IDENTIFICATION AND EXPRESSION OF NOVEL PKA GENES IN MUCOR CIRCINELLOIDES

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

Carlos Paz, Córdoba, Argentina

Congreso; XLIV Reunion Anual de SAIB; 2008

Sociedad Argentina de Bioquimica y Biologia Molecualr

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.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.