RESUMO
Expression of mouse mammary tumor virus in T lymphocytes appears to be required for accession of horizontally transmitted virus to the mammary gland. Further, deletions in the long terminal repeat which relax constraints on viral transcription promote T cell lymphoma. We have identified a polypurine transcriptional repressor element (NRE1) that is deleted from viruses that induce T cell lymphoma. NRE1 binding activity in nuclear extracts proved to be related to a growth inhibitory activity that represses c-myc expression in mature B cells. Mobility shift, DNA footprinting, and UV cross-linking identified three factors that interacted preferentially with double-stranded NRE1 or the separated single strands. NRE1 binding factor (NBF) (80 kDa) bound double and upper strand NRE1, apparently in concert with NRE1 associated factor (NAF) (95 kDa), which interacted directly with DNA only on the upper strand. NRE1 lower strand binding factor (NLF) (50 kDa) cross-linked specifically to lower strand NRE1. On sucrose gradients NBF, NAF, and NLF binding activities cosedimented at 8 S, implying an in vitro association of the 50-, 80-, and 95-kDa factors which precedes DNA binding. Therefore, NRE1 appears to be the site of action of a complex transcriptional repressor comprised of at least three factors that interact differentially with each DNA strand to repress steroid hormone-induced transcription of mouse mammary tumor virus in T cells.
Assuntos
Regulação Viral da Expressão Gênica , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/química , DNA Viral/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Técnicas In Vitro , Linfoma de Células T/genética , Linfoma de Células T/microbiologia , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
We demonstrate that DNA-binding protein extracts can be effectively prepared directly from tissue culture cells preserved under liquid nitrogen without returning the cells to culture. We prepared DNA-binding protein extracts directly upon thawing of T47D, Jurkat and CAC-L153S cell lines after storage in liquid nitrogen for periods of up to one year. Our results show that DNA binding of a repressor of mouse mammary tumor virus transcription in these extracts is indistinguishable from binding activity in similar extracts prepared from cells maintained in culture.
Assuntos
Criopreservação , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/isolamento & purificação , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaRESUMO
The gene for human apolipoprotein CII (APOCII) is located on the proximal long arm of chromosome 19. It has been established as a closely linked marker for myotonic dystrophy (DM), the most common form of adult muscular dystrophy. In the present linkage study, we have analysed 6 APOCII RFLPs in 213 haplotypes: TaqI, 3.8/3.5 kb; BgII, 12.0/9.0 kb; BanI, 2.5/1.6 kb; BamHI, 6.0/4.9 kb; NcoI, 14.5/11.5 kb, and AvaII, 0.6/0.4 kb. The polymorphic enzyme sites were determined to be present at the following frequencies: TaqI, 0.43; BglI, 0.51; BanI, 0.25; BamHI, 0.99; NcoI, 0.51, and AvaII, 0.52. Ordering of the polymorphic sites, 5'----3', has been determined to be (NcoI-BglI)-AvaII-BanI-TaqI. Significant disequilibrium was seen between 5 of the APOCII RFLPs.
Assuntos
Apolipoproteínas C/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , DNA/genética , Desequilíbrio de Ligação/genética , Polimorfismo Genético/genética , Alelos , Apolipoproteína C-II , Sequência de Bases , Humanos , Dados de Sequência Molecular , Polimorfismo de Fragmento de RestriçãoRESUMO
The gene for myotonic dystrophy (DM), the most common form of adult muscular dystrophy, is situated on the proximal long arm of chromosome 19. Although there exist markers that are tightly linked to the DM locus, its precise location is unknown. The identification and characterization of additional DNA probes closely linked to the DM locus continue to be priorities. In this study, we report on the linkage between a new DNA marker, designated p alpha 1.4P, and the DM locus in 50 families. The probe p alpha 1.4P was derived from a cloned breakpoint junction fragment from the chromosomal translocation t(14;19)(q32;q13.1). This translocation has been previously described in some cases of chronic lymphocytic leukemia. We have identified a BanI restriction fragment length polymorphism that is detected by p alpha 1.4P. Segregation analysis between this RFLP and DM revealed close linkage between the two loci (lod = 10.95, theta = 0). Furthermore, statistical evidence for linkage disequilibrium between p alpha 1.4P and the DM locus in a French Canadian population was found. Finally, by means of a somatic cell hybrid mapping panel, p alpha 1.4P was sublocalized to 19q12----19q13.2.
Assuntos
Cromossomos Humanos Par 19 , Distrofias Musculares/genética , Alelos , Mapeamento Cromossômico , Desoxirribonucleases de Sítio Específico do Tipo II , Ligação Genética , Marcadores Genéticos , Humanos , Polimorfismo de Fragmento de Restrição , Translocação GenéticaRESUMO
The gene for human apolipoprotein C2 (APOC2), situated on the proximal long arm of chromosome 19, is closely linked to the gene for the most common form of adult muscular dystrophy, myotonic dystrophy (DM). Six APOC2 RFLPs (TaqI, BglI, BanI, BamHI, NcoI, and AvaII) have been identified to date. We have conducted a comprehensive DM linkage study utilizing all six RFLPs and involving 50 families and 372 individuals. The most informative RFLPs are, in descending order, NcoI (lod = 6.64, theta = 0.05), BglI (lod = 6.12, theta = 0.05), AvaII (lod = 6.02, theta = 0.03), BanI (lod = 5.76, theta = 0.04), TaqI (lod = 4.29, theta = 0.06), and BamHI (lod = 1.75, theta = 0.01). A substantial increase in the lod scores over those seen with the individual RFLPs was obtained when the linkage of the entire APOC2 haplotype (composed of the six RFLPs) was studied (lod = 17.87, theta = 0.04). We have observed significant inter-APOC2 RFLP linkage disequilibrium. Consequently, the three most informative RFLPs have been found to be BanI, TaqI, and either BglI, AvaII, or NcoI polymorphisms. We also demonstrate linkage disequilibrium between DM and APOC2 in our French-Canadian population (standardized disequilibrium constant phi = .22, chi 2 = 5.12, df = 1, P less than 0.04). This represents the first evidence of linkage disequilibrium between APOC2 and the DM locus.
Assuntos
Apolipoproteínas C/genética , Cromossomos Humanos Par 19 , Ligação Genética , Distrofia Miotônica/genética , Alelos , Apolipoproteína C-II , Mapeamento Cromossômico , Frequência do Gene , Humanos , Ontário , Polimorfismo de Fragmento de RestriçãoRESUMO
A number of DNA probes from the short arm of the X chromosome have been used to study the inheritance of the translocation chromosomes in a girl with an X; autosome translocation and muscular dystrophy. The two translocation chromosomes were found to be derived from the father's single normal X chromosome, ruling out maternal inheritance of a pre-existent mutation and enhancing the concept that the de novo translocation is responsible for the dystrophic phenotype.