RESUMO
The protein encoded by the human testis determining gene, SRY, contains a high mobility group (HMG) box related to that present in the T cell-specific, DNA-binding protein TCF-1. Recombinant SRY protein was able to bind to the same core sequence AACAAAG recognized by TCF-1 in a sequence dependent manner. In five XY females point mutations were found in the region encoding the HMG box. In four cases DNA binding activity of mutant SRY protein was negligible; in the fifth case DNA binding was reduced. These results imply that the DNA binding activity of SRY is required for sex determination.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Masculino , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteína da Região Y Determinante do Sexo , Fatores de Transcrição/metabolismoRESUMO
The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.
Assuntos
Cartilagem/metabolismo , Elementos Facilitadores Genéticos , Proteínas de Grupo de Alta Mobilidade/genética , Pró-Colágeno/genética , Fatores de Transcrição/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Doenças do Desenvolvimento Ósseo/genética , Cartilagem/citologia , Diferenciação Celular/genética , Sondas de DNA/genética , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição SOX9 , Transativadores/genética , TransfecçãoRESUMO
BACKGROUND: While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody-antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. RESULTS: The crystal structure of the NC10 Fab-neuraminidase complex has been refined to a nominal resolution of 2.5A. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. CONCLUSIONS: The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein-protein complexes.
Assuntos
Complexo Antígeno-Anticorpo/química , Proteína HN/química , Orthomyxoviridae/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Sítios de Ligação , Cristalografia , Proteína HN/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Orthomyxoviridae/enzimologia , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
The SOX (sex-determining region [SRY]-type high mobility group [HMG] box) family of transcription factors play key roles in determining cell fate during organ development. In this study, we have identified a new human SOX gene, SOX13, as encoding the type 1 diabetes autoantigen, islet cell antigen 12 (ICA12). Sequence analysis showed that SOX13 belongs to the class D subgroup of SOX transcription factors, which contain a leucine zipper motif and a region rich in glutamine. SOX13 autoantibodies occurred at a significantly higher frequency among 188 people with type 1 diabetes (18%) than among 88 with type 2 diabetes (6%) or 175 healthy control subjects (4%). Deletion mapping of the antibody epitopes showed that the autoantibodies were primarily directed against an epitope requiring the majority of the protein. SOX13 RNA was detected in most human tissues, with the highest levels in the pancreas, placenta, and kidney. Immunohistochemistry on sections of human pancreas identified SOX13 in the islets of Langerhans, where staining was mostly cytoplasmic. In mouse pancreas, Sox13 was present in the nucleus and cytoplasm of beta-cells as well as other islet cell types. Recombinant SOX13 protein bound to the SOX consensus DNA motif AACAAT, and binding was inhibited by homodimer formation. These observations-along with the known molecular interactions of the closely related protein, rainbow trout Sox23-suggest that SOX13 may be activated for nuclear import and DNA binding through heterodimer formation. In conclusion, we have identified ICA12 as the putative transcription factor SOX13 and demonstrated an increased frequency of autoantibody reactivity in sera from type 1 diabetic subjects compared with type 2 diabetic and healthy control subjects.
Assuntos
Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/imunologia , Ilhotas Pancreáticas/imunologia , Sequência de Aminoácidos , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoantígenos/genética , DNA/metabolismo , Dimerização , Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/química , Humanos , Zíper de Leucina , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , Proteínas Recombinantes/metabolismo , Fatores de Transcrição SOXDRESUMO
SOX13 is the member of the SOX (Sry related HMG BOX) family of transcription factors which encodes the type-1 diabetes autoantigen, ICA12, and is expressed in a number of tissues including pancreatic islets and arterial walls. By fluorescence in situ hybridisation, radiation hybrid mapping and YAC analysis we determined that the human SOX13 gene maps to Chromosome 1q31.3-32.1 near the marker D1S504, a region associated with type-1 diabetes susceptibility and familial dilated cardiomyopathy. Mouse Sox13 maps to the syntenic region near the marker D1Mit57. The human SOX13 gene spans >15.5kb of genomic DNA and is composed of 14 exons with introns interrupting regions encoding the HMG DNA binding domain and the leucine zipper/glutamine-rich dimerisation domain. Comparison with the mouse Sox13 gene suggests the existence of long and short forms of the SOX13 protein which may arise by differential splicing during different stages in embryogenesis. The high sequence conservation between human SOX13 and mouse, Xenopus and trout orthologues implies a conserved function in vertebrates. SOX13 belongs to SOX Group D members which contain a leucine zipper/glutamine-rich region. Phylogenetic analyses of SOX proteins suggest that such domains were acquired after the initial divergence of groups A to G.
Assuntos
Autoantígenos , Genes/genética , Proteínas de Grupo de Alta Mobilidade/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento de Sequências Contíguas , DNA/química , DNA/genética , DNA/isolamento & purificação , Éxons , Humanos , Células Híbridas , Íntrons , Camundongos , Dados de Sequência Molecular , Filogenia , Fatores de Transcrição SOXD , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
We have constructed a temperature-inducible Escherichia coli expression vector (pPOW) for enhanced secretion of antibody (Ab) domains and other foreign proteins. The vector contains the lambda pRpL promoters in tandem, and the cI857 gene encoding the temperature-sensitive repressor which provide tight control over protein production. The PelB secretion signal directs the synthesized foreign protein through the cytoplasmic membrane. A mouse Ab fragment (the variable heavy (VH) domain of NC41) was synthesized efficiently by this vector and accumulated with the cell membranes (not as inclusion bodies) at levels of 30 mg/l. This represents the highest yields reported to date for Ab fragments with a native N terminus. An octapeptide (FLAG) tail was fused to the C terminus of the VH domain to aid in purification, and remained intact throughout the protein purification process. The optimum conditions for protein production were controlled by the type of culture medium used, the age of the bacterial population at the time of induction, and the period of synthesis of the protein product. The purified Ab VH fragment showed binding affinity (Ka less than 10(4)/M) to its target antigen (neuraminidase).
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Polissacarídeo-Liases , Sinais Direcionadores de Proteínas/genética , Animais , Sequência de Bases , Clonagem Molecular/métodos , Vetores Genéticos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , TemperaturaRESUMO
The campomelic dysplasia/autosomal sex reversal protein SOX9 is an important developmental transcription factor, required for correct bone and testis formation. Through in vitro and in vivo studies we have identified the heat shock protein HSP70 as an interacting partner for SOX9 in chondrocyte and testicular cell lines. HSP70 forms a ternary complex with DNA-bound SOX9. The interaction between HSP70 and SOX9 is ATP-independent and involves a highly conserved region of SOX9 hitherto of unknown function and the C-terminal region of HSP70. Our results implicate HSP70-SOX9 interactions in the assembly of multi-protein complexes during SOX9-mediated transcription.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Western Blotting , Osso e Ossos/embriologia , Linhagem Celular , Condrócitos/metabolismo , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Humanos , Imuno-Histoquímica , Masculino , Mutagênese , Testes de Precipitina , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Fatores de Transcrição SOX9 , Processos de Determinação Sexual , Testículo/embriologia , Transcrição Gênica , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. By affinity chromatography, native gel electrophoresis and fluorescence spectroscopy, we show the calcium-dependent binding of SRY to CaM. Binding occurs via the HMG box and an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM and we propose that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors.
Assuntos
Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Autorradiografia , Sequência de Bases , Sítios de Ligação , Cálcio/farmacologia , Cromatografia de Afinidade , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/química , Radioisótopos de Fósforo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sitios de Sequências Rotuladas , Proteína da Região Y Determinante do Sexo , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Testículo/metabolismoRESUMO
The decision of the bi-potential gonad to develop into either a testis or ovary is determined by the presence or absence of the Sex-determining Region gene on the Y chromosome (SRY). Since its discovery, almost 13 years ago, the molecular role that SRY plays in initiating the male sexual development cascade has proven difficult to ascertain. While biochemical studies of clinical mutants and mouse genetic models have helped in our understanding of SRY function, no direct downstream targets of SRY have yet been identified. There are, however, a number of other genes of equal importance in determining sexual phenotype, expressed before and after expression of SRY. Of these, one has proven of central importance to mammals and vertebrates, SOX9. This review describes our current knowledge of SRY and SOX9 structure and function in the light of recent key developments.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Mamíferos/genética , Proteínas Nucleares , Processos de Determinação Sexual , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Genes sry , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Masculino , Camundongos , Modelos Genéticos , Mutação , Fatores de Transcrição SOX9 , Proteína da Região Y Determinante do Sexo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Human mutations in the transcription factor gene, SOX9, cause campomelic dysplasia (CD), a severe dwarfism associated with brain abnormalities including dilation of lateral ventricles, hypoplasia of the corpus callosum and cerebellum defects. To improve our understanding of how SOX9 contributes to the molecular genetic pathway of brain development we sought to investigate the distribution of SOX9 protein in rat and mouse brain. The regions of SOX9 expression identified in this study correlated with the sites of reported brain abnormalities in CD patients. SOX9 immunoreactivity was observed in nuclei of scattered cells throughout the brain, in the ependymal layer and cells of the choroid plexus. In the forebrain most SOX9-immunoreactive nuclei co-localised with the glial astrocyte marker S-100. In the cerebellum, SOX9 was observed mostly in cells surrounding Purkinje cells, which were identified, by electron microscopy, as Golgi epithelial cells, also known as Bergmann glia. Using SOX9 antibody as a marker for the precursors of Bergmann glia, we traced their origin during mouse development. At embryonic day (E)14.5 and E16.5, SOX9 immunoreactivity was present mainly in the primordial choroid plexus, and ventricular zone. By E18.5, SOX9 was observed in the granular cell and Purkinje cell layers but no labelling was detectable in the external granular layer. These results suggest that SOX9 immunoreactivity is a marker for Bergmann cells during development and favour the proposed origin of the secondary glial scaffold arising from Bergmann cells derived exclusively from the ventricular zone.
Assuntos
Diferenciação Celular/fisiologia , Cerebelo/embriologia , Cerebelo/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Malformações do Sistema Nervoso/metabolismo , Neuroglia/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cerebelo/ultraestrutura , Plexo Corióideo/embriologia , Plexo Corióideo/metabolismo , Plexo Corióideo/ultraestrutura , Epêndima/embriologia , Epêndima/metabolismo , Epêndima/ultraestrutura , Feminino , Feto , Imuno-Histoquímica , Masculino , Camundongos , Microscopia Eletrônica , Malformações do Sistema Nervoso/patologia , Malformações do Sistema Nervoso/fisiopatologia , Neuroglia/ultraestrutura , Ratos , Fatores de Transcrição SOX9 , Células-Tronco/ultraestruturaRESUMO
Regulatory factors governing the formation of bone in the glenoid fossa in response to functional appliance therapy have not been identified. Therefore, the purpose of this study was to investigate the temporal pattern of expression of two key chondrogenesis markers-SOX9 and its target gene, type II collagen-in the glenoid fossa by immunostaining in a 35-day-old Sprague Dawley rat model during both natural growth and forward mandibular positioning. The expression of both factors was up-regulated when the mandible was positioned forward, indicating an enhancement of chondrocyte differentiation and chondroid matrix formation. Our results indicate that chondroid bone formation in the glenoid fossa in response to forward mandibular positioning is regulated by molecular markers indicative of endochondral ossification.
Assuntos
Colágeno Tipo II/análise , Proteínas de Grupo de Alta Mobilidade/análise , Mandíbula/anatomia & histologia , Aparelhos Ortodônticos Funcionais , Osso Temporal/metabolismo , Fatores de Transcrição/análise , Regulação para Cima , Análise de Variância , Animais , Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/genética , Matriz Extracelular/efeitos dos fármacos , Feminino , Proteínas de Grupo de Alta Mobilidade/genética , Mandíbula/crescimento & desenvolvimento , Modelos Animais , Osteogênese/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOX9 , Osso Temporal/crescimento & desenvolvimento , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
In an effort to identify novel candidate genes involved in testis determination, we previously used suppression subtraction hybridisation PCR on male and female whole embryonic (12.0-12.5 days post coitum) mouse gonads. One gene to emerge from our screen was Redd1. In the current study, we demonstrate by whole-mount in situ hybridisation that Redd1 is differentially expressed in the developing mouse gonad at the time of sex determination, with higher expression in testis than ovary. Furthermore, Redd1 expression was first detected as Sry expression peaks, immediately prior to morphological sex determination, suggesting a potential role for Redd1 during testis development. To determine the functional importance of this gene during testis development, we generated Redd1-deficient mice. Morphologically, Redd1-deficient mice were indistinguishable from control littermates and showed normal fertility. Our results show that Redd1 alone is not required for testis development or fertility in mice. The lack of a male reproductive phenotype in Redd1 mice may be due to functional compensation by the related gene Redd2.
Assuntos
Reprodução/fisiologia , Testículo/embriologia , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/metabolismo , Cruzamentos Genéticos , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fertilidade , Feto/embriologia , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Fenótipo , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Células de Sertoli/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Mammalian sex determination is a dynamic process involving balanced gene expression leading to the development of either a testis or an ovary. Candidate sex-determining genes have been identified through microarray-based studies of gonadal gene expression; however, few methods exist for validation. This study describes a new technique for transfecting gonads using nucleofection. Fifteen micrograms of expression plasmid DNA was transfected into E11.5 gonads, cultured for 3 days and gene expression analyzed. Following optimization, we consistently achieved cell transfection efficiencies of 11% of cells using pMax-GFP plasmid. To test the applicability of nucleofection to studies of gene function, a testis-determining gene was transfected into gonads and its ability to sex-reverse was examined. When Sry was transfected into female (XX) gonads, upregulation of its target gene Sox9 was observed, as well as a downregulation of the ovarian gene Foxl2. Conversely, when shSox9 was introduced into male (XY) gonads, reduction of Sox9 and its target gene, Amh was observed, with a concomitant upregulation of Foxl2. Nucleofection-based gene delivery can recapitulate in vivo events of gonadal development that demonstrates 'proof-of-principle' of the method as a screening tool to evaluate the cellular function of potential sex-determining and gonadal differentiation genes.
Assuntos
Ovário/embriologia , Ovário/metabolismo , Processos de Determinação Sexual/genética , Diferenciação Sexual/genética , Testículo/embriologia , Testículo/metabolismo , Animais , Técnicas de Cultura Embrionária , Feminino , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes sry , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição SOX9/antagonistas & inibidores , Fatores de Transcrição SOX9/genética , Proteína da Região Y Determinante do Sexo/genética , TransfecçãoRESUMO
During mouse sex determination, SRY upregulates the core testis-specific enhancer of Sox9, TESCO. Mutations in human SRY are found in one third of cases with XY pure gonadal dysgenesis (XY GD; Swyer syndrome), while two thirds remain unexplained. Heterozygous SOX9 mutations can cause XY GD in association with the skeletal malformation syndrome campomelic dysplasia. We hypothesized that human TESCO mutations could cause isolated XY GD. Sixty-six XY GD cases with an intact SRY were analyzed for TESCO point mutations or deletions. No mutations were identified. We conclude that TESCO mutations are not a common cause of XY GD.
Assuntos
Disgenesia Gonadal 46 XY/genética , Proteínas de Homeodomínio/genética , Mutação/genética , Proteína da Região Y Determinante do Sexo/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas do Citoesqueleto , Humanos , Proteínas com Domínio LIM , Masculino , Camundongos , Proteínas de Ligação a RNARESUMO
In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.
Assuntos
Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/embriologia , Gônadas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Animais , Primers do DNA/análise , Eletroforese em Gel de Ágar , Perfilação da Expressão Gênica , Mesonefro/embriologia , Mesonefro/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Padrões de ReferênciaRESUMO
The study of the mammalian sex-determining pathway has been hampered by the lack of cell culture systems to investigate the underlying molecular relationships between sex-determining genes. Recent approaches using high-throughput genome-wide studies have revealed a number of sexually dimorphic genes expressed in the developing mouse gonad. Here, we investigated a human testicular cell line in terms of its expression of known sex-determining genes and newly identified candidates. The human embryonal carcinoma cell line NT2/D1 was screened for the expression of 46 genes with known or potential roles in the sex-determining and differentiation pathway. Forty genes tested were expressed in NT2/D1 cells including the testis-determining genes SRY, SOX9, SF-1, DHH and FGF9. Genes not expressed included WT1, DAX1 and the ovary-specific genes FOXL2 and WNT4. Cell-specific markers demonstrate that NT2/D1 cells reflect a number of cell types in the gonad including Sertoli, Leydig and germ cells. Our results suggest that male pathways initiated by SRY, SOX9 and SF-1 remain intact in these cells. Lack of expression of ovary-specific genes is consistent with a commitment of these cells to the male lineage. Manipulation of gene expression in this cell line could be an important new in vitro tool for the discovery of new human sex-determining genes.
Assuntos
Perfilação da Expressão Gênica , Sistema Urogenital/metabolismo , Carcinoma Embrionário/genética , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Células Germinativas/metabolismo , Gônadas/metabolismo , Humanos , Células Intersticiais do Testículo/metabolismo , Masculino , Ovário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/metabolismo , Processos de Determinação Sexual , Diferenciação Sexual/genética , Transdução de Sinais/genética , Testículo/metabolismo , Sistema Urogenital/embriologiaRESUMO
The human sex-determining gene on the Y chromosome, termed SRY, has recently been isolated by positional cloning; compelling evidence now exists equating SRY with the testis-determining factor, TDF. The SRY gene product is an HMG box protein whose DNA-binding activity is vital for testis formation as sex-reversed patients with SRY mutations lack this activity in vitro. The in vivo DNA target for SRY, however, remains elusive. Here, we show, by gel retardation analysis, that SRY recognises specific DNA sequences and that such sequences exist upstream of the AMH promoter, a potential downstream target for SRY. We also describe the DNA bending and cruciform DNA-binding functions of SRY and propose a model for the potential action of SRY in the "HMG-1-rich" mammalian nucleus.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Análise para Determinação do Sexo , Cromossomo Y , Sequência de Aminoácidos , Animais , Diferenciação Celular , Mapeamento Cromossômico , Feminino , Cariotipagem , Masculino , Mamíferos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Aberrações dos Cromossomos Sexuais/genética , Proteína da Região Y Determinante do Sexo , Testículo/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Analysis of the NS and M genes of the archetype H6N5 influenza virus strain A/shearwater/Australia/1/72 shows it to be a typical example of the avian host reservoir, containing Old World/Eurasian internal proteins with divergent surface proteins, which is a potential source of new pandemic strains.
Assuntos
Genes Virais , Vírus da Influenza A/genética , Proteínas da Matriz Viral/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Surtos de Doenças , Reservatórios de Doenças , Evolução Molecular , Biblioteca Gênica , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The neuraminidase (NA) gene from the prototype N5 influenza virus, A/Shearwater/Australia/72, has been cloned and completely sequenced. An open reading frame of 1404 bp (468 amino acids) is flanked by 20-bp 5'- and 31-bp 3'-untranslated regions. The deduced amino acid sequence of the N5 gene was compared with sequences from N2, N1, N7, N8, and N9 subtypes. One hundred thirteen amino acid residues (24%) are completely conserved across subtypes and include active site residues, cysteines, potential glycosylation sites, and certain glycines which suggests that these subtypes share a common ancestor and adopt the same 3-D conformation. Three groups can be assigned from amino acid homologies: (i) N5, N8, N1; (ii) N7, N9; and (iii) N2 where the percentage identity within groups is 55-68% and between groups is 40-46%, the N5-N8 pair bearing the closest identity (68%). Phylogenetic analysis suggests that these groups diverged concurrently.
Assuntos
DNA Viral/genética , Vírus da Influenza A/genética , Neuraminidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sondas de DNA , Desoxirribonuclease EcoRI , Vírus da Influenza A/enzimologia , Dados de Sequência Molecular , FilogeniaRESUMO
Although sex determination starts in the gonads, this may not be the case for species with temperature sex determination (TSD). Since temperature affects the whole embryo, extragonadal thermosensitive cells may produce factors that induce gonadal sex determination as a secondary event. To establish if gonads of a species with TSD respond directly to temperature, pairs of gonads were cultured, one at female-promoting temperature (FPT) and the contralateral at male-promoting temperature (MPT). Histological and immunohistochemical detection of SOX9 revealed that the response to temperature of isolated gonads was similar to that of the gonads of whole embryos. While gonads cultured at MPT maintained SOX9 expression, it was downregulated in gonads at FPT. Downregulation of SOX9 took longer in gonads cultured at stage 23 than in gonads cultured at stage 24, suggesting that a developmental clock was already established at the onset of culture. To find out if sex commitment occurs in vitro, gonads were switched from FPT to MPT at different days. Results showed that the ovarian pathway was established after 4 days of culture. The present demonstration that gonads have an autonomous temperature detector that regulates SOX9 expression provides a useful starting point from which the molecular pathways underlying TSD can be elucidated.