RESUMO
Cytokines control a variety of cellular responses including proliferation, differentiation, survival and functional activation, via binding to specific receptors expressed on the surface of target cells [1]. The cytokine receptors of the haemopoietin family are defined by the presence of a conserved 200 amino acid extracellular domain known as the haemopoietin domain [2]. We report here the isolation of NR6, a haemopoietin receptor that, like the p40 subunit of interleukin-12 (IL-12) [3] and the EBI3 gene induced by Epstein-Barr virus infection in lymphocytes [4], contains a typical haemopoietin domain but lacks transmembrane and cytoplasmic domains. Although in situ hybridisation revealed NR6 expression at multiple sites in the developing embryo, mice lacking NR6 did not display obvious abnormalities and were born in the expected numbers. Neonatal NR6(-/-) mice failed to suckle, however, and died within 24 hours of birth, suggesting that NR6 is necessary for the recognition or processing of pheromonal signals or for the mechanics of suckling itself. In addition, NR6(-/-) mice had reduced numbers of haemopoietic progenitor cells, suggesting a potential role in the regulation of primitive haemopoiesis.
Assuntos
Animais Lactentes/fisiologia , Proteínas de Transporte/fisiologia , Hematopoese/fisiologia , Receptores de Superfície Celular , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Receptores para Leptina , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
max 41 transgenic mice consistently exhibit elevated numbers of mature granulocytes and monocytes in the peripheral blood and of immature and mature cells of these lineages in the marrow, spleen, lymph nodes and liver. The immature populations are not autonomous and exhibit a normal quantitative responsiveness to proliferative stimulation by the four colony-stimulating factors. The present studies examined three other candidate regulators of granulocyte formation and showed that max 41 cells exhibit normal quantitative responsiveness to stem cell factor, slightly enhanced responsiveness to IL-6 but reduced responsiveness to Flk-ligand. Serum levels of growth factors were not unusually elevated in max 41 mice before or after the injection of endotoxin nor were excessive levels of the four CSFs or IL-6 produced in cultures of max 41 organs. Responses to injected G-CSF were not unusually high in terms of fold-elevations in max 41 mice. Levels of mRNA for various growth factors were not abnormal in max 41 marrow populations although, in crowded cultures, max 41 marrow cells exhibited a higher level of endogenously stimulated colony formation than control cells. max 41 cells also exhibited elevated responsiveness to stimulation by mixtures of growth factors, particularly those in organ-conditioned media. The present observations suggest some possible mechanisms by which a max 41 mouse might achieve a sustained elevation of granulocyte and monocyte production but the data seem insufficient to provide a complete explanation and indicate persisting deficiencies in knowledge of how granulocyte and monocyte production is regulated.
Assuntos
Granulócitos/citologia , Hematopoese , Animais , Sequência de Bases , Células da Medula Óssea , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Fatores Estimuladores de Colônias/biossíntese , Fatores Estimuladores de Colônias/farmacologia , Feminino , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Interleucina-6/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/metabolismo , Fator de Células-Tronco/farmacologiaRESUMO
In this paper we report on the structure and expression of the genes encoding the alpha and beta chains of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in human leukemia. The alpha chain gene is highly polymorphic in normal individuals and no evidence for rearrangement within this locus was found in 47 hemopoietic, nine non-hemopoietic malignancies and five human cell lines. Using the polymerase chain reaction the gene for the alpha chain was shown to be expressed in 18/18 primary myeloid as well as 8/8 primary lymphoid leukemias analysed. To investigate the integrity of the mRNA, polymerase chain reactions (PCR) using a combination of oligonucleotides spanning the entire coding region of the alpha chain were performed. Normal sized fragments were generated with all combinations of oligonucleotides from all but one leukemia. One chronic lymphoid leukemia displayed an apparent alteration at the 3' end of the 3' untranslated region of the alpha chain mRNA. No polymorphisms were detected in the beta chain gene which was also not rearranged in any of the samples analysed. The beta chain mRNA was expressed in 17/18 primary myeloid and 7/8 primary lymphoid leukemias and in those leukemias there was no evidence for any lesions in the mRNA, as judged by PCR fragment size. Thus gross structural lesions in the genes encoding the GM-CSF receptor alpha and beta chains appear to be infrequent in hemopoietic neoplasms.
Assuntos
Leucemia/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Sequência de Bases , DNA de Neoplasias/genética , Expressão Gênica , Rearranjo Gênico , Genes , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
Factor-specific cell line bioassays were used to monitor the production in vitro by adult and fetal mouse organs of GM-CSF, G-CSF, M-CSF, Multi-CSF (IL-3), IL-6 and leukemia inhibitory factor (LIF). No tissue was observed to produce Multi-CSF. Highest producers of the other regulators were lung, muscle, thymus, heart and bone shaft and all tissues producing detectable growth factors produced all five with the same rank order of activity. Adult tissues produced more GM-CSF than G-CSF and less M-CSF than either, no differences being observed between male, female and pregnant female tissues. In contrast, the pregnant uterus produced high levels of M-CSF as did the fetal membranes and tissues with only low GM-CSF and no G-CSF production. Pre-irradiation did not alter the pattern of regulator production by adult tissues. The intravenous injection of endotoxin elevated serum levels of GM-CSF, G-CSF, M-CSF and IL-6 but the dominant rise was in G-CSF levels. The data indicating that multiple organs produce the regulators monitored in a common rank order suggest some overall linkage in their production with major differences between adult and fetal tissues.
Assuntos
Fatores Estimuladores de Colônias/biossíntese , Inibidores do Crescimento/biossíntese , Linfocinas/biossíntese , Animais , Sequência de Bases , Bioensaio , Linhagem Celular , Fatores Estimuladores de Colônias/sangue , Meios de Cultivo Condicionados , Feminino , Feto/metabolismo , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Inibidores do Crescimento/sangue , Interleucina-3/biossíntese , Interleucina-6/biossíntese , Fator Inibidor de Leucemia , Linfocinas/sangue , Fator Estimulador de Colônias de Macrófagos/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dados de Sequência MolecularRESUMO
SOCS-1 was originally identified as an inhibitor of interleukin-6 signal transduction and is a member of a family of proteins (SOCS-1 to SOCS-7 and CIS) that contain an SH2 domain and a conserved carboxyl-terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino-terminal and SH2 domains are essential for SOCS-1 and SOCS-3 to inhibit cytokine signaling. Inhibition of cytokine-dependent activation of STAT3 occurred in cells expressing either SOCS-1 or SOCS-3, but unlike SOCS-1, SOCS-3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS-1 and SOCS-3 action when overexpressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS-1(-/-) mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS-1(-/-) mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS-1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects.
Assuntos
Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Repressoras , Transdução de Sinais , Animais , Proteínas de Transporte/genética , Humanos , Camundongos , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , Domínios de Homologia de srcRESUMO
Activating NOTCH1 mutations occur in ~60% of human T-cell acute lymphoblastic leukemias (T-ALLs), and mutations disrupting the transcription factor IKZF1 (IKAROS) occur in ~5% of cases. To investigate the regulatory interplay between these driver genes, we have used a novel transgenic RNA interference mouse model to produce primary T-ALLs driven by reversible Ikaros knockdown. Restoring endogenous Ikaros expression in established T-ALL in vivo acutely represses Notch1 and its oncogenic target genes including Myc, and in multiple primary leukemias causes disease regression. In contrast, leukemias expressing high levels of endogenous or engineered forms of activated intracellular Notch1 (ICN1) resembling those found in human T-ALL rapidly relapse following Ikaros restoration, indicating that ICN1 functionally antagonizes Ikaros in established disease. Furthermore, we find that IKAROS mRNA expression is significantly reduced in a cohort of primary human T-ALL patient samples with activating NOTCH1/FBXW7 mutations, but is upregulated upon acute inhibition of aberrant NOTCH signaling across a panel of human T-ALL cell lines. These results demonstrate for the first time that aberrant NOTCH activity compromises IKAROS function in mouse and human T-ALL, and provide a potential explanation for the relative infrequency of IKAROS gene mutations in human T-ALL.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Fator de Transcrição Ikaros/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores Notch/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Biomarcadores Tumorais/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Imunoprecipitação da Cromatina , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fator de Transcrição Ikaros/antagonistas & inibidores , Fator de Transcrição Ikaros/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores Notch/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genéticaRESUMO
A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.
Assuntos
Fatores Biológicos/farmacologia , Osso e Ossos/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Interleucina-6 , Linfocinas , Animais , Reabsorção Óssea/fisiopatologia , Cálcio/metabolismo , Citocinas , Relação Dose-Resposta a Droga , Humanos , Indometacina/farmacologia , Fator Inibidor de Leucemia , Camundongos , Fenilalanina/metabolismo , Crânio/efeitos dos fármacos , Crânio/metabolismo , Timidina/metabolismoRESUMO
Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.
Assuntos
Repetição de Anquirina/genética , Proteínas de Transporte/genética , Genes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Expressão Gênica , Humanos , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
We have previously characterized, purified and cloned a novel murine and human regulator [leukaemia inhibitory factor, LIF] which induces the differentiation of certain murine and human myeloid leukaemic cells. Recently we have shown that there are specific LIF receptors on murine embryonic stem [ES] and embryonal carcinoma [EC] cells and that purified recombinant LIF can substitute for feeder cells and crude sources of differentiation inhibiting activity [DIA] [such as BRL-cell-conditioned medium] in the maintenance of ES cells in a pluripotential state in vitro. Furthermore, ES cells maintained in culture in recombinant LIF for a prolonged period can give rise to germline chimaeric mice. Thus, based on a number of biochemical and biological similarities, it is likely that LIF and DIA are the same molecule. The identification of LIF as a molecule, necessary and sufficient for the maintenance of ES cells in culture, should have a profound impact on the use of these cells for genetic manipulations.
Assuntos
Inibidores do Crescimento/farmacologia , Interleucina-6 , Leucemia Mieloide/patologia , Linfocinas , Células-Tronco/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Células-Tronco de Carcinoma Embrionário , Humanos , Fator Inibidor de Leucemia , Camundongos , Dados de Sequência Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Homologia de Sequência do Ácido NucleicoRESUMO
This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.
Assuntos
Adenosina Trifosfatases/análise , Aminoácidos/análise , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Mutação , Conformação ProteicaRESUMO
Leukaemia inhibitory factor (LIF) is a pleiotropic growth factor active in diverse cell systems in both the adult and the embryo. The LIF gene from a number of mammalian species is highly conserved. The ovine and porcine LIF genes were cloned, sequenced and compared to the previously published murine and human LIF gene sequences. While the coding regions of the LIF gene are highly conserved, the non-coding regions are largely non-conserved. In a region of approximately 340 bp, the 5' end of the translational initiation codon is highly conserved (84%). This region includes four conserved TATA boxes, two transcriptional start-sites identified in the murine gene and the minimal region required to function as the LIF promoter. A sequence in the murine gene adjacent to this highly conserved region which appears to contain a negative control element is, however, poorly conserved between the four species compared, except for a sequence of 16 conserved nucleotides. Within the largely non-conserved first intron, there is a block of approximately 150 nucleotides which is highly conserved between all four species (approximately 72%). However, a sequence in intron 1 of the murine LIF gene which corresponds to an alternative exon of a putative variant LIF transcript is very poorly conserved between species, with only relics of this exon evident in the other three species. A comparison of the five LIF protein sequences available (murine, rat, human, ovine and porcine) revealed that the protein displays a high degree of similarity, ranging from 74% between mouse and sheep to 92% between rat and mouse. Several large blocks of absolutely conserved amino acid sequence were identified. The ovine LIF gene was modified to allow production of recombinant ovine LIF in yeast cells, which was shown to be biologically active on murine cells.
Assuntos
Inibidores do Crescimento/genética , Interleucina-6 , Linfocinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Códon , DNA/química , Expressão Gênica , Inibidores do Crescimento/química , Humanos , Íntrons , Kluyveromyces/genética , Fator Inibidor de Leucemia , Linfocinas/química , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Ratos , Homologia de Sequência do Ácido Nucleico , Ovinos , Especificidade da Espécie , Suínos , Transformação GenéticaRESUMO
A series of yeast mitochondrial mit- mutants with defects in the oli2 gene, coding for subunit 6 of the mitochondrial ATPase complex, has been analyzed at the DNA sequence level. Fifteen of sixteen primary mit- mutants were shown to contain frameshift or nonsense mutations predicting truncated subunit 6 polypeptides, in various strains ranging from about 20% to 95% of the wild-type length of 259 amino acids. In only one strain could the defect in subunit 6 function be assigned to amino acid substitution in an otherwise full-length subunit 6. Many mutants carried multiple base substitutions or insertions/deletions, presumably arising from the manganese chloride mutagenesis treatment. Revertants from three of the mit- mutants were analyzed: all contained full-length subunit 6 proteins with one or more amino acid substitutions. The preponderance of truncated proteins as opposed to substituted full-length proteins in oli2 mit- mutants is suggested to reflect the ability of subunit 6 to accommodate amino acid substitutions at many locations, with little or no change in its functional properties in the membrane FO-sector of the ATPase complex.
Assuntos
Adenosina Trifosfatases/genética , DNA Fúngico/genética , DNA Mitocondrial/genética , Genes Fúngicos , Genes , Mitocôndrias/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Códon , Substâncias Macromoleculares , Saccharomyces cerevisiae/enzimologiaRESUMO
The gene encoding one subunit of the receptor for the hemopoietic growth factor, GM-CSF, has been previously localized to the short arm of the human sex chromosomes. By pulsed-field gel electrophoresis, the precise localization of this gene, CSF2RA, within the pseudoautosomal region has been determined. The gene is located 1180 to 1300 kb from the telomere, in close proximity to the CpG island B5. The CSF2RA gene spans at least 45 kb, and a representation of most of the gene on three overlapping cosmid clones has been obtained. The exon(s) encoding the first 35 bp of cDNA sequence lies outside these cosmids. The CSF2RA gene is characterized by abundant hypervariable sequences, and a number of informative restriction fragment length polymorphisms have been defined.
Assuntos
Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Cromossomo X , Cromossomo Y , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , DNA , Fosfatos de Dinucleosídeos/genética , Eletroforese em Gel de Campo Pulsado , Humanos , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Mapeamento por RestriçãoRESUMO
In this study the gene for the murine interleukin-11 receptor alpha chain (IL-11Ralpha) has been characterized. The gene spans 9 kilobase pairs of DNA, and the organization of its 14 exons conforms to the pattern observed for other members of the hematopoietin receptor family. Analysis of the 5' end of the cDNA using 5' RACE showed that the first two exons, designated exons 1a and 1b, are spliced to form alternate transcripts. Transcripts initiating from exon 1b were not found in adult tissues but were present in embryonic stem cells. S1 nuclease and 5' rapid amplification of cDNA ends assays demonstrated multiple major and minor sites of transcription initiation for each exon. The putative promoter regions of both exons lacked TATA boxes, although potential recognition sites for several transcription factors including Sp1, AP1, and AP2 were present. A comparison of the murine and human IL-11Ralpha revealed that the 5' sequence upstream of the major site of transcription initiation site for exon 1b is highly conserved. Northern analysis showed that IL-11Ralpha is expressed in many adult murine tissues. A second IL-11Ralpha-like locus containing a sequence homologous to exons 2-13 was also identified.
Assuntos
Receptores de Interleucina/genética , Processamento Alternativo , Animais , Sequência de Bases , Sítios de Ligação/genética , Mapeamento Cromossômico , Sequência Conservada , Primers do DNA/genética , DNA Complementar/genética , Éxons , Humanos , Subunidade alfa de Receptor de Interleucina-11 , Íntrons , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Estrutura Molecular , RNA Mensageiro/genética , Receptores de Interleucina/química , Receptores de Interleucina-11 , Homologia de Sequência do Ácido NucleicoRESUMO
The gene for the beta-chain of the human GM-CSF receptor (CSF2RB) has been mapped to chromosome 22 by PCR analysis of a series of human x rodent somatic cell hybrids. In situ hybridization to normal human chromosomes and two translocations involving chromosome 22 and the chromosome expressing the rare fragile site FRA22A place the gene in the region 22q12.2-->q13.1, proximal to the fragile site.
Assuntos
Cromossomos Humanos Par 22 , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Translocação Genética/genética , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da PolimeraseRESUMO
Leukemia inhibitory factor, LIF, is a glycoprotein with multiple activities in both the adult and the embryo. LIF appears to be encoded by a unique gene in both mouse and man, although the 3'-untranslated region of the mouse LIF gene gives a complex hybridization pattern on Southern blots. The complete nucleotide sequences of both the murine and human LIF genes and their flanking regions (8.7 and 7.6 kilobase pairs, respectively) were determined and compared. Both genes comprise three exons, two introns and an unusually long 3'-untranslated region (3.2 kilobase pairs), specificying a mRNA of approximately 4.1 kilobases. Two start sites of LIF-transcription were determined, by S1-nuclease protection and by a novel approach involving the polymerase chain reaction. S1-nuclease protection revealed a start site 60-64 base pairs upstream of the translational start codon and immediately downstream of a TATA box (TATATAAAT). The PCR approach identified a second transcriptional start site 160 base pairs 5' of the start codon and adjacent to a "TATA-like" element (CATAATTT). A comparison of the murine and human LIF gene sequences revealed a high degree of conservation in the coding regions and in segments of the untranslated and flanking regions. Seven segments displaying greater than 75% homology were identified, with the 5' and 3' ends of the transcription unit revealing the highest degree of homology. These conserved regions represents potential cis-acting control elements.
Assuntos
Evolução Biológica , Genes , Glicoproteínas/genética , Inibidores do Crescimento/genética , Interleucina-6 , Linfocinas , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Citoplasma/metabolismo , DNA/genética , DNA/isolamento & purificação , Éxons , Biblioteca Gênica , Humanos , Íntrons , Fator Inibidor de Leucemia , Camundongos , Dados de Sequência Molecular , Poli A/análise , Poli A/genética , Reação em Cadeia da Polimerase , RNA/análise , RNA/genética , RNA Mensageiro/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors.
Assuntos
Antígenos CD/química , Receptores de Interleucina/química , Receptores de Interleucina/genética , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Sequência de Bases , Divisão Celular , Células Cultivadas , DNA Complementar/genética , Genes , Interleucina-13/metabolismo , Subunidade alfa1 de Receptor de Interleucina-13 , Interleucina-4/metabolismo , Camundongos , Dados de Sequência Molecular , Receptores de Interleucina/metabolismo , Receptores de Interleucina-13 , Receptores de Interleucina-4 , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1(-/-) mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1(-/-) mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.
Assuntos
Proteínas de Transporte/fisiologia , Inibidores Enzimáticos , Interleucina-6/fisiologia , Fígado/patologia , Tecido Linfoide/patologia , Proteínas Repressoras , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Envelhecimento , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/fisiologia , Proteínas de Transporte/genética , Hematócrito , Contagem de Leucócitos , Fígado/anormalidades , Linfócitos/citologia , Linfócitos/imunologia , Tecido Linfoide/anormalidades , Camundongos , Camundongos Knockout , Fenótipo , Contagem de Plaquetas , Proteínas Tirosina Quinases/antagonistas & inibidores , Recombinação Genética , Mapeamento por Restrição , Transdução de Sinais , Baço/imunologia , Células-Tronco , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , Timo/imunologiaRESUMO
A cDNA library was constructed using mRNA from Krebs ascites tumor cells that was shown by Northern blot hybridization to contain mRNA for murine leukemia inhibitory factor (LIF). This library was screened with an oligonucleotide corresponding to the 3' end of a partial LIF cDNA clone, and an overlapping cDNA clone isolated. Nucleotide sequence analysis of this latter clone allowed the complete sequence of LIF to be derived. A cDNA molecule encoding the entire mature LIF protein was installed in a yeast expression vector, and LIF produced up to about 100 ng/ml in the growth medium. The LIF produced by yeast cells has the same biologic properties as native LIF and competes with native 125I-LIF for binding to specific cellular receptors. Two forms of native LIF, distinguishable by their chromatographic behavior on DEAE-Sepharose, were converted by neuraminidase treatment to a form with similar chromatographic behavior, suggesting that the major difference between these two species is the content of sialic acid on the carbohydrate portion. Moreover, yeast-derived recombinant LIF appears to display a different pattern of glycosylation to both forms of native LIF. From in vitro experiments, we conclude that the nature of the glycosylation is not crucial to biologic activity.
Assuntos
Carcinoma Krebs 2/genética , Inibidores do Crescimento/biossíntese , Interleucina-6 , Linfocinas , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/genética , Animais , Clonagem Molecular , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Glicosilação , Inibidores do Crescimento/fisiologia , Fator Inibidor de Leucemia , Camundongos , Transcrição GênicaRESUMO
A human homologue of the recently cloned murine leukemia-inhibitory factor (LIF) gene was isolated from a genomic library by using the murine cDNA as a hybridization probe. The nucleotide sequence of the human gene indicated that human LIF has 78% amino acid sequence identity with murine LIF, with no insertions or deletions, and that the region of the human gene encoding the mature protein has one intervening sequence. After oligonucleotide-mediated mutagenesis, the mature protein-coding region of the LIF gene was introduced into the yeast expression vector YEpsec1. Yeast cells transformed with the resulting recombinant could be induced with galactose to produce high levels of a factor that induced the differentiation of murine M1 leukemic cells in a manner analogous to murine LIF. This factor competed with 125I-labeled native murine LIF for binding to specific cellular receptors on murine cells, compatible with a high degree of structural similarity between the murine and human factors.