RESUMO
Receptors for immunoglobulin G immune complexes (Fc gamma RII and Fc gamma RIII) are expressed on most hematopoietic cells and show much structural and functional diversity. In order to determine the genetic basis for this diversity, a family of genes encoding the human and mouse receptors was isolated and characterized. Humans have five distinct genes for low-affinity Fc gamma Rs, in contrast to two in the mouse. With the use of yeast artificial chromosomes, the genes encoding the human receptors were oriented and linked, which established the structure of this complex locus. Comparison of the human and mouse genes generated a model for the evolutionary amplification of this locus.
Assuntos
Antígenos de Diferenciação/genética , Família Multigênica , Receptores Fc/genética , Animais , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Southern Blotting , Éxons , Genoma Humano , Humanos , Imunoglobulina G/metabolismo , Íntrons , Camundongos , Dados de Sequência Molecular , Mutação , Receptores Fc/metabolismo , Receptores de IgG , Recombinação Genética , Mapeamento por Restrição , Baço/imunologiaRESUMO
A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.
Assuntos
Clonagem Molecular , DNA/isolamento & purificação , Genoma Humano , Cromossomos Fúngicos , Enzimas de Restrição do DNA , Fator IX/genética , Biblioteca Gênica , Glicoproteínas/genética , Humanos , Peso Molecular , Inativadores de Plasminogênio , Saccharomyces cerevisiae/genéticaRESUMO
Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Fosfatos de Dinucleosídeos , Genes do Tumor de Wilms/genética , Tumor de Wilms/genética , Passeio de Cromossomo , Sondas de DNA , Humanos , Transcrição GênicaRESUMO
In vitro experiments with C3H 10T(1/2) mouse cells were performed to determine whether Frequency Division Multiple Access (FDMA) or Code Division Multiple Access (CDMA) modulated radiofrequency (RF) radiations induce changes in gene expression. After the cells were exposed to either modulation for 24 h at a specific absorption rate (SAR) of 5 W/ kg, RNA was extracted from both exposed and sham-exposed cells for gene expression analysis. As a positive control, cells were exposed to 0.68 Gy of X rays and gene expression was evaluated 4 h after exposure. Gene expression was evaluated using the Affymetrix U74Av2 GeneChip to detect changes in mRNA levels. Each exposure condition was repeated three times. The GeneChip data were analyzed using a two-tailed t test, and the expected number of false positives was estimated from t tests on 20 permutations of the six sham RF-field-exposed samples. For the X-ray-treated samples, there were more than 90 probe sets with expression changes greater than 1.3-fold beyond the number of expected false positives. Approximately one-third of these genes had previously been reported in the literature as being responsive to radiation. In contrast, for both CDMA and FDMA radiation, the number of probe sets with an expression change greater than 1.3-fold was less than or equal to the expected number of false positives. Thus the 24-h exposures to FDMA or CDMA RF radiation at 5 W/kg had no statistically significant effect on gene expression.
Assuntos
Telefone Celular , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Expressão Gênica/fisiologia , Expressão Gênica/efeitos da radiação , Micro-Ondas , Proteoma/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta à Radiação , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Doses de RadiaçãoRESUMO
The striking clinical response of hairy cell leukemia to alpha-interferon led us to investigate the effects of interferon on hairy cells in vitro. We examined the nature of protein induction by interferon in the target hairy cells. To do this, we analyzed whole cell lysates of hairy cells from 11 patients by one-dimensional polyacrylamide gel electrophoresis. With this method, we showed the induction of 16 proteins, which ranged from Mr 140,000 to 12,000. Exposure to alpha-interferon caused very rapid induction of specific proteins in hairy cells, and induction continued for at least 9 days. Proteins were induced at extremely low interferon concentrations, and a dose-response effect was seen with increasing concentrations. A larger number of proteins was induced in hairy cells than in other lymphoid cells under the same conditions. Induction of most proteins was inhibited by actinomycin D, showing that new messenger RNA synthesis was required for their induction to occur. The number or pattern of induced proteins, or their prolonged induction, may be relevant to a change in the target hairy cells which is part of the process resulting in the antiproliferative effect of interferon in hairy cell leukemia.
Assuntos
Interferon Tipo I/farmacologia , Leucemia de Células Pilosas/metabolismo , Biossíntese de Proteínas , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Interferon Tipo I/uso terapêutico , Leucemia de Células Pilosas/terapia , Peso Molecular , Fatores de TempoRESUMO
alpha-Interferon (IFN-alpha) induced unique ultrastructural alterations in peripheral blood and splenic hairy cell leukemia (HCL) cells (14 of 20 cases) treated in vitro. To further investigate the effects of B-cell growth factor (BCGF) and IFN-alpha on target hairy cells (HCs), we utilized immunogold labeling in conjunction with scanning electron microscopy. This methodology, in contrast to other immunological methods, facilitated direct view of the expression, density, and rearrangement of selected antigens/receptors on individual cells before and after BCGF or IFN-alpha treatment. In addition to inducing proliferation of HCL cells, BCGF enhanced the expression of interleukin 2 receptors (CD25; T-activated cell antigen) with no change in the expression of class I and class II human leukocyte antigen. On the other hand, IFN-alpha did not exert a noticeable proliferative effect on HCL cells but rather inhibited the proliferation of BCGF-treated cells. In addition, IFN-alpha treatment revealed an enhanced expression of class I (4 of 9) and class II (12 of 15) human leukocyte antigen on target HCs. Two-day exposure of HCs to IFN-alpha resulted in enhanced expression of CD25 (11 of 14), whereas a decrease in CD25 expression was recorded in 4 of 5 cases treated with IFN-alpha for 3 days. Also, no significant change in the expression of two other HCL-related surface antigens, CD22 (S-HCL-1; Leu-14) and CD11c(S-HCL-3; Leu-M5), was recorded following up to 3 days of IFN-alpha or BCGF treatment. However, a 5-day exposure to IFN-alpha resulted in a significant decrease in expression of CD11c on treated HCs. Finally, the IFN-alpha-induced immunoultrastructural changes in target HCs were primarily encountered in cells from HCL cases classified as responders to in vivo IFN-alpha therapy. Our data add support to the concept that the effect of IFN-alpha in HCL is mediated by impairment of the response to B-cell growth factors and induction of further differentiation of the target cells.
Assuntos
Antígenos de Neoplasias/metabolismo , Antígenos HLA/metabolismo , Interferon Tipo I/farmacologia , Interleucina-4/farmacologia , Leucemia de Células Pilosas/patologia , Receptores de Interleucina-2/metabolismo , Idoso , Antígenos de Superfície/metabolismo , Divisão Celular/efeitos dos fármacos , Humanos , Interferon Tipo I/uso terapêutico , Interleucina-4/antagonistas & inibidores , Leucemia de Células Pilosas/imunologia , Leucemia de Células Pilosas/metabolismo , Leucemia de Células Pilosas/terapia , Masculino , Microscopia Eletrônica , Pessoa de Meia-IdadeRESUMO
The structure, synthesis, and post-transcriptional modifications of 23-S and 16-S ribosomal RNAs (rRNAs) have been studied in the facultatively parasitic bacterium, Bdellovibrio bacteriovorus. The mature 23-S and 16-S type of rRNAs in Bdellovibrio are larger than the analogous molecules in Escherichia coli by at least 1.0 - 10(5) and 0.5 - 10(5) daltons, respectively, and have a conformation different from E. coli rRNAs as judged by relative electrophoretic mobilities in polyacrylamide gels with and without denaturing conditions. Studies on the kinetics of synthesis and maturation of ribosomal RNA in Bdellovibrio show that precursor forms analogous to p23-S and p16-S in E. coli are synthesized. In addition, some earlier precursor rRNAs in Bdellovibrio are seen that appear analogous to the 25S and 17.5-S pre-rRNAs that have only been observed in the RNAase III deficient mutant of E. coli strain AB301-105 (Nikolaev, Birenbaum, M. and Schlessinger, D. (1975) Biocheim, Biophys. Acta 395, 478-489). These early precursor stages have not been observed in other procaryotic species, including E. coli that have normal levels of RNAase III. The results from the Bdellovibrio system provide that the 25-s and 17.5-S pre-rRNAs are normal stages of rRNA modification and are part of a multiple step maturation process, and therefore are not aberrations associated with the RNase III deficient mutation.
Assuntos
Bdellovibrio/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Transcrição Gênica , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Formaldeído , Peso Molecular , Mutação , Desnaturação de Ácido Nucleico , Especificidade da EspécieRESUMO
We have investigated the direct effects of interferon (IFN) on hairy cells (HCs) isolated from patients with hairy cell leukemia using one- and two-dimensional gel electrophoresis. We have previously characterized the induction of synthesis of 10-16 specific proteins by IFN-alpha 2b in HCs, as analyzed by one-dimensional electrophoresis. By two-dimensional electrophoresis, we have now confirmed this induction and shown that the synthesis of the same number of specific proteins is down-regulated in HCs exposed to IFN-alpha 2b. When compared to HCs, fewer proteins are induced by IFN-alpha 2b in other normal, or neoplastic, lymphoid cells. We also report that protein induction occurs in HCs exposed in vivo to IFN-alpha 2b. We have demonstrated the presence of tubuloreticular structures in the cytoplasm of HCs exposed to IFN-alpha 2b in vitro, using transmission electron microscopy. We now report that these too are seen in HCs exposed to IFN in vivo during therapy. We investigated the effects of IFN-gamma on HCs and found that it also induces specific proteins. The pattern of induced proteins is distinctly different after IFN-gamma exposure in vitro. The fact that such induction occurs suggests that HCs possess also a receptor for IFN-gamma. These results demonstrate that there are direct effects of IFN-alpha on HCs and that such direct effects might be important in the antitumor activity of IFN-alpha in hairy cell leukemia.
Assuntos
Interferons/farmacologia , Leucemia de Células Pilosas/metabolismo , Proteínas de Neoplasias/biossíntese , Eletroforese em Gel de Poliacrilamida , Humanos , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Ponto Isoelétrico , Leucemia de Células Pilosas/patologia , Peso Molecular , Receptores Imunológicos/fisiologia , Receptores de Interferon , Células Tumorais CultivadasRESUMO
The nature of the interleukin 2 (IL-2) receptor on purified human B lymphocytes was examined. Both normal and malignant cells showed evidence of a 70-75,000 mol. wt (p75) IL-2 binding molecule as assessed by 125I-labeled IL-2 binding and receptor cross-linking. On normal, Tac-negative B lymphocytes the estimated number of p75 binding sites was 1100 per cell and the dissociation constant (Kd) was 1.7 nM. Consistent with this, cross-linking experiments demonstrated the presence of an IL-2 binding molecule of 70-75,000 mol. wt. Purified B cells from patients with hairy cell leukemia and chronic lymphocytic leukemia (CLL) also expressed the p75 IL-2 binding molecule. In the HCL samples, a small number of high-affinity IL-2 binding sites were detected (27-90) while the majority of binding sites (2100-10,800) were typical of low-affinity p55 Tac binding. IL-2 added to the purified normal and CLL B lymphocytes led to the induction of p55 Tac expression and the generation of high-affinity IL-2 receptors. This response to IL-2 was equivalent to the response observed when normal B lymphocytes were stimulated by Staphylococcus aureus Cowan I.
Assuntos
Linfócitos B/imunologia , Linfoma de Burkitt/imunologia , Interleucina-2/metabolismo , Receptores de Interleucina-2/metabolismo , Antígenos CD/análise , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Cinética , Peso Molecular , Receptores de Interleucina-2/isolamento & purificação , Succinimidas/farmacologiaRESUMO
The traditional approach to the study of biology employs small-scale experimentation that results in the description of a molecular sequence of known function or relevance. In the era of the genome the reverse is true, as large-scale cloning and gene sequencing come first, followed by the use of computational methods to systematically determine gene function and regulation. The overarching goal of this new approach is to translate the knowledge learned from a systematic, global analysis of genomic data into a complete understanding of biology. For investigators who study shock, the specific goal is to increase understanding of the adaptive response to injury at the level of the entire genome. This review describes our initial experience using DNA microarrays to profile stress-induced changes in gene expression. We conclude that efforts to apply genomics to the study of injury are best coordinated by multi-disciplinary groups, because of the extensive expertise required.
Assuntos
Genômica/tendências , Pesquisa/tendências , Ferimentos e Lesões/fisiopatologia , Previsões , Técnicas Genéticas , Genoma Fúngico , Genômica/métodos , Humanos , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/patologia , Projetos de Pesquisa , Saccharomyces cerevisiae/fisiologia , Baço/imunologia , Baço/lesões , Baço/fisiopatologia , Ferimentos e Lesões/genéticaRESUMO
We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.
Assuntos
Cromossomos Fúngicos , Genoma Humano , Cadeias kappa de Imunoglobulina/genética , Animais , Sequência de Bases , Blastocisto , Quimera , Biblioteca Gênica , Rearranjo Gênico , Marcadores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Protoplastos , TransfecçãoRESUMO
This unit provides a series of protocols describing the analysis and manipulation of an isolated YAC clone. The procedures are based upon the use of the YAC vector pYAC4. Once an isolated YAC clone has been obtained from a core laboratory, the clone can be analyzed as described herein. Methods for analysis involve growing and storing YAC-containing yeast strains and purifying YAC DNA in a form suitable for assessing the size of the artificial chromosome and for conventional Southern blotting. Preparation of yeast chromosomes in agarose plugs for subsequent analysis by pulsed-field gel electrophoresis is also described. Additional protocols are provided for recovering DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high-molecular-weight YAC DNA is described and a general method for subcloning YAC inserts into cosmid or lambda vectors for higher-resolution analysis is provided.
Assuntos
Passeio de Cromossomo , Cromossomos Artificiais de Levedura , DNA Fúngico/genética , Southern Blotting , Cromossomos Artificiais , Cromossomos Artificiais de Levedura/genética , Cromossomos Fúngicos , Clonagem Molecular , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Ágar , Biblioteca Genômica , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
This unit provides an introduction to the use of yeast artificial chromosome-bearing yeast clones (hereafter referred to as YAC clones) in genome analysis. It describes criteria for designing a polymerase chain reaction (PCR) assay to be used in screening a YAC core library and discusses the rationale for verification and characterization of YAC clones obtained from these core laboratories. Protocols for maintaining YAC clones, analyzing YAC insert structure, preparing YAC DNA, and subcloning YAC inserts into other vectors are presented elsewhere in this volume.
Assuntos
Cromossomos Artificiais de Levedura/genética , Biblioteca Gênica , Genoma Humano , Reação em Cadeia da Polimerase/métodos , Clonagem Molecular , Primers do DNA , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Humanos , Peso MolecularRESUMO
Pulse-labeled cells of Bacillus megaterium were converted to protoplasts, and lysates of the protoplasts were analyzed by sucrose gradient sedimentation. Precursor ribonucleoprotein (RNP) particles then appeared predominantly as 50S and 30S precursor ribosomal subunits. Polyacrylamide gel electrophoresis of the ribosomal ribonucleic acid from the 50S and 30S RNP particles confirmed their precursor nature since they were shown to contain precursor 23S and 16S ribosomal ribonucleic acid, respectively. Treatment of protoplast lysates with 0.5% deoxycholate prior to sedimentation analysis resulted in a markedly different radioactivity profile. The 50S RNP particles were no longer present, but 43S particles were observed in addition to increased amounts of pulse-labeled material sedimenting at 30S and slower. Extracts from cells broken in a French press showed a profile from sucrose gradient sedimentation similar to that of the deoxycholate-treated protoplast lysate. These data suggest that the nature of the precursor ribosomal particles appears to be a function of the method of cell disruption or detergent treatment of the cell extract preparation. The observed 50S and 30S RNP particles may be the major precursor ribosomal subunits in vivo; the slower-sedimenting species could result from some form of breakdown or change in the configuration of the 50S and 30S precursors.
Assuntos
Bacillus megaterium/análise , Proteínas de Bactérias/análise , Precursores de Proteínas/análise , RNA Bacteriano/análise , RNA Ribossômico/análise , Fracionamento Celular , Ácido Desoxicólico/farmacologia , Muramidase/farmacologia , Protoplastos/análise , Ribossomos/análiseRESUMO
Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.
Assuntos
Bacillus megaterium/efeitos dos fármacos , Detergentes/farmacologia , Ribossomos/efeitos dos fármacos , Bacillus megaterium/metabolismo , Fracionamento Celular , Precursores de Ácido Nucleico/metabolismo , Fosfolipídeos/metabolismo , Protoplastos/metabolismo , RNA Ribossômico/metabolismoRESUMO
The mechanism of the antineoplastic effect of interferon (IFN) is not known and may result from direct effects on the neoplastic cells themselves, activation of intermediary effector cells, or a combination of these effects. The synthesis of specific proteins is induced in hairy cells when they are exposed to alpha-IFN in vitro. In particular, the called p80, is markedly induced. We investigated this effect in the hairy cells of seven patients in the leukemic phase of hairy cell leukemia who were being treated with subcutaneous (SC) IFN alpha 2b (r-Hu-IFN-alpha 2). Polyacrylamide gel electrophoresis (PAGE) was carried out on [35S]methionine-labeled whole cell lysates visualized by autoradiography and silver staining. Within 2 days of starting IFN therapy, induction of specific protein synthesis, including p80 was seen by [35S]methionine labeling in freshly isolated circulating hairy cells from 6 of 6 patients tested. Therefore, alpha-IFN has a direct biochemical effect on hairy cells in vivo that is similar to in vitro effects, at least with regard to p80 synthesis, although the kinetics of this effect may vary in the two situations.
Assuntos
Interferon Tipo I/uso terapêutico , Leucemia de Células Pilosas/tratamento farmacológico , Proteínas de Neoplasias/biossíntese , Proteínas Recombinantes/uso terapêutico , HumanosRESUMO
Despite the striking antiproliferative effect of alpha-interferon (alpha-IFN) in hairy cell leukemia, gamma-interferon (gamma-IFN) does not appear to have such an effect. We have previously demonstrated the induction of synthesis of specific proteins by alpha-IFN, both in vitro and in vivo. We have now shown that gamma-IFN induces synthesis of specific proteins, but the pattern differs from that seen after alpha-IFN exposure. The prominent 80,000-dalton protein induced by alpha-IFN was not induced by gamma-IFN, and the prominent 62,000-dalton protein induced by gamma-IFN was only rarely induced by alpha-IFN. Two other proteins were induced by both alpha-IFN and gamma-IFN. The other proteins induced by alpha-IFN were not induced by gamma-IFN. These differences may be related to the different biological response of hairy cells to the two types of IFN. We also showed for both alpha-IFN and gamma-IFN that some IFN-induced proteins are probably transported to the nucleus of the hairy cell, although the majority of proteins induced by both alpha-IFN and gamma-IFN were in the cytosol/membrane fraction of the cell. We have therefore demonstrated that gamma-IFN does have a biochemical effect on hairy cells in terms of induction of specific protein synthesis, leading to the inference that hairy cells must possess another receptor at least functionally analogous to the type II IFN receptors on fibroblasts, with which gamma-IFN interacts.
Assuntos
Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Leucemia de Células Pilosas/metabolismo , Proteínas de Neoplasias/biossíntese , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas In Vitro , Peso MolecularRESUMO
We have studied the mode of transcription of the three double-stranded RNA segments found in bacteriophage phi 6. Stable transcription intermediates, isolated following in vitro incorporation of nucleoside triphosphates by phi 6 nucleocapsids, were examined by electron microscopy. Specimens were either spread and shadowed or deposited on polylysine film and stained. In either case, branched molecules with one or more single-stranded arms were seen. The single-stranded arm, in all molecules observed, has about half the contour length of one double-stranded arm. The branched molecules are stable in high salt or hot phenol, resistant to proteinase K, but sensitive to RNAase A in high salt, yielding fragments of double-stranded RNA. These results are consistent with a transcription mechanism in which each new transcript displaces one of the parental RNA strands. From the rate of movement of the branch point, we found transcription rates in vitro of similar to or approximately 25 nucleotides per sec at 30 degrees C and 19 nucleotides per sec at 25 degrees C. Based on the spacing between branches in multiply branched molecules, initiation occurs approximately once every 40 sec at 30 degrees C on M or S RNA templates and about 6 times less frequently on L RNA.
Assuntos
Bacteriófagos/genética , Capsídeo/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Transcrição Gênica , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Cinética , Microscopia Eletrônica , Peso Molecular , Pseudomonas , Ribonucleoproteínas/metabolismoRESUMO
The ends of the three dsRNA genome segments (L, M, and S) of bacteriophage phi 6 (strand separated and/or intact) and the 5' ends of the middle and small single-strand messenger RNAs have been sequenced by base-specific partial enzymatic digestion. Terminal sequences for the large and middle dsRNA strands extend about 60 bases. The three dsRNA segments have 18 homologous bases at the left end except for position 2, which differs in the L segment. A 17-base homology defines the right ends of L and M dsRNAs and probably S dsRNA as well. The 5' ends of middle and small messenger RNAs are identical to the corresponding viral (+) strands.
Assuntos
Bacteriófagos/genética , Genes Virais , Pseudomonas/genética , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , RNA Viral , Sequência de Bases , Genes Bacterianos , RNA de Cadeia Dupla/isolamento & purificação , Transcrição GênicaRESUMO
Heat shock proteins (HSPs) are though to represent a ubiquitous cellular response to heat or stress. We tested whether HSPs can be induced in hairy cells, other human leukemic cells, and normal lymphocytes, and whether there are additive or synergistic effects between heat shock and interferon-alpha 2b (IFN-alpha 2b) on these cells. We analyzed lysates of cells from 22 patients (6 with hairy cell leukemia, 12 with other acute and chronic leukemias and lymphocytes of 4 normals) after exposure to a heat shock and/or IFN-alpha 2b by one-dimensional polyacrylamide gel electrophoresis. In all cells a pattern of HSPs was readily induced with prominent bands identified at approximately 115, 90, and 65 kD. None of these major proteins appeared to represent the previously described IFN-alpha 2b induced band at 80 kD. IFN-alpha 2b by itself was not found to induce HSPs. We conclude that a pattern of HSPs can readily be induced in a variety of normal and leukemic human blood cells. IFN-alpha 2b is not a HSP inducer in these cells. The previously described IFN-induced p80 is apparently not a HSP.