ELSEVIER Biochimica et Biophysica Acta 1218 (1994) 439-442 BB Biochi~ic~a et Biophysica A~ta Short Sequence-Paper Isolation and characterization of the tandemly repeated U6 genes from the sea urchin Strongylocentrotus purpuratus Sameer A. Sakallah 1, Devon R. Norton, Wei Zhang, William F. Marzluff * Department of Biochemistry and Biophysics and Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Received 10 December 1993 Abstract The tandemly repeated U6 genes were isolated from the sea urchin Strongylocentrotuspurpuratus. Each 1.8 kb repeat unit contains a single U6 RNA sequence. There are no sequence similarities between the U6 promoter and other sea urchin snRNA genes, other than a long polypyrimidine tract 3' of the U6 sequence. Key words: Echinoderm; snRNA; U6 snRNA; (Sea urchin) The sea urchin U1 and U2 snRNA genes expressed in early sea urchin embryos are encoded in separate tandemly repeated units [1,2]. The tandemly repeated genes are expressed in oogenesis and early embryogen- esis and then are silenced between the blastula and gastrula stage [2-4]. All the spliceosomal snRNAs are synthesized at a high rate from the 32-cell stage until after hatching [5]. It is likely that each of the snRNAs is encoded in a tandemly repeated gene set and that these genes are coordinately regulated during embryo- genesis. Here we report the isolation of one of the tandemly repeated U6 snRNA genes from the sea urchin Strongylocentrotus purpuratus. Repeated screening of sea urchin genomic libraries, which had yielded U1 [6,7] and U2 [2] snRNA genes yielded only U6 pseudogenes, which contained trun- cated U6 RNA sequences (S.A.S and W.F.M., unpub- lished data). Since other sea urchin snRNAs are en- coded by tandemly repeated genes and tandemly re- peated genes may be under-represented in phage li- * Corresponding author. Fax: + 1 (919) 9666821. 1 Present address: Department of Pathology, Division of Medical Diagnostics, 1507W Biomedical Tower, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA. The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession numbers X76389 and X76546. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4781(94)00038-5 braries, we cloned the U6 repeat using the polymerase chain reaction. The sea urchin U6 RNA sequence was determined by dideoxysequencing using reverse tran- scriptase and a primer complementary to the conserved U6 3' end. The sequence was confirmed by amplifying U6 RNA by RT-PCR using primers complementary to the 5' and 3' ends of the deduced U6 sequence (S.A.S. and W.F.M., unpublished results). A repeat unit can be isolated using two primers from the U6 coding region, using the strategy shown in Fig. 1. The sequences of the primers are given in the legend to Fig. 1. Primer 1 is nts 86 to 105 of the U6 sequence, and primer 2 is complementary to nts 90 to 71 of the U6 sequence with an EcoRI site added at the 5' end to facilitate cloning. The amplified products were cloned into the plasmid pUC18, and bacteria containing the U6 gene were selected by colony hybridization using a 50 nt oligo- nucleotide containing the central conserved region of U6 snRNA as a probe. The complete sequence of the 1.82 kb insert (SpU6.1) was determined by dideoxyse- quencing [8] using Sequenase (U.S. Biochemicals) or by automated DNA sequencing. Since the amplified DNA did not contain the complete U6 coding region, a 440 nt fragment containing the U6 coding region was am- plified using primers located 5' and 3' of the coding region (primers 3 and 4 in Fig. 1A). Primer 3 contained nts -283 to -255 of the SpU6.1 repeat, including the ClaI site at -265. Primer 4 was complementary to nts +51 to + 32 and has an HindlII site added to facili-