RESUMO
A complementary DNA clone for an alpha-tubulin has been isolated from a mouse testis complementary DNA library. The untranslated 3' end of this complementary DNA is homologous to two RNA transcripts present in postmeiotic cells of the testis but absent from meiotic cells and from several tissues including brain. The temporal expression of this alpha-tubulin complementary DNA provides evidence for the haploid expression of a mammalian structural gene.
Assuntos
Testículo/metabolismo , Tubulina (Proteína)/genética , Animais , Encéfalo/metabolismo , Clonagem Molecular , DNA/genética , Drosophila , Regulação da Expressão Gênica , Haploidia , Masculino , Camundongos , Hibridização de Ácido Nucleico , Ratos , Espermátides/metabolismo , Espermatogênese , Espermatozoides/fisiologiaRESUMO
Fatty acid binding proteins (FABPs) are small cytoplasmic proteins that are expressed in a highly tissue-specific manner and bind to fatty acids such as oleic and retinoic acid. Mice with a null mutation in aP2, the gene encoding the adipocyte FABP, were developmentally and metabolically normal. The aP2-deficient mice developed dietary obesity but, unlike control mice, they did not develop insulin resistance or diabetes. Also unlike their obese wild-type counterparts, obese aP2-/- animals failed to express in adipose tissue tumor necrosis factor-alpha (TNF-alpha), a molecule implicated in obesity-related insulin resistance. These results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-alpha.
Assuntos
Tecido Adiposo/metabolismo , Proteínas de Transporte/fisiologia , Ácidos Graxos/metabolismo , Resistência à Insulina , Proteína P2 de Mielina/fisiologia , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Obesidade/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Glicemia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Gorduras na Dieta/administração & dosagem , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Feminino , Regulação da Expressão Gênica , Marcação de Genes , Teste de Tolerância a Glucose , Homeostase , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/genéticaRESUMO
Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.
Assuntos
Actinas/genética , Espermátides/fisiologia , Espermatócitos/fisiologia , Espermatogênese , Actinas/classificação , Animais , Regulação da Expressão Gênica , Masculino , Meiose , Camundongos , Peso Molecular , RNA Mensageiro/genética , Testículo/fisiologiaRESUMO
Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.
Assuntos
Encéfalo/metabolismo , Testículo/metabolismo , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Variação Genética , Masculino , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Tubulina (Proteína)/análiseAssuntos
Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Divisão Celular , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Família Multigênica , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-jun , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The ubiquitous cytokine transforming growth factor-beta1 (TGF-beta1) is one of the most potent metastatic inducers. Functional interactomic mapping using high-throughput proteomic and genomic data provides valuable insights into the regulation of tumor suppressive and metastatic attributes of TGF-beta1. Polarity changes of the TGF-beta1 interactome at a given time contributes to these contrasting effects. Differential expression profiles of pivotal interactomic nodes contribute to these polarity changes. These insights are of immense value in the development of effective cancer therapeutics. Moreover, TGF-beta1 interactomic nodes are useful in discovering novel cancer biomarkers. This review describes an initial version of the TGF-beta1 interactome in relation to tumor progression and metastasis. Thus, this review embodies an important step towards the mapping of comprehensive and individualized TGF-beta1 interactomes that will assist in the development of personalized cancer therapeutics.
Assuntos
Neoplasias/terapia , Proteoma/análise , Fator de Crescimento Transformador beta1/metabolismo , Animais , Humanos , Metástase Neoplásica/prevenção & controle , Neoplasias/irrigação sanguínea , Neoplasias/química , Neoplasias/metabolismo , Neovascularização Patológica , Transdução de SinaisAssuntos
Apoptose , Inibidores Enzimáticos/farmacologia , Janus Quinase 2/antagonistas & inibidores , Transtornos Mieloproliferativos/patologia , Transtornos Mieloproliferativos/prevenção & controle , Pimozida/farmacologia , Fator de Transcrição STAT5/antagonistas & inibidores , Antidiscinéticos/farmacologia , Sinergismo Farmacológico , Humanos , Transtornos Mieloproliferativos/metabolismo , Células Tumorais CultivadasRESUMO
The nucleotide sequence of a 404-base cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine has been determined. This insert, isolated from a mouse testis cDNA library, encodes a polypeptide of 50 amino acids of which 28 are arginine, 9 are cysteine, and 3 are tyrosine. The insert contains the complete 3' noncoding region of 151 bases and most of the 5' noncoding region. The predicted amino acid sequence of mouse protamine 1 is about 80% homologous to boar protamine and 67% homologous to bull protamine and contains the central, highly basic domain of four arginine clusters found in the trout protamines. The identification of a cDNA clone for a mouse protamine will facilitate studies of the evolution, regulation, and protein-DNA interaction of this nuclear protein unique to haploid spermatogenic cells.
Assuntos
Clonagem Molecular , DNA/isolamento & purificação , Protaminas/genética , Testículo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon , Enzimas de Restrição do DNA , Masculino , Camundongos , Hibridização de Ácido Nucleico , RNA Mensageiro/genéticaRESUMO
Fatty acids are important metabolic substrates and may also be involved in pathological syndromes such as the insulin resistance of diabetes and obesity. We demonstrate here that fatty acids can regulate specific gene expression; mRNAs encoding the fatty acid binding protein adipocyte P2 (aP2) and the Fos-related transcription factor Fra1 are specifically induced at least 20-fold upon treatment of preadipocytes with oleate. For aP2, the effect requires long chain fatty acids and occurs without a generalized activation of the genes linked to adipocyte differentiation. Other fibroblastic cells without preadipocyte characteristics do not induce aP2 mRNA in response to fatty acids. Unlike aP2, Fra1 induction by fatty acids also can be detected in NIH 3T3 and 3T3-C2 fibroblasts. Nuclear transcription assays in 3T3-F442A preadipocytes demonstrate that fatty acids elicit no transcriptional increase in the aP2 gene. Fra1, on the other hand, shows a 3-4-fold increase in transcription. These results demonstrate at least two distinct mechanisms by which fatty acids may influence gene expression.
Assuntos
Proteínas de Transporte/genética , Ácidos Graxos não Esterificados/farmacologia , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Células 3T3 , Animais , Northern Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , Expressão Gênica/efeitos dos fármacos , Cinética , Camundongos , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/metabolismo , Relação Estrutura-AtividadeRESUMO
We have isolated several cDNA clones encoding cytoplasmic poly(A)+ RNAs which are enriched in postmeiotic (haploid) spermatogenic cells in the mouse. Seventeen of 750 clones from a testis cDNA library hybridized more strongly to 32P-labeled cDNA copied from cytoplasmic poly(A) RNA of round spermatids than pachytene spermatocytes. Northern gel blots demonstrated that these 17 plasmids hybridized to RNA(s) approximately 0.5 kb (1 clone), 0.7 kb (13 clones), 0.8 kb (1 clone), and 0.9 kb (2 clones). Four plasmids hybridizing to RNAs 0.7 and 0.9 kb were further characterized by Northern blots. The levels of hybridization were about 10-fold greater with RNA from round spermatids, elongating spermatids and residual bodies than from pachytene spermatocytes from adult testis. These plasmids did not hybridize with cytoplasmic poly(A)+ RNA from sexually immature testis, adult liver, or brain, larger precursors in adult testis nuclear RNA, total RNA from cultured Sertoli cells, poly(A)- RNA from adult testis or the mouse mitochondrial genome. These results demonstrate that certain poly(A)+ RNAs are abundant in haploid cells but barely or not detectable in meiotic cells suggesting the accumulation of these RNAs in round spermatids requires transcription in haploid cells.
Assuntos
DNA/genética , RNA Mensageiro/genética , Espermatogênese , Animais , Diferenciação Celular , Separação Celular , Clonagem Molecular , Regulação da Expressão Gênica , Haploidia , Masculino , Meiose , Camundongos , Peso Molecular , Espermátides/fisiologia , Testículo/citologiaRESUMO
The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.
Assuntos
Protaminas/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Espermátides/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Testículo/metabolismo , Animais , Masculino , Camundongos , Peso Molecular , Poli A/análise , Polirribossomos/metabolismo , RNA Mensageiro/isolamento & purificaçãoRESUMO
We have analyzed the expression, through spermiogenesis, of a series of testicular cell polypeptides encoded by genes within the mouse t complex. Two of these polypeptides, TCP-3 and TCP-7, are synthesized in a testes-specific manner with highest levels of expression during haploid stages of spermatogenesis. A third, TCP-1, is also expressed at highest levels in haploid cells, and expression of this polypeptide continues until the last residual body stage of spermiogenesis. The genes that encode these polypeptides have been correlated with the t phenotype of transmission ratio distortion.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Associadas aos Microtúbulos , Proteínas Nucleares , Biossíntese de Proteínas , Espermatogênese , Animais , Linhagem Celular , Regulação da Expressão Gênica , Haploidia , Fígado/metabolismo , Masculino , Meiose , Camundongos , Fosfoglicerato Quinase/biossíntese , Proteínas/genética , Espermátides/metabolismo , Espermatozoides/metabolismo , Baço/metabolismo , Testículo/metabolismo , Ubiquitina-Proteína Ligases , Região do Complexo-t do GenomaRESUMO
The adipocyte P2 (aP2) gene contains a regulatory element, FSE2, that functions during adipocyte differentiation and binds a protein complex containing the product of the fos proto-oncogene (Fos). We show here that the quantitative and qualitative nature of the FSE2 binding complex closely reflects the status of Fos expression within a given cell type. There is a dramatic increase in the FSE2 binding complex when Fos levels are induced with serum, benzodiazepine, and nerve growth factor or are expressed from a v-fos gene. Immunoblotting analysis of DNA retardation gels indicates a comigration of FSE2 complex with the predominant Fos species. Using a combination of mutational analyses of FSE2 and competition for binding with related sequences, we show that the Fos complex recognizes DNA containing the sequence TGACTCA, previously identified as the consensus binding site for the transcription factors AP-1 in mammalian cells and GCN4 in yeast. The simultaneous presence in cell extracts of proteins related to both AP-1 and Fos with similar sequence recognition properties was demonstrated by photo-cross-linking to FSE2 DNA and immunoprecipitating with antibodies directed toward c-fos or v-jun. These results suggest a functional relationship between Fos and AP-1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Tecido Adiposo/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Humanos , Imunoquímica , Mutação , Fotoquímica , Ligação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-junRESUMO
We utilized a cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine, mouse protamine 1 (MP1), to detect the presence of several classes of differentiating germ cells in testicular extracts from wild-type and male sterile mutant mice. This assay is based on the changes in the poly (A) length of MP1-mRNA during spermatogenesis. Testicular extracts of sexually mature CD-1 mice contain a heterogeneous population of protamine-1 mRNA ranging in length from 450 to 580 nucleotides. When the protamine-1 probe was hybridized to testicular RNA preparations from 16- to 20-day-old animals, no MP1-mRNA was detected. Twenty-four-day-old mice contain only the 580-nucleotide form of MP1-mRNA. This size class of protamine mRNA is also present in purified populations of round spermatids, whereas elongating spermatids and residual bodies contain mRNAs ranging from 450 to 580 nucleotides in length, which are identical in size to those present in the testes of sexually mature animals. When the protamine cDNA probe was used to examine the progression of spermiogenesis in three male sterile mouse mutants, blind sterile (bs), quaking (qk) and testicular feminization (Tfm), the results demonstrated that each mutant is pathologically distinct. Analysis of the bs mutant revealed a diminution in the amount of both size classes of MP1-mRNA, in agreement with the cytological reports of reduced numbers of haploid spermatogenic cells in these animals. The presence of both size classes of protamine mRNA in the qk mutant indicates that germ-cell differentiation has proceeded at least to the step-12 spermatid in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Protaminas/genética , RNA Mensageiro/análise , Espermatogênese , Síndrome de Resistência a Andrógenos , Animais , Extratos Celulares , DNA , Masculino , Camundongos , Camundongos Mutantes , Camundongos Quaking , Mutação , Hibridização de Ácido Nucleico , Protaminas/análise , Protaminas/metabolismo , RNA Mensageiro/genética , Maturidade Sexual , Espermátides/análise , Espermátides/citologia , Testículo/citologia , Testículo/fisiologiaRESUMO
Following intratesticular injection of [35S]methionine, the multiple isoforms of actin and tubulin from highly purified mouse testicular meiotic and post-meiotic cells have been analysed by high resolution two-dimensional gel electrophoresis. In pachytene spermatocytes both beta and gamma actin are synthesized, gamma actin being made in a significantly greater amount. The relative proportion of synthesis of beta and gamma actin changes during spermiogenesis, beta actin increasing and gamma actin decreasing in round spermatids, elongating spermatids, and residual bodies. Both alpha and beta tubulin are synthesized in approximately equal proportion in pachytene spermatocytes. In addition to the tubulin isoforms synthesized during meiosis, at least one new form of both alpha and beta tubulin first appears in post-meiotic (haploid) cells. In elongating spermatids and residual bodies, the synthesis of alpha tubulin is drastically reduced.
Assuntos
Actinas/biossíntese , Espermatogênese , Testículo/fisiologia , Tubulina (Proteína)/biossíntese , Actinas/isolamento & purificação , Animais , Eletroforese em Gel de Poliacrilamida , Masculino , Meiose , Metionina/metabolismo , Camundongos , Radioisótopos de Enxofre , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
To evaluate whether the absence or modification of paternal mitochondrial DNA or methylation of the oocyte mitochondrial DNA could be the molecular basis for maternal inheritance of mitochondria in mammals, the mitochondrial genome has been analyzed in four meiotic and postmeiotic testicular cell types, and in oocytes from the mouse. All four testicular cell types including spermatozoa contain mitochondrial DNA. Between meiosis and the end of spermatogenesis the number of mitochondrial genomes per haploid genome decreases 8- to 10-fold with spermatozoa containing approximately one copy of the mitochondrial genome per mitochondrion. Restriction enzyme digestions with six different enzymes indicate no gross differences in DNA sequence in the testicular mitochondrial DNA from meiotic cells, early haploid cells, late haploid cells, and spermatozoa. By the criterion of differential digestion with the isoschizomers, MspI and HpaII, the mitochondrial DNA is not differentially methylated during spermatogenesis. No methylation differences were detected in mitochondrial DNA from sperm and oocytes following digestion with seven methylation-sensitive restriction enzymes.
Assuntos
DNA Mitocondrial/genética , Oócitos/análise , Espermatozoides/análise , Animais , Sequência de Bases , Enzimas de Restrição do DNA , DNA Mitocondrial/análise , DNA Mitocondrial/metabolismo , Feminino , Masculino , Metilação , Camundongos , Hibridização de Ácido Nucleico , EspermatogêneseRESUMO
Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including a putative lipid-binding protein termed adipocyte P2 (aP2). The aP2 gene contains a regulatory element (FSE2) 124 bases 5' to its start of transcription. This element binds nuclear factors in sequence-specific and differentiation-dependent fashion as determined by altered mobility in gel retardation assays. Deletion analysis of promoter-linked transfection assays and competition of these constructions in cells with a synthetic FSE2 element suggest that trans-acting factors bind to this region and act as negative regulators of aP2 gene activity in preadipocytes. c-fos appears to participate directly in this nucleoprotein complex, as demonstrated by the ability of antibodies to c-fos to disrupt specific binding of factors to the FSE2 sequence but not to factor-binding sequences from several other genes. Antibodies to c-fos specifically immunoprecipitate protein complexes covalently bound to FSE2 DNA via UV cross-linking.
Assuntos
Tecido Adiposo/fisiologia , Diferenciação Celular , Regulação da Expressão Gênica , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Nucleoproteínas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proto-Oncogenes , Serina Endopeptidases , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Proteínas de Transporte/genética , Mapeamento Cromossômico , Fator D do Complemento , Proteínas de Ligação a DNA/fisiologia , Endopeptidases/genética , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Glicerolfosfato Desidrogenase/genética , Camundongos , Regiões Promotoras GenéticasRESUMO
Hybridization of RNA blots of total testicular RNA from prepuberal and sexually mature CD1 mice with several mouse testicular cDNA probes reveals that the mRNA encoding the two mouse protamines, an actin sequence of 1.5 kb, and a post-meiotically expressed 620 nucleotide mRNA are first detected in the testes of mice 22 days of age. These experiments and other studies analysing RNA preparations from isolated populations of testicular cell types with cDNA probes [1, 2] demonstrate that haploid gene expression occurs in the mammalian testis.