RESUMO
A 4-kilobase complementary DNA (cDNA) encoding human macrophage-specific colony-stimulating factor (CSF-1) was isolated. When introduced into mammalian cells, this cDNA directs the expression of CSF-1 that is structurally and functionally indistinguishable from the natural human urinary CSF-1. Direct structural analysis of both the recombinant CSF-1 and the purified human urinary protein revealed that these species contain a sequence of at least 40 amino acids at their carboxyl termini which are not found in the coding region of a 1.6-kilobase CSF-1 cDNA that was previously described. These results demonstrate that the human CSF-1 gene can be expressed to yield at least two different messenger RNA species that encode distinct but related forms of CSF-1.
Assuntos
Fatores Estimuladores de Colônias/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Fatores Estimuladores de Colônias/urina , DNA/genética , Regulação da Expressão Gênica , Humanos , Macrófagos/fisiologia , Peso Molecular , Fragmentos de Peptídeos , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genéticaRESUMO
A novel hemopoietic CSF has been identified in the medium conditioned by lectin-stimulated human T cells. The cDNA clone encoding this factor, isolated by functional expression cloning in monkey cos-1 cells, proved to be identical with the cDNA encoding the cytokine B cell stimulatory factor-2/IFN-beta 2, a factor now known as IL-6. In the murine system, IL-6 indirectly supports the formation of several different types of hemopoietic colonies, including those derived from early blast cells, and directly supports the proliferation of granulocyte/macrophage progenitors. These results expand the range of known target cells of IL-6 to include hemopoietic progenitors in addition to B cells, T cells, and fibroblasts and provide further evidence that this cytokine plays an important role within a network of interacting cytokines that regulates many different biologic responses.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Fluoruracila/farmacologia , Substâncias de Crescimento/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-6 , Camundongos , Peso Molecular , Baço/citologiaRESUMO
A cDNA encoding a human macrophage migration inhibitory factor (MIF) was isolated, through functional expression cloning in COS-1 cells, from a cDNA library prepared from a lectin-stimulated T-cell hybridoma, T-CEMB. The 115-amino acid polypeptide encoded by the MIF cDNA (p7-1) was effectively released from the transfected COS-1 cells and yielded readily detectable MIF activity in the culture supernatant despite the apparent lack of a classical protein secretory sequence. Insertional mutational analysis and elution of MIF activity from polyacrylamide gel slices demonstrated that the Mr 12,000 protein with MIF activity released by the COS-1 cells is encoded by p7-1. The p7-1 cDNA hybridized with a 700-base mRNA expressed by Con-A-stimulated lymphocytes but not unstimulated lymphocytes. The availability of the MIF cDNA clone and recombinant MIF will facilitate the analysis of the role of this lymphokine in cell-mediated immunity, immunoregulation, and inflammation.
Assuntos
Clonagem Molecular , DNA/genética , Fatores Inibidores da Migração de Macrófagos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Elementos de DNA Transponíveis , Humanos , Dados de Sequência Molecular , Mutação , TransfecçãoRESUMO
A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene, which was readily identified because of its high degree of homology to the gibbon sequence, also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells, proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3.
Assuntos
Interleucina-3/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hylobates/genética , Interleucina-3/farmacologia , Camundongos/genética , RNA Mensageiro/análise , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Linfócitos T/análiseRESUMO
We have used a mammalian cell expression cloning system to identify cDNA clones encoding human granulocyte-macrophage colony stimulating factor. The human clone was used as a hybridization probe to identify the corresponding sequence from a cDNA library prepared from a gibbon T-cell line. The human cDNA has been used to produce recombinant GM-CSF in monkey COS-1 cells. The purified protein from COS cells is very similar to the GM-CSF isolated from a continuous human T-cell line.