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Embedded in the genomes of all vertebrates are the proviral remnants of previous retroviral infections. Although the overwhelming majority has suffered inactivating mutations, current research suggests that members of one family of human retroelements may still be capable of movement.
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Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in vivo. In hepatocyte nuclei, the incoming single-stranded (ss) vector genomes are converted into various forms of double-stranded (ds) genomes including extrachromosomal linear and circular monomers and concatemers, and a small portion of the vector genomes integrate into chromosomes. The mechanism of genome conversion is not well understood. In the present study, we analyzed the role of inverted terminal repeat (ITR) sequences of ds circular or linear rAAV vector intermediates in concatemerization. We synthesized supercoiled ds circular monomers with a double-D ITR (DDITR) (C+), and ds linear monomers with an ITR at each end (L+), and their control molecules, C- and L-, which lack the ITR-derived sequences, and transfected mouse hepatocytes with these molecules in vivo to assess their capacity for concatemerization. The transfected L+ or L-, but not C+ or C- molecules, concatemerized in vivo irrespective of the presence or absence of the ITRs. In addition, our results suggested that transfected C+ or C- species were not efficient substrates for integration. Based on these observations, we propose a model whereby ds linear molecules with free DNA ends, but not circular molecules, play an important role in rAAV vector genome concatemerization.
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Bone mineral density (BMD), the main determining risk factor for osteoporotic fractures, has a strong genetic component. Estrogen and its receptors play a critical role in both skeletal maturity and bone loss. We investigated the association between dinucleotide (cytosine-adenine; CA) repeat polymorphisms located in the flanking region of the estrogen receptor beta gene and bone mineral density (BMD) in 325 healthy southern Chinese women. BMD at the lumbar spine and hip region were measured using dual-energy X-ray absorptiometry (DEXA). The number of the repeats observed in our population ranged from 16 to 28. After adjusting for age, height, weight, and years of estrogen exposure, we observed that premenopausal subjects (n = 120) bearing at least one allele of 20 CA repeats had significantly higher BMD at the L2-4 lumbar spine (1.049 +/- 0.016 vs. 0.984 +/- 0.015; p = 0.01), total hip (0.836 +/- 0.014 vs. 0.813 +/- 0.013; p < 0.02), femoral neck (0.773 +/- 0.014 vs. 0.728 +/- 0.013; p = 0.02), trochanter (0.665 +/- 0.013 vs. 0.614 +/- 0.012; p = 0.01), and Ward's triangle (0.715 +/- 0.017 vs. 0.651 +/- 0.016; p = 0.02). There was no difference in the vertebral area of L-3 and femoral neck width in these premenopausal women with or without 20 CA repeats. However, in postmenopausal women (n = 205), Estrogen receptor beta (ER beta) gene polymorphisms were not related to BMD at any skeletal site. We conclude that ER beta gene polymorphisms are associated with higher BMD in premenopausal women, suggesting that the ER beta gene may have a modulatory role in bone metabolism in young adulthood.
Sudden mobilization of transposable elements in Drosophila is a well-reported phenomenon but one that usually affects no more than a few elements (one to four). We report here the existence of a D. simulans natural population (Canberra) from Australia, which had high copy numbers for various transposable elements (transposons, LTR retrotransposons and non-LTR retrotransposons). The impact of transposable elements on the host genome and populations is discussed.
The presence of mariner-like elements in four strains of the housefly, Musca domestica, was surveyed by PCR. Using the inverted terminal repeat (ITR) sequences of the Mos 1 element as primers, DNAs were successfully amplified from all strains of the housefly. Southern blot analysis indicated that these amplified DNAs were repetitive sequences in the genome of M. domestica. Sequence analyses of cloned PCR products showed that they were 45% identical to the Mos 1 element. These fragments appeared to be nonfunctional, because they contained no intact open reading frame (ORF) capable of encoding transposase. We conclude that these DNAs are degraded mariner-like elements (MLEs) in M. domestica. Because these endogenous MLEs in M. domestica do not encode any functional proteins, they probably would not affect the behavior of mariner-based vectors if such were introduced into this species as transformation vectors.
A new transposable element, Tcr3, was identified in the unicellular green alga Chlamydomonas reinhardtii. The Tcr3 element contained imperfect terminal inverted repeat sequences of 56 bp and created a 2 bp target site duplication upon insertion. Insertion of Tcr3 into the 3'-untranslated region of the NIT8 gene, which is essential for nitrate assimilation, prevented expression of the gene. Excision of the Tcr3 element correlated with reversion of the mutant phenotype and left behind a 3 bp footprint. Tcr3 was found in all Chlamydomonas isolates tested and should prove to be useful for transposon-tagging experiments in Chlamydomonas.
1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene constructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control. 3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts. 4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.
Recombinant plasmids that express human foamy virus (HFV) Bel 1 transactivator and human estrogen receptor (ER) fusion proteins were constructed. The HFV bel 1 gene was inserted up- and downstream of the ER gene. Recombinant Bel 1-ER and ER-Bel 1 fusion proteins were expressed in eukaryotic cells. In the absence of estrogen, the ER moiety of the fusion proteins suppressed Bel 1-mediated transactivation as measured in CAT reporter gene-based transactivation assays. However, transactivation of the HFV LTR and the HFV internal promoter by Bel 1-ER and ER-Bel 1 fusion proteins was recovered in the presence of estrogen. Thus, the transactivation function of the Bel 1 moiety of the chimeric Bel 1-ER fusion proteins can be efficiently, specifically, and intentionally activated and inactivated by simply adding the low-molecular weight effector estrogen.
Cre-loxP based gene deletion approaches hold great promise to enhance our understanding of molecular pathways controlling mammary development and breast cancer. We reported earlier the generation of transgenic mice that express the Cre recombinase under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). These mice have become a valuable research tool to delete genes specifically in the mammary gland, other secretory organs, and the female germline. We have now characterized in depth the expression of the MMTV-Cre transgene using the ROSA26-lox-Stop-lox-LacZ reporter strain to determine the temporal and spatial activation of Cre on the level of single cells. Our results show that MMTV-mediated Cre-activation is restricted to specific cell types of various secretory tissues and the hematopoietic system. Secondly, the timing of Cre expression varies between tissues and cell types. Some tissues express Cre during embryonic development, while other selected cell types highly activate Cre around puberty, suggesting a strong influence of steroid hormones on the transcriptional activation of the MMTV-LTR. Thirdly, Cre expression in the female germline is restricted to individual mouse lines and is therefore dependent on the site of integration of the transgene. Information provided by this study will guide the researcher to those cell types and developmental stages at which a phenotype can be expected upon deletion of relevant genes.
The terminal DNA arrays on chromosomes of Drosophila melanogaster are composed of two families of non-LTR retrotransposons, HeT-A and TART. Available evidence suggests that chromosome length in this species and its close relatives is maintained by targeted transposition of these elements, with attachment of the elements to the chromosome end by their 3' oligo(A) tails. However, the regulation of transposition of these elements and the control of telomere length are poorly understood. Here we present the hypothesis that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as telomeric position effect (TPE). Based on recent studies of TPE, which found that expression of a reporter gene is influenced by telomere structure in cis and trans, we propose that the subtelomeric satellite (TAS) in D. melanogaster plays an important role in controlling telomere elongation. Transcription of a HeT-A element is probably initiated at a promoter in the 3' UTR of an upstream element, and TAS may repress this transcriptional activity in cis and trans. A region of HeT-A not at the extreme 3' end of the element may act as a transcriptional enhancer that may be modulated by TAS.
Transposable elements (TEs) in the two sibling species, Drosophila melanogaster and D. simulans, differ considerably in amount and dynamics, with D. simulans having a smaller amount of TEs than D. melanogaster. Several hypotheses have been proposed to explain these differences, based on the evolutionary history of the two species, and claim differences either in the effective size of the population or in genome characteristics. Recent data suggest, however, that the higher amount of TEs in D. melanogaster could be associated with the worldwide invasion of D. melanogaster a long time ago while D. simulans is still under the process of such geographical spread. Stresses due to new environmental conditions and crosses between migrating populations could explain the mobilization of TEs while the flies colonize. Colonization and TE mobilization may be strong evolutionary forces that have shaped and are still shaping the eukaryote genomes.
A possible involvement of retroelements in the epigenetic regulation of human gene expression was considered by the example of methylation of long terminal repeats (LTRs) of the human endogenous retrovirus family K (HERV-K). The methylation status of six HERV-K LTRs was determined in various gene-enriched regions of the human genome. The methylation of four LTRs was shown to be tissue-specific. Our results correlated with published data on the tissue-specific changes in the expression level of human genes adjacent to the LTRs under study. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.
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FIS-2, a less oncogenic, immunosuppressive variant of the Friend murine leukaemia virus (F-MuLV), was used to explore whether the differences in biological features were related to early virus dissemination rates or sites of replication. We found that erythroblasts were the primary target cells for both F-MuLV and FIS-2, while B- and T-cells were infected later in the infection. Although FIS-2 replicated to similar titres as F-MuLV, we observed a delay in peak viraemia titre and in the number of virus-positive cells in bone marrow and spleen. Studies including the chimeric viruses RE3 (FIS-2LTR with a F-MuLV background) and RE4 (F-MuLV LTR with a FIS-2 background) indicated that the delay in dissemination was due to mutations in FIS-2 LTR. The kinetics for early virus replication correlated with previously reported mean latency time for virus-induced erythroleukaemia in mice inoculated as newborns and with the onset of immunosuppression in adult mice. In addition, F-MuLV-induced late immunosuppression coincided with signs of erythroleukaemia and persistent viraemia. FIS-2 induced a more moderate late immunosuppression without persistent viraemia or signs of erythroleukaemia. Overall, our findings indicated that early viral replication is a prognostic factor in murine retrovirus-induced pathogenesis.
Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.
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