RESUMO
We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Algoritmos , Animais , Sequência de Bases , Resinas de Troca de Cátion , Regulação para Baixo , Fluoresceína-5-Isotiocianato , Biblioteca Gênica , Genes Reporter/genética , Engenharia Genética , Células HeLa , Humanos , Lipídeos , Luciferases/genética , Metilação , Dados de Sequência Molecular , Ensaios de Proteção de Nucleases , Oligorribonucleotídeos/administração & dosagem , Oligorribonucleotídeos/química , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Estabilidade de RNA , RNA Catalítico/administração & dosagem , RNA Catalítico/química , RNA Catalítico/genética , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribonuclease H/metabolismo , Software , Especificidade por Substrato , Termodinâmica , TransfecçãoRESUMO
Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and growth arrest in myeloma cells. However, the molecular mechanisms behind these events are not known. The MYC oncogene is a master regulator of cell growth and protein synthesis and MYC overexpression has been proposed to be associated with the progression of multiple myeloma. Here, we show that BMP-induced apoptosis in myeloma cells is dependent on downregulation of MYC. Moreover, the results suggest that targeting the MYC addiction in multiple myeloma is an efficient way of killing a majority of primary myeloma clones. We also found that myeloma cells harboring immunoglobulin (IG)-MYC translocations evaded BMP-induced apoptosis, suggesting a novel way for myeloma cells to overcome potential tumor suppression by BMPs.
Assuntos
Apoptose/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Genes myc , Mieloma Múltiplo/patologia , Proteínas Smad/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported. The sequence data indicated that the gene product consists of 284 amino acids. This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.
Assuntos
Gluconacetobacter xylinus/genética , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Gluconacetobacter xylinus/enzimologia , Dados de Sequência Molecular , UTP-Glucose-1-Fosfato Uridililtransferase/biossínteseRESUMO
We report here the characterization of PSKH1, a novel human protein serine kinase with multiple intracellular localizations. The gene consists of three exons distributed over 35 kb of genomic DNA in region 16q22.1. The 3.4-kb cDNA predicts a protein of 424 amino acids with a calculated molecular mass of 48.1 kDa and pI of 9.6. PSKH1 is expressed in all tissues and cell lines tested as shown by Northern blots, with the highest level of abundance in testis. PSKH1 displays the highest level of similarity with rat CaM kinase I (50. 2%) over 259 amino acids in the conserved catalytic region, but lacks significant homology with proteins in the database outside the catalytic core. Polyclonal antibodies have been raised, and indirect immunofluorescence microscopy of untransfected COS-1 cells suggests that PSKH1 is localized in the Brefeldin A-sensitive Golgi compartment, at centrosomes, in the nucleus with a somewhat speckle-like presence, and more diffusely in the cytoplasm. The presence in the centrosome appears to be enhanced during osmotic stress. Immunoisolated PSKH1 does not phosphorylate any of the common kinase substrates in vitro, but autophosphorylates exclusively serines within its COOH-terminal region in an intermolecular fashion. Furthermore, autophosphorylation activity is repressed upon addition of Ca(2+)/CaM, suggesting that PSKH1 activity depends on Ca(2+) concentration in vivo.
Assuntos
Núcleo Celular/enzimologia , Centrossomo/enzimologia , Complexo de Golgi/enzimologia , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Sequência de Bases , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Compartimento Celular , Clonagem Molecular , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosforilação , Sinais Direcionadores de Proteínas , Transporte Proteico , Homologia de Sequência de Aminoácidos , Serina/metabolismo , Testículo/enzimologia , Distribuição TecidualRESUMO
A cDNA encoding a novel member of the helicase family, MDDX28, has been cloned from a human testis library. This apparently intronless gene was transcribed in all tissues studied. MDDX28 encodes a protein of 540 amino acids, with approximately 30% homology to other helicases over the core region, containing all the conserved DEAD-box helicase motifs. No homologue is known. MDDX28 has RNA and Mg(2+)-dependent ATPase activity. Subcellular localization studies of MDDX28 using oligoclonal antibodies raised against the protein as well as its enhanced green fluorescence protein (EGFP) demonstrated that the protein is localized in the mitochondria and the nucleus. To our knowledge, MDDX28 is the first member of the RNA helicase described with this dual location. The nuclear localization of MDDX28 depended on active RNA polymerase II transcription, suggesting that the protein could be transported to and from the nucleus. This was confirmed further in an interspecies heterokaryon assay, in which MDDX28 was seen to translocate to the nucleus and mitochondria. The mitochondrial uptake of the MDDX28-EGFP-N1 fusion protein was inhibited by carbonyl cyanide p-(trichloromethoxy)phenylhydrazone. Our results indicate that MDDX28 can be transported between the mitochondria and the nucleus.
Assuntos
Núcleo Celular/enzimologia , Mitocôndrias/enzimologia , RNA Helicases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células COS , Clonagem Molecular , RNA Helicases DEAD-box , DNA , Humanos , Dados de Sequência Molecular , RNA Helicases/química , RNA Helicases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Pulsed-field gel electrophoresis has been used to construct a long-range restriction map spanning more than 900 kb in the q22.1 region of human chromosome 16. The gene cluster containing the lecithin:cholesterol acyl transferase (LCAT) gene is located less than 480 kb from the anonymous DNA marker D16S124 in this map. The results suggest three putative CpG islands within 125 kb, in addition to the island previously shown to be located within the gene cluster. This implies a clustering of both genes and CpG islands in this chromosomal region.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 16 , Ligação Genética , Família Multigênica/genética , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Marcadores Genéticos , HumanosRESUMO
A pooled DNA probe from P1 artificial chromosome clones (PACs) containing the human lecithin:cholesterol acyl transferase (LCAT) gene cluster was used in fluorescence in situ hybridization (FISH) experiments assigning the genes to pig chromosome 6p13. In addition, probes derived from the coding regions in the human gene cluster were used in long range mapping experiments to show that the overall structures of the human and porcine LCAT gene clusters are identical. Both the linear order and the close physical distance of five apparently unrelated genes have been maintained throughout 90 million years of divergent evolution between human and pig. The extremely dense clustering of the genes in the LCAT gene cluster suggests that this gene organization has biological significance. The conservation of the gene cluster between human and pig supports this suggestion.
Assuntos
Mapeamento Cromossômico , Sequência Conservada , Família Multigênica , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Animais , Sequência de Bases , Cromossomos , DNA Complementar , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Mapeamento por Restrição , SuínosRESUMO
Host versus graft disease is the fatal syndrome of altered immunity that follows the perinatal inoculation of related F1 hybrid spleen cells to susceptible strains of inbred mice. The allogenic reaction results in severe depletion of T-lymphocytes, but causes hyperplasia and hypersecretion of B-cells. Among the long-term survivors of acute host versus graft reactions, there is a high incidence of nonthymic lymphomas associated with ecotropic murine leukemia virus that may be of donor F1 origin. The present studies were done to determine whether ecotropic murine leukemia virus played any role in the pathogenesis of acute host versus graft disease in RFM mice perinatally inoculated with (T6 X RFM)F1 spleen cells. In RFM/(T6 X RFM)F1 chimeras, N-tropic murine leukemia virus can be detected as early as 3 days. The progression of the disease was accompanied by increasing viral expression. The inoculation of N-tropic virus of F1 donor origin into RFM neonates failed to induce disease, although the virus proliferated. Detection of progressively rising titers of antibody to murine leukemia virus linked the virus to the development of hyperimmunoglobulinemia by virtue of its ability to serve as a replicating source of antigens. These and other studies provided evidence that the seemingly paradoxical appearance of hyperimmunoglobulinemia in T-cell-deficient mice with the host versus graft syndrome is due, at least in part, to the stimulation of presensitized F1 donor B-cells, which are not destroyed in the allogenic reaction, as are the T-cells. Another unusual finding was the detection of polytropic murine leukemia virus in 25-day-old RFM/(T6 X RFM)F1 chimeras. It is suggested that the allogenic host versus graft reaction favored the formation of recombinants.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Reação Hospedeiro-Enxerto , Leucemia Experimental/imunologia , Camundongos Endogâmicos/imunologia , Baço/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/análise , Linfócitos B/imunologia , Quimera , Genótipo , Células-Tronco Hematopoéticas/imunologia , Vírus da Leucemia Murina/imunologia , Vírus da Leucemia Murina/isolamento & purificação , Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/microbiologia , Camundongos , Linfócitos T/imunologiaRESUMO
Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.