RESUMEN
Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.
Asunto(s)
Células Epiteliales/citología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/fisiología , Glicoproteínas de Membrana/metabolismo , Animales , Secuencia de Bases , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/fisiología , Femenino , Factor de Crecimiento de Hepatocito/farmacología , Interleucina-2/genética , Lactancia , Mamíferos , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/biosíntesis , Ratones , Datos de Secuencia Molecular , Morfogénesis , Embarazo , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
A polymerase chain reaction-based method was used to generate a Drosophila melanogaster antennal cDNA library from which head cDNAs were subtracted. We identified five cDNAs that code for antennal proteins containing six cysteines in a conserved pattern shared with known moth antennal proteins, including pheromone-binding proteins. Another cDNA codes for a protein related to vertebrate brain proteins that bind hydrophobic ligands. In all, we describe seven antennal proteins which contain potential signal peptides, suggesting that, like pheromone-binding proteins, they may be secreted in the lumen of olfactory hairs. The expression patterns of these putative odorant-binding proteins define at least four different subsets of olfactory hairs and suggest that the Drosophila olfactory apparatus is functionally segregated.
Asunto(s)
Drosophila melanogaster/metabolismo , Receptores Odorantes/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario/biosíntesis , ADN Complementario/metabolismo , Drosophila melanogaster/genética , Expresión Génica , Biblioteca de Genes , Datos de Secuencia Molecular , Familia de Multigenes , Odorantes , Mucosa Olfatoria/metabolismo , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/biosíntesis , Homología de Secuencia de AminoácidoRESUMEN
The obese (ob) gene has recently been isolated through a positional cloning approach, the mutation of which causes a marked hereditary obesity and diabetes mellitus in mice. In the present study, we isolated rat ob cDNA and examined the tissue distribution of the ob gene expression in rats. We also studied the gene expression in genetically obese Zucker fatty (fa/fa) rats. The rat ob gene product, a 167 amino acid protein with a putative signal sequence, was 96 and 83% homologous to the mouse and human ob proteins, respectively. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kb in size in the adipose tissue, while no significant amount of ob mRNA was present in other tissues in rats. The ob gene was expressed in the adipose tissue with region specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue > or = interscapular brown adipose tissue. The ob gene expression occurred in mature adipocytes rather than in stromalvascular cells isolated from the rat adipose tissue. Expression of the ob gene was markedly augmented in all the adipose tissue examined in Zucker fatty (fa/fa) rats at the stage of established obesity. The present study leads to the better understanding of the physiologic and pathophysiologic roles of the ob gene.
Asunto(s)
Expresión Génica , Obesidad/genética , Proteínas/genética , Ratas Zucker/genética , Tejido Adiposo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Sondas de ADN , Humanos , Leptina , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Ratas , Homología de Secuencia de AminoácidoRESUMEN
In a screen for cell cycle-regulated genes in the yeast Saccharomyces cerevisiae, we have identified a gene, EGT2, which is involved in cell separation in the G1 stage of the cell cycle. Transcription of EGT2 is tightly regulated in a cell cycle-dependent manner. Transcriptional levels peak at the boundary of mitosis and early G1 The transcription factors responsible for EGT2 expression in early G1 are Swi5 and, to a lesser extent, Ace2. Swi5 is involved in the transcriptional activation of the HO gene during late G1 and early S phase, and Ace2 induces CTS1 transcription during early and late G1 We show that Swi5 activates EGT2 transcription as soon as it enters the nucleus at the end of mitosis in a concentration-dependent manner. Since Swi5 is unstable in the nucleus, its level drops rapidly, causing termination of EGT2 transcription before cells are committed to the next cell cycle. However, Swi5 is still able to activate transcription of HO in late G1 in conjunction with additional activators such as Swi4 and Swi6.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular/genética , Secuencia de Consenso , Proteínas de Unión al ADN/metabolismo , Fase G1 , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Genotipo , Datos de Secuencia Molecular , Fenotipo , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genéticaRESUMEN
The expression of the yeast gene CPA1, which encodes the small subunit of the arginine pathway carbamoylphosphate synthetase, is repressed by arginine at a translational level. CPA1 mRNA contains a 250-nucleotide-long leader which includes a 25-codon upstream open reading frame (uORF). Oligonucleotide site-directed mutagenesis of this uORF as well as sequencing of constitutive cis-dominant mutations has suggested that the leader peptide product of the CPA1 uORF is an essential negative element for repression of the CPA1 gene by arginine. In this work, a series of deletions affecting the regions 5' and 3' to the uORF in the leader sequence was constructed. The arginine-dependent repression of CPA1 was little affected in these constructions, indicating that these regions are not essential for the regulatory response. This conclusion was further supported by the finding that inserting the mRNA segment encoding the leader peptide sequence of CPA1 in the leader sequence of another gene, namely, GCN4, places this gene under arginine repression. Similarly, the behavior of fusions of the leader sequence of CPA1 with those of ARG4 or GAL10 confirmed that the regions of this leader located upstream and downstream from the uORF are dispensable for the regulation by arginine. Finally, a set of substitution mutations which modify the uORF nucleotide sequence while leaving unchanged the corresponding amino acid sequence was constructed. The mutations did not affect the repression of CPA1 by arginine. The data presented in this paper consequently agree with the conclusion that the leader peptide itself is the main element required for the translational repression of CPA1.
Asunto(s)
Arginina/farmacología , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/biosíntesis , Expresión Génica/efectos de los fármacos , Genes Fúngicos , Biosíntesis de Proteínas/efectos de los fármacos , Señales de Clasificación de Proteína/biosíntesis , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/efectos de los fármacos , Secuencia de Aminoácidos , Secuencia de Bases , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Clonación Molecular , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Represión Enzimática , Escherichia coli , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Mutagénesis Insercional , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Señales de Clasificación de Proteína/genética , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Saccharomyces cerevisiae/efectos de los fármacos , Eliminación de SecuenciaRESUMEN
Full-length clones encoding a novel member of the metalloproteinase-like, disintegrin-like, cysteine-rich (MDC) family of proteins have been isolated from a monkey (Macaca fascicularis) testicular cDNA library. The encoded putative 82 kDa transmembrane protein (tMDC I) shows striking sequence similarity to other members of the MDC family including rat and monkey EAP I, guinea-pig PH-30 and human MDC protein, as well as a number of snake venom components.
Asunto(s)
Expresión Génica , Macaca fascicularis/genética , Péptidos/genética , Proteínas/genética , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Desintegrinas , Biblioteca de Genes , Cobayas , Humanos , Masculino , Proteínas de la Membrana , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Ratas , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Sequences of avian retroviral RNAs suggest that short open reading frames in the putatively untranslated leader sequences might direct the synthesis of small peptides. Previous analyses indicate that translation of Rous sarcoma virus (RSV) RNA in vitro faithfully reflects translation of the viral RNA in the chick cell. Accordingly, we sought to determine if the heptapeptide LP1, encoded in the open reading frame closest to the 5' end of RSV RNA, could be synthesized in vitro since this would strongly suggest that it might also be synthesized in vivo. Here we confirm that RSV RNA directs the synthesis of LP1 in rabbit reticulocyte lysates. LP1 is rapidly degraded in the lysate by an aminopeptidase activity. On the basis of the following observations, we propose that the open reading frame encoding LP1 plays a role in the life cycle of avian retroviruses. The LP1 open reading frame is ubiquitous with respect to position and length in 12 strains of avian retrovirus. In the amino acid sequences of the 12 strains, only three of the seven residues are invariant. On the basis of the conservation of the -3 and +4 nucleotides flanking the AUG codon, the strengths of initiation for translation of LP1 are approximately the same in the different viruses. The LP1 open reading frame is positioned in front of sites on retrovirus RNA that are required for initiation of cDNA synthesis and for packaging of the RNA into mature virus.
Asunto(s)
Virus del Sarcoma Aviar/genética , Señales de Clasificación de Proteína/biosíntesis , ARN Viral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cinética , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/aislamiento & purificación , ARN Viral/metabolismo , ConejosRESUMEN
The 5' non-coding sequence of rabbit beta-globin mRNA was mutagenized in an attempt to identify structural features that might contribute to the ability to support translation in an homologous rabbit reticulocyte lysate. Translational efficiency was not reduced by substitutions introduced in nearly every position of the beta-globin leader sequence, suggesting that the 5' non-coding domain of this highly efficient mRNA contains no special effector motifs. Instead, efficient translation appears to require only a moderately long leader sequence devoid of secondary structure, especially near the 5' end. Consistent with that interpretation, substitutions in several positions actually improved translation relative to the wild-type beta-globin leader sequence; experimental assessment of the secondary structure of these derivatives revealed a perfect inverse correlation between secondary structure content and translational efficiency. Other experiments probed the structural basis for the long-noted difference in translational efficiency between rabbit alpha and beta-globin mRNAs, a difference that was reproduced here using only the 5' non-coding domains of those mRNAs. The possibility that translation of ribosomal protein mRNAs might be modulated by a mechanism similar to that of alpha-globin mRNA is discussed. Because the beta-globin leader sequence has been incorporated into some popular expression vectors, and because globin genes are targets for gene therapy, this analysis of how globin mRNA leader sequences function in translation and how they can be improved may have practical applications.
Asunto(s)
Globinas/biosíntesis , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , ARN Mensajero/química , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Globinas/química , Metionina/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Señales de Clasificación de Proteína/biosíntesis , Conejos , Mapeo Restrictivo , Transcripción Genética , XenopusRESUMEN
Expression of the threonine (thr) operon enzymes of Escherichia coli is regulated by an attenuation mechanism. The regulatory portion of the operon contains a region coding for a leader peptide that contains consecutive threonine and isoleucine codons. It is thought that translation of the leader peptide controls the frequency of transcription termination at the attenuator site. Using oligonucleotide-directed site-specific mutagenesis we have altered the putative control codons of the leader peptide coding region. In two of the mutants the threonine and isoleucine codons were changed to produce peptides containing histidine and tyrosine codons. Both mutants showed loss of regulation by threonine and isoleucine. A hisT mutation, which leads to an undermodification of tRNA(His), increased thr operon expression in the mutants threefold but did not affect expression of the wild-type thr operon. Two other mutants were constructed that contained two histidine codons early in the leader peptide. Expression in both of these mutants was unaltered by the presence of the hisT allele or by the addition of threonine and isoleucine to the growth medium. In addition, a wild-type strain containing a temperature-sensitive threonyl-tRNA synthetase mutation showed increased thr operon expression at the non-permissive temperature, whereas none of the mutants showed any change. Taken together these data indicate that the specificity of the attenuation response is effected by specific control codons within the thr leader peptide coding region. We have also directly demonstrated thr leader peptide synthesis in vitro using a plasmid encoding the wild-type thr leader region to direct the synthesis of a peptide of the appropriate molecular weight when labeled with [3H]threonine but not with [3H]histidine or [3H]tyrosine. Conversely, when extracts were incubated with templates containing the mutated DNAs, peptides were labeled that showed patterns consistent with the expected amino acid compositions. These data indicate that the thr leader RNA is translated into the predicted leader peptide.
Asunto(s)
Codón , Escherichia coli/genética , Isoleucina/genética , Operón , ARN Mensajero , Treonina/genética , Transcripción Genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Mutación , Plásmidos , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , TemperaturaRESUMEN
In sarcomagenesis in rats infected neonatally with feline sarcoma virus (ST-FeSV), v-fes product (P85) was previously shown by us to be a predictive and preventive determinant. In order to explore the part played by P85 in tumor suppression, DNA was extracted from precancerous granulomas and from slow or rapid growing sarcomas induced by neonatal injection of the virus. The v-fes signal from extracted DNA was analyzed by PCR-SSCP. The prototype v-fes gene signal was detected in most lesions and found to be generally amplified in rapid growing sarcomas and in some granulomas. Several v-fes homologs showing varying mobilities in gel were seen in most sarcomas and some granulomas with or without the prototype v-fes signal. In slow growing sarcomas and granulomas induced in hosts that were immunized with ST-FeSV induced syngeneic sarcoma and proved to carry IgG antibody to P85, the prototype v-fes gene was found to be down-regulated and v-fes homologs were found to be reduced in number or eliminated. These results suggest that the development of v-fes mutations is associated with the growth potential of cells carrying the v-fes gene, and that host immunity to v-onc product influences the development of virogene rearrangements and results in slow and suppressed growth of tumors caused by neonatal infection with retrovirus.
Asunto(s)
Proteínas de Fusión gag-onc/biosíntesis , Oncogenes , Virus del Sarcoma Felino/patogenicidad , Sarcoma Experimental/genética , Sarcoma Experimental/patología , Vacunas Virales , Animales , Animales Recién Nacidos , Secuencia de Bases , Gatos , Línea Celular , Cartilla de ADN , ADN Viral/análisis , Femenino , Proteínas de Fusión gag-onc/genética , Granuloma/patología , Granuloma/virología , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/biosíntesis , Ratas , Ratas Wistar , Virus del Sarcoma Felino/genética , Virus del Sarcoma Felino/aislamiento & purificación , Sarcoma Experimental/prevención & control , Sarcoma Experimental/virologíaRESUMEN
We have isolated and sequenced a novel lambda 1 constant region related cDNA clone which might represent an allelic variant of the recently described lambda 5 gene. This lambda 5 transcript is present in pre-B cell lines and bone marrow cells, but not in B cell lines, plasma cell lines or in spleen cells. In vitro translation studies show that the translation product contains a signal peptide of approx. 30 amino acids at its N-terminus.
Asunto(s)
Linfocitos B/metabolismo , Genes de Inmunoglobulinas , Cadenas Ligeras de Inmunoglobulina/aislamiento & purificación , Señales de Clasificación de Proteína/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Recombinante , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peso Molecular , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN , Translocación GenéticaRESUMEN
In this article, the authors isolate a gene encoding a neuropeptide in Drosophila melanogaster. The substance is called pigment-dispersing factor (PDF), based on one of the roles it plays in crustaceans (the arthropods in which this factor was initially discovered). The PDF-encoding Drosophila gene (pdf) is intronless and present in a single copy per haploid genome. The cytological location of pdf is 97B on the third chromosome. The putative 102-amino-acid precursor (prepro-PDF) consists of a signal peptide and a PDF-associated peptide, followed by the mature PDF. The PDF-associated peptide region of the precursor is highly diverged from those of the crustacean precursors, whereas the primary structure of the mature PDF is conserved in other members of the pigment-dispersing hormone family. A single pdf transcript (ca. 0.8 kb) is expressed predominantly in the head; the expression levels of pdf mRNA are consistently higher in males than in females. Putative pdf homologous transcripts are present in other Drosophila species, which exhibit similar sexual dimorphic expression patterns. Cyclic expression of pdf over the course of the day and night was assessed, but the mRNA exhibited at best very gentle cycling. The pdf expression in two behaviorally arrhythmic mutants were examined; the expression was intact in a period0 mutant but absent in the disconnected mutant.
Asunto(s)
Mapeo Cromosómico , Proteínas de Drosophila , Drosophila melanogaster/fisiología , Genes de Insecto , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Periodicidad , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Crustáceos , Drosophila melanogaster/genética , Femenino , Biblioteca Genómica , Masculino , Datos de Secuencia Molecular , Neuropéptidos/química , Señales de Clasificación de Proteína/biosíntesis , ARN Mensajero/biosíntesis , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.
Asunto(s)
Fosfatasa Alcalina/genética , Quimosina/genética , Clonación Molecular , Precursores Enzimáticos/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína/genética , Animales , Bovinos , Quimosina/biosíntesis , Precursores Enzimáticos/biosíntesis , Escherichia coli/enzimología , Plásmidos , Señales de Clasificación de Proteína/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Two different hybrid genes were constructed which fuse the Bacillus amyloliquefaciens alkaline protease gene (apr[BamP]) promoter and signal peptide coding region to a synthetic bpr gene coding for the mature bovine pancreatic RNase A. The first gene fusion (apr-bpr1) contained the apr[BamP] signal peptide coding region fused to mature bpr through a linker coded 3-amino acid region and retained the signal processing site ala-ala of the alkaline protease. The second fusion (apr-bpr2) joined the end of the apr[BamP] signal peptide coding sequence to the mature bpr resulting in a hybrid signal processing site ala-lys. B. subtilis strains harboring these gene fusions secreted bovine pancreatic RNase A into the growth medium. Cleavage at the hybrid signal processing site ala-lys resulted in the secretion of bovine pancreatic RNase A from B. subtilis which had an N-terminal amino acid sequence that was identical to the native RNase A. Bovine pancreatic RNase A contains four disulfide bonds and the proper formation of these bonds is required for activity. RNase activity could be detected in the culture supernatants of strains carrying the apr-bpr gene fusions, which suggests that the proper disulfide bonds have formed spontaneously.
Asunto(s)
Genes Sintéticos , Ribonucleasa Pancreática/biosíntesis , Serina Endopeptidasas , Secuencia de Aminoácidos , Animales , Bacillus subtilis/genética , Secuencia de Bases , Bovinos , Clonación Molecular , ADN/genética , Endopeptidasas/genética , Vectores Genéticos , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismoRESUMEN
The murine 4-1BB gene, encoding a member of the nerve growth factor/tumor necrosis factor (NGF/TNF) receptor family, is thought to be selectively expressed in T cells and is involved in the regulation of lymphocyte proliferation. We detected two forms of the 4-1BB mRNA by RT-PCR which were expressed in an activation-dependent pattern in splenocytes and thymocytes. cDNA sequencing showed that the smaller form was a mRNA splice variant lacking the transmembrane region (4-1BB delta TM), because of the deletion of exon 8. The two forms of mRNA are differentially expressed in murine T cells, macrophages, 3T3 fibroblasts and epitheloid cells. Northern blotting also identifies two forms of mRNA of 1.5 and 2.4 kb, and the cell-type-specific pattern correlated with the PCR results. These results identify a novel form of 4-1BB. This and the previously known membrane-associated form have a broad tissue distribution, suggesting a more diverse role in host defense.
Asunto(s)
Variación Genética , Ratones/genética , ARN Mensajero/biosíntesis , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Receptores de Factor de Crecimiento Nervioso/genética , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Antígenos CD , Secuencia de Bases , Northern Blotting , Cartilla de ADN , Exones , Expresión Génica , Activación de Linfocitos , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
The MID2 gene whose defect (the mid2-1 mutation) results in mating-pheromone-induced death in Saccharomyces cerevisiae was cloned and its nucleotide (nt) sequence determined. The sequence showed an open reading frame (ORF) coding for a 376-amino-acid (aa) protein with an estimated M(r) of 39,104, and six potential TATA boxes and two pheromone-response elements in its 5'-upstream region. The deduced aa sequence showed that the MID2 product (Mid2p) contains a putative N-terminal signal sequence followed by a long Ser-rich region that could contain O-glycosylation sites, a potential transmembrane domain and a conserved Ca(2+)-binding domain, with the latter two located in the C-terminal half. Northern blot analysis showed that the expression of MID2 is stimulated threefold by mating pheromone. Cells that lack MID2 were able to grow normally, but died when exposed to mating pheromone, like the original mid2-1 mutant.
Asunto(s)
Proteínas de Unión al Calcio/genética , Expresión Génica , Proteínas de la Membrana/genética , Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Northern Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Glicosilación , Péptidos y Proteínas de Señalización Intracelular , Cinética , Factor de Apareamiento , Glicoproteínas de Membrana , Proteínas de la Membrana/biosíntesis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Feromonas/metabolismo , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , TATA BoxRESUMEN
A hybrid gene consisting of the sequences coding for the signal peptide and N terminus of a type-I membrane protein, the neural cell adhesion molecule (N-CAM), and the extracellular domain of the adhesion molecule on glia (AMOG/beta 2), a type-II membrane protein, was constructed. The sequence was inserted into a eukaryotic expression vector containing the human cytomegalovirus promoter and the glutamine synthetase selection marker, and used to transfect Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of soluble recombinant AMOG/beta 2 (reAMOG/beta 2), which was secreted into the culture medium as a heavily glycosylated 40-55-kDa protein. N-terminal sequence analysis revealed that the protein is not cleaved at the natural signal peptide cleavage site of N-CAM, but two amino acids (aa) further downstream. Treatment of reAMOG/beta 2 with N-glycosidase F (GlycoF) reduced the molecular mass to 27 kDa, corresponding to the calculated mass of the unglycosylated form. In contrast to AMOG/beta 2 isolated from mouse brain, which is sensitive to endoglycosidase H, the immunoaffinity-purified re-protein is more resistant to this treatment, indicating that the sugars attached to reAMOG/beta 2 are mainly of the complex type. Our results demonstrate the feasibility of secreting the extracellular domain of a type-II membrane protein, which is usually inserted into the membrane with the C terminus facing the extracellular side.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Adenosina Trifosfatasas , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas de Transporte de Catión , Moléculas de Adhesión Celular Neuronal/biosíntesis , Moléculas de Adhesión Celular Neuronal/aislamiento & purificación , Línea Celular Transformada , Cricetinae , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/aislamiento & purificación , Vectores Genéticos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/aislamiento & purificación , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/aislamiento & purificación , Mapeo Restrictivo , TransfecciónRESUMEN
To search for the existence and distribution of a riboflavin-binding protein (RfBP), total RNA from estrogen-treated oviparous animals were screened by Northern hybridization using chicken RfBP cDNA as a probe. Besides avian livers and oviducts, RfBP mRNA was found in turtle liver, but not in the turtle oviduct. To elucidate the structure of the RfBP from a reptilian source, we constructed a cDNA library from estrogen-injected turtle liver, and a full-length turtle RfBP-encoding cDNA was cloned and sequenced. The open reading frame (ORF) encoded 242 amino acids (aa) including a signal peptide of 18 aa. There is an overall 71.3% aa identity between the deduced aa sequences of turtle and chicken. The aa sequence of turtle and chicken RfBP also show more than 30% similarity to a fragment of folate-binding protein (FBP). Six Trp and nine pairs of Cys residues are conserved between the two RfBPs with only one pair of Cys residues missing in FBP. The two Asn-linked glycosylation sites found in chicken RfBP are conserved in turtle RfBP, but only one of which is conserved in FBP. However, there is an additional potential N-glycosylation site in the turtle sequence and this may provide a better explanation for the greater molecular weight of the turtle protein than chicken RfBP. Turtle RfBP contains a region of nine Ser and five Glu residues which is present in mature chicken RfBP as eight phosphorylserine clusters forming a highly anionic region at the C terminus, but this region is not found in FBP.
Asunto(s)
Proteínas Portadoras/genética , Pollos/genética , Proteínas de Transporte de Membrana , Tortugas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Clonación Molecular , ADN Complementario , Estrógenos/farmacología , Biblioteca de Genes , Hígado/efectos de los fármacos , Hígado/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oviductos/metabolismo , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
A cDNA of rat C6 cells was cloned, which was considered to be involved in glial cell differentiation induced by dibutyryl cyclic AMP and theophylline. The cDNA fragment of the gene, termed r-gsp, was originally isolated by mRNA fingerprinting using arbitrarily primed polymerase chain reaction, and was homologous to complement C1s precursors of hamster and human. It encodes a protein of 694 amino acids containing a potential signal peptide, an epidermal growth factor-like domain surrounded by two complement C1r/C1s-related repeats, and a putative trypsin-type serine protease domain. Since the hamster and human C1s, and a protein encoded by r-gsp shared high similarity in primary structure, the r-gsp gene could encode a C1s counterpart of the rat. Messenger RNA expression of this gene was markedly increased during cyclic AMP-induced glial cell differentiation. Its expression profile was well correlated with those of glial fibrillary acidic protein (GFAP) and S100B, which are known as glial differentiation markers. It was, moreover, observed that the r-gsp expression in brain increased considerably after birth, like those of S100B and GFAP. The results presented here suggest that the rat C1s gene would be also implicated in glial differentiation besides the complement cascade.
Asunto(s)
Complemento C1s/química , Neuroglía/enzimología , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Clonación Molecular , Cricetinae , Cartilla de ADN , ADN Complementario , Factor de Crecimiento Epidérmico/química , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Neuroglía/citología , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/química , ARN Mensajero/biosíntesis , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genéticaRESUMEN
The novel gene named PSX1, encoding a new protopectinase with the polymethoxygalacturonase activity, was isolated from Trichosporon penicillatum. Nucleotide sequencing revealed that the PSX1 gene is composed of 1080 bases (360 amino acids, 38,747 Da). The N-terminal amino acid sequences of the open reading frame correspond to a signal peptide and propeptide processed by a Kex2-like proteinase. Mature PPase SX1 was composed of 334 amino acids (36,121 Da). PPase SX1 produced by a S. cerevisiae transformant harboring the PSX1 gene degraded methoxylated polygalacturonic acid as a substrate, but not degraded unmethoxylated polygalacturonic acid.