Cloning and sequencing of the cDNA encoding Pneumocystis carinii inosine monophosphate dehydrogenase (IMPDH).

Important note: Information in this article was accurate in 1994. The state of the art may have changed since the publication date.

Cloning and sequencing of the cDNA encoding Pneumocystis carinii inosine monophosphate dehydrogenase (IMPDH).

Abstr Gen Meet Am Soc Microbiol. 1994;94:590 (abstract no. F-13). Unique Identifier : AIDSLINE ASM94/94313113
O'Gara MJ; Queener SF; Lee CH; Indiana University School of Medicine, Indianapolis.

Abstract: Pneumocystis carinii is an opportunistic pathogen that causes pneumonia in immunocompromised hosts. Chemotherapy is available for treating P. carinii pneumonia, but severe toxicity is a significant problem with the compounds currently considered drugs of choice. IMP dehydrogenase (IMPDH; EC 1.1.1.205) may be a good alternative target for anti-Pneumocystis chemotherapy because IMPDH inhibitors have been used successfully to treat leukemia. Our lab found the IMPDH inhibitor, mycophenolic acid, to be effective in preventing the growth of P. carinii in culture. In order to characterize P. carinii IMPDH and assay potential inhibitors in vitro, we cloned part of the IMPDH gene. Initially, we amplified a 1.3 kb fragment of P. carinii IMPDH from genomic DNA using PCR and two degenerate oligonucleotides based on two conserved sequences identified in IMPDH from six different species. The DNA sequence of the 1.3 kb product indicated the presence of putative introns having the characteristic 5'-GT and AG-3' signal. Using a combination of RACE (rapid amplification of cDNA ends) and RT-PCR, we isolated and cloned the entire cDNA of the P. carinii IMPDH. The nucleotide and amino acid sequences (57-59% and 32-63%, respectively) revealed significant homology with published sequences of IMPDH genes. We are currently working to express P. carinii IMPDH which will be assayed to confirm catalytic activity.

Keywords: Amino Acid Sequence Base Sequence Cloning, Molecular/*METHODS Comparative Study DNA, Complementary/*ANALYSIS DNA, Fungal/ANALYSIS/GENETICS Gene Amplification IMP Dehydrogenase/*GENETICS Pneumocystis carinii/*ENZYMOLOGY/*GENETICS Polymerase Chain Reaction/METHODS Sequence Homology, Amino Acid Sequence Homology, Nucleic Acid ABSTRACT
941030
M94A0841

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