RESUMO
Neuronal growth factors regulate the expression of voltage-activated sodium current in differentiating sympathetic neurons and PC12 cells. We show that, in PC12 cells, the NGF- and FGF-induced sodium current results from increased expression of two distinct sodium channel types. Sodium current results from the rapid induction of a novel sodium channel transcript, also found in peripheral neurons, and from the long term induction of brain type II/IIA mRNA. Expression of the type II/IIA sodium channel requires activation of the cyclic AMP-dependent protein kinase (A-kinase), whereas induction of the peripheral neuron type sodium channel occurs through an A-kinase-independent signal transduction pathway. These findings suggest that the two sodium channel types act in concert to ensure the generation of action potentials during neuronal differentiation.
Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento Neural/fisiologia , Canais de Sódio/fisiologia , Animais , AMP Cíclico/fisiologia , Expressão Gênica , Técnicas In Vitro , Potenciais da Membrana , Células PC12 , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , Ratos , Transdução de Sinais , Sódio/fisiologia , Fatores de Tempo , Transcrição GênicaRESUMO
Phase variation in bacteria is regulated by homologous recombination at a specific DNA site. This recombinational event causes the inversion of a 970-base-pair DNA sequence that includes the promoter necessary for transcription of a flagellar gene. The invertible segment is flanked by two sites that are necessary for the inversion and contains a gene (hin) whose product mediates the inversion event. The hin gene shows extensive homology with the TnpR gene carried on the Tn3 transposon. It is also homologous with the gin gene carried on bacteriophage mu. These relationships suggest that the phase variation system may have evolved by the association of a transposon with a resident gene and the subsequent specialization of these elements to regulate flagellar antigen expression.
Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Flagelina/genética , Salmonella/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Genes , Recombinação GenéticaRESUMO
Fertilization initiates in the egg a dramatic increase in intracellular calcium that opens ion channels and causes exocytosis. To explore the possibility that these events might involve a receptor-mediated pathway, receptors for serotonin or acetylcholine (M1 muscarinic) were expressed in the Xenopus egg; serotonin or acetylcholine then could initiate a series of responses similar to those normally initiated by sperm. Thus, there may be an endogenous receptor in the egg membrane that is activated by sperm, and the serotonin or M1 muscarinic receptor may replace the sperm receptor in this pathway.
Assuntos
Fertilização , Receptores Muscarínicos/fisiologia , Receptores de Serotonina/fisiologia , Animais , Clonagem Molecular , Grânulos Citoplasmáticos/fisiologia , Endocitose , Exocitose , Feminino , Proteínas de Ligação ao GTP/fisiologia , Engenharia Genética , Fosfatos de Inositol/fisiologia , Masculino , Potenciais da Membrana , Interações Espermatozoide-Óvulo , Xenopus laevisRESUMO
To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.
Assuntos
Encéfalo/fisiologia , Precursores de Proteínas/fisiologia , Hormônio Liberador de Tireotropina/fisiologia , Sequência de Aminoácidos , Animais , DNA/genética , Hipotálamo/fisiologia , Peso Molecular , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/análogos & derivados , Ratos , Ratos Endogâmicos , Hormônio Liberador de Tireotropina/genéticaRESUMO
Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.
Assuntos
Metalotioneína/genética , Adeno-Hipófise/metabolismo , Somatostatina/genética , Animais , DNA Recombinante/metabolismo , Genes , Engenharia Genética , Humanos , Técnicas Imunoenzimáticas , Hormônio Luteinizante/metabolismo , Camundongos , Ratos , Somatostatina/metabolismoRESUMO
The deduced amino acid sequence of a Drosophila gene isolated with a vertebrate sodium channel complementary DNA probe revealed an organization virtually identical to the vertebrate sodium channel protein; four homologous domains containing all putative membrane-spanning regions are repeated in tandem with connecting linkers of various sizes. All areas of the protein presumed to be critical for channel function show high evolutionary conservation. These include those proposed to function in voltage-sensitive gating, inactivation, and ion selectivity. All 24 putative gating charges of the vertebrate protein are in identical positions in the Drosophila gene. Ten introns interrupt the coding regions of the four homology units; introns with positions conserved among homology units bracket a region hypothesized to be the selectivity filter for the channel. The Drosophila gene maps to the right arm of the second chromosome in region 60D-E. This position does not coincide with any known mutations that confer behavioral phenotypes, but is close to the seizure locus (60A-B), which has been hypothesized to code for a voltage-sensitive sodium channel.
Assuntos
Drosophila/genética , Canais Iônicos , Proteínas de Membrana/genética , Sódio/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , DNA/genética , Enzimas de Restrição do DNA , Drosophila melanogaster/genética , Electrophorus/genética , Éxons , Regulação da Expressão Gênica , Íntrons , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
The continuous presence of nerve growth factor (NGF) is thought to be required for the elaboration of neuronal-like traits in PC12 cells. Surprisingly, we find that a 1 min exposure to NGF is sufficient to engage a longer-term genetic program leading to the acquisition of membrane excitability. Whereas continuous exposure to NGF causes the induction of a family of sodium channels, the effect of a brief exposure is to induce selectively expression of the peripheral nerve-type sodium channel gene PN1, through a distinct signaling pathway requiring immediate-early genes. A 1 min exposure of PC12 cells to interferon-gamma also causes PN1 gene induction, suggesting that the "triggered" NGF and interferon-gamma signaling pathways share common molecular intermediates.
Assuntos
Expressão Gênica/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Neurônios/fisiologia , Canais de Sódio/biossíntese , Animais , Northern Blotting , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genes Precoces/efeitos dos fármacos , Interferon gama/farmacologia , Cinética , Neurônios/efeitos dos fármacos , Células PC12 , Técnicas de Patch-Clamp , Nervos Periféricos/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Genetic elements involved in cell-specific expression of the type II sodium channel gene were revealed using transient expression assays. A chimeric reporter gene containing 1051 bp of the sodium channel 5' flanking region was active in neuroblastoma and PC12 cells, but inactive in nonneuronal cell types. Deletion of upstream sequences resulted in an 80-fold increase in reporter gene activity in skeletal muscle cells, suggesting the presence of negative elements. Although no homologies were found between sequences in the type II 5' flanking region and other negative elements or "silencers," a small region common to the type II gene and other genes expressed in the nervous system was identified and may be involved in transcriptional regulation of neuronal genes.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Sequências Reguladoras de Ácido Nucleico , Canais de Sódio/metabolismo , Animais , Sequência de Bases , Quimera/genética , Genes , Dados de Sequência Molecular , Músculos/metabolismo , Mutação , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Ratos , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
In the protochordate Halocynthia roretzi, voltage-activated sodium current undergoes a change in kinetics within 48 hr of fertilization. Molecular cloning and microinjection of antisense DNA into single cells suggest that the kinetic changes are due to the increased expression of a putative neural-specific sodium channel gene, TuNa I. TuNa I gene transcription is first induced in late stage gastrulae, preceding the appearance of the rapidly inactivating sodium current unique to neural cells. In cleavage-arrested and intact embryos, cell interactions between specific animal and vegetal blastomeres are required for induction of TuNa I gene expression. Our results implicate cell contact, prior to neurulation, as a mechanism for selectively activating the TuNa I gene expressed in cells of the neural lineage.
Assuntos
Comunicação Celular , Expressão Gênica , Sistema Nervoso/embriologia , Canais de Sódio/genética , Urocordados/embriologia , Animais , Sequência de Bases , Blastômeros/citologia , Blastômeros/fisiologia , Clonagem Molecular , DNA Antissenso/farmacologia , DNA Complementar/química , Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Hibridização In Situ , Microinjeções , Dados de Sequência Molecular , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Canais de Sódio/fisiologia , Urocordados/crescimento & desenvolvimento , Urocordados/metabolismoRESUMO
Neural-specific expression of a sodium channel mini-gene has been shown to be mediated by a 28 bp silencer element, RE1, located in the 5' flanking region of the gene. This element is active exclusively in cell lines that do not express the endogenous brain type II sodium channel gene, including fibroblast, skeletal muscle, and certain neuronal cell lines. All of these non-type II expressing cells contain RE1-binding complexes. On the basis of mutational analysis and in vivo "repressor trap" experiments, we propose that cell-specific RE1-binding proteins are responsible, at least in part, for restricting expression of the type II sodium channel gene to specific neurons in the vertebrate nervous system.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genes Reguladores/genética , Neurônios/fisiologia , Canais de Sódio/genética , Neoplasias das Glândulas Suprarrenais/patologia , Neoplasias das Glândulas Suprarrenais/ultraestrutura , Animais , Sequência de Bases , Células Cultivadas , DNA/genética , DNA de Neoplasias/genética , Desoxirribonucleases , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Genes Reguladores/fisiologia , Dados de Sequência Molecular , Músculos/citologia , Músculos/ultraestrutura , Mutação/genética , Neurônios/citologia , Neurônios/ultraestrutura , Feocromocitoma/patologia , Feocromocitoma/ultraestrutura , Ratos , Sequências Reguladoras de Ácido Nucleico , Canais de Sódio/fisiologia , Transfecção , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/ultraestruturaRESUMO
Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.
Assuntos
Axônios/metabolismo , Bainha de Mielina/metabolismo , Nervo Óptico/crescimento & desenvolvimento , Canais de Sódio/metabolismo , Animais , Axônios/ultraestrutura , Imuno-Histoquímica , Camundongos , Camundongos Mutantes Neurológicos/anatomia & histologia , Camundongos Mutantes Neurológicos/crescimento & desenvolvimento , Camundongos Mutantes Neurológicos/metabolismo , Bainha de Mielina/ultraestrutura , Nervo Óptico/metabolismo , Nervo Óptico/ultraestrutura , Nervos Periféricos/crescimento & desenvolvimento , Nervos Periféricos/metabolismo , Nervos Periféricos/ultraestrutura , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/ultraestrutura , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/ultraestrutura , Canais de Sódio/genéticaRESUMO
Four critically positioned amino acids on each of the alpha, beta, delta, and gamma subunits of the Torpedo nicotinic acetylcholine receptor are determinants of channel conductance. Our results show that the gamma and epsilon subunits of Xenopus muscle receptors are identical at all four positions, despite the fact that alpha 2 beta delta epsilon receptors have a 50% greater conductance than alpha 2 beta delta gamma receptors. Instead, the functional difference is conferred by a single charged residue that lies extracellular to all four positions, corresponding to a location in the Torpedo receptor previously shown to have no influence on conductance. Substitution of a positively charged lysine residue in gamma by the neutral methionine in epsilon at this extra-cellular position is responsible for the increased conductance during maturation of the amphibian neuromuscular junction.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Receptores Colinérgicos/química , Receptores Colinérgicos/fisiologia , Xenopus/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Condutividade Elétrica , Eletroquímica , Feminino , Cinética , Larva/química , Lisina/química , Substâncias Macromoleculares , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Mutagênese , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Receptores Colinérgicos/genética , Relação Estrutura-Atividade , TorpedoRESUMO
The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.
Assuntos
Córtex Cerebral/fisiologia , Neurônios/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células COS , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Cromatina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Histona Desacetilase 2 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2 , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células PC12 , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Canais de Sódio/genética , Canais de Sódio/fisiologia , Fatores de Transcrição/genética , Transfecção , Dedos de ZincoRESUMO
This study examined the diversity of Na+ channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na+ channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na+ channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-1, and rat brain-2 Na+ channel, thereby identifying pCSC-1 as a related member of the family of Na+ channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na+ channel gene probes, expression of the rat brain-1 Na+ channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na+ channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na+ channel genes, which appears to be expressed in a cardiac-specific manner.
Assuntos
Regulação da Expressão Gênica , Miocárdio/metabolismo , RNA Mensageiro/genética , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Encéfalo/enzimologia , Células Cultivadas , Dados de Sequência Molecular , Músculos/metabolismo , Hibridização de Ácido Nucleico , Fosfotransferases/metabolismo , RatosRESUMO
To investigate the mechanisms responsible for urinary acidification in the terminal nephron, primary cultures of cells isolated from the renal papilla were grown as monolayers in a defined medium. Morphologically, cultured cells were epithelial in type, and similar to collecting duct principal cells. Cell pH measured fluorometrically in monolayers grown on glass slides showed recovery from acid loads in Na+-free media. Recovery was inhibited by cyanide, oligomycin A, and N-ethylmaleimide. Cyanide and oligomycin inhibited recovery less in the presence than in the absence of glucose. When cells were first acid loaded in a Na+-free medium and then exposed to external Na+, pH recovery also took place. This recovery exhibited first-order dependence on Na+ concentration and was inhibited by 5-(N-ethyl-N-isopropyl)amiloride. These studies demonstrate that in culture, collecting duct principal cells possess at least two mechanisms for acid extrusion: a proton ATP-ase and an Na+-H+ exchanger. The former may be responsible for some component of the urinary acidification observed in the papillary collecting duct in vivo; the role of the latter in acid-base transport remains uncertain.
Assuntos
Equilíbrio Ácido-Base , Medula Renal/fisiologia , Túbulos Renais Coletores/fisiologia , Túbulos Renais/fisiologia , Equilíbrio Ácido-Base/efeitos dos fármacos , Animais , Cianetos/farmacologia , Etilmaleimida/farmacologia , Concentração de Íons de Hidrogênio , Medula Renal/efeitos dos fármacos , Medula Renal/ultraestrutura , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/ultraestrutura , Microscopia Eletrônica , Oligomicinas/farmacologia , RatosRESUMO
Multiple Na+ channel types, differing in functional properties, have been identified in the nervous system. The role of distinct alpha subunits in generating this functional diversity is discussed in light of the recent finding that the beta 1 subunit modulates Na+ channel function. Possible mechanisms involved in the regulation of the genes coding for the different subunits are also discussed.
Assuntos
Fenômenos Fisiológicos do Sistema Nervoso , Canais de Sódio/fisiologia , Potenciais de Ação/fisiologia , Animais , HumanosRESUMO
The mechanisms underlying transcriptional activation and repression have become much clearer. Recent evidence suggests that transcription factors that do not bind DNA directly, the co-activators and co-repressors, mediate a large number of cell signaling events. Their association with histone acetylases, to mediate activation, or deacetylases, to mediate repression, provide a model for explaining how gene expression is regulated.
Assuntos
Regulação da Expressão Gênica , Fenômenos Fisiológicos do Sistema Nervoso , Transdução de Sinais/genética , Ativação Transcricional/fisiologia , Proteína de Ligação a CREB , Proteínas Nucleares/genética , Transativadores/genéticaRESUMO
The functional properties of most sodium channels are too similar to permit identification of specific sodium channel types underlying macroscopic current. Such discrimination would be particularly advantageous in the nervous system in which different sodium channel family isoforms are coexpressed in the same cell. To test whether members of the mu-conotoxin family can discriminate among known neuronal sodium channel types, we examined six toxins for their ability to block different types of heterologously expressed sodium channels. PIIIA mu-conotoxin blocked rat brain type II/IIA (rBII/IIA) and skeletal muscle sodium current at concentrations that resulted in only slight inhibition of rat peripheral nerve (rPN1) sodium current. Recordings from variant lines of PC12 cells, which selectively express either rBII/IIA or rPN1 channel subtypes, verified that the differential block by PIIIA also applied to native sodium current. The sensitivity to block by PIIIA toxin was then used to discriminate between rBII/IIA and rPN1 sodium currents in NGF-treated PC12 cells in which both mRNAs are induced. During the first 24 hr of NGF-treatment, PN1 sodium channels accounted for over 90% of the sodium current. However, over the ensuing 48 hr period, a sharp rise in the proportion of rBII/IIA sodium current occurred, confirming the idea, based on previous mRNA measurements, that two distinct sodium channel types appear sequentially during neuronal differentiation of PC12 cells.
Assuntos
Conotoxinas/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Neurônios/fisiologia , Canais de Sódio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Eletrofisiologia , Expressão Gênica/fisiologia , Ativação do Canal Iônico/fisiologia , Fator de Crescimento Neural/farmacologia , Neurônios/química , Neurônios/efeitos dos fármacos , Oócitos/fisiologia , Células PC12 , RNA Mensageiro/análise , Ratos , Sódio/metabolismo , Canais de Sódio/genética , XenopusRESUMO
Expression of a Moloney murine leukemia virus (MLV) rat somatotropin fusion gene was examined in a transgenic pig. The fusion gene was integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated MLV-rat somatotropin fusion gene produced high levels of circulating rat somatotropin and resulted in an elevation in the circulating levels of insulin-like growth factor I. Although there was no increase in the rate of growth of the transgenic animal during the rapid growth phase, several phenotypic changes were evident. Skeletal growth was markedly increased and fat deposition was reduced throughout the animal. Blood glucose levels were elevated without ketosis. Northern blot analyses of rat somatotropin RNA revealed that expression of the fusion gene was highest in the spleen, lung, intestine, lymph nodes, and bone marrow. These results show that the MLV promoter can be used to express high levels of biologically active rat somatotropin in transgenic swine.
Assuntos
Hormônio do Crescimento/genética , Vírus da Leucemia Murina de Moloney/genética , Animais , Animais Geneticamente Modificados , SuínosRESUMO
Plasmids encoding the amino terminal portion of an influenza virus hemagglutinin (HA) fused to polyoma virus middle T (mT) or large T (lT) sequences have been constructed. Stable expression of the chimeric proteins was obtained in established rat embryo fibroblasts following plasmid co-transfection and selection for G418 resistance. The synthesis and localization of the proteins was followed by metabolic labeling with [35S]methionine and [3H]mannose, cell fractionation, and immunoprecipitation with anti-polyoma T antibody. The HA leader and amino terminal peptide direct the synthesis of the lT and mT proteins into the endoplasmic reticulum where they undergo glycosylation, but this occurs with a very low efficiency. Most of the HA-mT and HA-lT fusion protein molecules do not enter completely into the endoplasmic reticulum, but rather achieve their normal locations in the cell as slightly higher molecular weight proteins, presumably due to the extra sequences derived from HA at their amino termini. HA-mT fusion protein is found to have associated tyrosine-specific protein kinase activity precipitable with anti-src as well as anti-T antibody, and cells expressing this fusion protein have a transformed phenotype.