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MOLECULAR CLONING AND IN VITRO CHARACTERIZATION OF THE ASPERGILLUS FUMIGATUS CAMP-DEPENDENT PROTEIN KINASE

by OLIVER, BRIAN G.

Abstract (Summary)
Cyclic AMP signaling has been shown to be essential for growth, morphology and virulence in fungal pathogens of plants and animals. However, the function of this pathway is unknown in the opportunistic pathogen, Aspergillus fumigatus. As a first step toward elucidating the function of this pathway in A. fumigatus the genes encoding both the regulatory and catalytic subunits were cloned and sequenced and the response to exogenous cAMP was characterized. The availability of pkaR and pkaC sequences was used to construct vectors, facilitating future studies into the function of these genes. The regulatory subunit gene, pkaR, is expressed from an intronless coding sequence and the predicted translation product shares 79% sequence identity with the regulatory subunit of Aspergillus nidulans, both of which are defined as a type II regulatory subunits by the presence of a conserved autoinhibition site and two cAMP-binding domains. The gene encoding the A. fumigatus catalytic subunit, pkaC, is interrupted by three introns. The predicted PkaC protein shares 83% sequence identify with A. nidulans PkaC, and contains domains that are characteristic of PkaC proteins. Both pkaR and pkaC mRNAs are expressed throughout the asexual lifecycle of A. fumigatus. In regard to the effects of exogenous cAMP, both cAMP and phosphodiesterase inhibitors markedly reduced radial growth rate of A. niger, which has previously been demonstrated to respond exogenous cAMP, after 48 hours on minimal media with glucose as the carbon source, whereas growth of A. fumigatus was not affected. However, when glycerol, which does not initiate carbon catabolite repression, was used as a carbon source, cAMP inhibited the radial growth rate of only A. fumigatus (p<0.05). The addition of cAMP to glycerol-minimal medium resulted in a two-fold increase in protein kinase A activity in A. fumigatus cell extracts when compared with negative controls. The PKA activity in A. fumigatus cell extracts from cultures grown in glucose did not change significantly with the addition of cAMP. This document reports the first analyses of the PKA pathway in A. fumigatus and provides the foundation for future analysis of PKA function in the growth, and potentially the virulence, of A. fumigatus.
Bibliographical Information:

Advisor:

School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:aspergillus camp pka fungus

ISBN:

Date of Publication:01/01/2001

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