Molecular cloning of a 2',3'-cyclic nucleotide 3'-phosphodiesterase: mRNAs with different 5' ends encode the same set of proteins in nervous and lymphoid tissues

Abstract

Antibodies raised to a mixture of the 46 and 48 kDa rat CNS 2',3'-cyclic nucleotide 3-phosphodiesterases (CNPs) recognized apparently identical proteins in peripheral nervous system (PNS), thymus, and circulating blood lymphocytes. These antibodies were used to identify, in a rat brain phage lambda gt11 expression library, cDNA clones encoding beta-galactosidase-CNP fusion proteins, some of which showed CNP activity. In RNA blots, the subcloned CNP cDNA inserts hybridized to mRNAs of approximately 2400 and approximately 2800 nucleotides (nts), and to a approximately 2500 nt mRNA from thymus. Several nonexpressing CNP cDNAs were identified by plaque hybridization, and the mRNA transcribed in vitro from one of these cDNAs (pCNP7) encoded a complete 46 kDa CNP polypeptide. Examination of the deduced amino acid sequence revealed an apparent homology to cAMP binding sites in several other proteins. A 373 bp segment from the 5' end of this pCNP7 hybridized only to the 2800 nt nervous system mRNAs, thus revealing that not all CNP mRNAs share the same 5'-ends. Genomic DNA blots probed with CNP cDNAs suggest that there is a single gene which can be alternatively spliced to produce the various mRNA transcripts in the nervous and lymphoid tissues.