~ Pergamon 0305-0491(94)00142-1 Comp. Biochem. Physiol. Vol. I10B,No. I, pp. 75-82, 1995 Copyright © 1995ElsevierScienceLtd Printed in Great Britain. All rights reserved 0305-0491/95$9.50+ 0.00 Sequence-independent detection of gene family homologs: identification of a transcript encoding a molluscan serine protease homologous to the pancreatic enzymes of vertebrates Jay C. Groppe* and Daniel E. Morse Marine Biotechnology Center and the Department of Biological Sciences, University of California, Santa Barbara, CA 93106, U.S.A. Autoradiography of 32p-labeled cDNA, fractionated at high resolution by eleetrophoresis through thin (0.8-1.5 ram) vertical alkaline agarose gels, provides a sequence-independent screening procedure for gene family homologs. A screen of tissues of a marine mollusc revealed a prominent intestine-specific cDNA encoding a pancreatic serine protease homolog, which was not detectable as a discrete poly(A) ÷ RNA species on formaldehyde agarose gels. Discrete cDNA products are authentic, non-truncated transcripts of tissue-specific mRNA. A band-sharpening effect is imparted to cDNA products due to (a) substitution of a uniform length 5'-oligo(dT) terminus for heterogeneous 3'-poly(A) termini and (b) the inherent superior resolution of alkaline-denatured DNA. Key words: cDNAs; Gene family homologs; Transcript; Molluscan serine protease; Pancreatic enzymes. Comp. Biochem. Physiol. llOB, 75-82, 1995. Introduction The first cDNAs to be isolated were synthesized from abundant, tissue-specific mRNAs and detected by sequence- independent methods, i.e. without the benefit of sequence specific probes or primers. Two sequence-independent ap- proaches were available: (a) detection of abundant clones in cDNA libraries by differential hybridization, and (b) identifi- cation of uniform-sized fragments from Correspondence to: D. E. Morse, Department of Biological Sciences, University of California, Santa Barbara, CA 93106, U.S.A. *Department of Cell Biology, Biocenter of the Uni- versity of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Received 8 April 1994; accepted 30 June 1994. initially heterogeneous populations of cDNAs by digestion with restriction endonucleases and fractionation by gel elec- trophoresis (Goodman and MacDonald, 1979). Because of the initial inability to synthesize and clone full-length cDNAs, both approaches required an enrichment step (for mRNA, cDNA or both); library screenings yielded random length, partial cDNA clones corresponding with any specific mRNA; and discrete-length cDNA products could not be detected by gel electrophoresis without prior treatment with restriction endonucleases. Subsequent improvements in the isolation of RNA, the preparation of reverse transcriptases, and the synthesis of double-stranded cDNA, together have led CBPB II0/J--V 75