Differential splicing of Pneumocystis carinii f. sp. carinii inosine 5 '-monophosphate dehydrogenase pre-mRNA

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in guanine nucleotide metabolism that has drawn attention as a drug target in several organisms. Pneumocystis carinii f. sp. carinii IMPDH mRNA (GeneBan k Accession No: U42442) previously identified from cultured organisms yield ed a predicted amino acid sequence about 70 amino acids shorter at the amin o terminus than IMPDH from other species. Recent research has shown that th e amino terminal region is important for enzyme activity, suggesting that t he previous putative P. carinii IMPDH might not represent full length, func tional enzyme. To test this hypothesis, RT-PCR was performed with total RNA isolated from P. carinii f. sp. carinii. Three IMPDH splicing variants wer e found and splicing preference was observed: P. carinii isolated from infe cted rat lung contained primarily splicing variant one (introns two and fou r deleted), but organisms from spinner flask culture contained primarily sp licing variant three tall four introns deleted). Importantly, splicing vari ant one (GeneBank Accession No: AF196975) contained an open reading frame f or 529 amino acids, a size comparable to that of other eukaryotic IMPDH for ms. The other variants contained the same open reading frame (454 amino aci ds) previously reported. Sequence analysis and complementation studies sugg est variant one represents the full length, catalytically active form of P. carinii IMPDH. The differential splicing of the enzyme may reflect a mecha nism by which the organism regulates the expression of IMPDH in response to environmental stresses. (C) 2001 Elsevier Science B.V. All rights reserved .