RESUMO
High-risk human papillomavirus types, especially type 16, are risk factors for cervical cancer. Preliminary studies suggest that HPV16 polymorphisms in the long control region or in the E6 gene may alter the oncogenic potential of the virus. This could partially explain why some lesions progress to cancer while others do not. A systematic study combining the long control region and E6 has not been undertaken. This prompted us to investigate the long control region and the E6 in northern European women infected with human papillomavirus 16. We identified the sequence variations of both regions and investigated the long control region promoter activity among various isolates. In addition, we correlated the distribution of long control region and E6 polymorphisms with disease status. We analyzed 45 samples from Swedish and Finnish women. The long control region and the E6 gene were sequenced after polymerase chain reaction long control region fragments of six European isolates covering the majority of polymorphisms in this region were ligated into the pALuc vector and used for luciferase assays. In European HPV16 isolates, polymorphisms in the long control region are more frequent than in the E6 gene. Nevertheless, the promoter function was slightly increased in only one of the tested European long control region variants. In addition, we found a specific European E6 variant, L83V, to be enriched in high-grade lesions and cancer rather than a specific European long control region variant. The difference in oncogenicity between European HPV16 genotypes is more probably due to an altered property of the corresponding E6 proteins rather than to an altered activity of the P97 promoter.
Assuntos
Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Infecções Tumorais por Vírus/complicações , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/virologia , Adulto , Europa (Continente)/epidemiologia , Feminino , Humanos , Incidência , Papillomaviridae/patogenicidade , Reação em Cadeia da Polimerase , Fatores de RiscoRESUMO
Infection with high-risk human papillomavirus (HPV) is necessary for the development of a cervical lesion, but only a fraction of precursor lesions progress to cancer. Additional factors, other than HPV type per se, are likely to increase the probability for progression. Intratype genome variations have been reported to be associated with viral persistence and the development of a major cervical disease. We have recently shown that the prevalence of specific HPV16-E6 variants in invasive cervical cancer (ICC) varies between Italian and Swedish women. To extend our initial study we have analyzed E6 variants in cervical lesions from Czech women, ranging from low-grade cervical intraepithelial neoplasia (LCIN) to ICC and scaled up the sample size of our initial study of Swedish and Italian women. In addition, we have correlated the cases of cancers with human leukocyte antigen (HLA) class II haplotypes. In line with our earlier observation, the distribution of specific HPV16-E6 genotypes in CIN and ICC varied in the 3 cohorts. For instance, the HPV16-E6 L83V variant, which has been found to be positively associated with ICC in Swedish women (p = 0.002), was more prevalent in LCIN than in ICC in Italian and Czech women (p = 0.01 and = 0.03, respectively). These data indicate that host genetic factors, such as HLA polymorphism, may determine the potential oncogenicity of the HPV16-E6 L83V variant. Indeed, the DR04-DQ03 haplotype, which is approximately 3-fold more abundant in the normal Swedish population than in those in Italy and the Czech Republic, was found to be positively associated with HPV16-E6 L83V in the 3 cohorts investigated (p = 0.01). This observation may explain why L83V is a risk factor more in Sweden than in the other 2 countries.
Assuntos
Genes MHC da Classe II , Haplótipos , Polimorfismo Genético , Proteínas Repressoras , Neoplasias do Colo do Útero/virologia , Estudos Transversais , República Tcheca , Feminino , Genótipo , Humanos , Itália , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Suécia , Linfócitos T Citotóxicos/imunologia , Neoplasias do Colo do Útero/imunologiaRESUMO
Risk factors other than human papillomavirus (HPV) infection per se for cervical cancer development have been investigated recently. It was suggested that HPV 16 E6 variants and the p53 codon 72 arginine polymorphism could be progression markers. Indeed, it has been demonstrated that specific E6 variants and p53 arginine were both enriched in cancer. However, especially with regard to the latter, divergent results have been reported. Our aim was thus to investigate whether p53 arginine is important for cervical carcinogenesis by scaling up samples of the two European cohorts, the initial results of which were reported previously. In addition, we have assessed the occurrence of p53 codon 72 arginine, in combination with specific HPV 16 E6 genotypes. We found p53 arginine to be increased in cancer of both cohorts, consistent with our previous concept. Although specific E6 genotypes increased gradually with the severity of the lesion, p53 arginine was enriched in cancer only. Moreover, the frequency of the arginine allele was similar in groups with different E6 genotypes. It is concluded that p53 arginine is a risk factor for cervical cancer but probably acts independently of E6 variants.
Assuntos
Proteínas Oncogênicas Virais/genética , Proteínas Repressoras , Proteína Supressora de Tumor p53/genética , Displasia do Colo do Útero/patologia , Neoplasias do Colo do Útero/patologia , Arginina/genética , Códon/genética , Estudos de Coortes , Estudos Transversais , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Genótipo , Humanos , Itália , Invasividade Neoplásica , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Fatores de Risco , Suécia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologiaRESUMO
The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.
Assuntos
Quimiocina CCL2/genética , Cromatina/fisiologia , Regulação da Expressão Gênica , Papillomaviridae/genética , Fator de Transcrição AP-1/metabolismo , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Mensageiro , Análise de Sequência de DNA , Fator de Transcrição AP-1/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Representational difference analysis of cDNA (cDNA-RDA) was used for a comparison of the global transcript level of tumor of the larynx and the corresponding normal epithelial tissue toward the end of detecting differentially expressed genes. Overall, some 130 gene fragments were identified. By sequence analysis and homology comparison, they could be put into several groups related to (potential) functions. Apart from genes whose overexpression was most likely a result of tumor growth or dedifferentiation of epithelial tissue, a lot of genes were isolated that play major roles in signal transduction pathways or apoptosis or act as oncogenes or tumor suppressor genes, in addition to new, entirely unknown genes. Moreover, some cDNAs of known genes were identified that derived from unconventional splicing activity or other transcript modifications. All identified fragments were arrayed on solid support and used for reverse Northern blot analyses. The use of preselected RDA fragments as targets in array-based profiling experiments circumvents many of the problems encountered when dealing with large clone libraries.
Assuntos
Perfilação da Expressão Gênica , Neoplasias/genética , DNA Complementar/análise , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Approximately 90% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for future tumor therapy. Using recombinant adeno-associated virus type 2 (AAV-2) vectors, two types of therapeutic genes were expressed in cervical carcinoma cells with the aim of suppressing the E6/E7 oncogenes: (a) antisense E6/E7 and ribozyme genes and (b) the monocyte chemoattractant protein-1 (MCP-1) gene encoding MCP-1. Previous studies have shown that the MCP-1 protein is able to indirectly repress E6/E7 gene expression and is consistently absent in tumorigenic HPV-positive cervical carcinoma cell lines. Here, the effect of these therapeutic genes on tumor formation is analyzed in nude mice after ex vivo gene transfer into a HPV16- or HPV18-positive cervical carcinoma cell line (HeLa or SiHa, respectively). Whereas AAV-2 vector-mediated transfer of antisense or even ribozyme genes did not significantly influence tumor formation from implanted SiHa cells, the transfer and expression of human MCP-1 strongly inhibited the development of tumors derived from either HeLa or SiHa cells. Similar results were also obtained after in vivo delivery of these genes into SiHa-derived tumors. This suggests that transfer of therapeutic genes mediating a systemic effect via recombinant AAV-2 vectors offers a promising approach for the development of gene therapies directed against papillomavirus-induced human cancers.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Proteínas Repressoras , Neoplasias do Colo do Útero/terapia , Animais , Northern Blotting , Divisão Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Vetores Genéticos , Células HeLa , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Nus , Modelos Genéticos , Oligonucleotídeos Antissenso/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Plasmídeos/genética , RNA Catalítico/genética , Fatores de Tempo , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise beta-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3-6], the effect of other Dkks on the Wnt/beta-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/beta-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/beta-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Transativadores , Animais , Linhagem Celular , Receptores Frizzled , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Microinjeções , Morfogênese , Proteínas/antagonistas & inibidores , Proteínas/genética , RNA Mensageiro/metabolismo , Proteínas Wnt , Proteínas de Xenopus , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Proteínas de Peixe-Zebra , beta CateninaRESUMO
Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.
Assuntos
Proteínas Pol1 do Complexo de Iniciação de Transcrição , RNA Ribossômico/biossíntese , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Genes Reporter/genética , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Ribossômico/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transfecção , Leveduras/genéticaRESUMO
Full-length genomes of the feline foamy virus (FFV or FeFV) isolate FUV were constructed. DNA clone pFeFV-7 stably directed the expression of infectious FFV progeny virus indistinguishable from wild-type, uncloned FFV isolate FUV. The env and bel 1 genes of pFeFV-7 were substituted for by corresponding sequences of the FFV serotype 951 since previous studies implicated a defined part of FFV Env protein as responsible for serotype-specific differences in serum neutralization (I. G. Winkler, R. M. Flügel, M. Löchelt, and R. L. P. Flower, 1998. Virology 247: 144-151). Recombinant virus derived from chimeric plasmid pFeFV-7/951 containing the hybrid env gene and the parental clone pFeFV-7 were used for neutralization studies. By means of a rapid titration assay for FFV infectivity, we show that progeny virus derived from plasmid pFeFV-7 was neutralized by FUV- but not by 951-specific antisera, whereas pFeFV-7/951-derived chimeric virus was neutralized by 951-specific antisera only. Both recombinant proviruses will be useful for repeated delivery of foreign genes for therapeutic gene applications into cats.
Assuntos
Anticorpos Antivirais/imunologia , Genes env , Genoma Viral , Spumavirus/genética , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Gatos , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologia , Proteínas dos Retroviridae/genética , Análise de Sequência de DNA , Spumavirus/imunologia , Transativadores/genética , Proteínas do Envelope Viral/genéticaRESUMO
The anterior endomesoderm of the early Xenopus gastrula is a part of Spemann's organizer and is important for head induction. Here we describe Xblimp1, which encodes a zinc finger transcriptional repressor expressed in the anterior endomesoderm. Xblimp1 represses trunk mesoderm and induces anterior endomesoderm in a cooperative manner with the pan-endodermal gene Mix.1. Furthermore, Xblimp1 can cooperate with the BMP inhibitor chordin to induce ectopic heads, while a dominant-negative Xblimp1 inhibits head formation. The head inducer cerberus is positively regulated by Xblimp1 and is able to rescue microcephalic embryos caused by dominant-negative Xblimp1. Our results indicate that Xblimp1 is required for anterior endomesodermal cell fate and head induction.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Mesoderma/metabolismo , Proteínas/genética , Proteínas Repressoras/genética , Proteínas de Xenopus , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Clonagem Molecular , Indução Embrionária/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/metabolismo , Cabeça/embriologia , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Microinjeções , Dados de Sequência Molecular , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/química , Alinhamento de Sequência , Xenopus/embriologiaRESUMO
Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.
Assuntos
Proteínas de Membrana/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus , Ativinas , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 4 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/metabolismo , Células COS , Técnicas de Cultura , Embrião não Mamífero/metabolismo , Expressão Gênica , Humanos , Inibinas/antagonistas & inibidores , Inibinas/metabolismo , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/química , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Homologia de Sequência , Células Tumorais Cultivadas , XenopusRESUMO
Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Ectoderma/metabolismo , Células Epiteliais/metabolismo , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
A systematic search for expressed sequences in the human Xq28 region resulted in the isolation of 8.5 kb large contigs of human and murine cDNAs with no apparent conserved open reading frames. These cDNAs were found to be derived from the 3"-untranslated region (3"-UTR) of the methyl-CpG-binding protein 2 gene ( MeCP2 ). This long 3"-UTR is part of an alternatively polyadenylated, 10.1 kb MeCP2 transcript which is differentially expressed in human brain and other tissues. RNA in situ hybridization to sections of mouse embryo and adult tissues of an Mecp2 3"-UTR probe showed ubiquitous low level expression in early organogenesis and enhanced expression in the hippocampus during formation of the differentiated brain. Sequence comparison between the human and mouse homologues revealed several blocks of very high conservation separated by less conserved sequences. Additional support for a domain-like conservation pattern of the long 3"-UTR of the MeCP2 gene was obtained by examining conservation in the chimpanzee, orangutan, macaque, hamster, rat and kangaroo. The minimum free energy distribution for the predicted RNA secondary structure was very similar in human and mouse sequences. In particular, the conserved blocks were predicted to be of high minimum free energy, which suggests weak secondary structure with respect to RNA folding. The fact that both the sequence and predicted secondary structure have been highly conserved during evolution suggests that both the primary sequence and the three-dimensional structure of the 3"-UTR may be important for its function in post-transcriptional regulation of MeCP2 expression.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Adenina/metabolismo , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Proteínas Repressoras , Animais , Sequência de Bases , Sequência Conservada , Cricetinae , Evolução Molecular , Variação Genética , Humanos , Proteína 2 de Ligação a Metil-CpG , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Ratos , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.
Assuntos
Dependovirus/classificação , Vírus Auxiliares/classificação , Parvovirus/classificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Capsídeo/genética , DNA Viral , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Genoma Viral , Células HeLa , Vírus Auxiliares/genética , Humanos , Dados de Sequência Molecular , Parvovirus/genética , Primatas , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
In a large-scale gene expression screen 1765 randomly picked cDNAs were analyzed by whole-mount in situ hybridization in Xenopus embryos. Two hundred and seventy three unique, differentially expressed genes were identified, 204 of which are novel in Xenopus. Partial DNA sequences and expression patterns were documented and assembled into a database, 'AXelDB'. Approximately 30% of cDNAs analyzed represent differentially expressed genes and about 5% show highly regionalized expression. Novel marker genes and potential developmental regulators were found. Differential expression of mitochondrial genes was observed. Marker genes were used to study regionalization of the entire gastrula as well as the tail forming region and the epidermis of the tailbud embryo. Four 'synexpression' groups representing genes with shared, complex expression pattern that predict molecular pathways involved in patterning and differentiation were identified. According to their probable functional significance these groups are designated as Delta1, Bmp4, ER-import and Chromatin group. Within synexpression groups, a likely function of genes without sequence similarity can be predicted. The results indicate that synexpression groups have strong prognostic value. A cluster analysis was made by comparing gene expression patterns to derive a novel parameter, 'tissue relatedness'. In conclusion, this study describes a semi-functional approach to investigate genes expressed during early development and provides global insight into embryonic patterning.
Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Xenopus/embriologia , Xenopus/genética , Animais , Bases de Dados Factuais , Ectoderma , Embrião não Mamífero , Indução Embrionária/genética , Endoderma , Epiderme/embriologia , Gástrula , Técnicas Genéticas , Hibridização In Situ/métodos , Cauda/embriologiaRESUMO
Feline foamy virus (FeFV) belongs to the group of spumaretroviruses that contain in addition to gag, pol, and env accessory genes collectively called bel genes. Primate FVs have been shown to utilize internal promoters in addition to the 5' LTR promoters. In contrast to other known retroviruses, the FV pol genes are expressed via spliced transcripts. Northern blot analysis and reverse transcription-coupled polymerase chain reactions (RT-PCR) were used to amplify, clone, and characterize cDNAs generated from subgenomic viral transcripts. Sequencing of the splice site junctions of the different FeFV mRNAs showed that singly and multiply spliced subgenomic transcripts were expressed in virus-infected cells. The relative amount of the spliced pol-specific transcripts was quantitated and FeFV pol mRNA found to be expressed at about one-half of that of the genomic mRNA. The major FeFV internal start site of transcription was identified at RNA position 7925. Comparison of the FeFV transcriptional patterns to those of the human foamy virus revealed that the FeFV bel 1 mRNA was expressed exclusively from the internal promoter in contrast to primate foamy viruses that use both the LTR and the internal promoter for Bel 1 expression. Unexpectedly, an env-bel 2 mRNA was identified in FeFV-infected cells. In addition, cDNAs from FeFV-infected cells were directly amplified by PCR without RT reactions and found to correspond to genomic and to a subset of different subgenomic FeFV mRNAs.
Assuntos
DNA Complementar/genética , DNA Viral/genética , RNA Mensageiro/genética , RNA Viral/genética , Spumavirus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Mapeamento Cromossômico , Primers do DNA/genética , DNA Recombinante , Genes Virais , Genes env , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
High-risk human papillomavirus (HPV) is a known risk factor in the etiology of cervical intraepithelial neoplasia (CIN) I-III and invasive cervical carcinoma (ICC). The most severe preinvasive lesion is CIN III, and it is still not entirely understood why some cases progress to invasion, whereas others do not. Our hypothesis that this could be predicted by intratype variation of the immortalizing and transforming early proteins E6 and E7 was tested. Because HPV16 is frequently detected in cervical neoplastic lesions, 25 CIN III and 17 ICC cases from Swedish women, all positive for this genotype, were selected to investigate the E6 and E7 genes for mutations. PCR-amplified products were sequenced by the fluorescent dideoxy termination method. ICC harbored almost exclusively HPV16 E6 variants (94%) and rarely harbored the prototype (6%), whereas CIN III demonstrated a more uniform distribution of variants (56%) and prototype (44%; P = 0.013). All variants contained variations that were identified in areas likely to be important for protein-protein interaction with p53 or in areas of immunological significance. The most frequent E6 variation was seen at residue 83. This polymorphism was detected alone or in combination with others in 88% of ICC and 44% of CIN III cases. E7 variations were extremely rare and were only detected together with E6 variations in 4% of CIN III and in 6% of ICC cases, suggesting that the HPV16 E7 but not the HPV16 E6 oncoprotein is highly conserved in vivo. This indicates that HPV16 E6 variants, specifically those containing the substitution at residue 83, may be more oncogenic than the prototype and thus carry a higher risk for the development of invasive cervical disease. This may be due to subtle differences in the type of transformation produced or to evasion of host immune defenses. These results might have implications for future in vitro studies, diagnostics, treatment, and vaccine design.
Assuntos
Proteínas de Ligação a DNA , Proteínas Oncogênicas Virais/genética , Displasia do Colo do Útero/genética , Neoplasias do Colo do Útero/genética , Sequência de Bases , Progressão da Doença , Feminino , Variação Genética , Humanos , Mutação , Papillomaviridae , Proteínas E7 de Papillomavirus , Proteínas Tirosina QuinasesRESUMO
The DNA genomes of four new human papillomaviruses, HPV 75, HPV 76, HPV 77, and HPV 80, have been cloned, sequenced, and characterized. HPV 75, HPV 76 (both HPV 49-related), and HPV 77 (HPV 29-related) were isolated from benign cutaneous warts and HPV 80 (HPV 15-related) from histologically normal skin. HPV 77 has also been demonstrated in dysplastic warts and squamous cell carcinomas of the skin. The sequence data presented in this study led to a proposed modification of the definition of a new HPV type. The high degree of DNA sequence similarity between the E7 ORF of HPV 77 and HPV 29 (97.7%), as opposed to the E6 (82.8%) and L1 (85.3%) ORFs, might suggest conservation of a specific function or a possible recombinational event. Only the E6 and L1 ORFs of HPV 75 and HPV 76 have a similarity lower than 90%, whereas the DNA sequences of their upstream regulatory regions (URRs) share a similarity of 93%. The E7, E1, and E4 ORFs, as well as the URR of HPV 15 and HPV 80, share sequence similarities higher than 90%. Such a divergence in the similarity between different segments of the virus genomes of closely related HPV types has not been noted to date. A detailed comparative sequence analysis was performed. HPV 75, HPV 76, and HPV 80 revealed features characteristic of truly cutaneous HPV types, whereas HPV 77 shared several characteristics with the mucosal HPV types, some of which may have functional consequences.
Assuntos
Genes Virais/genética , Glicoproteínas de Membrana/genética , Papillomaviridae/classificação , Papillomaviridae/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Genoma Viral , Humanos , Complexo Antígeno L1 Leucocitário , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/genética , Fases de Leitura Aberta , Papillomaviridae/isolamento & purificação , Proteínas E7 de Papillomavirus , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Integration of human papillomavirus (HPV) DNA into the host cell genome is an important step in cervical carcinogenesis. In tumour cells with integrated HPV DNA, transcription of viral oncogenes E6 and E7 continues into the flanking cellular sequences thereby producing viral-cellular fusion transcripts. Analysis of cellular sequences flanking the integrated HPV68 DNA in the cervical carcinoma cell line ME180 revealed homozygosity of the mutant allele in ME180 cells. We speculated that this could indicate the existence of a cellular tumour suppressor gene in the integration region. We report here the identification of a novel human gene, named APM-1, which is co-transcribed with the HPV68 E6 and E7 genes and is present in the 3'-cellular part of the ME180 viral-cellular fusion transcripts. The APM-1 gene encodes a protein with a BTB/POZ domain and four zinc fingers, and is located at chromosome 18q21. APM-1 transcripts are detected in normal cervical keratinocytes, but not in the majority of cervical carcinoma cell lines analysed. The APM-1 gene caused a reduction of clonal cell growth in vitro of HeLa and CaSki tumour cells. These characteristics make APM-1, the first novel human gene identified in a HPV integration region, a likely candidate for the postulated tumour suppressor gene.