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At least 1,099 records · Page 61Linked to original sources
PubMed2003

Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas.

The identification of tumor-inducing genes is a driving force for elucidating the molecular mechanisms underlying cancer. Many retroviruses induce tumors by insertion of viral DNA adjacent to cellular oncogenes, resulting in altered expression and/or structure of the encoded proteins. The availability of the mouse genome sequence now allows analysis of retroviral common integration sites in murine tumors to be used as a genetic screen for identification of large numbers of candidate cancer genes. By positioning the sequences of inverse PCR-amplified, virus-host junction fragments within the mouse genome, 19 target genes were identified in T-cell lymphomas induced by the retrovirus SL3-3. The candidate cancer genes included transcription factors (Fos, Gfi1, Lef1, Myb, Myc, Runx3, and Sox3), all three D cyclins, Ras signaling pathway components (Rras2/TC21 and Rasgrp1), and Cmkbr7/CCR7. The most frequent target was Rras2. Insertions as far as 57 kb away from the transcribed portion were associated with substantially increased transcription of Rras2, and no coding sequence mutations, including those typically involved in Ras activation, were detected. These studies demonstrate the power of genome-based analysis of retroviral insertion sites for cancer gene discovery, identify several new genes worth examining for a role in human cancer, and implicate the pathways in which those genes act in lymphomagenesis. They also provide strong genetic evidence that overexpression of unmutated Rras2 contributes to tumorigenesis, thus suggesting that it may also do so if it is inappropriately expressed in human tumors.

Animals↗
PubMed2001

Length analyses of mammalian G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) play a crucial role in mediating effects of extracellular messengers in a wide variety of biological systems, comprising the largest gene superfamily at least in mammals. Mammalian GPCRs are broadly classified into three families based on pharmacological properties and sequence similarities. These sequence similarities are largely confined to the seven transmembrane domains, and much less in the extracellular and intracellular loops and terminals (LTs). Together with the fact that the LTs vary considerably in length and sequence, the LT length of GPCRs has not been studied systematically. Here we have applied a statistical analysis to the length of the LTs of a wide variety of mammalian GPCRs in order to examine the existence of any trends in molecular architecture among a known mammalian GPCR population. Tree diagrams constructed by cluster analyses, using eight length factors in a given GPCR, revealed possible length relations among GPCRs and defined at least three groups. Most samples in Group J (joined) and Group M (minor) had an exceptionally long N-terminal and I3 loop, respectively; and other samples were considered as Group O (other/original). This length-based classification largely coincided with the conventional sequence- and pharmacology-based classification, suggesting that the LT length contains some biological information when analysed at the population level. Principle component analyses suggested the existence of inherent length differences between loops and terminals as well as between extracellular and intracellular LTs. Wilcoxon rank transformation tests unveiled statistically significant differences between Group O and Group J, not only in the N-terminal and I3 loop, but also in the E3 loop. Correlation analyses identified an E1-I2 length-correlation in Group O and Group J and an N-E3 length-correlation in Group J. Taken together, these results suggest a possible functional importance of LT length in the GPCR superfamily.

Animals↗
PubMed2004

pCLPG: a p53-driven retroviral system.

We have modified the pCL retroviral system by the insertion of a p53-responsive element, called PG, in the U3 region of the 3'-LTR, either in addition to or in place of the native negative control region/enhancer sequence. We show here that either endogenous or exogenous wild-type p53 may be used to drive expression from the pCLPG system in transduced cells. Upon genotoxic induction of endogenous p53, pCLPG expression surpassed that of the parental, nonmodified virus, specifically when the native promoter was removed and substituted by the p53-responsive element. We propose that the novel pCLPG system will prove to be a valuable tool whether used as a reporter system of p53 function or as an in vitro and in vivo gene transfer vehicle.

Cell Line↗
PubMed2006

Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis.

DRD1 is a SWI/SNF-like protein that cooperates with a plant-specific RNA polymerase, Pol IVb, to facilitate RNA-directed de novo methylation and silencing of homologous DNA. Screens to identify endogenous targets of this pathway in Arabidopsis revealed intergenic regions and plant genes located primarily in euchromatin. Many putative targets are near retrotransposon LTRs or other intergenic sequences that encode short RNAs, which might epigenetically regulate adjacent genes. Consistent with this, derepression of a solo LTR in drd1 and pol IVb mutants was accompanied by reduced cytosine methylation and transcriptional upregulation of neighboring sequences. The solo LTR and several other LTRs that flank reactivated targets are associated with euchromatic histone modifications but little or no H3K9 dimethylation, a hallmark of constitutive heterochromatin. By contrast, LTRs of retrotransposons that remain silent in the mutants despite reduced cytosine methylation lack euchromatic marks and have H3K9 dimethylation. We propose that DRD1 and Pol IVb establish a basal level of silencing that can potentially be reversed in euchromatin, and further reinforced in heterochromatin by other proteins that induce more stable modifications.

Arabidopsis↗
PubMed2006

[Sequence analysis of integration sites of reticuloendotheliosis virus LTR in fowlpox vaccine virus genomes].

By use of genomic DNA prepared from 5 fowlpox virus (FPV) vaccines made in China (from Shandong, Beijing, Liaoning, Zhejing and Shanghai respectively) as the templates, reticuloendotheliosis virus (REV) LTR was amplified in PCR with a pair of primers synthesized according to the sequences flanking the integrated REV-5'LTR in FPV genomes published in US and Australia. Sequence analysis indicated that the REV-5'LTR integration sites in genomes of all 5 Chinese PFV vaccine products were identical to American and Australian FPV vaccines with integrated REV-5' LTR. Among the 5 FPV vaccine products made in China, 3 productes Vac-B-Ch, Vac-D-Ch and Vac-E-Ch had the integrated REV-5' LTR sequences of 223bp with 100% homology to that in American vaccine Vac-3-Am and Australian vaccine Vac-M3-Au. The integrated 505bp REV-LTR sequences in another Chinese products Vac-A-Ch and Vac-C-Ch had 99.6% homology to the integrated REV-LTR of American vaccine Vac-1-Am and Australian vaccine Vac-S-Au. However, REV-5'LTR integrated in all 5 FPV vaccines made in China gave only 75.4%-91.5% homology to LTR of a Chinese field strain HA9901 of REV. Based on the above results, it is reasonable to speculate that the virus stocks for the 5 Chinese FPV vaccine products were rather originally imported with their integrated REV LTR than recombinated with LTR of REV local field strains in China.

China↗
PubMed2000

Evaluation of heterologous insulator function with regard to chromosomal position effect in the mouse blastocyst and fetus.

Insulators are located at the boundaries of differentially regulated genes and delimit their interactions by establishing independent chromatin structures. Recently, an insulator sequence has been found in the 5'-flanking region of arylsulfatase (ARS) gene from sea urchin. To investigate functional conservation of this ARS insulator in mice, we performed blastocyst assays to evaluate the effect of this insulator on the chromosomal position effect, quantitatively. We constructed transgenes that have a luciferase gene under the control of the CMV-IE enhancer and the human elongation factor 1 alpha promoter in the presence or absence of the ARS insulator in both flanking regions. These transgenes were microinjected into 1-cell mouse embryos and luciferase activity was measured at the blastocyst stage. We found that the presence of ARS insulator sequence doubled the number of luciferase-expressing blastocysts, and that the proportion of the blastocysts with high-level expression (> or = 1 x 10(4) relative light units (RLU)) was increased more than tenfold. In the case of transgenic fetuses, however, the presence of ARS insulator did not seem to improve transgene expression. These results suggest that the sea urchin ARS insulator confers position-independent expression driven by the human elongation factor 1 alpha promoter, at least in the blastocyst stage of the mouse.

Animals↗
PubMed2001

In vitro analysis of transcriptional repression of the mouse mammary tumor virus promoter.

Transcription of the proviral DNA of mouse mammary tumor virus (MMTV) is induced by several classes of hormone-activated steroid receptor proteins. Basal promoter activity in the absence of receptor-mediated activation is selectively repressed by a distal negative regulatory element (dNRE) centered approximately 400 bp upstream of the transcription initiation site. An in vitro transcription system based on synthetic T-free cassette templates was developed to assess MMTV promoter activity, and dNRE-mediated repression was partially reconstituted with this system. Repression was observed with templates in which the dNRE was present in several sequence contexts. The activity of transcription preinitiation complexes formed in vitro in the presence of the dNRE could not be distinguished from that of complexes formed in its absence as assessed by the kinetics of transcript accumulation after addition of nucleoside triphosphates to preformed preinitiation complexes. dNRE-mediated repression in vitro appeared to be the result of decreased efficiency of assembly of functional transcription complexes on the MMTV promoter. However, repression could not be explained by inhibition of assembly of TATA-binding protein or transcription factor IIB into transcription complexes, as neither protein decreased the extent of repression when supplied in excess as a purified recombinant protein.

Gene Expression Regulation, Viral↗
PubMed2005

The genomic organization of retrotransposons in Brassica oleracea.

We have investigated the copy numbers and genomic organization of five representative reverse transcriptase domains from retrotransposons in Brassica oleracea. Two non-homologous Pseudoviridae (Ty1/copia-like) elements, two Metaviridae (Ty3/gypsy-like) elements (one related to the Athila family) and one Retroposinae (LINE) element were hybridized to a gridded BAC library, "BoB". The results indicated that the individual LTR retrotransposons (copia and gypsy-like) were represented by between 90 and 320 copies in the haploid genome, with only evidence of a single location for the LINE. Sequence analysis of the same elements against genome survey sequence gave estimates of between 60 and 570, but no LINE was found. There was minimal evidence for clustering between any of these retroelements: only half the randomly expected number of BACs hybridized to both LTR-retrotransposon families. Fluorescent in situ hybridization showed that each of the retroelements had a characteristic genomic distribution. Our results suggest there are preferential sites and perhaps control mechanisms for the insertion or excision of different retrotransposon groups.

Arabidopsis↗
PubMed2001

Recombination breakpoints in the Charcot-Marie-Tooth 1A repeat sequence in Norwegian families.

OBJECTIVE: To investigate the recombination breakpoint in a 3.2 kb junction fragment of the 24 kb CMT1A repeat sequences (CMT1A-REPs) on chromosome 17p11.2-12. MATERIALS AND METHODS: Thirty-eight Norwegian CMT1 patients and 15 asymptomatic family members of 15 separate families including 10 normal controls were investigated using repeat (REP)-PCR. RESULTS: Twenty-six (68.4%) of the CMT1 patients from 9 (60%) families were positive for the CMT1A duplication which was not found in any of the controls. In 89.9% of the REP-PCR positive families the recombination breakpoint was mapped to a 1.7 kb "hot-spot" region, and in 11.1% of the families to a 1.5 kb region telomeric to the 1.7 kb region. CONCLUSION: The frequency and regions for CMT1A-REPs crossover events in Norwegian CMT1A cases are similar to what is found in other populations. REP-PCR is not, however, as sensitive as other diagnostic methods to detect the CMT1A duplication.

Charcot-Marie-Tooth Disease↗
PubMed2005

Telomere binding protein Taz1 establishes Swi6 heterochromatin independently of RNAi at telomeres.

BACKGROUND: The telomere is a specialized heterochromatin conserved among eukaryotes. However, it remains unknown how heterochromatin protein 1 (HP1) is recruited to telomeres and how telomere heterochromatin is formed. In fission yeast, the RNAi (RNA interference)-RITS (RNA-induced initiation of transcriptional silencing) pathway initiates heterochromatin formation at the centromeres and the silent mat locus by using common DNA sequences, the dg and dh repeats, as the templates for small interfering RNA (siRNA). RESULTS: We found that telomeric repeats are sufficient for the establishment of Swi6 (a fission-yeast HP1 homolog) heterochromatin, and the establishment requires Taz1, a telomere binding protein of the TRF family. Additionally, Swi6 heterochromatin is established by a part of the subtelomere that contains sequences highly homologous to that of the dh repeat, and it is strikingly destabilized by the deletion of both Taz1 and RNAi-RITS. Transcripts from the telomeric dh-homologous region were specifically associated with RITS, and deletion of the telomeric dh-homologous region showed the phenotype similar to that of the rnai mutant in terms of the telomeric silencing, indicating that the RNAi-RITS pathway acts at the telomeric dh-homologous region to establish Swi6 heterochromatin. Furthermore, we found that Taz1 establishes Swi6 heterochromatin independently of the telomeric repeats and the RNAi-RITS pathway at the subtelomeres. CONCLUSION: The telomere heterochromatin is regulated by at least two factors: One is Taz1, which is telomere specific, and the other is RNAi-RITS, which is commonly used at the constitutive heterochromatin regions.

Chromatin Immunoprecipitation↗
PubMed2001

DNase protection analysis of retrovirus integrase at the viral DNA ends for full-site integration in vitro.

Retrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating the DNA termini in a concerted fashion into exogenous target DNA (full site), mimicking integration in vivo. Molecular insights into the organization of avian myeloblastosis virus IN at the viral DNA ends were gained by reconstituting nucleoprotein complexes possessing intasome characteristics. Assembly of IN-4.5-kbp donor complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and other donors containing gain-of-function attachment site sequences are specifically protected by IN at low concentrations (<20 nM) with a defined outer boundary mapping ~20 nucleotides from the ends. A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DNase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 ends for full-site integration shows binding by IN at low concentrations probably occurs only at the very terminal nucleotides (<10 bp) on U5. The results suggest that assembly requires a defined number of avian IN subunits at each viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.

Attachment Sites, Microbiological↗
PubMed2004

Insertional polymorphism of a non-LTR mobile element (NLRCth1) in European populations of Chironomus riparius (Diptera, Chironomidae) as detected by transposon insertion display.

The midge Chironomus riparius is distributed all over the Palearctic region and is well characterized both at the morphological and cytogenetic levels. Here we describe a population study based on the insertional polymorphism of the retroposon NLRCth1, by means of a S-SAP (sequence-specific amplification polymorphism) derived technique (transposon insertion display; TID). While a previous study of allozyme polymorphism in Russian samples showed little variability, all the amplicons we identified are polymorphic. Genetic distances between 6 natural populations were calculated according to Nei and did not show a positive correlation with geographic distances. The genetic diversity detected among individuals of a given population was one order of magnitude higher than that among populations. However, the value of phi(ST) was significant (p < 0.001) and indicates that natural populations are more genetically differentiated than random samples of individuals.

Alleles↗
PubMed2004

Somatic transgenesis using retroviral vectors in the chicken embryo.

The avian embryo is an excellent model system for experimental studies because of its accessibility and ease of microsurgical manipulations. While the complete chicken genome sequence will soon be determined, a comprehensive germ cell transmission-based genetic approach is not available for this animal model. Several techniques of somatic cell transgenesis have been developed in the past decade. Of these, the retroviral shuttle vector system provides both (1) stable integration of exogenous genes into the host cell genome, and (2) constant expression levels in a target cell population over the course of development. This review summarizes retroviral vectors available for the avian model and outlines the uses of retroviral-mediated gene transfer for cell lineage analysis as well as functional studies of genes and proteins in the chick embryo.

Animals↗
PubMed2000

Genetic profile of insertion mutations in mouse leukemias and lymphomas.

Murine leukemia retroviruses (MuLVs) cause leukemia and lymphoma in susceptible strains of mice as a result of insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Using a novel approach to amplify and sequence viral insertion sites, we have sequenced >200 viral insertion sites from which we identify >35 genes altered by viral insertion in four AKXD mouse strains. The class of genes most frequently altered are transcription factors, however, insertions are found near genes involved in signal transduction, cell cycle control, DNA repair, cell division, hematopoietic differentiation, and near many ESTs and novel loci. Many of these mutations identify genes that have not been implicated in cancer. By isolating nearly all the somatic viral insertion mutations contributing to disease in these strains we show that each AKXD strain displays a unique mutation profile, suggesting strain-specific susceptibility to mutations in particular genetic pathways.

Animals↗
PubMed1983

Regions specifying transcriptional termination and pausing in the bacteriophage SP01 terminal repeat.

We have determined the nucleotide sequences of four termination sites recognized by Bacillus subtilis RNA polymerase. These sites are located in the terminally repeated segment of the bacteriophage SP01 genome, where most early phage transcription occurs. The SP01 terminators have structures that are similar to those recognized by Escherichia coli RNA polymerase, containing a region of dyad symmetry followed by a stretch of HMU residues in the noncoding DNA strand (HMU is substituted for T in SP01 DNA). We note that in a terminator that is only 60% efficient in vitro, there is a greater distance between these two conserved elements that exists in more efficient terminators. We also find that RNA polymerase molecules which elongate a transcript through a partial terminator often pause at this site.

Bacillus subtilis↗
PubMed2006

Stable gammaretroviral vector expression during embryonic stem cell-derived in vitro hematopoietic development.

Unlike conventional gammaretroviral vectors, the murine stem cell virus (MSCV) can efficiently express transgenes in undifferentiated embryonic stem cells (ESCs). However, a dramatic extinction of expression is observed when ESCs are subjected to in vitro hematopoietic differentiation. Here we report the construction of a self-inactivating vector from MSCV, MSinSB, which transmits an intron embedded within the internal transgene cassette to transduced cells. The internal transgene transcriptional unit in MSinSB comprises the composite cytomegalovirus immediate early enhancer-chicken beta-actin promoter and associated 5' splice site positioned upstream of the natural 3' splice site of the gammaretroviral envelope gene, and is configured such that the transgene translational initiation sequence is coincident with the envelope ATG. MSinSB could be produced at titers approaching 10(6) transducing units/ml and directed higher levels of transgene expression in ESCs than a splicing-optimized MSCV-derived vector, MSGV1. Moreover, when transduced ESCs were differentiated into hematopoietic cells in vitro, MSinSB remained transcriptionally active in greater than 90% of the cells, whereas MSGV1 expression was almost completely shut off. Persistent high-level expression of the MSinSB gammaretroviral vector was also demonstrated in murine bone marrow transplant recipients and following in vitro myelomonocytic differentiation of human CD34(+) cord blood stem/progenitor cells.

Animals↗
PubMed2006

A unique configuration of genome-wide DNA methylation patterns in the testis.

In the mammalian lifecycle, the two periods of genome-wide epigenetic reprogramming are in the early embryo, when somatic patterns are set, and during germ cell development. Although some differences between the reprogrammed states of somatic and germ cells have been reported, overall patterns of genomic methylation are considered to be similar. Using restriction landmark genomic scanning to examine approximately 2,600 loci distributed randomly throughout the genome, we find that the methylation status of testicular DNA is highly distinct, displaying eightfold the number of hypomethylated loci relative to somatic tissues. Identification and analysis of >300 loci show that these regions are generally located within nonrepetitive sequences that are away from CpG islands and 5' regions of genes. We show that a contributing factor for these differences is that the methylation state of non-CpG-island DNA is correlated with the regional level of GC content within chromosomes and that this relationship is inverted between the testis and somatic tissues. We also show that in Dnmt3L-deficient mice, which exhibit infertility associated with abnormal chromosomal structures in germ cells, this unique testicular DNA methylation pattern is not established. These special properties of testicular DNA point to a broad, distinct epigenetic state that may be involved in maintaining a unique chromosomal structure in male germ cells.

Animals↗
PubMed2003

Gene silencing triggered by non-LTR retrotransposons in the female germline of Drosophila melanogaster.

Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing.

Animals↗