RESUMO
Cloning of the mouse tag7 gene encoding a novel cytokine is described. The Tag7 protein consists of 182 amino acids. Genomic organization of the tag7 gene and its promoter region remind those of the genes of the tumor necrosis factor locus, although the tag7 gene is not linked to this locus. The gene is located on chromosome 7 at the area that corresponds to band 7A3, which has genetic linkage with lupus-like disease in mouse models. tag7 transcription is essential for lymphoid organs. It is also detected in certain areas of lungs, brain, and intestine and in some tumors. Tag7 protein is detectable in both cell-associated and soluble forms. The soluble form of Tag7 triggers apoptosis in mouse L929 cells in vitro and does not involve NF-kappaB activation. The relationship between Tag7 and tumor necrosis factor family of ligands is discussed.
Assuntos
Apoptose , Citocinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Citocinas/química , Células-Tronco Hematopoéticas/metabolismo , Ligantes , Tecido Linfoide/metabolismo , Camundongos , Dados de Sequência Molecular , Transplante de Neoplasias , Transcrição Gênica , Células Tumorais CultivadasAssuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/enzimologia , Metástase Neoplásica/genética , Proteínas Serina-Treonina Quinases/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , Primers do DNA , Neoplasias Mamárias Experimentais/genética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/biossíntese , Homologia de Sequência de Aminoácidos , Proteínas de XenopusRESUMO
Gene expression was compared in a metastatic (VMR-Liv) neoplastic cell line and a related nonmetastatic (VMR-O) neoplastic cell line by means of the differential display method. A fragment of cDNA corresponding to the tag7 gene, differentially expressed in the metastatic cell line, was isolated. The full-length tag7 cDNA was cloned and its nucleotide sequence was determined. No homology between the tag7 gene and known sequences was revealed. tag7 gene transcription was studied in some tumors, cell lines, and normal mouse organs.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Oncogenes , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Camundongos , Camundongos Endogâmicos A , Dados de Sequência Molecular , Metástase Neoplásica , Reação em Cadeia da Polimerase/métodos , Valores de Referência , Análise de Sequência de DNA/métodos , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Functional differentiation of a bovine mammary gland in the course of lactation is characterized by significant increase of tissue-specific expression of genes encoding milk proteins (caseins, lactoglobulin etc.). The NF1 is known as a ubiquitous transcription factor which modulates a tissue-specific transcription of different genes (including beta-casein) cooperating with tissue-specific and other ubiquitous transcription factors. We have observed a dramatic increase of NF1-binding activity in nuclear extracts of bovine mammary gland during lactation. The NF1 transcription factor appears to have a cytoplasmic precursor pool. This cytoplasmic precursor as well as NF-kappa B cytoplasmic precursor could be activated in vitro by deoxycholate (DOC) treatment which caused possibly dissociation of a complex of NF1 and its cytoplasmic inhibitor. There was an inverse proportion between concentrations of active nuclear NF1 factor and its cytoplasmic precursor. We have observed an increase of nuclear factor binding and a simultaneous decrease of the cytoplasmic precursor pool in the course of lactation. We have determined the NF1 protein subunit composition using UV-cross-linking 32P labeled NF1-oligonucleotide with nuclear and cytoplasmic proteins of mammary gland. The main subunits of NF1 factor were p50 and p20. The drastic increase of nuclear NF1 binding activity was correlated with significant increase of the p20 subunit concentration in nuclear protein during lactation.
Assuntos
Lactação , Glândulas Mamárias Animais/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Bovinos , Eletroforese em Gel de Poliacrilamida , Feminino , Glândulas Mamárias Animais/metabolismo , Proteínas do Leite/genética , Dados de Sequência Molecular , Especificidade por Substrato , Fatores de Transcrição/genéticaAssuntos
Proteínas Estimuladoras de Ligação a CCAAT , Caseínas/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Caseínas/metabolismo , Bovinos , DNA/genética , DNA/metabolismo , Impressões Digitais de DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Fígado/metabolismo , Glândulas Mamárias Animais/metabolismo , Dados de Sequência Molecular , Fatores de Transcrição NFI , Proteínas Nucleares , Proteína 1 de Ligação a Y-BoxRESUMO
Nucleotide(nt) sequences were determined for the toxin (SHT) operon present in the chromosome of Shigella dysenteriae 1 and for the shiga-like toxin (SLT) operon found in the lambdoid phage H30 genome. The coding sequences of the sht and slt genes differ in 4 nt with 1 nt change responsible for an amino acid replacement. The deduced amino acid sequence in the A chain of the toxins is highly homologous to that of the A chain of ricin, a plant toxin. SHT-coding mRNAs were detected by mapping the 5' termini and using blot-hybridisation; one of them was more abundant and coded only for the B subunit of SHT while the other (bi-cistronic mRNA) encoded both subunits. An IS element related to the IS3 element of Escherichia coli was found in the chromosome of S. dysenteriae near the sht operon.
Assuntos
Bacteriófagos/genética , Óperon , Shigella dysenteriae/genética , Toxinas Biológicas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Recombinante , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Transcrição GênicaRESUMO
A new effective method for purification of rabbit liver N-acetyltransferase to apparent homogeneity has been developed. The method consists in polymin P and ammonium sulfate fractionation, DEAE-Sephacel and Ultragel AcA 44 chromatography and chromatofocusing. In final preparations the enzyme was purified 2500-3000-fold and its specific activity was found to be about 3000-4000 units per mg of protein. During chromatofocusing of enzyme preparations on a middle pressure chromatograph FPLC (Pharmacia, Sweden) a partial separation of acetyltransferase allelic forms from fast and slow-acetylators took place. The supposed allelic acetyltransferase forms differ in some biochemical properties. In particular, the slow acetyltransferase form is much more sensitive towards 0.1 M KCl against the rapid enzyme form. It is assumed that the differences between the catalytic properties of acetyltransferase from rapid and slow acetylators may be explained by differences between their polypeptide primary structures.
Assuntos
Acetiltransferases/isolamento & purificação , Arilamina N-Acetiltransferase/isolamento & purificação , Isoenzimas/isolamento & purificação , Fígado/enzimologia , Acetilação , Alelos , Animais , Arilamina N-Acetiltransferase/genética , Catálise , Cromatografia DEAE-Celulose , Isoenzimas/genética , Cinética , Métodos , Fenótipo , CoelhosRESUMO
Four cytochromes P-450 induced by phenobarbital (PB-1--PB-4) and two cytochromes P-450 induced by S-methylcholanthrene (MC-1, MC-2) were purified to electrophoretic homogeneity from rat liver microsomes. The purification procedure involved sequential chromatography on n-aminooctyl-Sepharose 4B, DEAE-Sephacel and hydroxylapatite columns. The spectral and immunochemical properties of the cytochromes P-450 were estimated. All, but MC-1, cytochromes P-450 were found to exist in a low spin state. Using the Ouchterlony double diffusion method, it was shown that all cytochromes P-450 under study can be divided into two groups, i. e., PB-1--PB-2 and PB-3--PB-4, sharing common antigenic determinants inside the groups. High performance liquid chromatography of PB-3 and MC-2 on anion-exchangers yielded two additional peaks from the PB-induced major cytochrome P-450 PB-3 and three peaks from the MC-induced major cytochrome P-450 MC-2. The multiplicity of cytochrome P-450 forms is discussed.
Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Isoenzimas/isolamento & purificação , Fígado/enzimologia , Animais , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/imunologia , Indução Enzimática , Imunodifusão , Isoenzimas/biossíntese , Isoenzimas/imunologia , Masculino , Metilcolantreno/farmacologia , Fenobarbital/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Using two consecutive oligo(dT)-cellulose column chromatography steps, the total poly(A)RNA was isolated from the livers of rats injected with phenobarbital (PB) or 3-methylcholanthrene (MC). During translation of the PB-induced mRNA in the reticulocyte lysate cell-free protein-synthesizing system, a single polypeptide with an apparent molecular weight of 50,000 was synthesized which was specifically immunoprecipitated by antibodies to major PB-inducible cytochrome P-450 PB-3. In contrast, after completion of MC-mRNA translation, the antibodies to major MC-induced cytochrome MC-2 precipitated from the incubation mixture 4-5 polypeptides, of which the largest one with an apparent molecular weight of 58,000 corresponded to cytochrome P-450 MC-2. During sucrose density gradient centrifugation, the PB- and MS-mRNAs with sedimentation coefficients of about 18S and 20S, respectively, were precipitated.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Isoenzimas/biossíntese , Fígado/enzimologia , RNA Mensageiro/isolamento & purificação , Animais , Sistema Enzimático do Citocromo P-450/genética , Eletroforese em Gel de Poliacrilamida , Indução Enzimática , Isoenzimas/genética , Metilcolantreno/farmacologia , Fenobarbital/farmacologia , RNA Mensageiro/análise , RatosRESUMO
A fraction of hydrophobic proteins (9, 14, 18 kD) soluble in a chloroform-methanol mixture (2:1) has been isolated from Micrococcus lysodeikticus bacterial membranes. The proteins obtained were introduced into proteoliposomes at a protein/lipid weight ratio ranging from 0.1 to 0.25 in combination with the fluorescent probe pyrene or the spin probe 2-(14-carboxytetradecyl)-2-ethyl-4.4-dimethyl-3-oxasolidinyloxyl. The excimertization of pyrene upon direct excitation of its molecules (gamma excit.=338nm) and under conditions of energy transfer from the excited protein chromophores to pyrene (gamma excit.=286nm) and the spin-spin exchange between the spin probe molecules was investigated. The experimental results suggest that the hydrophobic protein molecules are surrounded by a structurally heterogenous lipid area containing up to 3.3 mg of lipid per l mg of protein. The maximal expression of structural heterogeneity was observed at the minimal content of protein in the proteoliposomes. Treatment with the membranotropic antibiotic gramicidin S resulted in disappearance of lateral heterogeneity of lipids in the constituted system and in lipid aggregation in bacterial membranes. It is assumed that the aggregability of membrane proteins depends on the structural rearrangement of some part of lipid bilayer around them.