Gene, 141 (1994) 307-308 0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00 GENE 07809 Cloning and sequencing of isoform-specific regions of human Ca2+-independent protein kinase C (PKC)-encoding genes* (Recombinant DNA; signal transduction; neuroblastoma; isoenzymes) M.V. Corriasa, F. Guarnacciab, P. Cornaglia-Ferrarisb and M. Ponzonia ‘Oncology Research Laboratory; and blVPediatric Division. G. Gaslini Children’s Hospital, 16148 Genoa, Italy Received by R.W. Davies: 18 June 1993; Revised/Accepted: 24 August/l October 1993; Received at publishers: 3 January 1994 307 SUMMARY The cloning and sequencing of the isoform-specific regions of the human Ca2+ . -independent protein kinase C-encoding genes is described. These clones will serve as correct size probes for screening human genomic or cDNA libraries and isolating full-length clones. We recently demonstrated that three sets of primers, designed to match the inside rat sequences of PKC iso- forms 6, E and { (Ono et al., 1988) were able to specifically amplify retro-transcribed total RNA from a human neu- roblastoma cell line, LAN-5 (Ponzoni et al., 1993). These primers were chosen, by means of the oligo 4.0-1346 pro- gram (Copyright 1989 Wojciech Rychlik), inside the re- gions between nt 1155 and nt 1470 of the 6 isoform, between nt 1150 and 1470 of the E isoform and between nt 210 and 525 of the 6 isoform to achieve the highest isoform specificity during RT-PCR amplification, be- cause a high degree of conservation through species was expected. Sequences and positions on the rat genes are given in the legend to Fig. 1. Since it has been suggested that the various isoforms play different roles in cell regu- lation and evidence has been provided for the role of some of these isoforms in the manteinance and/or induc- tion of the differentiation process, at least in the neuronal Correspondence to: Dr. M.V. Corrias, Oncology Research Laboratory, G. Gaslini Children’s Hospital, L.go G. Gaslini, 16148 Genoa, Italy. Tel. (39-10) 5636-342. Fax (39-10) 3776-590. *On request the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; PKC, protein kinase C; PKC, gene encoding PKC; RT, reverse transcriptase. SSDI 0378-l 119(94)00020-S model (Leli et al., 1993; Parrow et al., 1992; Ponzoni et al., 1993; Wada et al., 1989), it would be of great interest to isolate the human genes of the Ca2+-independent PKC isoforms. We therefore decided to clone and sequence the amplified fragments to obtain a correct size probe to screen a cDNA or a genomic human library. Genomic DNA was extracted from LAN-5 cells, amplified with the isoform-specific primers and cloned as described in the legend to Fig. 1. Three independent clones for each iso- form were then completely sequenced on both strands and sequences of isoform 6, E and c are shown in Fig. la, b and c, respectively. The human PKC-S fragment, EMBL accession No. X72972, being 210 bp, is shorter than the corresponding rat sequence of 237 bp. The nt homology between the two species, calculated by the DNASIS program (Hitachi Software Engineering) was 55%. In the human sequence only one ORF was found, the same as in the rat. The distribution of uncharged and charged aa was very sim- ilar in the two species. The human PKC-E fragment, EMBL accession No. X72974, is only 219 bp in length vs. the 264 bp of the rat sequence. The 45-bp gap seems to be located at the 5’ end because when the 3’ end was aligned to the rat sequence, 56% homology was found. Comparison of the aa sequence, deduced from the ORF, showed a significant decrease in acidic aa residues in the human isoform.