RESUMO
Recently, we reported that a 315 bp enhancer, located over 55 kilobases (kb) upstream of the transcriptional start site of the human apolipoprotein B (apoB) gene, was sufficient to direct high-level expression of human apoB transgenes in mice. In this report, we expand our analysis of the distant apoB intestinal control region (ICR), by examining the function of segments in the vicinity of the 315 bp intestinal enhancer (315 IE). DNaseI hypersensitivity (DH) studies of a 4.8 kb segment from the ICR revealed three new DH sites, in addition to the previously described DH1 region present within the 315 IE. DH2 mapped to a 485 bp segment (485 IE) immediately upstream of the 315 IE that exhibited strong intestinal enhancer activity in transient transfection experiments with intestine-derived CaCo-2 cells. Within the DH2 region, an HNF-4/ARP-1 binding site was demonstrated by gel retardation experiments. A 1.8 kb segment incorporating the 485 IE was capable of driving expression of human apoB transgenes in the intestines of mice. Additionally, a third component of the apoB ICR was found about 1.2 kb downstream of the 315 IE, within a 1031 bp segment (1031 IE) that also harbored two DH sites, DH3 and DH4. This segment did not display enhancer activity but was capable of driving transgene expression in the intestine. The three components of the ICR displayed a similar pattern of apoB mRNA expression along the horizontal axis of the intestine. The previously characterized in vivo liver-specific elements of the apoB gene, namely, the second intron enhancer and the 5' upstream liver enhancer, did not play a role in intestinal expression of apoB transgenes in mice.
Assuntos
Apolipoproteínas B/genética , Elementos Facilitadores Genéticos , Intestino Delgado/metabolismo , Transgenes , Animais , Apolipoproteínas B/biossíntese , Fusão Gênica Artificial/métodos , Composição de Bases , Sequência de Bases , Células CACO-2 , Desoxirribonuclease I/genética , Regulação da Expressão Gênica , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microinjeções , Dados de Sequência Molecular , Plasmídeos/administração & dosagem , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
We recently reported that an 8-kilobase (kb) region, spanning from -54 to -62 kb 5' of the human apolipoprotein B (apoB) gene, contains intestine-specific regulatory elements that control apoB expression in the intestines of transgenic mice. In this study, we further localized the apoB intestinal control region to a 3-kb segment (-54 to -57 kb). DNaseI hypersensitivity studies uncovered a prominent DNaseI hypersensitivity site, located within a 315-base pair (bp) fragment at the 5'-end of the 3-kb segment, in transcriptionally active CaCo-2 cells but not in transcriptionally inactive HeLa cells. Transient transfection experiments with CaCo-2 and HepG2 cells indicated that the 315-bp fragment contained an intestine-specific enhancer, and analysis of the DNA sequence revealed putative binding sites for the tissue-specific transcription factors hepatocyte nuclear factor 3beta, hepatocyte nuclear factor 4, and CAAT enhancer-binding protein beta. Binding of these factors to the 315-bp enhancer was demonstrated in gel retardation experiments. Transfection of deletion mutants of the 315-bp enhancer revealed the relative contributions of these transcription factors in the activity of the apoB intestinal enhancer. The corresponding segment of the mouse apoB gene (located -40 to -83 kb 5' of the structural gene) exhibited a high degree of sequence conservation in the binding sites for the key transcriptional activators and also exhibited enhancer activity in transient transfection assays with CaCo-2 cells. In transgenic mouse expression studies, the 315-bp enhancer conferred intestinal expression to human apoB transgenes.
Assuntos
Apolipoproteínas B/genética , Elementos Facilitadores Genéticos , Mucosa Intestinal/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonuclease HindIII/metabolismo , Desoxirribonuclease I/metabolismo , Eletroforese em Gel de Poliacrilamida , Fator 3-beta Nuclear de Hepatócito , Fator 4 Nuclear de Hepatócito , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Mapeamento por Restrição , Ribonucleases/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/metabolismo , TransgenesRESUMO
A number of DNaseI-hypersensitive (DH) sites have been mapped within a regulatory region situated upstream of the human apolipoprotein B (apoB) promoter (-5262 to -899) that is required for high level expression of human apoB transgenes in the livers of mice. These DH sites were observed in nuclei from transcriptionally active liver-derived HepG2 cells, but were absent from transcriptionally inactive HeLa cell nuclei. Several nuclear protein binding sites were detected in the DNaseI-hypersensitive region by DNaseI footprinting with HepG2 nuclear extracts, representing putative binding sites for the liver-specific activators. The locations of binding sites for these transcription factors were revealed via computer analysis of the DNA sequence of this region against a transcription factor database. Many micrococcal nuclease hypersensitive (MH) sites were also observed in nuclei from HepG2 cells but not in HeLa cell nuclei, implying that in hepatic cells, nucleosomes are either absent or have been displaced from this region by the liver-specific transcriptional activators, as inferred by the correspondence between the DH sites, the MH sites and the footprints.
Assuntos
Apolipoproteínas B/biossíntese , Apolipoproteínas B/genética , Cromatina/química , Elementos Facilitadores Genéticos/fisiologia , Fígado/metabolismo , Transgenes/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação/fisiologia , Núcleo Celular/química , Pegada de DNA , Desoxirribonuclease I/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Especificidade de Órgãos/genética , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
We have generated transgenic mice expressing human CYP7A1 transgenes. Only 1.5 kilobases (kb) of 5' upstream sequence and 6.5 kb of 3' sequence were sufficient for hepatic transcription of the transgenes. However, the 5' end segment alone was not sufficient to direct liver expression, suggesting that additional hepatic regulatory elements reside in the 3' extension or within introns. The level of expression of these transgenes was low in comparison to the levels of the endogenous mouse CYP7A1 mRNA. To generate mice expressing higher levels of CYP7A1 mRNA, we injected a large human genomic PAC clone, extending up to -105 kb 5' of the structural gene and about 50 kb 3' of the gene. These transgenic mice expressed CYP7A1 mRNA at higher levels, suggesting that additional hepatic regulatory elements are found either 5' of -1520 or beyond 6.5 kb 3' of the gene.
Assuntos
Colesterol 7-alfa-Hidroxilase/genética , Regulação Enzimológica da Expressão Gênica/genética , Animais , Linhagem Celular , Feminino , Dosagem de Genes , Humanos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fluorescence in situ hybridization analysis was performed to characterize a complex pericentric inversion involving chromosome 5 in a mother and son. The mother had hypertelorism, epicanthal folds, and mild mental deficiency while the son had additional anomalies that have been observed in patients with cri-du-chat syndrome. Both individuals were found to have an identical double pericentric inversion [inv5(p15.1q31(inv5(p14q12)))]. Neither inversion breakpoint mapped near the chromosomal regions implicated in the cri-du-chat syndrome. The phenotype of the son suggests that the inversion process may have affected the expression of some of the cri-du-chat syndrome genes, suggestive of a genomic imprinting or penetrance effect.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 5/genética , Síndrome de Cri-du-Chat/genética , Adulto , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Cariotipagem , Masculino , FenótipoAssuntos
Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cromossomos Humanos Par 5 , Cromossomos Humanos Par 9 , Interferon gama/farmacologia , Proteínas/genética , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Deleção Cromossômica , Mapeamento Cromossômico , Sondas de DNA , Proteínas Quinases Associadas com Morte Celular , Genes Supressores de Tumor , Humanos , Células Híbridas , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Roedores , Neoplasias da Bexiga Urinária/genéticaRESUMO
The cri-du-chat syndrome is a contiguous gene syndrome that results from a deletion of the short arm of chromosome 5 (5p). Patients present with a cat-like cry at birth, which is usually considered diagnostic of this syndrome. Additional features of the syndrome include failure to thrive, microcephaly, hypertelorism, epicanthal folds, hypotonia, and severe mental retardation. We report on four families in which patients with 5p deletions have only the characteristic cat-like cry, with normal to mildly delayed development. The precise locations of the deletions in each family were determined by FISH using lambda phage and cosmid clones. All of the deletion breakpoints map distal to a chromosomal region that is implicated with the facial features and severe mental and developmental delay in the cri-du-chat syndrome. DNA clones mapping in the chromosomal region associated with the cat-like cry feature will be useful diagnostic tools. They will allow for the distinction between 5p deletions that will result in the severe delay observed in most cri-du-chat syndrome patients and those deletions that result in the isolated cat-like cry feature, which is associated with a better prognosis.
Assuntos
Cromossomos Humanos Par 5/genética , Síndrome de Cri-du-Chat/genética , Deleção de Sequência , Distúrbios da Voz/genética , Adulto , Mapeamento Cromossômico , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Cariotipagem , Masculino , Modelos Genéticos , LinhagemRESUMO
Cri-du-chat is a well described partial aneusomy resulting from deletion of the short arm of chromosome 5. The hallmark clinical feature of cri-du-chat, a high-pitched monochromatic cry, has recently been localized to 5p15.3, separate from the remaining clinical features of the syndrome, which have been localized to 5p15.2. Five chromosome 5-specific probes from the latter region, designated the cri-du-chat critical region (CDCCR), were used to isolate 30 cosmids from the LANL chromosome 5 specific cosmid library. The 30 framework cosmids were used in a direct selection with three cDNA sources to isolate an initial set of expressed sequences. Nine unique cDNAs were found that hybridized to four discrete sets of cosmids in the CDCCR. The nine cDNAs are novel by sequence database comparisons, and conservatively represent four transcription units. More recently, we have also constructed a YAC contig of the CDCCR which spans approximately 2 Mb. As expected, ESTs derived from the nine novel cDNAs map back to the contig. Limited expression profiles of these cDNAs have been obtained. Two cDNAs that map to one discrete set of cosmids have different expression patterns, suggesting that they represent two different genes and increasing the number of putative genes to five. Further characterization of these genes and the estimated 100 additional genes deleted in cri-du-chat should lead to better diagnostic markers and an understanding of the molecular mechanisms of the disease.
Assuntos
Cromossomos Humanos Par 5 , Síndrome de Cri-du-Chat/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Cosmídeos , DNA Complementar , Expressão Gênica , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The deletion of the short arm of chromosome 5 is associated with the cri-du-chat syndrome. In addition, loss of this portion of a chromosome is a common cytogenetic marker in a number of malignancies. However, to date, no genes associated with these disorders have been identified. Physical maps are the first step in isolating causative genes, and genes involved in autosomal recessive disorders are now routinely mapped through the identification of linked markers. Extensive genetic maps based upon polymorphic short tandem repeats (STRs) have provided researchers with a large number of markers to which such disorders can be genetically mapped. However, the physical locations of many of these STRs have not been determined. Toward the goal of integrating the human genetic maps with the physical maps, a 5p somatic cell hybrid deletion mapping panel that was derived from patients with 5p deletions or translocations was used to physically map 47 STRs that have been used to construct genetic maps of 5p. These data will be useful in the localization of disease genes that map to 5p and may be involved in the etiology of the cri-du-chat syndrome.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 5 , Marcadores Genéticos , Animais , Deleção Cromossômica , Cromossomos Humanos Par 5/ultraestrutura , Síndrome de Cri-du-Chat/genética , Cricetinae , Primers do DNA , Ligação Genética , Humanos , Células Híbridas , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Translocação GenéticaRESUMO
Cri-du-chat is a chromosomal deletion syndrome characterized by partial deletion of the short arm of chromosome 5. The clinical symptoms include growth and mental retardation, microcephaly, hypertelorism, epicanthal folds, hypotonia, and a high-pitched monochromatic cry that is usually considered diagnostic for the syndrome. Recently, a correlation between clinical features and the extent of the chromosome 5 deletions has identified two regions of the short arm that appear to be critical for the abnormal development manifested in this syndrome. Loss of a small region in 5p15.2 correlates with all of the clinical features of cri-du-chat with the exception of the cat-like cry, which maps to 5p15.3. Here we report the construction of a YAC contig that spans the chromosomal region in 5p15.2 that plays a major role in the etiology of the cri-du-chat syndrome. YACs that span the 2-Mb cri-du-chat critical region have been identified and characterized. This YAC contig lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in the clinical features associated with the cri-du-chat syndrome. It also provides a new diagnostic tool for cri-du-chat in the shape of a YAC clone that may span the entire critical region.
Assuntos
Cromossomos Artificiais de Levedura , Síndrome de Cri-du-Chat/genética , Sequência de Bases , Deleção Cromossômica , Cromossomos Humanos Par 5 , Primers do DNA , Humanos , Células Híbridas , Dados de Sequência Molecular , Sitios de Sequências RotuladasRESUMO
Most patients who present with the 18q- syndrome have an apparent terminal deletion of the long arm of chromosome 18. For precise phenotypic mapping of this syndrome, it is important to determine whether the deletions are terminal deletions or interstitial deletions. A human telomeric YAC clone has been identified that hybridizes specifically to the telomeric end of 18q. This clone was characterized and used to analyze seven patients with 18q deletions. By FISH and Southern blotting analysis, all patients were found to lack this chromosomal region on their deleted chromosome, demonstrating that the patients do not have cryptic interstitial deletions.
Assuntos
Aberrações Cromossômicas/genética , Deleção Cromossômica , Cromossomos Humanos Par 18 , Sequência de Bases , Southern Blotting , Transtornos Cromossômicos , Cromossomos Artificiais de Levedura , Feminino , Marcadores Genéticos , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Dados de Sequência Molecular , SíndromeRESUMO
Transient expression assays with the herpes simplex virus type 1 (HSV-1) promoter/leader controlling the beta gamma (leaky-late) VP5 (UL19) mRNA encoding the major capsid protein showed that no more than 36 to 72 bases of VP5 leader are required for full-level expression. Constructs lacking the viral leader and the transcription initiation site expressed the reporter gene at about 20% of the maximum level. We confirmed this observation by using recombinant viruses in which VP5 promoter/leader deletions controlling the bacterial beta-galactosidase gene were inserted into the nonessential glycoprotein C (UL44) locus of the genome. Sequences within +36 are required for full-level expression, and removal of all leader sequences including the cap site resulted in a 10-fold decrease in reporter mRNA accumulation. The removal of the leader sequence had a measurable effect upon the kinetics of reporter mRNA accumulation, but insertion of the entire VP5 leader and cap site into a construct in which the reporter gene was controlled by the kinetically early (beta) dUTPase (UL50) promoter did not result in any significant change in the kinetics of dUTPase promoter expression. These results suggest that DNA sequences both 5' and 3' of the TATA box are important determinants of the beta gamma kinetics and levels of VP5 mRNA accumulation in the infected cell.
Assuntos
Capsídeo/genética , Análise Mutacional de DNA , Genes Virais , Regiões Promotoras Genéticas , Simplexvirus/genética , TATA Box , Animais , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Fibroblastos , Regulação Viral da Expressão Gênica , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/biossíntese , Coelhos , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Deleção de Sequência , Pele , TransfecçãoRESUMO
To examine the effect of genomic location on the details of expression of selected herpes simplex virus promoters, we have constructed recombination vectors for placing such promoters controlling the beta-galactosidase reporter gene into two regions of the viral genome lacking any nearby promoter or regulatory elements. The first vector generates the promoter-beta-galactosidase reporter gene inverted within the locus of the gC (UL44) translational reading frame; the second replaces the LAT promoter and the first 600 bases of the primary transcript in both copies of the RL region. These locations were chosen to obviate any possible influence of upstream but noncontiguous heterologous or homologous DNA sequence elements upon promoter activity. When the reporter gene controlled by the strict late (gamma) UL38 promoter was placed in the gC location, it was significantly less active than in its normal location; in contrast, promoter activity was comparable to wild-type values when the promoter was recombined into the RL region. The low level of activity in the gC location could be partially alleviated by the incorporation of additional DNA sequences upstream of the UL38 promoter. Despite the effect of genomic location upon the level of expression, the kinetics of expression in either location mirrors the wild-type UL38 strict late kinetics of expression. Finally, we used deletional analysis to demonstrate that no more than 29 bases of DNA sequence 5' of the mRNA cap site are required for promoter activity in either location; this result is consistent with earlier results of transient-expression assays and indicates that the UL38 promoter shares general features with other strict late (gamma) herpes simplex virus promoters.
Assuntos
Regulação Viral da Expressão Gênica , Herpes Simples/genética , Regiões Promotoras Genéticas/genética , Simplexvirus/genética , Transcrição Gênica , Animais , Análise Mutacional de DNA , DNA Recombinante , Fibroblastos , Vetores Genéticos , Genoma Viral , RNA Mensageiro/biossíntese , Células Vero , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
RNA from the region of the genome encoding herpes simplex virus type 1 latency-associated transcripts (LATs) expressed during lytic infection yields low abundances of both polyadenylated and nonpolyadenylated forms. As has been previously shown for latent infection (A. T. Dobson, F. Sedarati, G. Devi-Rao, W. M. Flanagan, M. J. Farrell, J. G. Stevens, E. K. Wagner, and L. T. Feldman. J. Virol. 63:3844-3851, 1989), all lytic-phase expression of such transcripts requires promoter elements situated approximately 600 bases 5' of the previously mapped 5' end of the poly(A)- forms of LAT. Transient expression experiments revealed no other clear promoter elements within this region, and relatively small amounts of latent-phase transcripts initiating at the same site as observed for lytic-phase LAT could be detected by RNase protection assays. In the lytic phase of infection, the most abundant forms of polyadenylated LAT extended 1,600 bases from the initiation site near the LAT promoter to a potential splice donor site. Poly(A)- LAT species were not recovered in significant amounts from lytically infected neuroblastoma cells, but such RNA from lytically infected rabbit skin cells comapped with poly(A)- LAT from latently infected sensory neurons. Both map between canonical 5' splice donor and 3' splice acceptor site 1,950 bases apart. Poly(A)- LAT cochromatographed with uncapped rRNA on m-aminophenyl boronate agarose under conditions in which capped mRNA was bound. All of these data confirm the previously presented scheme for the expression of poly(A)- LAT as a stable intron derived from the splicing of a large primary transcript; however, we were unable to detect the spliced polyadenylated product of this splicing reaction.