Methylation pattern of the putative tumor-suppressor gene LRRC3B promoter in clear cell renal cell carcinomas.

The leucine rich repeat containing 3B (LRRC3B) gene is a putative tumor suppressor located on human chromosome 3 in the 3p24 region. LRRC3B is frequently altered in colon and gastric cancers and also in leukaemias. In this study we investigated the promoter region methylation as a possible mechanism of LRRC3B gene inactivation in clear cell renal cell carcinomas. We found that the LRRC3B gene promoter was methylated in 43% of clear cell renal carcinoma samples. However, no correlation between DNA methylation and LRRC3B expression was found.

Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas.

UNLABELLED: Renal cell carcinoma (RCC) is the most common malignant tumor of kidney associated with the worst clinical outcome. No molecular markers for RCC diagnostics and prognosis that could be applied in clinics were described yet. Large-scale screening of 3p human chromosome genes/loci in RCC and histologically normal tissues surrounding the tumors using NotI-microarray approach demonstrated that NKIRAS1 gene contained the largest percent of genetic/epigenetic changes in RCC tumor cells. AIM: To validate the results of NotI microarray analysis and study genetic, epigenetic changes, and the expression level of NKIRAS1 gene in human RCC samples. METHODS: DNA and RNA were isolated from freshly-frozen renal tumors' samples (n = 12) and from normal tissues surrounding the tumors. Epigenetic changes (methylation status) of NKIRAS1 were detected by bisulfite sequencing. Genetic changes and expression level were analyzed by Quantitative real-time PCR (qPCR) with SYBR Green. For relative quantification 2-(DeltaDeltaCP) method was used. Nonparametric tests (Wilcoxon, Kruskal - Wallis and Mann - Whitney) were applied for statistical data analysis using the BioStat software. RESULTS: NKIRAS1 expression was downregulated in 75% of RCC samples (9 of 12) compared with surrounding normal tissue. High grade tumors (3 and 4) showed lower expression of NKIRAS1 at the mRNA level than tumors of low grade (1 and 2). No significant association was found between gene expression level and gender or age. Analysis of NKIRAS1 gene copy number was performed in 19 tumor samples. Changes in the copy number of NKIRAS1 gene were observed in 64% (9 of 14) of cRCC samples. 9 samples displayed ratio (< 0.85 and >or= 0.35), thus were considered as hemizygous deletions. 3 samples showed ratio (> 0.85) and were considered as normal copy number. Changes in NKIRAS1 gene copy number were detected in all 3 benign oncocytomas, 1 papillary cancer and 1 sarcoma, where hemizygous deletion was observed. No changes in methylation status of NKIRAS1 were found in RCC. CONCLUSIONS: We have validated the results of NotI microarray analysis of NKIRAS1 gene in RCC. It was shown the decreased expression level of NKIRAS1 in this type of tumor.

Screening of epigenetic and genetic disturbances of human chromosome 3 genes in colorectal cancer.

DNA microarray technology comprising NotI-linking clones was used in a large-scale study of genetic and epigenetic changes in colorectal cancer. Analysis of samples from 24 patients revealed methylation, deletions, and amplifications in 137 of 181 NotI clones. For 27 genes/loci, these changes occurred in more than 30% of the tumor samples, suggesting that these genes are involved in the development of colorectal cancer. An analysis of the methylation status of CpG island of the ITGA9 gene/loci by bisulfite sequencing confirmed the NotI microarray data on the gene/loci methylation in colorectal cancer. Aberrations in 19 genes/loci were unknown previously. Their characterization may help ascertain the mechanisms responsible for colorectal cancer development and identify novel diagnostic and prognostic markers.

Complete nucleotide sequences of ALV-related endogenous retroviruses available from the draft chicken genome sequence.

Complete nucleotide sequences of chicken endogenous retroviruses belonging to E33/E51 and EAV-0 groups have been analysed on the basis of the recently available draft genome sequence of red jungle fowl (Gallus gallus), the progenitor of domestic chicken (G.g. domesticus). It was shown that all these proviruses have deletions in the SU-coding domain of the env gene, involved in receptor recognition, whereas gag and pol genes appear to be intact. Phylogenetic analysis demonstrated that E33/E51 and EAV-0 groups are related to the ALV genus. An analysis of expression using chicken EST databases showed that these proviruses are transcriptionally active.

Distribution of HIV-1 in the genomes of AIDS patients.

The localization of HIV-1 proviruses in compositional DNA fractions from 27 AIDS patients during the chronic phase of the disease with depletion of CD4+ and different levels of viremia showed the following. (1) At low viremia, proviruses are predominantly localized in the GC-richest isochores, which are characterized by an open chromatin structure; this result mimics findings on HIV-1 integration in early infected cells in culture. (2) At higher viremia, an increased distribution of proviruses in GC-poor isochores (which match the GC poorness of HIV-1) was found; this suggests a selection of cells in which the 'isopycnic' localization leads to a higher expression of proviruses and, in turn, to higher viremia. (3) At the highest viremia, integrations in GC-rich isochores are often predominant again, but generally not at the same level as in (1); this may be the consequence of new integrations from the extremely abundant RNA copies.

Initial isolation and analysis of the human Kv1.7 (KCNA7) gene, a member of the voltage-gated potassium channel gene family.

A novel human potassium channel gene was identified and isolated. The maximal open reading frame encodes a protein of 456 amino acids. The predicted product exhibits 91% amino acid identity to the murine voltage-gated potassium channel protein Kv1.7 (Kcna7), which plays an important role in the repolarization of cell membranes. Based on the high similarity, the human gene has been classified as the ortholog of the mouse Kcna7 and given the name Kv1.7 (KCNA7). A structural prediction identified a pore region characteristic of potassium channels and six membrane-spanning domains. Northern expression analysis revealed the gene is expressed preferentially in skeletal muscle, heart and kidney. However, it is expressed at lower level in other tissues, including liver. A single mRNA isoform was observed, with a size of approximately 4.5 kb. Using fluorescence in situ hybridization, the gene was mapped to chromosomal band 19q13.4 (269.13 cR(3000)). A genomic sequence was identified in the database from this region, and the KCNA7 gene structure determined. Computational analysis of the genomic sequence reveals the location of a putative promoter and a likely muscle-specific regulatory region. Initial comparison to the published murine Kcna7 cDNA suggested a different N-terminal sequence for the human protein, however, further analysis suggests that the original mouse sequence contained an error or an unusual polymorphism.

Localization of HTLV-1 and HIV-1 proviral sequences in chromosomes of persistently infected cells.

Integration sites for HTLV-1 and HIV-1proviruses were detected by FISH on the chromosomes of HTHIV27 cells persistently infected by HIV-1 (strain IIIB). HTLV-1 signals were found on 9 loci of chromosomes 4, 6, 9, 15 and 16. Integration sites of GC-rich HTLV-1 provirus are located in GC-rich isochores, confirming an 'isopycnic' integration, namely an integration in which the GC level of the host sequences around the integration site match the GC level of the provirus. This conclusion is not only derived from the compositional map of human chromosomes, but also from HTLV-1 hybridization on compositional fractions of human DNA. Integration of GC-poor HIV-1 provirus was found on 4 loci of chromosomes 2, 7, 17 and 19. One copy of a complete HIV-1 provirus, which is active, was integrated in H1 isochores, whereas other defective copies were located in GC-poor L isochores. These results are discussed in terms of regional integration of retroviral sequences.

The regional integration of retroviral sequences into the mosaic genomes of mammals.

We have reviewed here three sets of data concerning the integration of retroviral sequences in the mammalian genome: (i) our experimental localization of a number of proviruses integrated in isochores characterized by different GC levels; (ii) results from other laboratories on the localization of retroviral sequences in open chromatin regions and/or next to CpG islands; and (iii) our compositional analysis of genes located in the neighborhood of integrated retroviral sequences. The three sets of data have provided a very consistent picture in that a compartmentalized, isopycnic integration of expressed proviruses appears to be the rule ('isopycnic' refers to the compositional match between viral and host sequences around the integration site). The results reviewed here suggest that: (i) integration of proviral sequences is targeted initially towards 'open chromatin regions'; while these exist in both GC-rich and GC-poor isochores, the 'open chromatin regions' of GC-rich isochores are the main targets for integration of retroviral sequences because of their much greater abundance; (ii) isopycnicity is associated with stability of integration; indeed, even non-expressed integrated retroviral sequences tend to show an isopycnic localization in the genome; (iii) transcription of integrated viral sequences (like transcription of host genes) appears to be associated, as a rule, with an isopycnic localization, as indicated by transcribed sequences that show an isopycnic integration and act in trans; (iv) selection plays a role in the choice of specific sites within an isopycnic region; in exceptional cases [such as mouse mammary tumor virus (MMTV) activating GC-rich oncogenes], selection may override isopycnicity.

Origin and evolution of the c-src-transducing avian sarcoma virus PR2257.

Avian sarcoma virus PR2257 transduced de novo the c-src gene and about 900 bp of 3' non-coding sequences belonging to the src locus. This virus contains only one mutation in the c-src coding sequence causing a reading frame shift after Pro-525. The molecular clone studied was derived from a cell line of transformed quail fibroblasts, C7. It contains endogenous virus (ev) derived sequences in the U5 and 3' non-coding regions, indicating that multiple recombination occurred with endogenous virus. Here we investigated the possible evolution of PR2257 when the original tumour was repeatedly passaged in vivo. After 16 passages a new virus, designated PR2257/16, appeared with a tenfold higher titre. The sequence of PR2257/16 was determined and showed that PR2257/16 resulted from recombination of PR2257 with the env gene of the helper virus (td daPR-C). This recombination expanded the env gene content in PR2257/16 and, in addition, five point mutations occurred in its genome. Because we thought that an endogenous virus might be involved in the mechanism of c-src transduction, we also reinvestigated the presence of ev sequences in PR2257 proviruses from several early passages of the original tumour. We found that in contrast with the first isolate from the C7 cell line, the provirus in these tumours did not contain such sequences. These results do not support the hypothesis that endogenous sequences were involved in the transduction process.

The 3' region of c-src gene mRNA is entirely included in exon 12 and does not encode another protein.

We previously reported the isolation of PR2257, a novel replication defective avian sarcoma virus which transduced the entire c-src coding region together with about 900 bp of c-src 3' non coding sequences. This virus originated from a chicken sarcoma induced by inoculation of a transformation-defective Rous sarcoma virus carrying only replicative genes. The 5' end of PR2257 was formed by a splice junction between viral leader sequences and the first exon of c-src. To understand the mechanism of 3' recombination between viral and cellular sequences, we analyzed the genomic organization of the 3' region in chicken and quail src DNA. We report that this sequence is colinear with that of a chicken src cDNA. Therefore, exon 12, which encodes the last 68 amino acids of c-src, also contains all 3' non coding sequences present in c-src mRNA and consequently, appears to be the last and largest (about 2 kbp) exon of c-src. We also show that the 3' regions of chicken and quail c-src genes does not contain the additional open reading frame (orf) which was previously reported (Dorai et al. (1991) Mol. Cell. Biol. 11, 4165-4176), and that no other significant conserved open reading frames could be found in this region for both species. Therefore, this region of src does not code for another protein. Taken together, our results suggest that PR2257 was generated by recombination at the RNA level. However, because of the absence of introns in this region of c-src, we cannot formally rule out the possibility that this recombination took place at the DNA level.

New case of c-src gene transduction: the generation of virus PR2257.

PR2257 is a new replication-defective avian sarcoma virus which harbours in addition to the spliced version of the c-src gene also about 950 bp of no-coding cellular sequences located downstream from the c-src stop codon (Geryk et al., 1989). Comparison of the 950 bp region transduced by PR2257 with the chicken c-src cDNA (Dorai et al., 1991) and genomic sequences of the c-src 3' non-coding region from chicken and quail has shown that there are no additional introns. The c-src 3' non-coding region represents the largest c-src exon (No. 12) comprising about 2 kb. Absence of conserved open reading frames within this region in chicken and quail genomic DNAs excludes the possibility for coding a protein by these sequences. Also, the possibility was excluded that numerous endogenous virus-derived sequences identified in molecularly cloned PR2257 provirus played a role in the c-src transduction. After serial passaging of PR2257 virus in vivo a variant PR2257/16 was isolated. In PR2257/16, the size of the env gene was increased due to homologous recombination with a helper virus. In addition to mutations in the viral leader and the v-src coding region, a large deletion in transduced c-src 3' non-coding sequences was found in the PR2257/16 genome. The significance of genome modifications for selective advantage of this viral variant in vivo is discussed.

Role of replaced v-src and env genes in the duck-adapted variant of Rous sarcoma virus.

The env and v-src genes of a duck-adapted variant of Rous sarcoma virus were replaced for corresponding genes from parental chick-derived virus. The generation of viral constructs with replaced genes is described. DNAs of viruses with replaced genes were assayed on chick and duck embryo fibroblasts by transfection assays. Transformation efficiency was measured by the focus assay and multiplication of virus by the reverse transcriptase assay. Duck-adapted virus with replaced env gene lost the higher transformation efficiency for duck cells, whereas replacement of the v-src gene had no effect on its host-specific transformation activity.

Biology of Rous sarcoma virus adapted for replication and transformation of duck cells.

A duck-adapted variant of the Prague strain of Rous sarcoma virus and its molecularly cloned derivatives were characterized biologically. The virus replicated and transformed with the same efficiency both duck and chicken embryo fibroblasts. The daPR-RSV-C was also used for induction of tumours in newborn hamsters. The virus from virogenic hamster tumours could be rescued using the duck cells as indicator cells. The virus rescue was influenced by restricted replication of the virus in the indicator cells.

Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.

Genome structure of a new avian sarcoma virus PR2257 determined by restriction analysis.

The genome structure of a new avian src-containing acute transforming virus, designated PR2257, was studied by restriction analysis. PR2257 was isolated from a sarcoma grown in a 155-day-old chick inoculated intraembryonally with a transformation-defective mutant of daPR-C retaining no src sequences. The 4.4-kb genome of PR2257 consists of the following regions: 5' LTR--spliced version of chicken c-src--incomplete env gene--3' LTR. This structure clearly indicates that PR2257 was generated by recombination between a td daPR-C and chicken c-src.