Identification and expression of novel PKA genes in Mucor circinelloides

LUCAS FERNÁNDEZ NUÑEZ; JOSEFINA OCAMPO; PAULA GIOINO; NICOLÁS BARDECI; SILVIA ROSSI; SILVIA MORENO

Villa Carlos Paz

Congreso; SAIB; 2008

SAIB

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  (MUstrain); 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 PKAC. 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 the mutant, 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.