RESUMO
Membrane lymphotoxin (LT) is heterotrimer LT alpha 1 beta 2, and its production depends on two genes. Northern blotting was employed in studying their transcription in B- and T-lymphoma cell lines and in peripheral blood lymphocytes before and after induction with phorbol myristate acetate (PMA). Transcription of either gene proved similarly regulated in several cell lines and in blood lymphocytes. Activation of the LT alpha gene was associated with induction of transcription factor NF-kappa B (p50/p65) upon cell treatment with PMA. On evidence of RT-PCR, two transcripts of the LT beta gene occurred in equimolar amounts in all lymphoid cells. A product of alternative splicing contained an open reading frame coding for the cytoplasmic portion of LT beta.
Assuntos
Linfócitos B/metabolismo , Linfotoxina-alfa/genética , Isoformas de Proteínas/genética , Processamento Alternativo , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Primers do DNA , Regulação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Genes encoding fragments of polypeptide chains of murine lymphotoxins (LT), namely, LT-alpha truncated from the N-terminus and the LT-beta extracellular domain, containing N-terminal hepta- and hexahistidine epitopes, respectively, were expressed in E. coli cells. The recombinant proteins purified by metallochelate chromatography were used to obtain polyclonal antibodies that specifically recognize murine LT.
Assuntos
Anticorpos/isolamento & purificação , Epitopos/genética , Escherichia coli/genética , Expressão Gênica , Linfotoxina-alfa/genética , Animais , Clonagem Molecular , Epitopos/imunologia , Histidina/genética , Histidina/imunologia , Immunoblotting , Linfotoxina-alfa/imunologia , Camundongos , PlasmídeosAssuntos
Cromatina/genética , Cromatina/ultraestrutura , Fígado/metabolismo , Baço/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular , Mapeamento Cromossômico , Humanos , Fígado/ultraestrutura , Camundongos , Camundongos Endogâmicos CBA , Especificidade de Órgãos , Baço/ultraestrutura , Fator de Necrose Tumoral alfa/biossínteseAssuntos
Cromossomos Humanos Par 13 , Cosmídeos , Marcadores Genéticos , Biblioteca Genômica , Oligonucleotídeos , Sequência de Bases , Southern Blotting , DNA , Humanos , Hibridização in Situ Fluorescente , Metáfase , Repetições de Microssatélites , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , Sitios de Sequências RotuladasRESUMO
Patients' genotyping of Russian ethnic group with multiple sclerosis (MS) was performed for the first time in two loci of the main complex of histocompatibility: in DR HLA class II (gene DRB1) and in the locus of tumor necrosis factors (TNF). There was no difference in the incidence of alleles groups which correlated with some specificity of DR. Meanwhile TNF-a1 and TNF-a9 alleles were encountered significantly more frequently in patients and TNF-a7 in control group. When all the patients and controls examined were divided into groups in dependence on combination of DR and TNF it was found that relations observed between MS and TNF-a7 and TNF-a9 alleles were displayed much more in individuals which carried alleles of gene DRB1, corresponding to DR15 specificity. These are alleles which are known as the main risk factor of MS in Caucasians. The patients with "protective" TNF-a7 allele were characterized by more favorable course of the disease. Thus highly significant genetic markers were revealed for the first time in region of TNF genes which were associated with increased or decreased risk of MS development at least in Russian ethnic group. There was also possibility of their interaction with group of DR15 alleles of DRB1 gene. One of the markers revealed (TNF-a7) occurred to be bound both with decreased risk of MS and with favorable clinical course, which was observed for genetic markers of MS for the first time. Manifestation of one or another property of TNF-a7 marker depends on the presence of alleles of DRB1 gene which corresponds to DR15 specificity.
Assuntos
Esclerose Múltipla/etnologia , Esclerose Múltipla/genética , Adulto , Alelos , Doença Crônica , Suscetibilidade a Doenças , Feminino , Frequência do Gene , Genes MHC da Classe II/genética , Genótipo , Antígenos HLA-DR/genética , Haplótipos , Humanos , Masculino , Moscou/epidemiologia , Esclerose Múltipla/imunologia , Fatores de Risco , Federação Russa/etnologia , Fator de Necrose Tumoral alfa/genéticaAssuntos
Antígenos CD/genética , Doenças Autoimunes/genética , Ligação Genética , Marcadores Genéticos , Polimorfismo Genético , Receptores do Fator de Necrose Tumoral/genética , Sequência de Bases , Mapeamento Cromossômico , DNA Satélite , Humanos , Dados de Sequência Molecular , Receptores Tipo II do Fator de Necrose TumoralRESUMO
Using polymerase chain reaction, a number of mutant genes encoding human tumor necrosis factor (TNF-alpha) with amino acid substitutions and a deletion were obtained. The mutant proteins (muteins) contained point mutations R32H, A33S, F144L, I118M, and I118A; double mutation R32H-F144L; and deletion of four amino acid residues 67-70. The mutant genes were expressed in E. coli under the control of constitutive promoters. A simple purification method for the muteins was developed and their physicochemical properties were studied. All the muteins obtained, except F144L and I118A, were shown by CD and cross-linking to from a spatial structure similar to that of the native TNF-alpha. The collection of muteins was characterized by their biological activity. Mutants R32H and A33S exerted a decreased cytotoxicity against murine fibroblast cell line L929, whereas point mutant F144L and double mutant R32H-F144L were essentially inactive.
Assuntos
Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Mutagênese , Plasmídeos , Mutação Puntual , Reação em Cadeia da Polimerase , Deleção de Sequência , Fator de Necrose Tumoral alfa/isolamento & purificaçãoAssuntos
Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , NF-kappa B/fisiologia , Transcrição Gênica/fisiologia , Fator de Necrose Tumoral alfa/genética , Animais , Sequência de Bases , Linhagem Celular Transformada , Humanos , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacosAssuntos
Mapeamento Cromossômico , Linfotoxina-alfa/genética , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Celular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Alinhamento de SequênciaAssuntos
Dano ao DNA/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA/metabolismo , TATA Box , Proteínas GADD45Assuntos
Linfotoxina-alfa/metabolismo , Receptores de Superfície Celular/genética , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Humanos , Dados de Sequência Molecular , Receptores do Fator de Necrose Tumoral , Mapeamento por RestriçãoAssuntos
Cromossomos Humanos , Clonagem Molecular/métodos , Genes/genética , Código Genético , Receptores de Superfície Celular/genética , Fator de Necrose Tumoral alfa/genética , Sequência de Bases , DNA Recombinante/genética , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Receptores do Fator de Necrose Tumoral , Mapeamento por RestriçãoRESUMO
Genes, coding for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta), have been cloned from the rabbit genomic library. The two genes are tandemly arranged and separated only by 1 kb of DNA as previously observed in human and mouse genomes. We have sequenced the entire rabbit lymphotoxin gene (LT) and calculated the amino acid sequence of the rabbit LT whose cDNA is not yet cloned. We also analyzed the upstream sequences of this gene and revealed a number of recognition sites for the known transcriptional factors. The rabbit TNF gene comprised in the cloned genomic region has been sequenced earlier.
Assuntos
Clonagem Molecular , Linfotoxina-alfa/genética , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Ligação Genética , Dados de Sequência Molecular , Coelhos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
We have developed a technique for restriction nuclease sites mapping in genomic DNA cloned into phage lambda vectors. Synthetic oligonucleotides homologous to the vector sequences and adjacent to the cloning site were used as hybridisation probes for indirect end labelling procedure. In addition, a number of oligonucleotides homologous to the sequences of tumour necrosis factor genes were used for mapping from the internal sites. As a result, a map of 35 kb genomic region on the chromosome 17 inside major histocompatibility complex and surrounding mouse tumour necrosis factor genes was constructed.
Assuntos
Oligonucleotídeos/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Mapeamento Cromossômico , Clonagem Molecular , Camundongos , Hibridização de Ácido NucleicoRESUMO
Seven cloned hybrid cell lines were obtained by fusion of OAV2a GFRT cells with lymphocytes of syngeneic rats. Three of the cell lines showed a reduced malignity when injected subcutaneously and were incapable of infiltrative growth when injected intraperitoneally. They were partially or totally devoid of colony forming activity in soft agar. Tumors which formed after injection of clone 2 cells revealed degenerative alterations and were infiltrated with lymphocytes. Separately floating rejected tumor fragments were vigorously attacked by macrophages.
