Cloning of the S-Adenosylhomocysteine Hydrolase Gene of Pneumocystis carinii

Biologic methylations play key roles in control of gene expression [2], protein h c t i o n [6] , and lipid structure and function [4]. In eukaryotes and many prokaryotes, these methylations are mediated by the activated methyl cycle, a pathway of reactions catalyzing the transfer of methyl groups from S-adenosylmethionine ( S A M ) to other cellular constituents and the regeneration of SAM. Sadenosylmethionine dependent methyltransferases catalyze the transfer of methyl groups from SAM converting it to Sadenosylhomocysteine (SAH) . This product works as a feedback inhibitor of the transfer reaction, so it must be removed from the cellular environment. This is achieved through the action of Sadenosylhomocysteine hydrolase (SAHH), which converts SAH to adenosine and L-homocysteine (L-HC). Inhibitors of SAHH action, such as 3deazaadenosinc and 2deoxyadenosine, increase the cellular levels of SAH or its analogue by reversing the direction of the hydrolase reaction, thereby preventing transmethylations [5,8]. In this short communication we report the cloning and sequencing of an SAHH gene from a P. curinii cDNA library. MATERIALS AND METHODS. Thirty cycles of polymerase chain reaction using P. curinii genomic DNA and primers designed to amplify a portion of the cysteine proteinase gene yielded a 297 bp fragment of DNA homologous to the SAHH gene of several sources. The PCR primers, CP3-1 and 458-CPF, were designed by aligning the sequences of the putative 5' and 3' active sites of three cysteine proteinase genes, those of D. farinue, S. cerevisiue, and S. thermophifus. Degeneracy of the primers was limited to a factor of 512, and the wobble position of codons was biased to A or T, in agreement With many P. curinii genes [3]. A P. curinii cDNA library constructed in the hZAPII (Stratagene) vector was screened using the PCR product as a probe. A single positive plaque was purified and the plasmid pBK-CMV was isolated by in vivo excision utilizing fl helper phage. Insert positive plasmids were then sequenced from both directions. Internal sequencing primers were designed to enable primer walking in order to sequence the entire fragment of cDNA. RESULTS AND DISCUSSION. Northern blot analysis confirmed the P. cun'nii origin of a 297 bp fragment of the P. curinii SAHH gene obtained by PCR designed to amplify a portion of P. curinii cysteine proteinase gene. Hybridization of a randomly primed probe of the PCR product showed signal only in those lanes corresponding to P. curinii infected HEL cells or rat lung. No signal was detected in lanes of total RNA from uninfected cells or uninfected rat lung. Screening of a P. curinii cDNA library yielded a clone of 1442 bp, coding for a 441 amino acid fragment of the P. curinii SAHH (Fig. 1). This library clone is 60% homologous with the rat SAHH gene at the nucleic acid level, and has 70% identity with the rat amino acid sequence for SAHH. In agreement with other P. curinii ruttus genes, the SAHH contains a G+C content of 35%, and 78% of codons in the open reading frame show an A or T bias in the wobble position. A single open reading frame is noted from position '10 to a putative stop codon at position '1332. No putative start codon is noted, attempts to amplify this region using PCR primers complementary to a portion of the library vector and a sequence with the SAHH cDNA fragment using WII library DNA as a template were unsuccessful. Alignment of DNA sequences of several SAHH genes and the P. curinii SAHN we isolated show significant homology in the putative NAD co-enzyme binding site [7], as well as the putative catalytic site [I], as underlined in Fig. 1. I