(1/416) Genetic localization and molecular characterization of two key genes (mitAB) required for biosynthesis of the antitumor antibiotic mitomycin C.
Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, and carbamoyl phosphate. A gene (mitA) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus, mrd, in Streptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the S. lavendulae chromosomal copy of mitA completely blocked the production of MC. The function of mitA was confirmed by complementation of an S. lavendulae strain containing a K191A mutation in MitA with AHBA. A second gene (mitB) encoding a 272-amino-acid protein (related to a group of glycosyltransferases) was identified immediately downstream of mitA that upon disruption resulted in abrogation of MC synthesis. This work has localized a cluster of key genes that mediate assembly of the unique mitosane class of natural products. (+info)
(2/416) Intrastrain variants of herpes simplex virus type 1 isolated from a neonate with fatal disseminated infection differ in the ICP34.5 gene, glycoprotein processing, and neuroinvasiveness.
Two intrastrain variants of herpes simplex virus type 1 (HSV-1) were isolated from a newborn with fatal disseminated infection. A small-plaque-producing variant (SP7) was the predominant virus (>99%) in the brain, and a large-plaque-producing variant (LP5) was the predominant virus (>99%) in the lung and gastrointestinal tract. EcoRI and BamHI restriction fragment patterns indicated that SP7 and LP5 are related strains. The large-plaque variants produced plaques similar in size to those produced by HSV-1 KOS. Unlike LP5 or KOS, SP7 was highly cell associated and processing of glycoprotein C and glycoprotein D was limited to precursor forms in infected Vero cells. The large-plaque phenotype from KOS could be transferred into SP7 by cotransfection of plasmids containing the EK or JK EcoRI fragment or a 3-kb plasmid with the UL34.5 gene of HSV-1 KOS together with SP7 DNA. PCR analysis using primers from within the ICP34.5 gene indicated differences for SP7, LP5, and KOS. Sequencing data indicated two sets of deletions in the UL34.5 gene that distinguish SP7 from LP5. Both SP7 and LP5 variants were neurovirulent (lethal following intracranial inoculation of young BALB/c mice); however, the LP5 variant was much less able to cause lethal neuroinvasive disease (footpad inoculation) whereas KOS caused no disease. Passage of SP7 selected for viruses (SLP-5 and SLP-10) which were attenuated for lethal neuroinvasive disease, were not cell-associated, and differed in the UL34.5 gene. UL34.5 from SLP-5 or SLP-10 resembled that of KOS. These findings support a role for UL34.5 in promoting virus egress and for neuroinvasive disease. (+info)
(3/416) Expression of Epstein-Barr virus BamHI-A rightward transcripts in latently infected B cells from peripheral blood.
In addition to the Epstein-Barr virus (EBV) EBNA and LMP latency genes, there is a family of alternatively spliced BamHI-A rightward transcripts (BARTs). These latency transcripts are highly expressed in the EBV-associated malignancies nasopharyngeal carcinoma and Burkitt's lymphoma, and are expressed at lower levels in latently EBV-infected B-cell lines. The contribution of the BARTs to EBV biology or pathogenesis is unknown. Resting B cells have recently been recognized as a reservoir for EBV persistence in the peripheral blood. In these cells, EBV gene expression is tightly restricted and the only viral gene known to be consistently expressed is LMP2A. We used cell sorting and reverse-transcriptase polymerase chain reaction (RT-PCR) to examine whether BARTs are expressed in the restricted form of in vivo latency. Our results demonstrated that RNAs with splicing diagnostic for transcripts containing the BART RPMS1 and BARFO open-reading frames (ORFs) were expressed in CD19(+) but not in CD23(+) B cells isolated from peripheral blood of healthy individuals. The product of the proximal RPMS1 ORF has not previously been characterized. The RPMS1 ORF was shown to encode a 15-kD protein that localized to the nucleus of transfected cells. Expression of the BARTs in peripheral blood B cells suggests that the proteins encoded by these transcripts are likely to be important for maintenance of in vivo latency. (+info)
(4/416) A combined biochemical and cytogenetic study of thioridazine-induced damage to nucleic acids.
In this work the biochemical effects of thioridazine, a commonly used phenothiazine, have been studied upon native double- and single-stranded DNA and also upon a supercoiled plasmid. The results indicate that thioridazine causes damage and scissions to these nucleic acids but only at concentrations much higher than the one used in our cytogenetic experiments and that the damage seems to depend on the concentrations used. Furthermore, we studied the action of thioridazine alone or in combination with caffeine and/or melphalan upon human lymphocytes in vitro. Thioridazine and caffeine (a well-known inhibitor of cellular repair mechanisms) were shown to act synergistically to potentiate the cytogenetic effect of melphalan on human lymphocytes. It is suggested that thioridazine alone or in combination with caffeine may exert its synergistic effect on melphalan cytotoxicity to cultured human lymphocytes not only indirectly, i.e. as a strong calmodulin inhibitor by facilitating the intracellular retention of melphalan, but also directly by reaction with nucleic acids and by causing scissions in and damage to them. Therefore, thioridazine (as chlorpromazine) has some potential as an adjuvant chemotherapeutic agent for the treatment of human cancer. (+info)
(5/416) Antisense oligonucleotide complementary to the BamHI-H gene family of Marek's disease virus induced growth arrest of MDCC-MSB1 cells in the S-phase.
DNA synthesis was effectively inhibited by antisense oligonucleotide A1 complementary to the BamHI-H gene family in Marek's disease virus (MDV)-derived lymphoblastoid MDCC-MSB1 cells. When a cell cycle distribution of a total cell population was analyzed by flow cytometry, the proportion of S-phase cells increased in the cell populations by treatment with oligonucleotide A1. Approximately 60-70% of the cells appeared in the S phase for 24 and 36 hr of incubation in the presence of oligonucleotide A1 (20-30% in the untreated control cells). The inhibition of cell cycle progression by treatment with oligonucleotide A1 was reversible. When the cells were treated with 5 microM aphidicolin for 12 hr, a similar pattern of cell cycle distribution was observed to that obtained after treatment with oligonucleotide A1. Aphidicolin is an inhibitor of cellular DNA polymerase alpha, and it halts progression of the cell cycle at the G1/S border or early S phase. When the cells were treated with aphidicolin for 12 hr and subsequently incubated with oligonucleotide A1, no significant difference was observed in the cycle phase distribution of cells in the presence and absence of oligonucleotide A1. In contrast, when the cells were treated with oligonucleotide A1 for 12 hr and subsequently incubated with aphidicolin, the cell cycle did not progress from the G1/S border or early S phase to the next phase. (+info)
(6/416) Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.
The MunI restriction enzyme recognizes the palindromic hexanucleotide sequence C/AATTG (the '/' indicates the cleavage site). The crystal structure of its active site mutant D83A bound to cognate DNA has been determined at 1.7 A resolution. Base-specific contacts between MunI and DNA occur exclusively in the major groove. While DNA-binding sites of most other restriction enzymes are comprised of discontinuous sequence segments, MunI combines all residues involved in the base-specific contacts within one short stretch (residues R115-R121) located at the N-terminal region of the 3(10)4 helix. The outer CG base pair of the recognition sequence is recognized solely by R115 through hydrogen bonds made by backbone and side chain atoms to both bases. The mechanism of recognition of the central AATT nucleotides by MunI is similar to that of EcoRI, which recognizes the G/AATTC sequence. The local conformation of AATT deviates from the typical B-DNA form and is remarkably similar to EcoRI-DNA. It appears to be essential for specific hydrogen bonding and recognition by MunI and EcoRI. (+info)
(7/416) Promoter interference in a bacteriophage lambda control region: effects of a range of interpromoter distances.
The p(R) and p(RM) promoters of bacteriophage lambda direct transcription in divergent directions from start sites separated by 83 phosphodiester bonds. We had previously shown that the presence of an RNA polymerase at p(R) interfered with open complex formation at p(RM) and that this effect was alleviated by the deletion of 10 bp between the two promoters. Here we present a detailed characterization of the dependence of the interference on the interpromoter distance. It was found that the reduced interference between the two promoters is unique to the 10-bp deletion. The relief of interference was demonstrated to be due to the facilitation of a step subsequent to RNA polymerase binding to the p(RM) promoter. A model to explain these observations is proposed. A search of known Escherichia coli promoters identified three pairs of divergent promoters with similar separations to those investigated here. (+info)
(8/416) The activity of the Epstein-Barr virus BamHI W promoter in B cells is dependent on the binding of CREB/ATF factors.
The programme of Epstein-Barr virus (EBV) gene expression that leads to virus-induced growth transformation of resting B lymphocytes is initiated through activation of the BamHI W promoter, Wp. The factors regulating Wp, and the basis of its preferential activity in B cells, remain poorly understood. Previous work has identified a B cell-specific enhancer region which is critical for Wp function and which contains three binding sites for cellular factors. Here we focus on one of these sites and show, using bandshift assays, that it interacts with three members of the CREB/ATF family of cell transcription factors, CREB1, ATF1 and ATFa. A mutation which abrogates the binding of these factors reduces Wp reporter activity specifically in B cell lines, whereas a mutation which converts the site to a consensus CREB-binding sequence maintains wild-type promoter function. Furthermore Wp activity in B cell, but not in non-B cell, lines could be inhibited by cotransfection of expression plasmids expressing dominant negative forms of CREB1 and ATF1. Increasing the basal activity of CREB/ATF proteins in cells by treatment with protein kinase A or protein kinase C agonists led to small increases in Wp activity in B cell lines, but did not restore promoter activity in non-B cell lines up to B cell levels. We conclude that CREB/ATF factors are important activators of Wp in a B cell environment but require additional B cell-specific factors in order to mediate their effects. (+info)