with HIV disease progression, we performed genome-wide association studies on a total of 556 treatment-naive HIV-infected individuals in Botswana. We characterized the pattern of HIV disease progression using a novel functional principal component analysis, which can better capture longitudinal CD4 and viral load (VL) trajectories. Two single-nucleotide polymorphisms (SNPs) near HCG22 (chr6, peak variant rs2535307, combined p = 3.72 × 10-7, minor allele as risky allele) and CCNG1 (chr5, peak variant kgp22385164, combined p = 1.88 × 10-6, minor allele as risky allele) were significantly associated with CD4 and VL dynamics. Inspection of SNPs in these gene regions in a third Botswana cohort (using GWATCH) also revealed a strong association of HCG22 with HIV-1C acquisition, suggesting that this region is associated with infection as well as disease progression. Our study uncovered two genetic regions that are significant and have specific effects on HIV-1C acquisition or progression in sub-Saharan Africans, and the result suggested new potential targets for AIDS prevention and treatment. In addition, our results also indicate the possibility of using genetic markers as HIV disease progression indicators in sub-Saharan Africans to prioritize fast progressors for antiretroviral treatment.

GWATCH: a web platform for automated gene association discovery analysis.

BACKGROUND: As genome-wide sequence analyses for complex human disease determinants are expanding, it is increasingly necessary to develop strategies to promote discovery and validation of potential disease-gene associations. FINDINGS: Here we present a dynamic web-based platform - GWATCH - that automates and facilitates four steps in genetic epidemiological discovery: 1) Rapid gene association search and discovery analysis of large genome-wide datasets; 2) Expanded visual display of gene associations for genome-wide variants (SNPs, indels, CNVs), including Manhattan plots, 2D and 3D snapshots of any gene region, and a dynamic genome browser illustrating gene association chromosomal regions; 3) Real-time validation/replication of candidate or putative genes suggested from other sources, limiting Bonferroni genome-wide association study (GWAS) penalties; 4) Open data release and sharing by eliminating privacy constraints (The National Human Genome Research Institute (NHGRI) Institutional Review Board (IRB), informed consent, The Health Insurance Portability and Accountability Act (HIPAA) of 1996 etc.) on unabridged results, which allows for open access comparative and meta-analysis. CONCLUSIONS: GWATCH is suitable for both GWAS and whole genome sequence association datasets. We illustrate the utility of GWATCH with three large genome-wide association studies for HIV-AIDS resistance genes screened in large multicenter cohorts; however, association datasets from any study can be uploaded and analyzed by GWATCH.

Annotated features of domestic cat - Felis catus genome.

BACKGROUND: Domestic cats enjoy an extensive veterinary medical surveillance which has described nearly 250 genetic diseases analogous to human disorders. Feline infectious agents offer powerful natural models of deadly human diseases, which include feline immunodeficiency virus, feline sarcoma virus and feline leukemia virus. A rich veterinary literature of feline disease pathogenesis and the demonstration of a highly conserved ancestral mammal genome organization make the cat genome annotation a highly informative resource that facilitates multifaceted research endeavors. FINDINGS: Here we report a preliminary annotation of the whole genome sequence of Cinnamon, a domestic cat living in Columbia (MO, USA), bisulfite sequencing of Boris, a male cat from St. Petersburg (Russia), and light 30× sequencing of Sylvester, a European wildcat progenitor of cat domestication. The annotation includes 21,865 protein-coding genes identified by a comparative approach, 217 loci of endogenous retrovirus-like elements, repetitive elements which comprise about 55.7% of the whole genome, 99,494 new SNVs, 8,355 new indels, 743,326 evolutionary constrained elements, and 3,182 microRNA homologues. The methylation sites study shows that 10.5% of cat genome cytosines are methylated. An assisted assembly of a European wildcat, Felis silvestris silvestris, was performed; variants between F. silvestris and F. catus genomes were derived and compared to F. catus. CONCLUSIONS: The presented genome annotation extends beyond earlier ones by closing gaps of sequence that were unavoidable with previous low-coverage shotgun genome sequencing. The assembly and its annotation offer an important resource for connecting the rich veterinary and natural history of cats to genome discovery.

Study of DNA replication in Drosophila using cell free in vitro system.

Using Drosophila early egg extracts we have developed an optimized cell free system to study DNA replication. The efficiency of replication depends on a cold treatment of Drosophila embryos before the extract preparation and a formation of nuclei facilitated by the addition of membrane fractions to the extracts. In vitro DNA replication is ORC and CDC6 dependent, as a removal of these proteins from the extracts abolishes DNA replication. The N-terminal part of Orc1 protein, which is important for non-replicative functions of ORC, is dispensable for the replication in vitro. We also show that the conserved ATP ase motif of CDC6 is crucial for the replication. Our studies indicate that a Drosophila cell free system proves to be an extremely useful tool for a functional dissection of the processes and factors involved in DNA replication in metazoans.

Drosophila Orc6 facilitates GTPase activity and filament formation of the septin complex.

The origin recognition complex or ORC is a six-subunit protein important for DNA replication and other cell functions. Orc6, the smallest subunit of ORC, is essential for both replication and cytokinesis in Drosophila, and interacts with the septin protein Pnut, which is part of the Drosophila septin complex. In this study, we describe the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.

Novel non-Mendelian determinant involved in the control of translation accuracy in Saccharomyces cerevisiae.

Two cytoplasmically inherited determinants related by their manifestation to the control of translation accuracy were previously described in yeast. Cells carrying one of them, [PSI(+)], display a nonsense suppressor phenotype and contain a prion form of the Sup35 protein. Another element, [PIN(+)], determines the probability of de novo generation of [PSI(+)] and results from a prion form of several proteins, which can be functionally unrelated to Sup35p. Here we describe a novel nonchromosomal determinant related to the SUP35 gene. This determinant, designated [ISP(+)], was identified as an antisuppressor of certain sup35 mutations. We observed its loss upon growth on guanidine hydrochloride and subsequent spontaneous reappearance with high frequency. The reversible curability of [ISP(+)] resembles the behavior of yeast prions. However, in contrast to known prions, [ISP(+)] does not depend on the chaperone protein Hsp104. Though manifestation of both [ISP(+)] and [PSI(+)] is related to the SUP35 gene, the maintenance of [ISP(+)] does not depend on the prionogenic N-terminal domain of Sup35p and Sup35p is not aggregated in [ISP(+)] cells, thus ruling out the possibility that [ISP(+)] is a specific form of [PSI(+)]. We hypothesize that [ISP(+)] is a novel prion involved in the control of translation accuracy in yeast.