Assuntos
Linfoma Difuso de Grandes Células B/patologia , Animais , Animais Congênicos , Biomarcadores Tumorais/análise , Cruzamentos Genéticos , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Rearranjo Gênico do Linfócito B , Humanos , Vírus da Leucemia Murina/patogenicidade , Linfoma Difuso de Grandes Células B/química , Linfoma Difuso de Grandes Células B/genética , Camundongos , Camundongos Endogâmicos , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Especificidade da Espécie , Fatores de Transcrição/genética , Translocação GenéticaRESUMO
Chromosomal translocations juxtaposing the MYC protooncogene with regulatory sequences of immunoglobulin (Ig) H chain or kappa (Ig kappa) or lambda (Ig lambda) L chain genes and effecting deregulated expression of MYC are the hallmarks of human Burkitt lymphoma (BL). Here we report that lymphomas with striking similarities to BL develop in mice bearing a mutated human MYC gene controlled by a reconstructed Ig lambda locus encompassing all the elements required for establishment of locus control in vitro. Diffusely infiltrating lymphomas with a typical starry sky appearance occurred in multiple founders and an established line, indicating independence from positional effects. Monoclonal IgM(+)CD5(-)CD23(-) tumors developed from an initially polyclonal population of B cells. These results demonstrate that the phenotype of B lineage lymphomas induced by MYC dysregulation is highly dependent on cooperativity among the regulatory elements that govern expression of the protooncogene and provide a new system for studying the pathogenesis of BL.
Assuntos
Linfoma de Burkitt/imunologia , Genes myc , Animais , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Modelos Animais de Doenças , Éxons , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/análise , Proteínas Proto-Oncogênicas c-myc/genética , Baço/imunologia , Baço/patologiaRESUMO
A combination of RT-PCR with cDNA library screening led to the identification of the human homologue of murine KIF4 (mKIF4), a microtubule-based motor protein. The nucleotide sequence of human KIF4 (hKIF4) comprised part of the 5' untranslated region (UTR), a long open reading frame (ORF) encoding 1232 amino acids, and the entire 3' UTR. Homologies with mKIF4 were 82% and 85% for nucleotides and amino acids, respectively. A single transcript of 5. 0 kb was highly expressed in hematopoietic tissues including fetal liver, spleen, and thymus, and adult thymus and bone marrow. Comparison of the ORF with that of mKIF4 revealed some dissimilarities in the putative cargo binding domain which might affect binding specificities.
Assuntos
Cinesinas/genética , Proteínas do Tecido Nervoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Feto , Biblioteca Gênica , Células HeLa , Humanos , Cinesinas/química , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
BCL6 encodes a transcription factor deregulated by chromosomal translocations in human diffuse large cell B lymphomas (DLCL). This study was designed to determine whether Bcl6 might also be involved in lymphomas of mice. BCL6 protein was expressed at high levels in 90% or more of DLCL but not in low grade B lymphomas. Southern hybridisation studies demonstrated altered organisation of Bcl6 in three primary DLCL and the WEHI 231 B-cell lymphoma cell line but not in low grade tumours. Chromosomal painting and fluorescence in situ hybridisation (FISH) analyses of the WEHI 231 metaphase spreads revealed a T(5;16) translocation with Bcl6 on Chromosome 16 at the translocation breakpoint. Deregulated expression of BCL6 is thus likely to contribute to the genesis of DLCL of mice as well as of humans.
Assuntos
Proteínas de Ligação a DNA/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Animais , Mapeamento Cromossômico , Genoma , Humanos , Cariotipagem , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6 , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4-Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.
Assuntos
Produtos do Gene gag/metabolismo , Cinesinas/metabolismo , Retroviridae/metabolismo , Animais , Vírus do Sarcoma Aviário , Produtos do Gene gag/genética , HIV-1/metabolismo , Células HeLa , Humanos , Vírus dos Macacos de Mason-Pfizer , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Imunodeficiência SímiaRESUMO
Germinal centers (GC) are the sites of antigen-driven B cell switch recombination, V(D)J gene hypermutation, and selection to generate high-afinity CD38+ memory B cells. A marked expansion of GC associated with hypergammaglobulinemia followed by complete disruption of normal splenic architecture and a striking drop in immunoglobulin levels are prominent features of the murine retrovirus-induced immunodeficiency syndrome, MAIDS. B cell lymphomas are frequent in long-term infected mice. Normal GC formation is critically dependent on a number of genes including the transcription factor, Bcl6. Deregulated expression of BCL6 protein has been implicated in the development of human and mouse B cell lymphomas. Another nuclear protein, SWAP-70, has been identified as a subunit of the protein complex, SWAP, that recombines switch regions in vitro. To develop a fuller understanding of B cell biology in MAIDS, we examined the characteristics of BCL6, SWAP-70, CD38, and peanut agglutinin (PNA)-staining cells during the course of the disease. The levels of both nuclear proteins increased rapidly until 6-8 weeks after infection. During this time frame, BCL6 was expressed at highest levels in the usually rare CD4+ Thyl- T cell subset as well as in B cells. At later times. BCL6 levels dropped to undetectable levels while SWAP-70 levels continued to increase. Changes in the levels of either protein could not be ascribed to transcriptional regulation. PNA-reactive cells decreased in concert with BCL6 while CD38 staining increased with SWAP-70. These results demonstrate that progression of MAIDS results in the massive accumulation of B cells with the morphology of secretory cells that behave like post-GC cells for expression of BCL6 and CD38, and for PNA-staining but with abnormally high-level expression of SWAP-70.
Assuntos
Antígenos CD , Proteínas de Ligação a DNA/genética , Centro Germinativo/imunologia , Fatores de Troca do Nucleotídeo Guanina , Síndrome de Imunodeficiência Adquirida Murina/genética , Síndrome de Imunodeficiência Adquirida Murina/imunologia , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos de Diferenciação/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linhagem Celular , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica , Genes de Troca , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Síndrome de Imunodeficiência Adquirida Murina/patologia , NAD+ Nucleosidase/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Recombinação GenéticaRESUMO
A cDNA clone encoding a cellular protein that interacts with murine leukemia virus (MuLV) Gag proteins was isolated from a T-cell lymphoma library. The sequence of the clone is identical to the C terminus of a cellular protein, KIF4, a microtubule-associated motor protein that belongs to the kinesin superfamily. KIF4-MuLV Gag associations have been detected in vitro and in vivo in mammalian cells. We suggest that KIF4 could be involved in Gag polyprotein translocation from the cytoplasm to the cell membrane.
Assuntos
Produtos do Gene gag/metabolismo , Cinesinas/metabolismo , Vírus da Leucemia Murina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso , Proteínas Virais/metabolismo , Animais , Produtos do Gene gag/genética , Cinesinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas/metabolismo , Saccharomyces cerevisiae , Proteínas Virais/genéticaRESUMO
Numerous genes contain promoter elements that are nuclease hypersensitive. These elements frequently possess polypurine/polypyrimidine stretches and are usually associated with altered chromatin structure. We have previously isolated a clone that binds a class of CT-rich promoter elements. We have further characterized this clone, termed the nuclease-sensitive element protein-1, or NSEP-1. NSEP-1 binds both duplex CT elements and the CT-rich strand of these elements in a 'generic' sequence specific manner and has overlapping but distinct single-and double-strand DNA binding domains. The minimal peptide region sufficient for both duplex and single-strand DNA binding includes two regions rich in basic amino acids flanking an RNP-CS-1 like octapeptide motif. Deletion analysis shows that the single-strand DNA binding activity is mediated by the RNP-CS-1 like octapeptide motif and is the key peptide region necessary for single-strand binding. NSEP-1's affinity for CT rich promoter elements with strand asymmetry in addition to its double- and single-strand DNA binding properties suggests that it may be a member of a class of DNA binding proteins that modulate gene expression by their ability to recognize DNA with unusual secondary structure.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas/genética , Sequência de Bases , DNA/genética , DNA/metabolismo , Análise Mutacional de DNA , DNA de Cadeia Simples/genética , Dados de Sequência MolecularRESUMO
Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA+ gene product. The requirement of SDR for recA+ can be suppressed by rin mutations (for recA+-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA+-dependent SDR seen in rnh- rin+ lexA+ strains, recA+-independent in rnh- rin- lexA+, and recA+-independent in rnh- rin+ lexA(Def). The expression of SDR in rin- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA+ requirement by rin mutations was shown to depend on some new function of the recF+ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF+. The lexA3 mutation inhibited recA+-dependent SDR via reducing the amount of recA+ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh- rin- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA+-regulated gene product in the recA+-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA+, rin+ and recF+ genes.
Assuntos
Replicação do DNA , Endorribonucleases/genética , Escherichia coli/genética , Mutação , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Genótipo , Cinética , Ribonuclease H , Especificidade da Espécie , TemperaturaRESUMO
The possible allelic relationship between dasF (dnaA suppressor) and sdrA/rnh (stable DNA replication/RNase H) mutations was examined. dasF mutations could not only suppress various dnaA(ts) mutations, but also the insertional inactivation of the dnaA gene or deletion of the oriC sequence, as could sdrA mutations. dasF mutants were found to exhibit the stable DNA replication phenotype, and the sensitivity to rich media, of sdrA mutants. The dasF and sdrA mutations were mapped very closely between metD and proA on the E. coli genetic map. The mutations were recessive to the wild-type allele for all the above phenotypes. It was concluded that dasF is allelic to sdrA/mh.
Assuntos
Proteínas de Bactérias/genética , Replicação do DNA , Escherichia coli/genética , Supressão Genética , Alelos , Mapeamento Cromossômico , Cromossomos Bacterianos , DNA Bacteriano/biossíntese , DNA Bacteriano/genéticaRESUMO
The sdrA102 mutation confers upon cells the ability to replicate DNA in the absence of protein synthesis. This mutation was combined with the recA200 mutation, which renders the recA protein thermolabile, and had little effect on normal replication. However, the sdrA102 recA200 double mutant exhibited temperature-sensitive stable DNA replication: it replicated DNA continuously in the presence of chloramphenicol at 30 degrees C, whereas at 42 degrees C DNA replication ceased after the DNA content increased only 40-45%. Suppressor mutants (rin; recA-independent) capable of stable DNA replication at 42 degrees C were isolated from the double mutant. The suppressor mutant retained all other recA- characteristics, i.e., deficient general recombination, severe UV-sensitivity, and incapability of prophage induction in lysogens. This indicates that the rin mutation specifically suppresses the recA+ dependency of stable DNA replication. It is suggested that the recA+ protein stabilizes a specific structure, similar to an intermediate in recombination, which may function in the initiation of stable DNA replication.
Assuntos
Proteínas de Bactérias/genética , Replicação do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Supressão Genética , Cloranfenicol/farmacologia , Recombinases Rec A , TemperaturaRESUMO
The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA+ lexA+-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication. The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with malidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3. Induced stable DNA replication was found to be considerably more resistant to UV irradiation than normal replication both in a uvrA6 strain and a uvr+ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.