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PubMed2004

Reduced proviral loads during primo-infection of sheep by Bovine Leukemia virus attenuated mutants.

BACKGROUND: The early stages consecutive to infection of sheep (e.g. primo-infection) by Bovine leukemia virus mutants are largely unknown. In order to better understand the mechanisms associated with this period, we aimed at analyzing simultaneously three parameters: B-lymphocytosis, cell proliferation and viral replication. RESULTS: Sheep were experimentally infected either with a wild type BLV provirus or with selected mutants among which: a virus harboring an optimalized LTR promoter with consensus cyclic AMP-responsive elements, two deletants of the R3 or the G4 accessory genes and a fusion-deficient transmembrane recombinant. Seroconversion, as revealed by the onset of an anti-viral antibody response, was detected at 3 to 11 weeks after inoculation. At seroconversion, all sheep exhibited a marked increase in the numbers of circulating B lymphocytes expressing the CD5 and CD11b cluster of differentiation markers and, interestingly, this phenomenon occurred independently of the type of virus. The net increase of the absolute number of B cells was at least partially due to accelerated proliferation as revealed, after intravenous injection of bromodeoxyuridine, by the higher proportion of circulating BrdU+ B lymphocytes. BLV proviral DNA was detected by polymerase chain reaction in the leucocytes of all sheep, as expected. However, at seroconversion, the proviral loads were lower in sheep infected by the attenuated proviruses despite similar levels of B cell lymphocytosis. CONCLUSIONS: We conclude that the proviral loads are not directly linked to the extent of B cell proliferation observed during primo-infection of BLV-infected sheep. We propose a model of opportunistic replication of the virus supported by a general activation process of B lymphocytes.

Animals↗
PubMed2006

SUV39H1 interacts with HTLV-1 Tax and abrogates Tax transactivation of HTLV-1 LTR.

BACKGROUND: Tax is the oncoprotein of HTLV-1 which deregulates signal transduction pathways, transcription of genes and cell cycle regulation of host cells. Transacting function of Tax is mainly mediated by its protein-protein interactions with host cellular factors. As to Tax-mediated regulation of gene expression of HTLV-1 and cellular genes, Tax was shown to regulate histone acetylation through its physical interaction with histone acetylases and deacetylases. However, functional interaction of Tax with histone methyltransferases (HMTase) has not been studied. Here we examined the ability of Tax to interact with a histone methyltransferase SUV39H1 that methylates histone H3 lysine 9 (H3K9) and represses transcription of genes, and studied the functional effects of the interaction on HTLV-1 gene expression. RESULTS: Tax was shown to interact with SUV39H1 in vitro, and the interaction is largely dependent on the C-terminal half of SUV39H1 containing the SET domain. Tax does not affect the methyltransferase activity of SUV39H1 but tethers SUV39H1 to a Tax containing complex in the nuclei. In reporter gene assays, co-expression of SUV39H1 represses Tax transactivation of HTLV-1 LTR promoter activity, which was dependent on the methyltransferase activity of SUV39H1. Furthermore, SUV39H1 expression is induced along with Tax in JPX9 cells. Chromatin immunoprecipitation (ChIP) analysis shows localization of SUV39H1 on the LTR after Tax induction, but not in the absence of Tax induction, in JPX9 transformants retaining HTLV-1-Luc plasmid. Immunoblotting shows higher levels of SUV39H1 expression in HTLV-1 transformed and latently infected cell lines. CONCLUSION: Our study revealed for the first time the interaction between Tax and SUV39H1 and apparent tethering of SUV39H1 by Tax to the HTLV-1 LTR. It is speculated that Tax-mediated tethering of SUV39H1 to the LTR and induction of the repressive histone modification on the chromatin through H3 K9 methylation may be the basis for the dose-dependent repression of Tax transactivation of LTR by SUV39H1. Tax-induced SUV39H1 expression, Tax-SUV39H1 interaction and tethering to the LTR may provide a support for an idea that the above sequence of events may form a negative feedback loop that self-limits HTLV-1 viral gene expression in infected cells.

Base Sequence↗
PubMed2006

The evolution of retrotransposon regulatory regions and its consequences on the Drosophila melanogaster and Homo sapiens host genomes.

It has now been established that transposable elements (TEs) make up a variable, but significant proportion of the genomes of all organisms, from Bacteria to Vertebrates. However, in addition to their quantitative importance, there is increasing evidence that TEs also play a functional role within the genome. In particular, TE regulatory regions can be viewed as a large pool of potential promoter sequences for host genes. Studying the evolution of regulatory region of TEs in different genomic contexts is therefore a fundamental aspect of understanding how a genome works. In this paper, we first briefly describe what is currently known about the regulation of TE copy number and activity in genomes, and then focus on TE regulatory regions and their evolution. We restrict ourselves to retrotransposons, which are the most abundant class of eukaryotic TEs, and analyze their evolution and the subsequent consequences for host genomes. Particular attention is paid to much-studied representatives of the Vertebrates and Invertebrates, Homo sapiens and Drosophila melanogaster, respectively, for which high quality sequenced genomes are available.

Animals↗
PubMed2000

Intron 1 rather than 5' flanking sequence mediates cell type-specific expression of c-myb at level of transcription elongation.

Previous studies have shown that expression of steady-state c-myb mRNA was regulated primarily by a block in intron 1 during transcription elongation. This study shows that the block site maps approximately 1700 bp from the start of the intron. Studies based on a reporter construct containing c-myb flanking region and intron 1 suggest that the flanking region is not important in the regulation of the cell type-specific expression of c-myb. RNA splicing of intron 1 may enhance the expression in a non-cell type-specific manner. A conserved intron domain comprising the block site is required for defining this site, but this function of the domain is independent of cell type. The cell type-specific regulation of c-myb transcription elongation is mediated by a 5' intron sequence. A mechanism for down regulation of c-myb gene expression by the block to transcription elongation has been proposed.

3T3 Cells↗
PubMed1999

Sequence patterns observed in 5' flanking regions of primate Alu elements.

Retrotransposons have generally been known to integrate randomly into host genomes. Jurka, however, showed some consensus sequence patterns at insertion sites of some mammalian retrotransposons and proposed enzymatic involvement that mediates integration. Jurka used about 400 complete human Alu and rodent ID sequences which retain full length with identical flanking sequences at both ends. In our study, more than 25,000 Alu sequences longer than 250 bp were used for comprehensive analysis to identify any consensus sequence(s) preceding the 5' end of Alu elements. "Entropy" at each nucleotide position within 500 bases of the 5' Alu end was computed. Significant drop of entropy was observed between position -20 and -10 of the 5' end of Alu, suggesting the existence of certain consensus sequence(s) in the region. Frequencies of all possible triplets (total of 64) were measured in the same region. Observation that frequencies of triplets "aaa," "taa," and "tta" in the 5' flanking sequences were high is consistent with Jurka. However, frequencies of triplets "att" and "aca" were also significantly high, which are not the primary candidates for nick site in Jurka.

Alu Elements↗
PubMed2002

Selection for c-myc integration sites in polyclonal T-cell lymphomas.

Type B leukemogenic virus (TBLV) is highly related to mouse mammary tumor virus but induces rapidly appearing T-cell lymphomas in mice. Unlike other T-cell tumors induced by retroviruses, only 5 to 10% of TBLV-induced lymphomas have detectable viral integrations near c-myc by Southern blotting, whereas Northern blotting has shown that most tumors have two- to sixfold overexpression of c-myc RNA. In this report, PCR was used to demonstrate that at least 30% of these lymphomas have TBLV insertions near c-myc. Some tumors contained multiple TBLV proviruses in different locations and orientations, suggesting that the tumors are polyclonal. The integrated proviruses near c-myc had different numbers (two to four) of long terminal repeat (LTR) enhancer repeats, although LTRs with three-repeat enhancers dominated the proviral population. Passage of polyclonal tumors in immunocompetent mice and semiquantitative PCR revealed that only cells with particular integrations were selected for growth. In three of six tumors tested, proviruses containing four-repeat enhancers near c-myc were selected during tumor passage. Since tumor cell selection may be accomplished by overexpression of c-myc RNA due to proximity to the unique TBLV LTR enhancer, we inserted LTRs at various locations within a plasmid containing the entire c-myc locus and cellular flanking sequences. To quantitatively measure effects on transcription, the Renilla luciferase gene was substituted for most of c-myc exon 2, and transient transfections were performed with c-myc reporter constructs in two different T-cell lines. As expected, insertion of a TBLV LTR with three-repeat enhancers in either orientation, 5" and 3", of the myc gene elevated reporter activity from 2- to 160-fold, consistent with enhancer function, but four-repeat LTRs had lower levels of expression compared to three-repeat LTRs. Surprisingly, LTR insertions that gave maximal c-myc expression in transient-transfection assays declined in tumor cells selected for growth in vivo. Selection for clonal growth may occur in tumor cells that have modest c-myc overexpression after proviral insertion to prevent apoptosis.

Animals↗
PubMed1995

Molecular cloning of glycoprotein antigens MGP57/53 recognized by monoclonal antibodies raised against bovine milk fat globule membrane.

A cDNA encoding 57 kDa and 53 kDa antigens (MGP57/53) recognized by monoclonal antibodies raised against bovine milk fat globule membrane (MFGM) (Biochim. Biophys. Acta 1199 (1994) 87-95) was cloned from lactating bovine mammary gland by a combination of reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and 3'-rapid amplification of cDNA ends (3'-RACE). The deduced amino-acid sequence showed that mature MGP57/53 consists of 409 amino-acid residues and the calculated molecular weight and isoelectric point are 45,544 and 6.42, respectively. Computer analysis reveals that it has a significant similarity to mouse mammary epithelial cell surface protein, MFG-E8 and a human breast tumor-associated glycoprotein antigen, BA46-1. An N-terminal cysteine-rich domain and a C-terminal tandemly repeated sequence were highly conserved among them, but bovine MGP57/53 lacks 36 amino-acid residues containing a cluster of 5 prolines found in mouse MFG-E8. Northern blot analysis showed that the cDNA hybridized to about 2.0 kb mRNA of lactating bovine mammary gland. These results strongly support our previous report that the two MFGM antigens originate from a single gene and are isoforms with different N-linked sugar chains.

Amino Acid Sequence↗
PubMed1988

Bovine leukaemia: facts and hypotheses derived from the study of an infectious cancer.

Bovine leukaemia virus (BLV) is the etiological agent of chronic lymphatic leukaemia/lymphoma in cows, sheep and goats. Infection without neoplastic transformation was also obtained in pigs, rhesus monkeys, chimpanzees, rabbits and observed in capybaras and water-buffaloes. Structurally and functionally, BLV is a relative of human T lymphotropic viruses 1 and 2 (HTLV-I and HTLV-II) In humans, HTLV-I induces a T-cell leukaemia and its type 2 counterpart has been found in dermatopathic lymphadenopathy, hairy T-cell leukaemia and prolymphocytic leukaemia cases. At variance with HTLV-I, BLV has not been associated with neurological diseases of the degenerative type. Bovine leukaemia virus, HTLV-I and HTLV-II show clearcut sequence homologies. The pathology of the BLV-induced disease, most notably the absence of chronic viraemia, a long latency period and lack of preferred proviral integration sites in tumours, is similar to that of adult T-cell leukaemia/lymphoma induced by HTLV-I. The most striking feature of these three naturally transmitted leukaemia viruses is the X region located between the env gene and the long terminal repeat (LTR) sequence. The X region contains several overlapping long open reading frames. One of them, designated XBL-I, encodes a trans-activator function capable of increasing the level of gene expression directed by BLV-LTR and most probably is involved in "genetic instability" of BLV-infected cells of the B cell lineage. The "genetic instability" renders the infected cell susceptible to move, along a number of stages, towards full malignancy. Little is known about these events and their causes; we present some theoretical possibilities. Bovine leukaemia virus infection has a worldwide distribution. In temperate climates, the virus spreads mostly via iatrogenic transfer of infected lymphocytes. In warm climates and in areas heavily populated by haematophagous insects, there are indications of insect-borne propagation of the virus.

Animals↗
PubMed1982

Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova.

On lysis of macronuclei from the ciliated protozoan Oxytricha at 0.5-2 M NaCl, the DNA, which is normally found as discrete molecules ranging from 0.5 to 20 kilobases, appears in high molecular weight aggregates. Various treatments of the macronuclear lysate (i.e., nucleases, proteases, variation of salt, pH, and temperature) indicate that preservation of the aggregate structure depends on both nucleic acid-nucleic acid and nucleic acid-protein interactions. Purification of the DNA-protein complex after lysing the nuclei in 2 M NaCl shows that one major nuclear protein copurifies with the DNA. As shown by DNA-protein binding experiments, this protein has a high affinity for DNA; however, no evidence for sequence specificity of the protein binding was obtained. Chromatin reconstitution experiments suggest that the protein in itself is not sufficient for DNA aggregation in nuclei, but other factors, possibly the native chromatin structure, are required. Electron microscopy of the purified DNA-protein complex showed structures similar to those observed previously with in vitro-aggregated purified macronuclear DNA (14). A model is presented in which the terminal inverted repeat sequences found on all macronuclear DNA molecules interact with each other forming multistranded DNA complexes. The formation of these structures may be accelerated and stabilized by a protein in vivo.

Animals↗
PubMed1987

Bovine leukaemia: facts and hypotheses derived from the study of an infectious cancer.

Bovine leukaemia virus (BLV) is the aetiological agent of a chronic lymphatic leukaemia/lymphoma in cows, sheep and goats. Infection without neoplastic transformation has also been demonstrated in pigs, rhesus monkeys, chimpanzees and rabbits and observed in capybaras and water buffaloes. Structurally and functionally, BLV is a relative of human T lymphotropic viruses 1 and 2 (HTLV-I and HTLV-II) since all three viruses show clear-cut sequence homologies. The pathology of the BLV-induced disease, most notably the absence of chronic viraemia, a long latency period and lack of preferred proviral integration sites in tumours, is similar to that of adult T-cell leukaemia/lymphoma induced by HTLV-I. The most striking feature of the three naturally transmitted leukaemia viruses is the X region located between the env gene and the long terminal repeat (LTR) sequence. The X region contains several overlapping long open reading frames, one of which, designated XBL-1, encodes a trans-activator function capable of increasing the level of gene expression directed by BLV-LTR and is most probably involved in genetic instability of BLV-infected cells of the B-cell lineage. The 'genetic instability' may put the cell into a state of fragility, ready to move along a number of stages towards full malignancy. Little is known about these events and their causes and we present some theoretical possibilities. BLV infection has a worldwide distribution. In temperate climates the virus spreads mostly through iatrogenic transfer of infected lymphocytes. In warm climates and in areas heavily populated by haematophagous insects, there are indications of insect-borne propagation of the virus.

Animals↗
PubMed2003

Sequence divergence within transposable element families in the Drosophila melanogaster genome.

The availability of the sequenced Drosophila melanogaster genome provides an opportunity to study sequence variation between copies within transposable element families. In this study,we analyzed the 624 copies of 22 transposable element (TE) families (14 LTR retrotransposons, five non-LTR retrotransposons, and three transposons). LTR and non-LTR retrotransposons possessed far fewer divergent elements than the transposons,suggesting that the difference depends on the transposition mechanism. However,there was not a continuous range of divergence of the copies in each class,which were either very similar to the canonical elements,or very divergent from them. This sequence homogeneity among TE family copies matches the theoretical models of the dynamics of these repeated sequences. The sequenced Drosophila genome thus appears to be composed of a mixture of TEs that are still active and of ancient relics that have degenerated and the distribution of which along the chromosomes results from natural selection. This clearly demonstrates that the TEs are highly active within the genome,suggesting that the genetic variability of the Drosophila genome is still being renewed by the action of TEs.

Animals↗
PubMed1999

The dominant mutation Glazed is a gain-of-function allele of wingless that, similar to loss of APC, interferes with normal eye development.

Dominant mutations have served as invaluable tools for Drosophila geneticists. Here we analyze the dominant eye mutation Glazed (Gla) that was described by T. H. Morgan more than 50 years ago. We show that Gla causes the loss of photoreceptor cells during pupal stages, in a process reminiscent of apoptosis, with a concomitant overproduction of eye pigment. This phenotype is very similar to that caused by the loss of D-APC, a negative regulator of Wingless (Wg) signal transduction. Genetic analyses reveal however that the Gla gain-of-function phenotype can be reverted to wild-type. By generating a P-element-induced revertant of Gla we demonstrate that Gla is allelic to wg. The molecular lesion in Gla indicates that the insertion of a roo retrotransposon leads to ectopic expression of wg during pupal stages. We show that the Gla phenotype is similar to that caused by ectopic expression of Wg driven by the sevenless (sev) enhancer. In both cases Wg exerts its effect, at least in part, by negatively regulating the expression of the Pax2 homolog sparkling (spa). Gla represents not only the first dominant allele of wg, but it may also be the first allele ever described for wg.

Alleles↗
PubMed2000

Human T-lymphotropic virus type 1 p30(II) functions as a transcription factor and differentially modulates CREB-responsive promoters.

Human T-lymphotropic virus type 1 (HTLV-1), a complex retrovirus, causes adult T-cell lymphoma/leukemia and is linked to a variety of immune-mediated disorders. The roles of proteins encoded in the pX open reading frame (ORF) II gene region in HTLV-1 replication or in mediating virus-associated diseases remain to be defined. A nucleus-localizing 30-kDa protein, p30(II), encoded within pX ORF II has limited homology with the POU family of transcription factors. Recently, we reported that selected mutations in pX ORF II diminish the ability of HTLV-1 to maintain high viral loads in infected rabbits. Herein we have tested the transcriptional ability of p30(II) in mammalian cells by using yeast Gal4 fusion protein vectors and transfection of luciferase reporter genes driven by CREB-responsive promoters. p30(II) as a Gal4 DNA-binding domain (DBD) fusion protein transactivates Gal4-driven luciferase reporter gene activity up to 25-fold in 293 and HeLa-tat cells. We confirmed nuclear localization of p30(II) and demonstrate dose-dependent binding of p30(II)-Gal4(DBD) to Gal4 DNA-binding sites. The transcriptional activity of p30(II)-Gal4(DBD) was independent of TATA box flanking sequences, as shown by using two different Gal4 reporter systems. Studies of selected p30(II) mutants indicated that domains that mediate transcription are restricted to a central core region of the protein between amino acids 62 and 220. Transfection of a p30(II)-expressing plasmid repressed cellular CRE-driven reporter gene activity, with or without Tax expression. In contrast, p30(II) at lower concentrations enhanced HTLV-1 long terminal repeat-driven reporter gene activity independent of Tax expression. These data are the first to demonstrate a transcriptional function for p30(II) and suggest a mechanism by which this nuclear protein may influence HTLV-1 replication or cellular gene expression in vivo.

Animals↗
PubMed2003

Effect of additional N-glycosylation signal in the N-terminal region on intracellular function of the human gonadotropin alpha-subunit.

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. The alpha-subunit has two N-glycosylation sites at Asn52 and Asn78. To obtain more information on the relationship between the structure and function of the alpha-subunit, we introduced a novel N-glycosylation site in the N-terminal region by mutating Asp3 and Gln5 into Asn and Thr, respectively. Glycosylation mutants were expressed alone or with hCGbeta-subunit in Chinese hamster ovary cells. New N-linked oligosaccharides were efficiently added to the wild-type and mutant alpha-subunits lacking N-glycan at Asn52 (alpha deltaAsn1), Asn78 (alpha deltaAsn2), and both (alpha deltaAsn(1 + 2)). The new sugar chain did not affect secretion and assembly except that 1) it increased the intracellular degradation of alpha deltaAsn(1 + 2), and 2) it augmented the assembly of alpha deltaAsn1 with hCGbeta-subunit. Amino acid changes generated the attachment of O-glycosylation in free alpha-subunit but not in assembled form. These data indicate that the newly introduced N-glycosylation consensus sequence is functional, and that the N-terminal region of the alpha-subunit is flexible and can be modified without affecting the intracellular function. Furthermore, amino acid sequences in the N-terminus are involved in the O-glycosylation in free alpha-subunit.

Amino Acid Sequence↗
PubMed2005

LINEs, SINEs and processed pseudogenes: parasitic strategies for genome modeling.

Two major classes of retrotransposons have invaded eukaryotic genomes: the LTR retrotransposons closely resembling the proviral integrated form of infectious retroviruses, and the non-LTR retrotransposons including the widespread, autonomous LINE elements. Here, we review the modeling effects of the latter class of elements, which are the most active in humans, and whose enzymatic machinery is subverted to generate a large series of "secondary" retroelements. These include the processed pseudogenes, naturally present in all eukaryotic genomes possessing non-LTR retroelements, and the very successful SINE elements such as the human Alu sequences which have evolved refined parasitic strategies to efficiently bypass the original "protectionist" cis-preference of LINEs for their own retrotransposition.

Animals↗
PubMed2002

Heterogeneous yet similar introns reside in identical positions of the rRNA genes in natural isolates of the archaeon Aeropyrum pernix.

Some archaeal ribosomal DNA (rDNA) introns carry homing endonuclease-like genes and are therefore assumed to propagate by "intron homing". A previous study demonstrated that three introns are located within the rRNA operon (arnSL) of Aeropyrum pernix strain K1, two of which, Ialpha and Igamma, harbor open reading frames (ORFs) encoding putative LAGLIDADG-type endonucleases. In an effort to understand further the rDNA intron distribution in natural A. pernix populations, 11 A. pernix strains were isolated from marine hydrothermal biotopes, and comparative nucleotide sequence analysis of the arnSL alleles was performed. Of the 11 isolates, eight contained multiple introns, and three patterns of intron insertion were found. Three novel introns, Idelta (62 bp in length), Ivarepsilon (122 bp) and Izeta (57 bp) were identified. They were all ORF-less, but their predicted RNA secondary structure at the exon-intron junctions was consistent with the bulge-helix-bulge motif. The insertion positions and the terminal inverted repeat sequences of Idelta and Izeta were in agreement with those of Ialpha and Igamma, respectively. This suggests that these intron variants were generated by large indels (insertions/deletions) during their evolution.

Amino Acid Sequence↗
PubMed1984

Heterogeneity in subregions of the terminal repeats of herpes simplex virus type 2 DNA.

We constructed recombinant plasmids from the 0.5 molar terminal fragments of herpes simplex virus type 2 (HSV2) DNA. Physical mapping of three different L-terminal clones demonstrated one, two or three copies of the terminally redundant a repeat. The nucleotide sequence at the end of the terminal a repeats was shown to be identical in these three cloned HSV2 genomic L-termini and closely resembled the internal a' repeat sequence reported previously by Davison and Wilkie. Virtually all clones that were obtained from the S-termini differed in a specific subregion of the inserts. This heterogeneous region of the HSV2 genome, that varied in size from 0.2 to 0.85 M daltons, seemed to result from local deletions or amplifications and mapped near the junctions between US and c repeats.

Base Sequence↗
PubMed2006

Identification of transplanted human cells in animal tissues.

The potential of cell and gene therapy has generated extensive interest over the past several years. More recently, identification of stem cells of various types, especially embryonic stem cells, reinforced this interest. Systematic studies are now being launched to define the biology of various stem cells, including after transplantation of cells in mmunodeficient animals. This requires robust and unequivocal means to identify transplanted cells. Ideally, it should be possible to screen animal tissues for human cells with relatively simpler methods, followed by more precise localization of transplanted cells. We describe the application of conserved primate Charcot-Marie-Tooth disease type 1A repeat element for polymerase chain reaction-based screening of animal tissues for human cells. Similarly, direct polymerase chain reaction labeling of pancentromeric human alphoid sequences with digoxigenin-UTP generates in situ hybridization probes for identifying transplanted human cells. This pancentromeric probe identifies human cells irrespective of the original tissue source and can be combined with additional in situ methods to analyze cell differentiation. Incorporation of these strategies will facilitate translational studies aimed at understanding mechanisms concerning the trafficking, engraftment, proliferation, differentiation and function of human stem cells in animals.

Animals↗