RESUMEN
We have isolated and characterized cDNA and genomic DNA clones encoding the catalytic subunit (C) of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. The C-subunit amino acid sequence derived from the nucleotide sequence predicts a basic polypeptide of 424 residues, excluding the initiator methionine, which by amino-terminal sequence analysis has been shown to be absent from the mature protein. The Blastocladiella C presents a 70-amino-acid extension at the amino terminus, when aligned to the mouse C alpha subunit, being one of the largest C subunits already characterized. The B. emersonii C-gene-coding region is interrupted by three introns, ranging in size over 57-69 bp. The positions of the introns are quite different from those found in other species, suggesting a considerable amount of evolutionary drift in the gene structure. The 5'-flanking region lacks recognizable TATA or CCAAT sequences, is remarkably high in GC content (70%), and primer extension experiments indicate that transcription initiates from multiple sites. Several sequence motifs were identified in the promoter region which could be involved in the developmental control of this gene.
Asunto(s)
Blastocladiella/enzimología , Blastocladiella/genética , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Genes Fúngicos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Cartilla de ADN , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , ADN Complementario/metabolismo , Exones , Biblioteca Genómica , Intrones , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene.
Asunto(s)
Blastocladiella/genética , Genes Fúngicos , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Blastocladiella/enzimología , Southern Blotting , Clonación Molecular , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Biblioteca Genómica , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Proteínas Quinasas/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.
Asunto(s)
Blastocladiella/enzimología , Regulación Fúngica de la Expresión Génica/genética , Proteínas Quinasas/genética , Blastocladiella/genética , Blastocladiella/crecimiento & desarrollo , Northern Blotting , Western Blotting , Diferenciación Celular/genética , AMP Cíclico/metabolismo , Sustancias Macromoleculares , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/metabolismoRESUMEN
A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo.