ELSEVIER Gene 176 (1996)17-21 GENE AN INTERNATIONAL JOURNAL ON GENES AND GENOME5 Translational inhibition by 5'-polycytidine tracts in Xenopus embryos and in vitro John S. Kuo a,b, Robin Veale 1'c, Bridey Maxwell a, Hazel Sive a,. a Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA b Harvard-MITDivision of Health Sciences and Technology, Harvard Medical School, 25 Shattuck St., Boston, MA 02115, USA c Department of Zoology, University of Witwatersrand, Johannesburg 2001, South Africa Received 24 July 1995; revised 30 January 1996; accepted 30 January 1996 Abstract Introduction of in vitro transcribed mRNA into Xenopus laevis embryos is a useful technique for analyzing gene function. In order to optimize expression of cDNA constructs from in vitro transcribed mRNAs, we examined the translational efficiency of reporter genes that simulated cDNAs synthesized by tailing with deoxyguanosine (dG) before second strand cDNA synthesis. When transcribed in vitro, these cDNAs give rise to RNAs containing a 5'-homopolymeric cytidine (poly(C)) stretch. We observed that the presence of a 5'-poly(C) tract depressed translation of a CAT reporter gene at least 100-fold in Xenopus embryos and up to 5-fold in vitro. This effect was not seen when a 5'-polyadenosine tract was tested. Translational depression was dependent on the phage polymerase used for in vitro transcription: RNAs transcribed by T7 polymerase translated far more poorly than those transcribed by SP6 polymerase. These results have general implications for optimizing expression of cDNA constructs. Keywords: Recombinant DNA; mRNA expression; cDNA library; SP6 RNA polymerase; T7 RNA polymerase; 5'-Leader sequences 1. Introduction Embryos of the frog Xenopus laevis are invaluable for defining principles of vertebrate development. In most studies, gene function in embryos has been analyzed by misexpression of mRNAs transcribed in vitro by phage polymerases (Melton et al., 1984; reviewed in Vize et al., 1991). For this approach to be effective, it is essential that injected mRNAs are efficiently translated, since a high concentration of exogenous nucleic acid is toxic to the embryos. The 5'-untranslated region (UTR) of an mRNA can have profound effects on translation during development or under various metabolic conditions (reviewed in Kozak, 1991; Melefors and Hentze, 1993; Richter, 1993). * Corresponding author. Tel. + 1 617 2588242; Fax + 1 617 2586505; e-mail: sive@wi.mit.edu i The first two authors contributed equally to this work Abbreviations: bp, base pair(s); CAT, chloramphenicol acetyltransfer- ase; cDNA, complementary deoxyribonucleic acid; dG, deoxyguanos- ine; kb, kilobase(s) or 1000bp; nt, nucleotide(s); PAGE, polyacrylamide-gel electrophoresis; RNA, ribonucleic acid; UTR, untranslated region(s). Although anecdotal evidence for the deleterious effects of 5' leader sequences on translation in Xenopus embryos has been reported (see Vize et al., 1991), no systematic study of the effects of leader sequences, or phage polymer- ase promoters, on translational efficiency has been made. It is generally desirable that cDNAs tested for their function contain full length coding regions. The most efficient method for obtaining full length cDNAs involves tailing first strand cDNA products with deoxyguanosine triphosphate (dGTP), then priming second strand syn- thesis with oligodeoxycytidine (oligo(dC)) (Sambrook et al., 1989). This method allows the extreme 5' end of an mRNA to be copied into cDNA, whereas the other commonly used method (using RNAse H) does not (Sambrook et al., 1989). Tailing is also used in PCR- based RACE cloning to isolate the 5' end of mRNAs (Frohman et al., 1988). Tailing is usually performed with dGTP rather than another nucleotide since dG-tailing reactions self-limit at 15-30 nt per cDNA molecule by formation of quadruple helices (Okayama et al., 1987). mRNAs that are transcribed from dG-tailed cDNA templates will therefore contain a stretch of 15-30 cytidine (poly(C)) residues 5' to the original mRNA start site. In this study, we asked whether 5'-poly(C) leader 0378-1119/96/$15.00 Copyright © 1996. Published by Elsevier Science B.V. All rights reserved PH S0378-1119(96)00201-6