RESUMO
Caveolin-1 traffics cholesterol between the endoplasmic reticulum and cell surface caveolae in a non-vesicle chaperone complex which contains heat shock protein 56, cyclophilin 40, and cyclophilin A. Recent studies demonstrate that endothelial nitric oxide synthase (eNOS), caveolin, hetero-trimeric G-protein coupled receptors, and a calcium channel form an activation complex that is associated with cholesterol-rich caveolae. Oxidized LDL depletes caveolae of cholesterol and prevents agonist stimulation of eNOS by disrupting the activation complex. HDL antagonizes the effects of oxLDL by donating cholesterol to caveolae, thereby preserving the structure and function of caveolae. These findings and others provide a possible mechanistic basis for some of the molecular changes observed in vascular disease.
Assuntos
Caveolinas/farmacologia , Colesterol/farmacologia , Lipoproteínas/farmacologia , Óxido Nítrico Sintase/metabolismo , Doenças Vasculares/metabolismo , Caveolina 1 , Humanos , Óxido Nítrico Sintase Tipo III , Receptores de Lipoproteínas/metabolismoRESUMO
Prolyl 4-hydroxylase (EC 1.14.11.2) converts peptidyl proline to peptidyl hydroxyproline in procollagen polypeptides during collagen biosynthesis. The active enzyme is a tetramer which is composed of two pairs of non-identical subunits in a molecular form of alpha 2 beta 2. In addition to the tetrameric prolyl 4-hydroxylase (alpha 2 beta 2), the free beta-subunit is also found inside cells. Recently it was shown that the beta-subunit of prolyl 4-hydroxylase is identical to the protein disulfide isomerase and cellular thyroid hormone-binding protein. We previously isolated and characterized cDNAs of the beta-subunit of chicken prolyl 4-hydroxylase. The cDNA of beta-subunit was used to screen a chicken genomic DNA library constructed with the lambda EMBL-3 vector. Two clones, lambda gCPH beta-22 and beta-50, were isolated and characterized by restriction enzyme analysis, heteroduplex analysis, and nucleotide sequencing. The results showed that the 2.5-kb mRNA of the beta-subunit is divided into eleven exons and that the gene is 9.0 kb long. The gene contains consensus sequence for TATA at -24 bp and four CAAT at -57, -157, -194 and -223 bp in the 5' end flanking sequence of the transcription start point. In addition, there are three GC boxes upstream from the TATA box and four GC boxes in the first intron. This is similar to the structure of the alpha 1(I) collagen coding gene (COL1A1). These elements may interact with nuclear factors and play important roles in expression regulation of the beta-subunit gene as has been described in COL1A1.
Assuntos
Genes , Pró-Colágeno-Prolina Dioxigenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , DNA/genética , Endonucleases/antagonistas & inibidores , Éxons , Íntrons , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes/análise , Regiões Promotoras Genéticas , Mapeamento por Restrição , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transcrição GênicaRESUMO
Preeclampsia is associated with altered placental production of several end-products of cyclooxygenase activity. Thromboxane (TX) is one of these end-products, and trophoblast is a source of villous thromboxane. We cultured term trophoblast in the presence or absence of fibrin to study how differentiation and epithelial-matrix interactions regulate cyclooxygenase expression. The cellular trophoblast present during the first 24 h of culture on uncoated plastic produced TXB2, but little or no TX was produced in cultures grown longer than 24 h when differentiation into syncytial trophoblast occurred. Growth of cells on a fibrin matrix enhanced cellular trophoblast TX production five-fold. Medium containing 10 mumol/l arachidonic acid maximized thromboxane production in cells cultured for less than 24 h, regardless of growth surface, but this medium had little or no effect on TX production by cultures grown for more than 24 h. In contrast, exogenous arachidonic acid enhanced prostaglandin E2 (PGE2) production by both cellular and syncytial trophoblast. Cytochemical staining indicated that changes in cyclooxygenase content occurred with trophoblast differentiation. Western immunoblot analysis of cells cultured in the presence or absence of a fibrin matrix showed cyclooxygenase was induced under both growth conditions. Detectable cyclooxygenase protein disappeared beyond 24 h in cells grown on uncoated plastic. In contrast, cells grown beyond 24 h on fibrin showed sustained expression of cyclooxygenase by Western immunoblotting, and this enzyme protein expression correlated with increased PGE2 production by the differentiated trophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Diferenciação Celular , Divisão Celular , Fibrina/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Tromboxanos/biossíntese , Trofoblastos/citologia , Ácido Araquidônico/farmacologia , Células Cultivadas , Colágeno/farmacologia , Meios de Cultura , Feminino , Humanos , Gravidez , Tromboxano B2/biossíntese , Tromboxano B2/farmacologia , Trofoblastos/efeitos dos fármacosRESUMO
OBJECTIVE: Annexins are a superfamily of proteins that are thought to inhibit phospholipase A2 activity and hence inhibit prostaglandin production. The purpose of this study was to test the hypothesis that annexin I concentration in human amnion is reduced with labor and that this reduction is mediated by a decrease in annexin I messenger ribonucleic acid expression. STUDY DESIGN: Amnion and choriodecidua were collected from term singleton pregnancies, eight after spontaneous vaginal delivery and eight from elective cesarean section without labor. Annexin I protein was quantitated by Western blotting. Ribonucleic acid was isolated from amnion, and then annexin I messenger ribonucleic acid was identified by Northern hybridization and quantitated by slot blotting. RESULTS: Annexin I (35 kd) was identified in amnion tissue. The concentration in the group undergoing labor (320 +/- 45 integrated optical density units, mean +/- SE) was significantly reduced (p < 0.05) compared with that in the group not undergoing labor (635 +/- 65 units). The size of the annexin I messenger ribonucleic acid was approximately 1.8 kb. The mean integrated optical density for the labor group (840 +/- 139 units, mean +/- SE) was significantly reduced (p < 0.05) compared with that of the nonlabor group (1912 +/- 464 units). CONCLUSION: There is a significant decrease in annexin I messenger ribonucleic acid expression in human amnion with labor, corresponding to a significant decrease in annexin I protein concentration. This may contribute to the increased phospholipase A2 activity, arachidonic acid mobilization, and prostaglandin production at labor in humans.
Assuntos
Âmnio/metabolismo , Anexina A1/genética , Trabalho de Parto/metabolismo , RNA Mensageiro/metabolismo , Anexina A1/metabolismo , Northern Blotting , Western Blotting , Feminino , Humanos , GravidezRESUMO
Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.
Assuntos
Âmnio/metabolismo , Anexinas/metabolismo , Córion/metabolismo , Trabalho de Parto/fisiologia , Anticorpos Monoclonais , Western Blotting , Cálcio/farmacologia , Decídua/metabolismo , Feminino , Humanos , Fosfolipases/metabolismo , Fosfolipídeos/metabolismo , Gravidez , Prostaglandinas/biossínteseRESUMO
The role of perfusate amino acid concentrations in regulating rates of protein synthesis was investigated using the perfused rat liver. Livers from fed rats were perfused with a nonrecirculating medium and the incorporation of [3H]leucine into albumin and total protein was determined under conditions where the leucyl-tRNALeu and perfusate leucine specific activities were equal and constant. During perfusions of less than 1 h, rates of total protein synthesis were sensitive to the concentrations of amino acids in the perfusate. When no exogenous amino acids were provided, rates of synthesis of albumin and total protein were 40% of the maximal rates which were achieved when the medium was supplemented with 5 times the normal plasma concentrations of amino acids. However, rates of synthesis in livers perfused with amino acid-deficient medium rose with extension of the duration of perfusion to 95 min. The defect induced by amino acid deficiency did not appear to result from reductions in the charging of tRNA since no change in the quantities of amino acids bound to tRNA occurred in the amino acid-deficient perfusion. The recovery of protein synthesis with time was prevented by inhibitors of proteolysis suggesting a role for protein degradation in this phenomenon.
Assuntos
Aminoácidos/metabolismo , Fígado/metabolismo , Biossíntese de Proteínas , Animais , Radioisótopos de Carbono , Cinética , Masculino , Perfusão , Técnica de Diluição de Radioisótopos , Ratos , Ratos EndogâmicosRESUMO
The extent of phosphorylation of protein phosphatase inhibitor-1 in skeletal muscle rose about 2.5-fold during 60 min of perfusion of the rat hemicorpus preparation and then did not change over the following 30 min. Addition of insulin at 60 min resulted in a 35% fall in inhibitor-1 phosphorylation by 90 min. The rise in inhibitor-1 phosphorylation was due to the presence of catecholamines as evidenced by an accumulation of epinephrine in the perfusate. Removal of the adrenal glands or cannulation of the vena cava prevented the accumulation of epinephrine and the rise in inhibitor-1 phosphorylation. Insulin did not alter the phosphorylation state of inhibitor-1 in the presence of the beta-adrenergic antagonist propranolol where the degree of phosphorylation was low (less than 10%) or at concentrations of isoproterenol (10 nM) where inhibitor-1 was highly phosphorylated (greater than 60%). In preparations with the adrenal glands removed, 0.5 nM isoproterenol produced a 2-fold rise in inhibitor-1 phosphorylation, an effect that was completely prevented by the addition of insulin. The antagonism of 0.5 nM idoproterenol by insulin correlated with a decrease in the muscle content of cyclic AMP. These results suggest that the dephosphorylation of inhibitor-1 may play an important role in the metabolic effects of insulin in vivo.
Assuntos
Proteínas de Transporte , Inibidores Enzimáticos/metabolismo , Epinefrina/farmacologia , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Isoproterenol/farmacologia , Músculos/metabolismo , Propranolol/farmacologia , Proteínas/metabolismo , Animais , AMP Cíclico/metabolismo , Cinética , Perfusão , RatosRESUMO
A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.
Assuntos
Fígado/metabolismo , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sistema Livre de Células , Cicloeximida/farmacologia , Edeína/farmacologia , Fator de Iniciação 2 em Eucariotos , Fatores de Troca do Nucleotídeo Guanina , Guanosina Trifosfato/farmacologia , Cinética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/farmacologia , Fosforilação , Polivinil/farmacologia , Proteínas/farmacologia , RNA/metabolismo , RatosRESUMO
Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (alpha, beta, and gamma), and the other contained only the alpha- and gamma-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the alpha- and gamma-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the alpha-subunits using antibodies against liver eIF-2. In contrast, the beta-subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-2 beta did not recognize the beta-subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The T1/2 for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with three-subunit eIF-2. In addition, the KD for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the beta-subunit of eIF-2 and suggest a crucial role for the beta-subunit in the functional interaction of eIF-2 and GEF.
Assuntos
Fígado/análise , Fatores de Iniciação de Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Animais , Eletroforese em Gel de Poliacrilamida , Fator de Iniciação 2 em Eucariotos , Fatores de Troca do Nucleotídeo Guanina , Guanosina Difosfato/metabolismo , Magnésio/metabolismo , Masculino , Peso Molecular , Biossíntese de Proteínas , Coelhos , Ratos , Ratos EndogâmicosRESUMO
OBJECTIVES: We examined whether pregnancy modulates the expression of prostaglandin endoperoxide synthase, prostacyclin synthase, and thromboxane A2 synthase in the systemic vasculature. Further, we examined whether elevated lipid peroxidation during pregnancy (induced by vitamin E deprivation) affects the normal adaptive process to pregnancy. STUDY DESIGN: Western immunoblotting was performed on aortas from normal and vitamin E-deprived late pregnant (18 to 19 days) and age-matched virgin control rats. RESULTS: Normal pregnancy resulted in an increased expression of prostaglandin endoperoxide synthase (2.91 vs 1.06 fmol/ng deoxyribonucleic acid, p < 0.05). Surprisingly, the expression for both prostacyclin and thromboxane A2 synthases were significantly decreased by pregnancy: prostacyclin synthase 2.60 versus 13.82 units/ng deoxyribonucleic acid and thromboxane A2 synthase 1.32 versus 9.85 units/ng of deoxyribonucleic acid. Elevation of endogenous lipid peroxidation partially reversed this normal pregnancy trend in enzyme expression: prostaglandin endoperoxide synthase 1.85 fmol/ng deoxyribonucleic acid, prostacyclin synthase 9.38 units/ng deoxyribonucleic acid, thromboxane A2 synthase 4.36 units/ng deoxyribonucleic acid. CONCLUSION: Changes in prostanoid activity in the systemic vasculature during pregnancy may be mediated by concerted induction and down-regulation of specific enzymes. Increased lipid peroxidation interferes with this normal pregnant pattern. Further studies on the cell-specific expression of these genes will help to define the cardiovascular role of prostaglandins in pregnancy and in preeclampsia.
Assuntos
Aorta/enzimologia , Eicosanoides/metabolismo , Oxirredutases Intramoleculares , Peróxidos Lipídicos/farmacologia , Prenhez/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Técnicas In Vitro , Isomerases/metabolismo , Gravidez , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Sprague-Dawley , Tromboxano A2/metabolismo , Tromboxano-A Sintase/metabolismoRESUMO
A two-site ELISA and a bioassay were used to detect NGF-like activity in human brain tissue. Both assays detected mouse NGF and recombinant human NGF with approximately equal sensitivity, whereas the antibodies showed little cross-reactivity with the recombinant human proteins NT-3 and brain-derived neurotrophic factor. NGF-like activity was detected in fresh human cortical samples obtained from epileptic patients, with the highest activity observed in the right hemisphere of men. NGF-like activity was subsequently measured in autopsy samples of frontal and occipital cortex from patients with Alzheimer's disease (AD) and from individuals with no history or pathological evidence of AD. Based on both the ELISA and the bioassay measurements, NGF-like activity was significantly elevated in both brain regions in AD. These results demonstrate the feasibility of detecting NGF-like activity in both fresh and postmortem human brain tissue and further suggest that AD is characterized by increased, rather than decreased, levels of cortical beta-NGF. The AD-related increase in NGF may be a consequence of degenerative changes in the basal forebrain cholinergic system.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Fatores de Crescimento Neural/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bioensaio , Biópsia , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo , Cadáver , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: We tested the hypothesis that aspirin affects trophoblast like other epithelial cells do, by inhibiting prostanoid production, inducing prostaglandin H synthase-2 expression, and enhancing secretion of 15-hydroxyeicosatetraenoic acid. STUDY DESIGN: Cytotrophoblast from placentas (n = 15) of uncomplicated singleton pregnancies were cultured in medium 199 for 4 to 72 hours in the presence or absence of aspirin. RESULTS: Aspirin (10(-4) M) inhibited (p < 0.01) average trophoblast prostaglandin E2 release by 60% and thromboxane B2 by 86%. Western immunoblotting showed the prostaglandin H synthase-1 was constitutively expressed in cytotrophoblast, and aspirin treatment caused a twofold increase in prostaglandin H synthase-1 expression. Prostaglandin H synthase-2 was also constitutively expressed in untreated cytotrophoblast but at lower levels than prostaglandin H synthase-1. Aspirin enhanced prostaglandin H synthase-2 expression in trophoblast cultures, but prostaglandin H synthase-2 contributed a range of only 10% to 33% (n = 4) of the total cellular prostaglandin H synthase protein pool even after aspirin induction. The increased prostaglandin H synthase expression depended on both transcription and translation because actinomycin D and cycloheximide each inhibited the increased prostaglandin H synthase protein expression after aspirin treatment. The aspirin induction of prostaglandin H synthase was accompanied by decreased release of 15-hydroxyeicosatetraenoic acid. CONCLUSIONS: Trophoblast differs from other cells studied because aspirin enhances expression of both prostaglandin H synthase-1 and prostaglandin H synthase-2 isozymes while decreasing, instead of increasing, the secretion of 15-hydroxyeicosatetraenoic acid. The aspirin effects on prostaglandin H synthase synthesis and 15-hydroxyeicosatetraenoic acid release in trophoblast suggest that the mechanisms of action for aspirin in the prophylaxis of preeclampsia may be more diverse than simply altering platelet thromboxane production.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , Trofoblastos/enzimologia , Western Blotting , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Isomerismo , Inibidores da Agregação Plaquetária/farmacologia , Gravidez , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/biossíntese , Inibidores da Síntese de Proteínas/farmacologia , Radioimunoensaio , Tromboxano B2/metabolismo , Fatores de Tempo , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacosRESUMO
Conditions were defined for maintaining optimal protein synthetic activity in suspensions of freshly isolated rat hepatocytes. Under these conditions, isolated hepatocytes exhibited rates of protein synthesis and levels of polysomal aggregation equivalent to those observed in vivo and in perfused liver. Deprivation of total amino acids or single, essential amino acids resulted in a rapid decrease in the rate of protein synthesis, which was readily reversed by readdition of the deficient amino acid(s). The decrease was accompanied by a disaggregation of polysomes and an inhibition of 43S initiation complex formation, which was indicative of a limitation in the rate of initiation of protein synthesis. Extracts prepared from perfused liver deprived of amino acids were inhibitory to initiation of protein synthesis in reticulocyte lysate. The inhibition in reticulocyte lysate was accompanied by an increase in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2), suggesting activation of an eIF-2 alpha kinase or inhibition of a phosphatase in amino acid-deprived hepatocytes. This suggestion was confirmed by prelabeling hepatocytes with 32Pi before amino acid deprivation. Incorporation of 32Pi into eIF-2 alpha was two- to threefold higher in lysine-deprived cells than in hepatocytes incubated in fully supplemented medium. Overall, the results indicated that an increase in eIF-2 alpha phosphorylation was responsible for the defect in initiation of protein synthesis caused by amino acid deprivation.
Assuntos
Aminoácidos/deficiência , Fígado/metabolismo , Biossíntese de Proteínas , Trifosfato de Adenosina/metabolismo , Aminoácidos/fisiologia , Animais , Centrifugação com Gradiente de Concentração , Fator de Iniciação 2 em Eucariotos , Cinética , Lisina/fisiologia , Metionina/fisiologia , Fatores de Iniciação de Peptídeos/metabolismo , Fosfatos/metabolismo , Radioisótopos de Fósforo , Fosforilação , Polirribossomos/metabolismo , Proteínas/metabolismo , Ratos , Triptofano/fisiologiaRESUMO
Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.