RESUMEN
Several new loci were identified by a comprehensive analysis of loss of heterozygosity (LOH) using a subtraction library between matched normal and renal cell carcinoma (RCC) tissues. A total of 187 clones from the library, with a complexity of 1x10(4), were mapped, and 44 clusters of the mapped loci were subjected to LOH analysis using microsatellite markers. A total of 27 loci, which exhibited frequencies of LOH of at least 10% among 44 tumors, mostly clear-cell RCC, included several loci that were reported previously, such as, the von Hippel-Lindau gene, adenomatous polyposis coli, and interferon regulatory factor-1, as well as new loci, at 5q32-q34, 6q21-q22, 8p12, and others. These loci exhibited LOH among 11.8-93.8% of tumors, and most, if not all, were derived from the sites of hemizygous deletions. The minimum regions of LOH of chromosomes 5, 6, and 8 were 9.0, 10.3, and 0.775 Mb, respectively. The average distance between the cloned fragments on the chromosomes was 2.2 Mb in 187 clones, indicating that the minimum LOH size expected from this subtraction analysis was roughly 50 kb. Therefore, the strategy described here provides comprehensive analysis of LOH sites, which were mostly caused by hemizygous deletions.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Eliminación de Gen , Biblioteca de Genes , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Pérdida de Heterocigocidad , Mapeo Cromosómico , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 5 , Cromosomas Humanos Par 6 , Cromosomas Humanos Par 8 , Clonación Molecular , Marcadores Genéticos , Humanos , Repeticiones de Microsatélite , Modelos GenéticosRESUMEN
We have isolated a genomic DNA fragment spanning the 5'-end of the gene encoding the catalytic subunit of mouse DNA polymerase alpha. The nucleotide sequence of the upstream region was G/C-rich and lacked a TATA box. Transient expression assays in cycling NIH 3T3 cells demonstrated that the GC box of 20 bp (at nucleotides -112/-93 with respect to the transcription initiation site) and the palindromic sequence of 14 bp (at nucleotides -71/-58) were essential for basal promoter activity. Electrophoretic mobility shift assays showed that Sp1 binds to the GC box. We also purified a protein capable of binding to the palindrome and identified it as GA-binding protein (GABP), an Ets- and Notch-related transcription factor. Transient expression assays in synchronized NIH 3T3 cells revealed that three variant E2F sites near the transcription initiation site (at nucleotides -23/-16, -1/+7 and +17/+29) had no basal promoter activity by themselves, but were essential for growth-dependent stimulation of the gene expression. These data indicate that E2F, GABP and Sp1 regulate the gene expression of this principal replication enzyme.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , ADN Polimerasa I/genética , Proteínas de Unión al ADN , Regulación Enzimológica de la Expresión Génica , Proteínas Oncogénicas , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Animales , Secuencia de Bases , Catálisis , Ciclo Celular/genética , ADN Polimerasa I/aislamiento & purificación , ADN Polimerasa I/metabolismo , Factores de Transcripción E2F , Genes Reguladores , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-ets , Proteína 1 de Unión a Retinoblastoma , Homología de Secuencia de Ácido Nucleico , Factor de Transcripción DP1 , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
We have isolated the genomic DNA fragment spanning the 5-end and the first four exons encoding the 68 kDa subunit (p68) of the mouse DNA polymerase alpha-primase complex [corrected]. The p68 promoter region lacks TATA and CAAT boxes, but contains a GC-rich sequence, two palindrome sequences and two putative E2F-binding sites [corrected]. A series of transient expression assays using a luciferase reporter gene indicated that a region from nucleotide position -89 to -30 (-89/-30) with respect to the transcription initiation site is crucial for basal transcription of the p68 gene in proliferating NIH 3T3 cells. In particular, part of the GC-rich sequence (-57/-46) and the palindrome (-81/-62) elements were necessary for promoter activity, both of which share homology with the E-box sequence. Gel mobility shift assays using NIH 3T3 nuclear extracts revealed that the upstream stimulatory factor, known as an E-box-binding protein, binds to these sites. Moreover, we observed binding of E2F to two sites near the transcription initiation site (-11/-3 and +9/+16). A transient luciferase expression assay using synchronized NIH 3T3 cells in G(0)phase revealed that these E2F sites are essential for transcription induction of the p68 gene after serum stimulation, but are dispensable for basal transcription. These results indicate that growth-dependent regulation of transcription of the mouse p68 and p180 genes is mediated by a common factor, E2F; however, basal transcription of the genes, interestingly, is regulated by different transcription factors.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , ADN Polimerasa I/química , ADN Polimerasa I/genética , Proteínas de Unión al ADN , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión/genética , Ciclo Celular/genética , Clonación Molecular , ADN/genética , ADN/metabolismo , Sondas de ADN/genética , Factores de Transcripción E2F , Expresión Génica , Genes Reporteros , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Estructura Cuaternaria de Proteína , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Factores Estimuladores hacia 5'RESUMEN
We report here the locations of curved DNA in the human erythropoietin receptor gene. A total of 13 DNA bend sites were mapped by circular permutation assays, appearing at an average interval of 651.2+/-214.6 (S.D.) in the 8-kb region. The bend centers in these 13 bend sites were confirmed by oligonucleotide-based assays where most of these centers had bend angles higher than that shown by (AAACCGGGCC) x (A)20 and lower than that shown by (AAACCGGGCC)2 x (A)10. DNA curvature mapping by TRIF software, which is based on the distribution of dinucleotides, primarily AA and TT, provided a highly accurate prediction for the locations of the bend sites. They showed approximately 20 degrees to 40 degrees of bend angles demonstrated by the oligonucleotide assays and by computer analysis.