Identification and expression of novel PKA genes in Mucor circinelloides

FERNANDEZ NUÑEZ,L., OCAMPO, J., GIOINO, P., BARDECI, N., ROSSI, S. AND MORENO, S

Córdoba, Carlos Paz

Congreso; XLIV Reunión Anual SAIB; 2008

Sociedad Argentina de Bioquímica

ST-P03. IDENTIFICATION AND EXPRESSION OF NOVEL PKA GENES IN Mucor circinelloides Fernández Núñez L, Ocampo J, Gioino P, Bardeci N, Rossi S, Moreno S. Departamento de Quimica Biológica, Facultad de Ciencias Exactas y Naturales, UBA E-mail: lfnunez@qb.fcen.uba.ar We are interested in the PKA (R2C2) of the fungus Mucor circinelloides. Until recently its genome was unknown. A sequence for the C subunit (pkaC) and R subunit (pkaR) from M.circinelloides was available. In a previous work we had disrupted the pkaR gene from M.circinelloides (MUDR strain); however this K.O. resulted in a decreased but not null cAMP binding activity and in a PKA still dependent on cAMP. The genome of this fungus is now cloned and the sequence available to us. A BLAST search of the genome using the already known sequences from this fungus and from mammals, resulted in the identification of 4 genes for R and 6 genes for C. The already described pkaC appears not to be a bonafide PKA C. The protein sequences were predicted and bioinformatic comparisons performed. Sq-RT PCR of a wt strain, in different stages of growth indicated that all the subunits are expressed in at least one stage of growth, including pkaC; the introns were confirmed. Three of the four R were identified by MALDI-TOF MS in purified preparations of R. The analysis of the expression of C and R in MUDR, in which only one R isoform is deleted, suggests that the other three R genes tend to compensate the lack of pkaR1. It is the first time that a fungal system with more than one R is described. The differential expression of some of the subunits could indicate specific functions for each stage.