Expression pattern analysis of transcribed HERV sequences is complicated by ex vivo recombination

<p>Abstract</p> <p>Background</p> <p>The human genome comprises numerous human endogenous retroviruses (HERVs) that formed millions of years ago in ancestral species. A number of loci of the HERV-K(HML-2) family are evolutionarily much younger. A recent study suggested an infectious HERV-K(HML-2) variant in humans and other primates. Isolating such a variant from human individuals would be a significant finding for human biology.</p> <p>Results</p> <p>When investigating expression patterns of specific HML-2 proviruses we encountered HERV-K(HML-2) cDNA sequences without proviral homologues in the human genome, named HERV-KX, that could very well support recently suggested infectious HML-2 variants. However, detailed sequence analysis, using the software RECCO, suggested that HERV-KX sequences were produced by recombination, possibly arising <it>ex vivo</it>, between transcripts from different HML-2 proviral loci.</p> <p>Conclusion</p> <p>As RT-PCR probably will be instrumental for isolating an infectious HERV-K(HML-2) variant, generation of "new" HERV-K(HML-2) sequences by <it>ex vivo </it>recombination seems inevitable. Further complicated by an unknown amount of allelic sequence variation in HERV-K(HML-2) proviruses, newly identified HERV-K(HML-2) variants should be interpreted very cautiously.</p

Analysis of transcribed human endogenous retrovirus W env loci clarifies the origin of multiple sclerosis-associated retrovirus env sequences

<p>Abstract</p> <p>Background</p> <p>Multiple sclerosis-associated retrovirus (MSRV) RNA sequences have been detected in patients with multiple sclerosis (MS) and are related to the multi-copy human endogenous retrovirus family type W (HERV-W). Only one HERV-W locus (ERVWE1) codes for a complete HERV-W Env protein (Syncytin-1). Syncytin-1 and the putative MSRV Env protein have been involved in the pathogenesis of MS. The origin of MSRV and its precise relation to HERV-W were hitherto unknown.</p> <p>Results</p> <p>By mapping HERV-W <it>env </it>cDNA sequences (n = 332) from peripheral blood mononuclear cells of patients with MS and healthy controls onto individual genomic HERV-W <it>env </it>elements, we identified seven transcribed HERV-W <it>env </it>loci in these cells, including ERVWE1. Transcriptional activity of individual HERV-W <it>env </it>elements did not significantly differ between patients with MS and controls. Remarkably, almost 30% of HERV-W <it>env </it>cDNAs were recombined sequences that most likely arose <it>in vitro </it>between transcripts from different HERV-W <it>env </it>elements. Re-analysis of published MSRV <it>env </it>sequences revealed that all of them can be explained as originating from genomic HERV-W <it>env </it>loci or recombinations among them. In particular, a MSRV <it>env </it>clone previously used for the generation of monoclonal antibody 6A2B2, detecting an antigen in MS brain lesions, appears to be derived from a HERV-W <it>env </it>locus on chromosome Xq22.3. This locus harbors a long open reading frame for an N-terminally truncated HERV-W Env protein.</p> <p>Conclusion</p> <p>Our data clarify the origin of MSRV <it>env </it>sequences, have important implications for the status of MSRV, and open the possibility that a protein encoded by a HERV-W <it>env </it>element on chromosome Xq22.3 may be expressed in MS brain lesions.</p

Characterization and Cloning of MHC Class I Sequences From Choriocarcinoma JEG-3 Cells

1. The use of sera and monoclonal antibodies has permitted the serological definition of three polymorphic genetic loci HLA-A, HLA-B, and HLA-C, which encode the 45kDa heavy chains of the classical class I transplantation antigens. These molecules were identified because they elicited strong allograft rejection. Their true function is to serve as restriction elements by presenting peptidic antigens for recognition by cytotoxic T lymphocytes in MHC restricted responses. Molecular genetic analyses revealed that the vertebrate class I gene family was larger than expected, and there is evidence that at least some of these genes encode polypeptides whose structure and perhaps function is different from those of the classical class I loci

Polyoma virus : polyadenylation and recombination

Imperial Users onl

Analysis of Sonic Hedgehog signalling pathway gene expression in Basal Cell Carcinoma and in GLII induced systems

PhDEmbryonic development is regulated by a number of signalling pathways, Which are critical for normal growth. Many of these genes then continue to play an important role in the regulation of cell growth and differentiation in adult. One such pathway is the sonic hedgehog (SHH) pathway; SHH protein is secreted which binds to its receptor patched (PTCH), leading to the activation and repression of target genes via zinc-finger GLI family transcription factors. Deregulation of this pathway, leads to a number of human birth defects and diseases such as Basal Cell Carcinoma (BCC) of the skin. In transgenic mouse model systems activation of GLI1 by SHH-signalling is a key step in initiating BCC formation. However, there is limited understanding of the molecular mechanisms involved in response to hedgehog signalling and GLI activity in human BCC formation and how this pathway interacts with other pathways. The aim of this thesis was to establish in vitro and in vivo model systems to investigate the molecular events leading to BCC formation. I have shown that Gl.Il , Gi12, Gi13, PTCH, SMO and KlF4 were induced and a-TUB was repressed in BCC relative to normal skin. Using an in vitro model I further showed that Gl.ll , Gi12, Gi13, PTCH, SMO and a-TUB were induced and KlF4 was repressed in GLII expressing keratinocytes. Collaborative work with Dr Fritz Aberger's laboratory in Salzburg showed that Gl.Il and FOXEI are direct targets of GLI2 and I showed that Gl.I? and FOXEI were expressed in the interfollicular epidermis and the outer root sheath of hair follicles in normal skin as well as in BCC tumour islands suggesting a possible link between hair follicle and BCe. I further showed that epidermal growth factor (EGF) signalling reduces transcription activity of GLI1 by shuttling GLII out of nucleus and altering the expression of PTCH, SMO, Gil2 and Gl.B SHH genes. In addition, I demonstrated that whilst EGF induced Vimentin and Snail2 expression and GLII repressed their expression suggesting that GLI is able to counter epithelial-mesenchymal transition associated with EGF and this may in part explain why Bee very rarely metastasise. Furthermore, GLI1 appears to upregulate stem cell like signature and EGF downregulates this signature. Finally, we were able to generate a KRTI4- Floxed-GFP-Gill transgenic mouse but were unable to activate the target gene (KRTI4-GFP-GiIl). In conclusion, I have identified possible targets of GLI activity and shown interactions between EGF signalling and GLI that will help us to understand the potential molecular actions of SHH signalling with the goal of developing better therapeutic strategies

The role of GLI2 in human basal cell carcinoma tumourigenesis

PhDAbnormal Sonic Hedgehog (SHH) signalling leads to increased transcriptional activation of its downstream effector, GLI2, which is implicated in the pathogenesis of a variety of human tumours, including human basal cell carcinoma (BCC). However, little is known about the molecular mechanisms underlying the tumourigenic role of GLI2 in human skin keratinocytes. This study examines the effects of inducible and stable expression of constitutively active GLI2 (GLI2:N) oncogenic transcription factor, on immortalised human epidermal keratinocytes. It is shown here that GLI2:N overexpressing N/TERT keratinocytes display gene expression patterns and phenotypic characteristics reminiscent of those observed in human BCC in vivo. It is also shown for first time, that expression of GLI2:N in N/TERT keratinocytes is sufficient to induce accumulation of binucleated/tetraploid cells as evidenced by an increase in G2/M phase of the cell cycle and binucleate cell counting, and to promote polyploidy and aneuploidy, in the absence of increased cell death or apoptosis. This cell cycle deregulation is accompanied by strong activation of anti-apoptotic protein BCL-2 and simultaneous suppression of important cell-cycle regulators such as 14-3-3σ and CDK inhibitor p21WAF1/CIP1, with no change in p53 protein levels, indicating uncontrolled proliferation of cells with ploidy abnormalities and/or DNA damage. Consistently, it is shown that p21WAF1/CIP1 protein is also absent in human BCC tumours and that forced overexpression of GLI2:N renders human keratinocytes resistant to apoptosis mediated by ultraviolet B (UVB, 290-320 nm), one of the most important etiological factors in BCC formation. Karyotype analysis of GLI2:N N/TERT keratinocytes further demonstrates that overexpression of GLI2:N induces numerical (tetraploidy, polyploidy, aneuploidy) and structural instability in N/TERT keratinocytes including chromosomal translocations and double minute chromosomes. Furthermore, β-catenin activation is the most common alteration observed during aberrant WNT signalling, and is often implicated in the development of human carcinogenesis and metastasis. In this study it is shown that GLI2:N induction induces nuclear accumulation of β-catenin in keratinocyte cell culture and in the basal layer of organotypic skin rafts, similar to human BCCs. In addition, several WNT genes were found to be upregulated upon GLI2:N induction, while β-catenin transcriptional activity is increased upon stable and conditional expression of GLI2:N. Overall these data give new insights for the possible mechanisms that mediate the tumourigenic potential of GLI2