R subunits isoforms of PKA from Mucor circinelloides: functional and biochemical characterization

OCAMPO J; PEREYRA, E; MORENO, S; GARRE, V; ROSSI, S

San Miguel de Tucuman, Tucuman, Argentina

Congreso; XLV Reunion Nacional de SAIB; 2009

Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular

In M. circinelloides PKA is a tetramer composed of two Regulatory subunits (R) and two Catalytic subunits (C).We demonstrated the existence of 4 genes for R subunit and 10 for C subunits.We disrupted pkaR1 and pkaR2 genes to study their role in regulation of morphological and cellular development. pkaR1D showed a reduction in growth and alterations in germination rates, cell volume, germ tube length, and asexual sporulation. In pkaR2D the only difference observed was that the germ tube emission was earlier than in the wild-type strain. The expression of all genes was detected by semiquantitative RT-PCR and they showed differential expression at different developmental stages.The expressions of the enes were also analyzed in pkaR1D which showed no variations in the expression of the other genes, and in pkaR2D which showed no expression of pkaR3 and an increased expression of pkaR4. Analysis of the nucleotide sequences of M. circinelloides pkaR genes reveled that they arose by gene duplication events, pkaR3 gene being less closely related to the other three.Our results suggest that these duplication events occurred very early in the zygomycete lineage. The results confirm that PKA is involved in M. circinelloides physiology and due to the existence of four R and ten C subunits there would be a multiplicity of possible PKA holoenzymes which could have different functional significanceD showed a reduction in growth and alterations in germination rates, cell volume, germ tube length, and asexual sporulation. In pkaR2D the only difference observed was that the germ tube emission was earlier than in the wild-type strain. The expression of all genes was detected by semiquantitative RT-PCR and they showed differential expression at different developmental stages.The expressions of the enes were also analyzed in pkaR1D which showed no variations in the expression of the other genes, and in pkaR2D which showed no expression of pkaR3 and an increased expression of pkaR4. Analysis of the nucleotide sequences of M. circinelloides pkaR genes reveled that they arose by gene duplication events, pkaR3 gene being less closely related to the other three.Our results suggest that these duplication events occurred very early in the zygomycete lineage. The results confirm that PKA is involved in M. circinelloides physiology and due to the existence of four R and ten C subunits there would be a multiplicity of possible PKA holoenzymes which could have different functional significance