RESUMO
Cre-mediated deletion of exons 3 and 4 of the mouse Brca2 gene occurring specifically in mammary epithelial cells of conditional female mutants carrying a combination of loxP-modified and null Brca2 alleles resulted in a high incidence (77%) of breast tumors that were often palpable and developed in one or more glands after long latency (time for median tumor-free survival of approximately 1.4 years; total of 40 tumors in 20 animals). These invasive adenocarcinomas were histologically quite uniform, exhibiting predominantly a solid, nodular tumor pattern with very few variants, in striking contrast to the morphological heterogeneity of analogous Brca1-associated tumors. The karyotypes of tumor cells lacking Brca2 had various chromosomal aberrations and ranged from diploid to hypertetraploid, but this wide variability was incongruous with the histological appearance of carcinomas that was comparable between specimens. The implications of these observations in the context of models positing that Brca2 is involved in the maintenance of genomic stability are discussed.
Assuntos
Adenocarcinoma/genética , Genes Supressores de Tumor , Neoplasias Mamárias Experimentais/genética , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Adenocarcinoma/patologia , Fatores Etários , Alelos , Animais , Proteína BRCA2 , Feminino , Marcação de Genes , Integrases/metabolismo , Cariotipagem , Masculino , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas de Neoplasias/deficiência , Especificidade de Órgãos , Fatores de Transcrição/deficiência , Proteínas Virais/metabolismoRESUMO
We generated mouse mutants carrying in the Brca1 locus a modification (Brca1(tr)) that eliminates the C-terminal half of the protein product and obtained results indicating that, depending on genetic background, the missing BRCT and/or other domains are dispensable for survival, but essential for tumor suppression. Most of the apparently hypomorphic Brca1(tr/tr) mutants developed various tumors. Lymphomas were detected at all ages, whereas sarcomas and carcinomas, including breast cancer, appeared after a long latency. The mammary tumors showed striking variability in histopathological patterns suggesting stochastic engagement of tumorigenic pathways in their progression, to which the Brca1(tr/tr) mutation was apparently a late participant.
Assuntos
Proteína BRCA1/genética , Neoplasias Experimentais/genética , Animais , Proteína BRCA1/metabolismo , Northern Blotting , Feminino , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Mutagênese , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologia , Deleção de SequênciaRESUMO
Deletions of the 13q14 chromosome region are associated with B-cell chronic lymphocytic leukemia (B-CLL) and several other types of cancer, suggesting the presence of a tumor suppressor gene. In previous studies the minimal region of deletion (MDR) was mapped to a less than 300-kilobase (kb) interval bordered by the markers 173a12-82 and 138G4/1.3R. For the identification of the putative tumor suppressor gene, the entire MDR (approximately 347 kb) has been sequenced, and transcribed regions have been identified by exon trapping, EST-based full-length complementary DNA cloning, database homology searches, and computer-assisted gene prediction analyses. The MDR contains 2 pseudogenes and 3 transcribed genes: CAR, encoding a putative RING-finger containing protein; 1B4/Leu2, generating noncoding transcripts; and EST70/Leu1, probably representing another noncoding gene (longest open reading frame of 78 codons). These genes have been sequenced in 20 B-CLL cases with 13q14 hemizygous deletion, and no mutations were found. Moreover, no somatic variants were found in the entire MDR analyzed for nucleotide substitutions by a combination of direct sequencing and fluorescence-assisted mismatch analysis in 5 B-CLL cases displaying 13q14-monoallelic deletion. The nondeleted allele of the CAR and EST70/Leu1 genes was expressed in B-CLL specimens, including those with monoallelic loss, whereas no expression of 1B4/Leu2 was detectable in B-CLL, regardless of the 13q14 status. These results indicate that allelic loss and mutation of a gene within the MDR is an unlikely pathogenetic mechanism for B-CLL. However, haplo-insufficiency of one of the identified genes may contribute to tumorigenesis. (Blood. 2001;97:2098-2104)
Assuntos
Cromossomos Humanos Par 13/genética , DNA de Neoplasias/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Animais , Sequência de Bases , Transformação Celular Neoplásica/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 13/ultraestrutura , Análise Mutacional de DNA , DNA Complementar/genética , Etiquetas de Sequências Expressas , Genes Supressores de Tumor , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas/genética , Pseudogenes , RNA Longo não Codificante , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Deleção de Sequência , Transcrição Gênica , Transferases , Proteínas Supressoras de TumorAssuntos
Deleção Cromossômica , Cromossomos Humanos Par 13/ultraestrutura , Genes Supressores de Tumor , Leucemia Linfocítica Crônica de Células B/genética , Alelos , Antígenos de Neoplasias/análise , Antígenos CD5/análise , Linhagem Celular Transformada , Mapeamento Cromossômico , Cromossomos Humanos Par 13/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Mutação , Proteínas de Neoplasias/genética , Proto-OncogenesRESUMO
Frequent deletions and loss of heterozygosity in a segment of chromosome 13 (13q14) in cases of B-cell chronic lymphocytic leukemia (CLL) have suggested that this malignancy is caused by inactivation of an unknown tumor suppressor gene located in this region. Toward the identification of the putative CLL tumor suppressor, we have constructed a high-resolution physical map of YAC, PAC, and cosmid contigs covering 600 kb of the 13q14 genomic region. In addition to densely positioned genetic markers and STSs, this map was further annotated by localization of 32 transcribed sequences (ESTs) using a combination of exon trapping, direct cDNA selection, sample sequencing of cosmids and PACs, and homology searches. On the basis of these mapping data, allelic loss analyses at 13q14 using CLL tumor samples allowed narrowing of the genomic segment encompassing the putative CLL gene to <300 kb. Twenty-three ESTs located within this minimally deleted region are candidate exons for the CLL-associated tumor suppressor gene.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 13 , Leucemia Linfocítica Crônica de Células B/genética , Alelos , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , DNA , Humanos , Células Híbridas , Dados de Sequência Molecular , Transcrição GênicaRESUMO
Mutations of the human BRCA1 and BRCA2 genes encoding tumor suppressors have been implicated in inherited predisposition to breast and other cancers. Disruption of the homologous mouse genes Brca1 and Brca2 by targeting showed that they both have indispensable roles during embryogenesis, because nullizygous embryos become developmentally retarded and disorganized, and die early in development. In Brca1 mutants, the onset of abnormalities is earlier by one day and their phenotypic features and time of death are highly variable, whereas the phenotype of Brca2 null embryos is more uniform, and they all survive for at least 8.5 embryonic days. Observations with Brca1/Brca2 double nullizygotes raise the possibility that the two developmental pathways could be linked. Interestingly, the impact of the Brca1 or Brca2 null mutation is less severe in a p53 null background.
Assuntos
Genes BRCA1 , Genes p53 , Neoplasias Mamárias Experimentais/genética , Mutação , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Animais , Proteína BRCA2 , Divisão Celular/genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Heterozigoto , Neoplasias Mamárias Experimentais/patologia , Camundongos , FenótipoRESUMO
Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.
Assuntos
Neoplasias da Mama/genética , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 13/genética , Proteínas de Neoplasias/genética , Software , Fatores de Transcrição/genética , Proteína BRCA2 , Sequência de Bases , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 13/ultraestrutura , Cosmídeos/genética , DNA Complementar/genética , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Seleção GenéticaRESUMO
We have developed a general method for en masse isolation of cDNAs present in a normalized library by hybridization to arrayed chromosome-specific phage lambda clones; we have used this approach to initiate exon-mapping of human chromosome 13. An advantage of the simultaneous isolation of cDNA/lambda pairs is that it allows cytogenetic assignment of a bona fide genomic clone by in situ hybridization, which also verifies that the corresponding cDNA or a homologous expressed sequence resides on chromosome 13. This information is enriched by partial sequencing of a selected cDNA from both ends. The sequence of the 3' noncoding region provides an 'identifier' that is used to develop STSs, while the sequence from the 5' end, often corresponding to a coding region, is used for homology searches in databases that occasionally reveal gene functions.
Assuntos
Cromossomos Humanos Par 13 , DNA Complementar/genética , Sequência de Aminoácidos , Animais , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , Cricetinae , Gliceraldeído-3-Fosfato Desidrogenases/genética , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Células Híbridas , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/genética , Proteínas Quinases/genética , Retroviridae/genética , Sitios de Sequências Rotuladas , Tubulina (Proteína)/genéticaRESUMO
Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.
Assuntos
Ciclo Celular , Divisão Celular/fisiologia , Substâncias de Crescimento/farmacologia , Mutação , Receptor IGF Tipo 1/genética , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Transformação Celular Neoplásica , Células Cultivadas , Embrião de Mamíferos , Fator de Crescimento Epidérmico/farmacologia , Genes ras , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Cinética , Camundongos , Fenótipo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor IGF Tipo 1/biossíntese , Transdução de Sinais , TransfecçãoRESUMO
Fibroblast cell lines were established from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 receptor for insulin-like growth factor I (IGF-I) and from their wild-type littermates. The cells from the wild-type embryos (W cells) grow in serum-free medium supplemented with platelet-derived growth factor, epidermal growth factor, and IGF-I, whereas the cells from Igf1r(-/-) embryos (R- cells) do not, although they grow at a reduced rate in 10% fetal calf serum. The simian virus 40 (SV40) large T antigen, expressed from a transfected plasmid, can transform W cells, which form foci in monolayer cultures and colonies in soft agar (anchorage-independent growth). In contrast, the SV40 large tumor antigen, although normally expressed from the transfected template, is unable to transform R- cells, which remain contact-inhibited and fail to grow in soft agar. The transformed phenotype is restored if the R- cells carrying the SV40 large tumor antigen are stably transfected with a plasmid expressing the human IGF-I receptor. These results demonstrate that signaling via the IGF-I receptor is an indispensable component of the SV40 transformation pathway. This conclusion is further supported from the results of antisense RNA experiments with tumor cell lines showing that interference with the function of the IGF-I receptor has a profound effect on anchorage-independent growth, even under conditions that only modestly affect growth in monolayers.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Transformação Celular Viral , Regulação Viral da Expressão Gênica , Receptor IGF Tipo 1/genética , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Divisão Celular , Células Cultivadas , Imunofluorescência , Glioblastoma/patologia , Técnicas In Vitro , Camundongos/embriologia , Células Tumorais CultivadasRESUMO
The mouse insulin-like growth factor II (IGF-II) gene encodes a polypeptide that plays a role in embryonic growth. We have examined the temporal and spatial pattern of expression of this gene in sections of the mouse conceptus between embryonic days 4.0 and 8.5 by in situ hybridization. Abundant IGF-II transcripts were detected in all the trophectodermal derivatives, after implantation. Labeling was then observed in primitive endoderm, but was transient and disappeared after formation of the yolk sac. Expression was next detected in extraembryonic mesoderm at the early primitive streak stage. Labeling in the embryo proper appeared first at the late primitive streak/neural plate stage in lateral mesoderm and in anterior-proximal cells located between the visceral endoderm and the most cranial region of the embryonic ectoderm. The position of the latter cells suggests that their descendants are likely to participate in the formation of the heart and the epithelium of the ventral and lateral walls of the foregut, where intense labeling was observed at the neural fold stage. Hybridization was also detected in cranial mesenchyme, including neural crest cells. The intensity of hybridization signal increased progressively in paraxial (presomitic and somitic) mesoderm, while declining in the ectoplacental cone. The neuroectoderm and surface ectoderm did not exhibit hybridization at any stage. Immunohistochemical analysis indicated co-localization of IGF-II transcripts, translated pre-pro-IGF-II, and the cognate IGF-II/mannose-6-phosphate receptor. These correlations are consistent with the hypothesis that IGF-II has an autocrine function.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Expressão Gênica/fisiologia , Fator de Crescimento Insulin-Like II/genética , Animais , Embrião de Mamíferos/química , Imunofluorescência , Camundongos , Hibridização de Ácido Nucleico , Precursores de Proteínas/análise , Receptor IGF Tipo 2 , Receptores de Superfície Celular/análiseRESUMO
Growth factors are thought to function as pivotal autocrine-paracrine regulatory signals during embryonic development. Insulin-like growth factor II (IGF-II), a mitogenic polypeptide for a variety of cell lines, could have such a role, as indicated by the pattern of expression of its gene during rodent development. The IGF-II gene uses at least three promoters and expresses several transcripts in many tissues during the embryonic and neonatal periods, whereas expression in adult animals is confined to the choroid plexus and the leptomeninges. To examine the developmental role of IGF-II, we have begun to study the consequences of introducing mutations at the IGF-II gene locus in the mouse germ line. We have disrupted one of the IGF-II alleles in cultured mouse embryonic stem (ES) cells by gene targeting and constructed chimaeric animals. Germ-line transmission of the inactivated IGF-II gene from male chimaeras yielded heterozygous progeny that were smaller than their ES cell-derived wild-type littermates (about 60% of normal body weight). These growth-deficient animals were otherwise apparently normal and fertile. The effect of the mutation was exerted during the embryonic period. These results provide the first direct evidence for a physiological role of IGF-II in embryonic growth.
Assuntos
Transtornos do Crescimento/genética , Heterozigoto , Fator de Crescimento Insulin-Like II/genética , Somatomedinas/genética , Animais , Sequência de Bases , Blastocisto , Células Cultivadas , Quimera , Desenvolvimento Embrionário e Fetal , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Pigmentação/genética , Reação em Cadeia da Polimerase , Células-TroncoRESUMO
We describe the development and application of a system of in vitro-assembled splicing complexes that can be used for the identification of protein splicing factors which become associated with the spliceosome at the end of the assembly process ("late" splicing components). A splicing reaction performed in the presence of polyvinyl alcohol is interrupted after 15 to 20 minutes, before the appearance of splicing intermediates and products in significant amounts. Following low-speed centrifugation, a pellet is obtained containing splicing complexes that can be solubilized with 0.6 M-KCl. These complexes can be rapidly complemented for splicing in the presence of ATP and Mg2+ with protein factors that are present in HeLa cell nuclear extracts or in chromatographic extract fractions. Biochemical features of the complementation reactions, and conditions for reversible uncoupling of the two splicing steps, are described and discussed. These conditions are used to generate fully assembled spliceosomes in which splicing of the pre-mRNA can occur in the presence of ATP and Mg2+, but in the absence of nuclear extract ("autonomous splicing").
Assuntos
Splicing de RNA , Trifosfato de Adenosina/metabolismo , Eletroforese , Células HeLa , Cinética , Magnésio/metabolismo , Modelos Genéticos , Álcool de Polivinil , RNA Mensageiro/genéticaRESUMO
We have used an in vivo system generating assayable amounts of a specific pre-mRNA to study the relationship between splicing and an operationally defined nuclear matrix preparation (NM). When NM is prepared by extraction of DNase I-treated nuclei with an approximately physiological concentration of KCl (0.1 M), a portion of NM-associated precursor can be spliced in vitro in the presence of ATP and Mg2+ and in the absence of splicing extract ("autonomous splicing"). We propose that the autonomous reaction, which does not exhibit a temporal lag and is half-complete in 5 min, occurs in fully assembled, matrix-bound ribonucleoprotein complexes (in vivo spliceosomes). Extraction of the NM with concentrations of KCl greater than 0.4 M eliminates autonomous splicing but leaves behind preassembled complexes that can be complemented for splicing with HeLa cell nuclear extract. The splicing complementing factor, representing one or more activities present in the nuclear extract and also in the cytoplasmic S100 fraction, is relatively heat resistant, devoid of an RNA component, and does not bind to DEAE-Sepharose in 0.1 M KCl. It exists in the nucleus in two forms; bound to autonomous spliceosomes and free in the nucleoplasm. Biochemical features of the complementation reaction, and conditions for reversible uncoupling of the two splicing steps are described and discussed.
Assuntos
Núcleo Celular/fisiologia , Precursores de RNA/genética , Splicing de RNA , Trifosfato de Adenosina/farmacologia , Cinética , Magnésio/farmacologia , Cloreto de Potássio/farmacologia , Precursores de RNA/metabolismo , Ribonucleoproteínas/fisiologiaRESUMO
We have constructed a series of compound cloning vectors (lambda ZD vectors), each consisting of phage lambda arms carrying a modified version of the retroviral expression vector pZIP-neoSV (x)1. cDNA, inserted into a cloning site present in the retroviral vector component, is cloned with high efficiency using the lambda system. A cDNA library in plasmids is then released by homologous recombination between the retroviral long terminal repeats. Retroviral transduction is achieved by transient expression of the released library in a cell line containing a packaging mutant of Moloney murine leukemia virus, followed by cocultivation of these producers with recipient cells. Transcription of cDNAs in the recipient cells is driven by the strong long terminal repeat promoter, and the transcripts, even from truncated cDNAs, can be expressed because translational start sites have been provided in all three reading frames (tri-initiator). Sequences conferring a recognizable phenotype can be rescued by cell fusion. The functionality of the tri-initiator and the rescue of a rare cDNA have been successfully tested using model systems.
Assuntos
Clonagem Molecular/métodos , DNA/genética , Vetores Genéticos , Animais , Células Cultivadas , Chlorocebus aethiops , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Ratos , Sequências Reguladoras de Ácido Nucleico , Timidina Quinase/genéticaAssuntos
Elementos de DNA Transponíveis , Genes , RNA/genética , Animais , Evolução Biológica , DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Retroviridae/genética , Transcrição GênicaRESUMO
Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome I but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes. DNA sequence comparisons of the gene-flanking regions in rats and mice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.
Assuntos
Replicação do DNA , Genes , Proinsulina/genética , Precursores de Proteínas/genética , RNA/genética , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Enzimas de Restrição do DNA , Globinas/genética , Insulina , Cariotipagem , Camundongos , Hibridização de Ácido Nucleico , Ratos , Transcrição GênicaRESUMO
We have constructed a cDNA library from the mRNA of a rat liver cell line (BRL-3A) and characterized cDNA clones encoding the protein precursor of the rat insulin-like growth factor II (pre-pro-rIGF-II). This precursor, inferred from the nucleotide sequence, consists of a signal peptide, the rIGF-II sequence, and a trailer polypeptide of unknown significance. The characterized cDNA sequence (1016 nt) is part of a 3.4 kb mRNA species. Northern analysis reveals that a probe containing the extreme 5' noncoding region hybridizes to a second RNA (1.6 kb), while a probe corresponding to the 5' noncoding region proximal to the coding region hybridizes to two other RNA species (1.75 and 1.1 kb). All four RNAs are differentially expressed in all of the neonatal tissues that were examined, while the 3.4 kb pre-pro-rIGF-II mRNA and the 1.1 kb transcript are absent from adult tissues.
Assuntos
Insulina/genética , Peptídeos/genética , Somatomedinas/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica , Precursores de Proteínas/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Ratos , Distribuição Tecidual , Transcrição GênicaRESUMO
We have examined by fine mapping the S1 nuclease-hypersensitivity of the 5' flanking regions of the human beta-globin and rat preproinsulin II genes and of the SV40 origin/enhancer region. In all cases S1-hypersensitive sites are located in known or presumed promoter/regulatory regions. Though a consensus DNA sequence is not evident, all of these sites reside in predominantly homopurine-homopyrimidine stretches. The alternate (non-B) DNA structure which is revealed by the enzymatic probe is a sequence-dependent feature of a short stretch of DNA, which is retained upon transplantation into a foreign environment. The alternate structure exhibits S1-nicking patterns uniquely different from those associated with the presence of Z-DNA.
Assuntos
Endonucleases/metabolismo , Genes , Globinas/genética , Proinsulina/genética , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Animais , Enzimas de Restrição do DNA , Humanos , Insulina , Plasmídeos , Ratos , Vírus 40 dos Símios/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Especificidade por SubstratoRESUMO
We have inserted into a retroviral vector the structural information encoding the selectable bacterial genes neo and gpt linked, respectively, to the promoters of the herpesvirus thymidine kinase gene and the rat preproinsulin II gene. We have used this recombinant retrovirus construction to transduce insulinoma cell lines that are positive or negative for insulin expression. Selection of the transductants for neo yielded resistant colonies from all of the cell lines, while selection for gpt yielded resistant transductants in relatively high frequency only from insulin-producing cells. The 5' end of the gpt transcripts maps to the authentic preproinsulin capping site of the construction.