RESUMEN
Noonan syndrome is characterized by short stature, facial dysmorphia and a wide spectrum of congenital heart defects. Mutations of PTPN11, KRAS and SOS1 in the RAS-MAPK pathway cause approximately 60% of cases of Noonan syndrome. However, the gene(s) responsible for the remainder are unknown. We have identified five different mutations in RAF1 in ten individuals with Noonan syndrome; those with any of four mutations causing changes in the CR2 domain of RAF1 had hypertrophic cardiomyopathy (HCM), whereas affected individuals with mutations leading to changes in the CR3 domain did not. Cells transfected with constructs containing Noonan syndrome-associated RAF1 mutations showed increased in vitro kinase and ERK activation, and zebrafish embryos with morpholino knockdown of raf1 demonstrated the need for raf1 for the development of normal myocardial structure and function. Thus, our findings implicate RAF1 gain-of-function mutations as a causative agent of a human developmental disorder, representing a new genetic mechanism for the activation of the MAPK pathway.
Asunto(s)
Mutación Missense , Síndrome de Noonan/genética , Proteínas Proto-Oncogénicas c-raf/genética , Animales , Línea Celular , Línea Celular Transformada , Femenino , Corazón/embriología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miocardio/metabolismo , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Noonan syndrome (NS) is the most common non-chromosomal syndrome seen in children and is characterized by short stature, dysmorphic facial features, chest deformity, a wide range of congenital heart defects and developmental delay of variable degree. Mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathways cause about 70% of NS cases with a KRAS mutation present in about 2%. In a cohort of 65 clinically confirmed NS patients of Japanese origin, we screened for mutations in the RAS genes by direct sequencing. We found a novel mutation in KRAS with an amino acid substitution of asparagine to serine at codon 116 (N116S). We analyzed the biological activity of this mutant by ectopic expression of wild-type or mutant KRAS. NS-associated KRAS mutation resulted in Erk activation and active Ras-GTP levels, and exhibited mild cell proliferation. In addition, kras-targeted morpholino knocked-down zebrafish embryos caused heart and craniofacial malformations, while the expression of mutated kras resulted in maldevelopment of the heart. Our findings implicate that N116S change in KRAS is a hyperactive mutation which is a causative agent of NS through maldevelopment of the heart.
Asunto(s)
Genes ras , Mutación , Síndrome de Noonan/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Estudios de Cohortes , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Hibridación in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de Aminoácido , Pez CebraRESUMEN
The left-right (LR) axis is essential for the proper function of internal organs. In mammals and fish, left-sided Nodal expression governs LR patterning. Here, we show that the Polycomb group protein Ezh1, which is highly conserved from fish to human, participates in LR patterning. Knockdown of olezh1, a medaka homologue of Ezh1, led to LR reversal of internal organs. It was shown that OLEZH1 acts in silencing the expression of Spaw (a medaka homolog of Nodal) via a previously unknown pathway. Furthermore, coimmunoprecipitation showed physical interaction of Ezh1 with FoxH1, a Nodal regulator. This represents a novel mechanism for LR patterning and implies that Ezh1 has developmental importance.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Peces/genética , Proteína Nodal/genética , Oryzias/embriología , Proteínas Represoras/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Humanos , Oryzias/metabolismo , Proteínas del Grupo PolycombRESUMEN
A synthetic microbial consortium called Effective Microorganisms (EM) consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects of EMâXGOLD, a health drink produced by EM, on life cycle of Dictyostelium discoideum were described previously. Here, we report our attempt to identify the active principle, termed EMF, that brought about the observed effects. Throughout the purification processes, the presence of the active principle was monitored by promoted fruiting body formation. By liquid-liquid separation the activity was recovered in aqueous phase, which, after concentration, was further subjected to reverse-phase column chromatography. No activity was detected in any eluant, while almost all the activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures to the residual fraction did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, the residue revealed spherical particles of µm size range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium. In separate experiments, acid treatment and alkali neutralization of EMâXGOLD completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticles to be an active principle of EM culture extract. How these particles exert their effect is currently under intensive investigation.
Asunto(s)
Bacterias/crecimiento & desarrollo , Medios de Cultivo/análisis , Hongos/crecimiento & desarrollo , Magnesio/química , Potasio/química , Bacterias/química , Cromatografía de Fase Inversa , Hongos/química , Ácido Láctico/química , Extracción Líquido-Líquido , Consorcios Microbianos , Microscopía de Fuerza Atómica , Tamaño de la PartículaRESUMEN
Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder presenting with an elfin-like face, supravalvular aortic stenosis, a specific cognitive-behavioral profile, and infantile hypercalcemia. We encountered two WBS patients presenting with infantile spasms, which is extremely rare in WBS. Array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) analyses revealed atypical 5.7-Mb and 4.1-Mb deletions at 7q11.23 in the two patients, including the WBS critical region and expanding into the proximal side and the telomeric side, respectively. On the proximal side, AUTS2 and CALN1 may contribute to the phenotype. On the telomeric side, there are two candidate genes HIP1 and YWHAG. Because detailed information of them was unavailable, we investigated their functions using gene knockdowns of zebrafish. When zebrafish ywhag1 was knocked down, reduced brain size and increased diameter of the heart tube were observed, indicating that the infantile spasms and cardiomegaly seen in the patient with the telomeric deletion may be derived from haploinsufficiency of YWHAG.
Asunto(s)
Proteínas 14-3-3/genética , Cardiomegalia/genética , Espasmos Infantiles/genética , Síndrome de Williams/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Proteínas 14-3-3/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Calmodulina/genética , Calmodulina/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patología , Deleción Cromosómica , Cromosomas Humanos Par 7/genética , Cromosomas Humanos Par 7/metabolismo , Hibridación Genómica Comparativa , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Lactante , Recién Nacido , Masculino , Miocardio/metabolismo , Miocardio/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos/genética , Espasmos Infantiles/metabolismo , Espasmos Infantiles/patología , Telómero/genética , Telómero/metabolismo , Telómero/patología , Síndrome de Williams/metabolismo , Síndrome de Williams/patología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The chromatin organizer modifier domain (chromodomain) is present in proteins that contribute to chromatin organization and mediates their binding to methylated histone H3. Despite a high level of sequence conservation, individual chromodomains manifest substantial differences in binding preference for methylated forms of histone H3, suggesting that posttranslational modification of the chromodomain might be an important determinant of binding specificity. We now show that mouse Cbx2 (also known as M33), a homolog of Drosophila Polycomb protein, is highly phosphorylated in some cell lines. A low-mobility band of Cbx2 observed on SDS-polyacrylamide gel electrophoresis was thus converted to a higher-mobility band by treatment with alkaline phosphatase. Mass spectrometric analysis revealed serine-42, a conserved amino acid in the chromodomain, as a phosphorylation site of Cbx2. Phosphorylation of the chromodomain of Cbx2 on this residue in vitro resulted in a reduced level of binding to an H3 peptide containing trimethylated lysine-9 as well as an increase in the extent of binding to an H3 peptide containing trimethylated lysine-27, suggesting that such phosphorylation changes the binding specificity of Cbx2 for modified histone H3. Phosphorylation of the chromodomain of Cbx2 may therefore serve as a molecular switch that affects the reading of the histone modification code and thereby controls epigenetic cellular memory.
Asunto(s)
Histonas/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Lisina/genética , Lisina/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Células 3T3 NIH , Fosforilación , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Estructura Terciaria de Proteína , Proteínas Represoras/genéticaRESUMEN
Left-right (LR) patterning is an essential part of the animal body plan. Primary cilia are known to play a pivotal role in this process. In humans, genetic disorders of ciliogenesis cause serious congenital diseases. A comprehensive mechanism that regulates ciliogenesis has not been proposed so far. Here, we show that EED, a core member of the Polycomb group (PcG) genes and a presumed player in many epigenetic processes, is required for ciliogenesis and subsequent LR patterning in the medaka fish, Oryzias latipes. Moderate knockdown of oleed, a medaka homolog of EED, preferentially caused situs inversus. In the affected embryo, the cilia in Kupffer's vesicle showed various defects in their structure, position and motility. Furthermore, we demonstrated that oleed maintains the expression of Noto, which, in mice, regulates ciliogenesis and LR patterning. This study provides the first evidence for the involvement of epigenetic plasticity in LR patterning through ciliogenesis.
Asunto(s)
Tipificación del Cuerpo/fisiología , Cilios/fisiología , Embrión no Mamífero/metabolismo , Proteínas de Peces/fisiología , Oryzias/embriología , Proteínas Represoras/fisiología , Animales , Embrión no Mamífero/citología , Proteínas de Peces/antagonistas & inhibidores , Técnicas para Inmunoenzimas , Hibridación in Situ , Factores de Determinación Derecha-Izquierda , Ratones , Morfogénesis , Oryzias/metabolismo , Proteínas del Grupo Polycomb , Proteínas Represoras/antagonistas & inhibidoresRESUMEN
Array-based comparative genomic hybridization identified a 2.3-Mb microdeletion of 17p13.2p13.1 in a boy presenting with moderate mental retardation, intractable epilepsy and dysmorphic features. This deletion region was overlapped with the previously proposed shortest region overlapped for microdeletion of 17p13.1 in patients with mental retardation, microcephaly, microretrognathia and abnormal magnetic resonance imaging (MRI) findings of cerebral white matter, in which at least 17 known genes are included. Among them, DLG4/PSD95, GPS2, GABARAP and KCTD11 have a function in neuronal development. Because of the functional importance, we paid attention to DLG4/PSD95 and GABARAP, and analyzed zebrafish in which the zebrafish homolog of human DLG4/PSD95 and GABARAP was knocked down and found that gabarap knockdown resulted in small head and hypoplastic mandible. This finding would be similar to the common findings of the patients with 17p13.1 deletions. Although there were no pathogenic mutations in DLG4/PSD95 or GABARAP in a cohort study with 142 patients with idiopathic developmental delay with/without epilepsy, further studies would be required for genes included in this region.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Cromosomas Humanos Par 17/genética , Técnicas de Silenciamiento del Gen , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Animales , Proteínas Reguladoras de la Apoptosis , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Proteínas Portadoras/genética , Niño , Deleción Cromosómica , Análisis Citogenético , Análisis Mutacional de ADN , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Oligonucleótidos Antisentido/farmacología , Embarazo , Proteínas de Pez Cebra/genéticaRESUMEN
A novel microdeletion of 14q13.1q13.3 was identified in a patient with developmental delay and intractable epilepsy. The 2.2-Mb deletion included 15 genes, of which TULIP1 (approved gene symbol: RALGAPA1)was the only gene highly expressed in the brain. Western blotting revealed reduced amount of TULIP1 in cell lysates derived from immortalized lymphocytes of the patient, suggesting the association between TULIP1 haploinsufficiency and the patient's phenotype, then 140 patients were screened for TULIP1 mutations and four missense mutations were identified. Although all four missense mutations were common with parents, reduced TULIP1 was observed in the cell lysates with a P297T mutation identified in a conserved region among species. A full-length homolog of human TULIP1 was identified in zebrafish with 72% identity to human. Tulip1 was highly expressed in zebrafish brain, and knockdown of which resulted in brain developmental delay. Therefore, we suggest that TULIP1 is a candidate gene for developmental delay.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 14/genética , Discapacidades del Desarrollo/genética , Epilepsia Generalizada/genética , Proteínas Activadoras de GTPasa/deficiencia , Mutación Missense , Proteínas del Tejido Nervioso/deficiencia , Secuencia de Aminoácidos , Animales , Encéfalo/anomalías , Encéfalo/embriología , Niño , Cromosomas Humanos Par 14/ultraestructura , Codón/genética , Secuencia Conservada , Femenino , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Discapacidad Intelectual/genética , Masculino , Datos de Secuencia Molecular , Hipotonía Muscular/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Linaje , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
Three histone H1 variants were identified in medaka fish and their sequence characteristics were analyzed. This paper reports one of these variants, termed H1-2, because of its possible implication in erythrocyte maturation. The amino acid sequence of H1-2 was phylogenetically similar to that of other replication-dependent histones. The mRNA transcribed from the h1-2 gene, however, possessed a poly(A) tail without a stem-loop structure, indicating that H1-2 may represent a replication-independent (RI) histone. Transcripts from the h1-2 gene were largely localized in erythrocytes, and knock-down of the h1-2 gene with morpholino antisense oligos resulted in failure to develop mature erythrocytes. In the morphants, residual erythrocytes showed severely impaired nuclear compaction. Although not structurally related to chicken RI histone H5, which is required for erythrocyte maturation, H1-2 may constitute its functional counterpart. Our findings may offer evolutionary insights into the function of H1 variants.
Asunto(s)
Eritrocitos/metabolismo , Histonas/metabolismo , Oryzias/metabolismo , Secuencia de Aminoácidos , Animales , Embrión no Mamífero , Perfilación de la Expresión Génica , Histonas/química , Histonas/genética , Datos de Secuencia Molecular , Oryzias/genética , Filogenia , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
A cellular slime mold, Dictyostelium discoideum, is an amoeboid organism that has a unique life cycle consisting of distinctly separated vegetative and developmental phases. Thus, this organism presents a rare opportunity in which to examine the effects of bioactive substances on separate cellular activities. In this research, we investigated the effect of a culture extract, termed EMXG, produced by a synthetic microbial consortium. EMXG promoted proliferative response of amoeba cells. It further accelerated the developmental phase, leading to the preferred fruiting body formation from fewer cells. Furthermore, EMXG modulated biological rhythm of this organism, that is, interval of oscillation of cAMP level observed in suspension starvation was significantly shortened. Concomitantly, the level of ERKB, a MAP kinase, was found to oscillate in a similar fashion to that of cAMP. Additionally, ErkB-deficient mutant amoeboid cells did not respond to proliferative stimulation by EMXG. These lines of evidence point to a likelihood that MAP kinase cascade is involved and further that ErkB could be the molecular target of EMXG.
Asunto(s)
Dictyostelium/citología , Sistema de Señalización de MAP Quinasas , Consorcios Microbianos , Proliferación Celular , AMP Cíclico/análisisRESUMEN
Latexin, the endogenous protein inhibitor of the A/B subfamily of metallocarboxypeptidases, is expressed in small nociceptive neurons in sensory ganglia and in a subset of neurons in the telencephalon. In this study, we generated latexin-deficient mice that exhibited increased tail-flick latency compared to wild-type animals upon noxious heat stimulation. The reduced pain sensitivity in the mutants was rescued by the systemic administration of a plant carboxypeptidase inhibitor that inhibits the A/B subfamily of metallocarboxypeptidases. These findings suggest that latexin is involved in the transmission of pain.
Asunto(s)
Antígenos/fisiología , Carboxipeptidasas/antagonistas & inhibidores , Dolor/fisiopatología , Animales , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Dolor/prevención & control , Tiempo de ReacciónRESUMEN
How animals adaptively respond to a cold or hot environment has been questioned for a long time. Recently, with the aid of microarray analysis, various temperature-sensitive genes have been identified in several species. However, a definitive hypothesis regarding the mechanism of adaptation has not been proposed. In the present study, we surveyed, in medaka (Oryzias latipes), genes for which the level of expression changes depending on the surrounding temperature. A messenger RNA differential display of medaka muscle total RNA revealed one such gene encoding transcription enhancer factor-1 (TEF-1). In medaka muscle, the TEF-1 gene produces two splicing variants, TEF-1A and TEF-1B mRNAs. During cold acclimation, the mRNA level of TEF-1A decreased, whereas that of TEF-1B increased. We also found that three putative downstream genes of TEF-1, two for myosin heavy chain (MyHC) and one for troponin T (TnT), a specific group of muscle proteins, were transcribed in a temperature-dependent manner. These results suggest that the transcription of MyHC and/or TnT is regulated by TEF-1 and that these molecules participate in muscle reconstruction during temperature adaptation in fish.
Asunto(s)
Aclimatación/genética , Frío , Proteínas de Unión al ADN/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica , Oryzias/genética , Factores de Transcripción/genética , Aclimatación/fisiología , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Oryzias/fisiología , Factores de TiempoRESUMEN
The mouse Polycomb group (PcG) protein M33 forms nuclear complexes with the products of other PcG members and maintains repressed states of developmentally important genes, including homeotic genes. In this context, nuclear localization is a prerequisite for M33 to exert its function. However, we previously found that M33 in mouse liver shuttles dynamically between the nucleus and the cytoplasm, depending on the proliferative states of cells, coupled with phosphorylation and dephosphorylation of M33 protein. To understand the mechanism and significance of this phenomenon, we identified the functional nuclear localization signal (NLS) of M33 protein. Deletion mutants that lack a particular one of three putative NLS motifs failed to localize in the nucleus. Green fluorescent protein (GFP) fused to this motif specifically localized in the nucleus. We conclude that this amino-acid stretch in M33 acts as the functional NLS for this protein.
Asunto(s)
Ratones/genética , Señales de Localización Nuclear/genética , Proteínas Represoras/genética , Animales , Análisis Mutacional de ADN/métodos , Orden Génico/genética , Proteínas Fluorescentes Verdes/análisis , Ratones/fisiología , Microscopía Fluorescente/métodos , Proteínas Mutantes/análisis , Proteínas Mutantes/biosíntesis , Células 3T3 NIH , Señales de Localización Nuclear/fisiología , Plásmidos , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Represoras/fisiologíaRESUMEN
We describe the use of a DNA display system for in vitro selection of peptide ligands from a large library of peptides displayed on their encoding DNAs. The method permits completely in vitro construction of a DNA-tagged peptide library by using a wheat germ in vitro transcription/translation system compartmentalized in water-in-oil emulsions. Starting with a library of 10(9)-10(10) random decapeptides, 21 different peptide ligands were isolated for monoclonal antibody anti-FLAG M2. DNA display selected more diverse peptides with a DYKXXD consensus motif than previously reported phage display systems. Binding and recovery rates of three peptides were significantly higher than those of the original FLAG peptide, implying that these peptides would be superior to the FLAG peptide for purification of tagged proteins. The simplicity of DNA display enables two selection rounds per day to be conducted. Further, DNA display can overcome the limitations of previous display technologies by avoiding the use of bacterial cells and RNA tags. Thus, DNA display is expected to be useful for rapid screening of a wide variety of peptide ligands for corresponding receptors.
Asunto(s)
ADN/análisis , Biblioteca de Péptidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Biotinilación , Clonación Molecular , ADN/metabolismo , Ligandos , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligopéptidos , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Biosíntesis de Proteínas , Estreptavidina/química , Transcripción GenéticaRESUMEN
A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [35 S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140-kDa, 105-kDa, 43-kDa, 32-kDa, and 28-kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32-kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo, several hours earlier than the onset of spicule formation. The synthesis of 32-kDa protein was paralleled to active spicule formation and the uptake of Ca2+ . Cell-free translation products directed by poly (A)+ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.
RESUMEN
Previously we showed that the arylsulfatase (Ars) activity increases markedly after the mesenchyme blastula stage, and that the increase in its activity is due to initiation of the Ars gene transcription. In order to detect cells responsible for Ars mRNA production in the sea urchin embryo, we carried out in situ hybridization with the use of 35 S-labeled Ars RNA probe transcribed from Ars cDNA in a vector pT7T3-19U. The Ars gene is uniformly expressed in the cells of blastula, and the expression becomes restricted to aboral ectoderm during development by gastrula stage.
RESUMEN
In vitro display technologies are powerful tools for screening peptides with desired functions. We previously proposed a DNA display system in which streptavidin-fused peptides are linked with their encoding DNAs via biotin labels in emulsion compartments and successfully applied it to the screening of random peptide libraries. Here we describe its application to functional and folded proteins. By introducing peptide linkers between streptavidin and fused proteins, we achieved highly efficient (>95%) formation of DNA-protein conjugates. Furthermore, we successfully enriched a glutathione-S-transferase gene by a factor of 20-30-fold per round on glutathione-coupled beads. Thus, DNA display should be useful for rapidly screening or evolving proteins based on affinity selection.
Asunto(s)
ADN/genética , ADN/metabolismo , Biblioteca de Péptidos , Proteínas/genética , Proteínas/metabolismo , Biotina/genética , Biotina/metabolismo , Evolución Molecular Dirigida , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Unión Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estreptavidina/genética , Estreptavidina/metabolismo , Transcripción GenéticaRESUMEN
We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.
Asunto(s)
Aves/genética , Hormona Folículo Estimulante de Subunidad beta/genética , Hormonas Glicoproteicas de Subunidad alfa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Hormona Folículo Estimulante de Subunidad beta/química , Hormonas Glicoproteicas de Subunidad alfa/química , Intrones/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Alineación de SecuenciaRESUMEN
We isolated a putative gene for the thyrotropin beta subunit (TSHbeta) from two types of genomic libraries of the Japanese crested ibis, Nipponia nippon. Exon-intron structure was deduced by comparing the determined sequence with those of TSH beta cDNA of other birds. The deduced amino acid sequence shows extensive similarities to those of the other birds, which assures our assumption that the acquired nucleotide sequence represents the TSHbeta gene. The assembled genomic fragment is 4192 bp in size and consists of 1937 bp of putative 5' flanking region followed by exon-intron structure with three exons and two introns, similar to those observed in rat, human and goldfish counterparts. Locations of introns are also similar to those in mammals and goldfish. Comparison of the 5' flanking region of the ibis TSHbeta gene with those of mammals reveals that several regulatory sequences, such as negative thyroid hormone responsive element (nTRE), Pit-1 responsive element, and AP-1 responsive element, which were characterized in mammalian TSHbeta genes, are also found in the promoter region. This is the first report on the exon-intron structure and 5' flanking region of the TSHbeta gene in an avian species.