Involvement of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system in regulation of transcription of catabolic genes. (65/18933)

Synthesis of catabolite-sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic AMP-receptor protein (crp mutation) Measurements of the differential rate of beta-galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains. It was concluded that mutational damage to the general components of the phosphoenolpyruvate-dependent phosphotransferase system diminishes activity of the lac promoter. The results obtained led to the conclusion that pts gene products (apparently phospho approximately HPr) are necessary for the initiation of transcription of catabolite-sensitive operons in E. coli.  (+info)

The 5' noncoding region of grapevine chrome mosaic nepovirus RNA-2 triggers a necrotic response on three Nicotiana spp. (66/18933)

The 5' noncoding region (NCR) of grapevine chrome mosaic nepovirus (GCMV) was cloned in a viral vector derived from potato virus X (PVX). The recombinant virus obtained was inoculated to Nicotiana benthamiana, N. clevelandii, and N. tabacum plants. Infected plants developed necrotic symptoms in place of the vein clearing and mosaic typically observed after inoculation with PVX. Northern (RNA) blot analysis showed that the replication of PVX was not specifically altered by the presence of the GCMV 5' NCR. Inoculation of recombinant PVX harboring deleted forms of the GCMV 5' NCR showed that the three stem-loop structures at the 3' end of the 5' NCR (nucleotides 153 to 206) are dispensable for the induction of necrosis. Further deletion analysis indicated that neither the 5'-most 70 nucleotides of the 5' NCR nor the downstream region (nucleotides 71 to 217) alone is able to induce the necrotic symptoms. In the presence of both the sequence encoding the GCMV coat protein and the GCMV 3' NCR, the GCMV 5' NCR failed to induce necrosis in the PVX background. The mechanisms by which the expression of the 5' NCR might modify PVX symptoms are discussed.  (+info)

Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue. (67/18933)

Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.  (+info)

Targeted deletions created in yeast vectors by recombinational excision. (68/18933)

We have developed a simple method for creating defined deletions in yeast vectors by utilizing the ability of Saccharomyces cerevisiae to perform homologous recombination. Two complementary single-stranded oligonucleotides are designed so that the 5' and 3' halves of the resulting double-stranded oligonucleotide are homologous to the 5' and 3' side of a desired deletion junction, respectively. The sequence to be deleted is cleaved by restriction endonuclease digestion, followed by co-transformation of the linearized plasmid and the oligonucleotide into yeast. By homologous recombination in vivo, a subset of the plasmids will recircularize and simultaneously acquire the deletion as defined by the oligonucleotide.  (+info)

Evidence for mitochondrial DNA recombination in a human population of island Melanesia. (69/18933)

Mitochondrial DNA (mtDNA) analysis has proved useful in studies of recent human evolution and the genetic affinities of human groups of different geographical regions. As part of an extensive survey of mtDNA diversity in present-day Pacific populations, we obtained sequence information of the hypervariable mtDNA control region of 452 individuals from various localities in the western Pacific. The mtDNA types fell into three major groups which reflect the settlement history of the area. Interestingly, we detected an extremely rare point mutation at high frequency in the small island of Nguna in the Melanesian archipelago of Vanuatu. Phylogenetic analysis of the mtDNA data indicated that the mutation was present in individuals of separate mtDNA lineages. We propose that the multiple occurrence of a rare mutation event in one isolated locality is highly improbable, and that recombination between different mtDNA types is a more likely explanation for our observation. If correct, this conclusion has important implications for the use of mtDNA in phylogenetic and evolutionary studies.  (+info)

Z/AP, a double reporter for cre-mediated recombination. (70/18933)

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.  (+info)

Analysis of human lymphotropic T-cell virus type II-like particle production by recombinant baculovirus-infected insect cells. (71/18933)

The molecular processes involved in retrovirus assembly and budding formation remain poorly understood. The gag-pro-pol genes of human lymphotropic T-cell virus type II (HTLV-II) are translated into Gag, Gag-Pro, or Gag-Pro-Pol by frameshift events. In the present study, we investigated the roles of the gag, pro, and pol regions of HTLV-II in viral particle formation using recombinant baculoviruses. In this study we could successfully produce mature HTLV-II viral particles containing core structures using a construct expressing the entire gag-pro-pol region. We also investigated the role of the pol region in particle formation. Deletion of the pol region affects viral particle assembly or release very little, indicating that the gag-pro region is sufficient for viral particle formation and maturation. Expression of the Gag proteins alone or Gag proteins with inactivated viral proteases (Pro) resulted in the formation of viral particles; however, these particles did not contain core structures. These results suggest the intracellular expression of Gag with Pro of HTLV-II is essential for the production of mature virus particles, whereas that of Pol is not.  (+info)

Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells. (72/18933)

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.  (+info)