Abstract
Phospholipase C-beta 1 (PLC-beta 1) exists as two immunologically indistinguishable polypeptides of 150 and 140 kDa and is encoded in rat brain by two distinct transcripts of 5.4 and 7.2 kilobases (kb). cDNA corresponding to the entire 5.4-kb transcript as reported previously reveals an open reading frame that is capable of coding a 1216-amino acid polypeptide (Suh, P. G., Ryu, S. H., Moon, K. H., Suh, H. W., and Rhee, S. G. (1988) Cell 54, 161-169). We have now isolated cDNAs corresponding to the entire 7.2-kb transcript from a rat brain cDNA library. The 7.2-kb transcript differs from the previously reported 5.4-kb transcript by possessing both an additional 118 nucleotides located near the end of the coding sequence and a 1738-nucleotide extension of the 3'-flanking region. The presence of the 118-nucleotide insert in the cumulative 7.2-kb sequence gives rise to an open reading frame that is capable of coding a 1173-amino acid polypeptide (PLC-beta 1b), the carboxyl-terminal sequence of which differs from that of the 1216-amino acid polypeptide (PLC-beta 1a) derived from the 5.4-kb transcript. Antibodies were raised against synthetic peptides corresponding to the carboxyl-terminal portions of PLC-beta 1a and PLC-beta 1b. Immunoblot analysis with these isozyme-specific antibodies revealed that both PLC-beta 1a and PLC-beta 1b are expressed in rat brain and C6Bu-1 glioma cells and that PLC-beta 1a and PLC-beta 1b correspond to the previously identified 150- and 140-kDa PLC-beta 1 enzymes, respectively. Analysis of PLC-beta 1 genomic DNA indicates that PLC-beta 1a and PLC-beta 1b are derived from a single gene by alternative RNA splicing.
Highlights
Young Yil Bahk, Young Han Lee, Taehoon G
On the basis of deduced amino acid sequence similarity and immunological cross-reactivity, these mammalian PLCs have nucleotides locatednear the end of the coding sequence been divided into three type(sPLC-p, PLC-y, and PLC-61, each and a 1738-nucleotide extension of the 3'-flanking re- of which comprises more than one subtype: PLC-p1, 4 2, -p3, gion. "he presence of the 118-nucleotideinsert in the -/34, -71, -72, -61, and -62 have been identified [15,16,17,18,19,20,21,22]
This work provides evidenctehat PLC-pl is composed of two subtypes, 150-kDa PLC-fila and 140-kDa PLC-plb, which are encoded by 5.4- and 7.2-kb mRNAs, respectively
Summary
&NA CZoning of PLC-PI-A rat brain cDNA library was constructed in thevector hgtll cloning system Filters were washed twicea t room temperature with 2 x SSC containing 0.1%SDSand once at 68 "C with 1x SSCcontaining0.1%SDS and exposed to x-ray film (KodakXAR-2).Two clones (pl-2 and pl-5) that hybridized strongly and were >3.5 kb in size were subcloned into a pBluescript KS* vector (Stratagene) a t the EcoRI site, and the sequences were determined These clones contained an additional 118 nucleotides in the vicinity of the 3'-end of the PLC-p1 open reading frame previously described [12].The 118-nucleotidefragment was excised from clonepl-5 with the restriction endonucleasesSty and PstI, random primer-labeled with [s2P]dCTP,and used to rescreen the same cDNA library. Poly(A)+RNA was preparedfrom total RNA by oligo(dT)-cellulosechro-
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