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At least 379 records · Page 21Linked to original sources
PubMed2003

Rescue of the adeno-associated virus genome from a plasmid vector: evidence for rescue by replication.

In cultured cells, adeno-associated virus (AAV) replication requires coinfection with a helper virus, either adenovirus or herpesvirus. In the absence of helper virus coinfection AAV can integrate its genome site specifically into the AAVS1 region of chromosome 19. Upon subsequent infection with a helper virus, the AAV genome is released from chromosome 19 by a process termed rescue, and productive replication ensues. The AAV genome cloned into a plasmid vector can also serve to initiate productive AAV replication. When such constructs are transfected into cells and those cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid. This process is thought to serve as a model for rescue from the human genomic site. In this report we present a model for rescue of AAV genomes by replication. A hallmark of this model is the production of a partially single-stranded and partially double-stranded molecule. We show that the AAV2 Rep 68 protein, together with the UL30/UL42 herpes simplex virus type 1 DNA polymerase and the UL29 single-strand DNA binding protein ICP8, is sufficient to efficiently and precisely rescue AAV from a plasmid in a way that is dependent on the AAV inverted terminal repeat sequence.

DNA Replication↗
PubMed2005

Transcriptional analysis of latent and inducible Kaposi's sarcoma-associated herpesvirus transcripts in the K4 to K7 region.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-2 herpesvirus with a genome containing a long unique coding region (LUR) flanked by GC-rich terminal repeat sequences. The LUR encodes approximately 90 annotated open reading frames (ORFs) with complex patterns of gene expression during viral latency, reactivation, and de novo infection. To identify unannotated KSHV genes, we examined the region between 21,500 and 30,000 bp of the KSHV LUR, representing approximately 8.5 kb of sequence. This region encodes seven known single-exon ORFs (K4, K4.1, K4.2, K5, K6, K7, and PAN), but previous computer analyses have failed to identify additional likely genes in the remaining 5.2 kb. We identified four novel transcripts using Northern blotting, phage library screening, and 5' rapid amplification of cDNA ends analysis in the region between ORFs K4.2 and K7. In vitro analysis of KSHV-infected primary effusion lymphoma cell lines in the presence of 12-O-tetradecanoylphorbol-13-acetate and phosphonoformic acid suggests that one latent transcript is coterminal with the previously annotated K3 gene encoding an ubiquitin-ligase known to downregulate major histocompatibility complex class I expression. This alternatively spliced transcript may contribute to KSHV adaptive immune evasion during latent infection. Other transcripts are inducible, including a 6.1-kb transcript that is the largest transcript found in the KSHV genome to date.

Cell Line↗
PubMed1990

Characterization of a novel promoter insertion in the c-rel locus.

Avian leukosis virus (ALV)-induced neoplasias are commonly found associated with integrations of proviral DNA in proximity to the myc gene. However, studies suggest that other genetic events are necessary for the complete neoplastic phenotype. A cell line (HP46) derived from an ALV-induced tumor has been analyzed and found to contain, in addition to an alteration in the myc gene, a promoter insertion in the c-rel locus. Both loci expressed large amounts of mRNA coding for their respective proteins. Several rel-related transcripts were expressed in the HP46 line, and four rel-related proteins of lower molecular weight than the wild-type p68c-rel product were detected. At least two of these transcripts contained U5 long terminal repeat sequences on the 5' end of the RNA. Structural data suggest that the messages may have evolved by an alternative splicing mechanism. This is the first example of a promoter insertion in the c-rel locus, a gene whose viral counterpart v-rel is responsible for the induction of lymphoid tumors.

Animals↗
PubMedDate not supplied

Expression of gene-specific RNA in cultured cells exposed to rotating 60-Hz magnetic fields.

Replicate sets of cultures of mouse and of human cells were exposed to cyclic, sine wave, 60-Hz rotating magnetic fields of 1.0 Gs (1 Gs [symbol: see text] 0.1 mT) for 24-, 48-, or 72-h periods. Total RNA extracted from unexposed control and from magnetic field exposed cells was dot blot hybridized to a number of oncogene probes (including v-myc, v-fos, v-raf, and v-Ha-ras), a probe for 3611 murine sarcoma virus, a probe for the 70,000 dalton heat shock protein (hsp70), and a probe for the long terminal repeat sequence of mouse mammary tumor virus. Comparisons of levels of RNA in unexposed and magnetic field exposed cells measured by densitometer readings of resulting autoradiographs revealed no significant increases or decreases in RNA levels in magnetic field exposed cells with the seven probes tested.

Animals↗
PubMed2001

Differential gene expression in a murine model of cancer cachexia.

Murine adenocarcinoma 16 (MAC16) tumors and cell lines induce cachexia in NMRI nude mice, whereas histologically similar MAC13 tumors do not. After confirming these findings in BALB/c nude mice, we demonstrated that this tissue wasting was not related to decreased food intake or increased total body oxidative metabolism. Previous studies have suggested that MAC16's cachexigenic properties may involve the production of tumor-specific factors. We therefore screened for genes having increased expression in the MAC16 compared with the MAC13 cell line by performing hybridization to a murine cDNA expression array, by generation and comparison of cDNA libraries from each cell line, and by PCR-based subtractive hybridization. Northern blot hybridization was performed to confirm differences in transcript expression. Transcripts encoding insulin-like growth factor binding protein-4, cathepsin B, ferritin light and heavy chain, endogenous long-terminal repeat sequences, and a viral envelope glycoprotein demonstrated increased expression in the MAC16 cell line. The roles of a number of these genes in known metabolic pathways identify them as potential participants in the induction of cachexia.

Adenocarcinoma↗
PubMed2003

Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.

AIM: To construct an AAV based vector carrying human endothelial nitric-oxide synthase (eNOS) cDNA and study its expression in vitro for future gene therapy. METHODS: eNOS cDNA was inserted into the EcoR I site of pSNAV-1 containing the cytomegalovirus (CMV) promoter and inverted terminal repeat sequences of adeno-associated virus. The constructed vector was transfected into BHK and C2C12 cells. eNOS cDNA and mRNA were detected by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), respectively. RESULTS: By restriction enzyme digestion analysis, it was proved that eNOS cDNA was inserted into pSNAV-1 in a proper direction. PCR detection demonstrated that pSNAV-eNOS was transferred into both BHK and C2C12 cells. RT-PCR analysis showed that these pSNAV-eNOS transfected cells could express eNOS mRNA. CONCLUSION: pSNAV-eNOS was successfully constructed with the ability to express human eNOS mRNA in cultured mammalian cells.

Animals↗
PubMed1992

EBV-induced human B cell lymphomas in hu-PBL-SCID mice.

Epstein-Barr virus (EBV) infection is associated with Burkitt's lymphoma (BL) in normal individuals and immunoblastic B cell lymphomas in immunosuppressed or HIV-infected individuals. SCID mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) from EBV-seropositive donors also may develop spontaneous B cell lymphomas which histologically and phenotypically resemble post-transplant tumors, and are distinct from BL. These tumors always contain EBV DNA. We have noted three different reproducible outcomes depending upon the EBV-seropositive donor used for generation of hu-PBL-SCID mice: (i) no tumors appear; (ii) tumors appear in a fraction of hu-PBL-SCID mice with a 10-20 wk. latent period; or (iii) tumors appear in all hu-PBL-SCID mice within 6-10 wk. Southern blot analysis of late versus early tumors using a probe specific for the EBV terminal repeat sequences (BamNJ), which allows distinction between circular latent and linear replicating genomes, shows that late tumors do not involve active EBV replication but that early tumors do show replicating genomes. In addition, EBV genomes were monoclonal in late tumors but polyclonal in early tumors. These data suggest two mechanisms for EBV lymphomagenesis, slow outgrowth of rare latently-infected B cells, and more rapid transformation of uninfected bystander B cells by replicating virus. The latter process may be highly amenable to therapy in patients at risk for EBV-related lymphomas. In addition, prospective screening of EBV-seropositive transplant recipients in the hu-PBL-SCID model may predict the risk of post-transplant lymphoma development.

Animals↗
PubMed1989

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

Adult↗
PubMed1989

HTLV-I tax gene product activates transcription via pre-existing cellular factors and cAMP responsive element.

Human T-cell leukemia virus type I (HTLV-I) is the etiological agent of adult T-cell leukemia. The 3' end of HTLV-I proviral DNA encodes the synthesis of two regulatory proteins, tax and rex. The 40-kDa tax protein is a nuclear protein which positively stimulates transcription from the U3 region of the viral long terminal repeat sequence. Three 21-base pair sequences in the U3 region have been found to serve as the cis-element for tax-mediated trans-activation. We now report that the tax protein can trans-activate HTLV-I LTR in the absence of de novo cellular protein synthesis. Saturated mutagenesis of the 21-base pair repeat sequence showed that specific mutations clustered in sequences homologous to the cAMP responsive element (TGACGTCA) abolish trans-activation by tax. Furthermore, although the TGACGTCN element is nearly palindromic, the mutations that abolish trans-activation are localized exclusively in the 5' 6 bases, suggesting the orientation of this element may play a role in transcription. That the purified tax protein does not bind the 21-base pair repeats or nonspecific DNA lends further support to the notion that tax protein does not directly interact with the 21-base pair repeats to activate transcription. Instead, tax most likely acts via cellular transcriptional factor(s) to bring about trans-activation.

Base Sequence↗
PubMed1988

Effect of radiofrequency radiation on mRNA expression in cultured rodent cells.

Four rodent cell lines were exposed to 2450 MHz microwave radiation at a Specific Absorption Rate (SAR) of 103.5 +/- 4.2 W/kg for varying lengths of time at 37 degrees, 40 degrees, 42 degrees and 45 degrees C. mRNA was extracted from microwave-exposed and sham-exposed cells and dot blotted or Northern blotted to nitrocellulose. Radioisotope labelled DNA probes of oncogenes, heat shock protein or long terminal repeat sequences were hybridized to the mRNA, and the resulting autoradiographs analyzed for differences in levels of mRNA expression between exposed and nonexposed samples. With the cell lines and probes used in this study no significant differences in mRNA expression were observed after microwave exposure.

Animals↗
PubMed1984

Correlation of undermethylation of intracisternal A-particle genes with expression in murine plasmacytomas but not in NIH/3T3 embryo fibroblasts.

We report here comparative studies of DNA methylation and expression of intracisternal A-particle (IAP) genes in murine plasmacytomas, TEPC-15 and MOPC-315, in NIH/3T3 embryo fibroblasts, and in normal liver from young adult BALB/c mice. In Southern blot analysis using methylation-sensitive restriction enzymes, we observed hypomethylation of 4.7- and 5.3-kilobase pair IAP proviruses, and partial undermethylation of other IAP DNA fragments in TEPC-15, MOPC-315, and 3T3 cells, while most of these DNA fragments were hypermethylated in liver. The extent of undermethylation at 12 different sites including the long terminal repeat sequence region and 5'-flanking sequence within one specific IAP gene was found to be similar between 3T3 cells and plasmacytomas. Little or no undermethylation of these sites was found in liver. Hypomethylation of IAP genes, however, is not correlated with their expression in 3T3 cells (as it is found for plasmacytomas), since 3T3 cells, like liver cells, have no detectable transcripts. We also observed hypomethylation of the kappa-light chain gene in 3T3 cells, suggesting that undermethylation may be a generalized phenomenon in these cells. The relationships between gene undermethylation and the control of gene expression in 3T3 cells is discussed.

Animals↗
PubMed1993

Detection of Epstein-Barr virus in human tissues by molecular genetic techniques.

In the past few years, there has been an explosion of new data on the association of Epstein-Barr virus (EBV) with human disease. Many of these discoveries have come as a direct result of the application of DNA technology. The nucleic acid hybridization techniques most commonly used to detect EBV in human tissues include Southern blot analysis, in situ hybridization to viral DNA or RNA, and polymerase chain reaction. An advantage of Southern blotting is the ability to distinguish latent from infectious EBV and to determine the clonality of infected tumors with respect to the structure of the viral terminal repeat sequences. In situ hybridization has the advantage of precise localization of the virus in infected tissues or tumors. Polymerase chain reaction is exquisitely sensitive in detecting viral DNA, perhaps too sensitive for disease-specific purposes given the ubiquitous nature of EBV. Each of these molecular genetic methods of EBV analysis is currently used in research laboratories, while some methods have found their way into routine diagnostic pathology because they are faster, more sensitive, or more informative than previous assays.

Blotting, Southern↗
PubMed1996

A purified mariner transposase is sufficient to mediate transposition in vitro.

Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5' and 3' cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.

Animals↗
PubMed1997

Molecular subtyping of human T-lymphotropic virus type I (HTLV-I) by a nested polymerase chain reaction-restriction fragment length polymorphism analysis of the envelope gene: two distinct lineages of HTLV-I in Taiwan.

The major type of human T-lymphotropic virus type I (HTLV-I), generally referred to as the cosmopolitan type, has been grouped into three subtypes (A, B, and C) by phylogenetic analysis of the long terminal repeat sequences of the viral genome. Twelve subtype-specific nucleotide variations have been deduced by comparison between the envelope (env) sequences of 16 HTLV-I strains with defined subtypes and 9 Taiwanese HTLV-I strains. To gain further insights into the molecular epidemiology of HTLV-I and the origin of this virus in Taiwan, a rapid method of identification for the cosmopolitan subtypes was developed by using a nested polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) studies using the two subtype B-specific and four subtype C-specific nucleotides located within the positions 5708 to 6320 of the envgene. The nested PCR-RFLP method was used to subtype HTLV-I from four virus-positive cell lines derived from 1 Japanese and 3 North American patients, as well as 41 blood-unrelated Taiwan Chinese. The sequences of PCR products were determined and the six positions of subtype-specific nucleotide variations were examined. The sequence data completely supported the subtyping data via the nested PCR-RFLP method. The results demonstrated that, as is the case in Japan, at least two distinct cosmopolitan subtypes (A and B) of HTLV-I were present in Taiwan, but the more prevalent subtype in Taiwan is A in contrast to subtype B in Japan. Furthermore, rapid subtyping could facilitate molecular epidemiological studies of HTLV-I infection and linkage between HTLV-I subtypes and virus-associated diseases.

Base Sequence↗
PubMed1997

Human T-lymphotropic virus type II infection in Vietnamese thalassemic patients.

Anti-human T-lymphotropic virus type I/II (HTLV-I/II) antibodies were screened by particle agglutination test in a total of 66 patients with thalassemia major who received multiple transfusion from paid donors at the Blood Transfusion Hematology Center of Ho Chi Minh City in South Vietnam. HTLV-II infection was confirmed in 6 patients (9.1%) by Western blot analysis and/or polymerase chain reaction. Phylogenetic analysis revealed that long terminal repeat sequences of HTLV-II proviruses from 5 thalassemic patients in Vietnam belonged to the same phylogenetic subgroup of HTLV-IIb as those from intravenous drug abusers in North America and Europe. These data shed light on the route of introducing HTLV-II into Vietnam.

Adolescent↗
PubMed1987

Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats.

The insertion sequence IS903 has perfect, 18-base-pair terminal repeats that are the presumed binding sites of its transposase. We have isolated mutations throughout this inverted repeat and analyzed their effect on transposition. We show that every position in the inverted repeat (with the possible exception of position 4) is important for efficient transposition. Furthermore, various substitutions at a single position can have a wide range of effects. Analysis of these hierarchical effects suggests that transposase contacts the minor groove in the region from position 13 to position 16 but makes major groove (or more complex) interactions with the outer portion of the inverted repeat. Our data indicate that the transposase exhibits relaxed specificity for the "second" end of a transposed segment; the defect in transposition of virtually all mutant inverted repeats can be rescued by a wild-type end. However, this rescue exhibits a pronounced position effect; in most cases, it is efficient only when the wild-type end is close to the 3' end of the transposase gene. This confirms the cis-acting nature of the transposase protein and suggests the initial transposase-inverted repeat interaction is the rate-limiting step in transposition. From the behavior of transposons with one mutant and one wild-type end, we infer that the inverted repeat contains two functional domains--one for initial complex formation with transposase and the other for effective completion of transpositional recombination. To support this hypothesis we show that an end with a mutation in one domain can significantly rescue an end with a mutation in the other domain.

Binding Sites↗
PubMed2006

Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus interacts with origin recognition complexes at the LANA binding sequence within the terminal repeats.

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the cis-acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G(1)/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.

Antigens, Viral↗
PubMed1984

Nucleotide sequence analysis of the long terminal repeat of integrated bovine leukemia provirus DNA and of adjacent viral and host sequences.

The nucleotide sequence of the 3' long terminal repeat and adjacent viral and host sequences was determined for a bovine leukemia provirus cloned from a bovine tumor. The long terminal repeat was found to comprise 535 nucleotides and to harbor at both ends an imperfect inverted repeat of 7 bases. Promoter-like sequences (Hogness box and CAT box), an mRNA capping site, and a core enhancer-related sequence were tentatively located. No kinship was detected between this bovine leukemia proviral fragment and other retroviral long terminal repeats, including that of human T-cell leukemia virus.

Animals↗