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We have cloned and partially characterized 24 loci from the human genome which are complementary to U1, U2, or U3, the three major species of small nuclear RNA (snRNA) in HeLa cells. When compared to the known Ul (human) and U2 (rat) snRNA sequences, the DNA sequences we report here for the complementary regions from two of the clones, Ul Among the various classes of molecules found in all eukaryotic cells, the small nuclear RNAs (snRNAs) have received relatively little attention until recently. The sequences of eight of these small homogeneous RNA species from mammalian cells (Ul, U2, U3A, U3B, U4, U5, U6, and 4.5S RNA) have been determined (1-6). U3 snRNA is found in the nucleolus, and other snRNA species are associated with either the nucleoskeleton or the nucleoplasm (7). At least three of the snRNAs are subject to considerable evolutionary conservation: Ul and U2 snRNA from chicken, rat, mouse, and human cells yield identical ribonuclease Ti fingerprints (1,8,9), and the RNA sequences of U1 from HeLa cells and Ula from rat Novikoff hepatoma differ in only 2 of 165 positions (1). We have also shown that a snRNA from the lower eukaryotic cellular slime mold Dictyostelium discoideum is over 40% homologous to U3 snRNA from the rat (10).Two recent developments have prompted new interest in the intracellular packaging and function of the snRNAs. First, antibodies produced by patients with the autoimmune disease systemic lupus erythematosus have been shown to recognize discrete cellular components that contain snRNAs complexed with a defined set of proteins (8), thus opening the way for detailed structural studies of small nuclear ribonucleoprotein particles. Second, Lerner et al. (9) (kb) (17).We originally decided to clone the genes encoding snRNAs in the hope that a systematic investigation of their organization and expression would also open up new avenues for understanding the function of the snRNAs themselves. We report here the surprising result that most, if not all, of the genomic loci complementary to snRNAs U1, U2, and U3 that we have examined contain divergent, and in some cases truncated, gene copies when compared with the corresponding HeLa cell snRNA species. MATERIALS AND METHODSA library of 15-kb partial EcoRI fragments of human placental DNA in the A vector Charon 4A (18) was kindly supplied by A. Biro, P. V. Choudary, J. T. Elder, and S. M. Weissman. Plaques were screened by the method of Benton and Davis (19); we used as probes U1, U2, and U3 snRNAs isolated from HeLa cells and labeled in vitro at the 3' end with 5'-32P-labeled pCp and T4 RNA ligase as described (14). As little as 106 cpm of snRNA at a specific activity of 106 cpm/,ug was sufficient to screen six 140-mm nitrocellulose filters, each bearing 8000 plaques. With each probe, 0. 1% of the plaques were scored as positive and repurified for further study. Small quantities of recombinant DNA were prepared from 4-ml NZY cultures (20); larger quantities were prepared from recombinant phage grown and purified as described (18)....

Human U1 small nuclear RNA is encoded by approximately 30 gene copies. All of the U1 genes share several kilobases of essentially perfect flanking homology both upstream and downstream from the U1 coding region, but remarkably, for many U1 genes excellent flanking homology extends at least 24 kilobases upstream and 20 kilobases downstream. Class I U1 RNA pseudogenes are abundant in the human genome. These pseudogenes contain a complete but imperfect U1 coding region and possess extensive flanking homology to the true U1 genes. We mapped four class I pseudogenes by in situ hybridization to the long arm of chromosome 1, bands q12-q22, a region distinct from the site on the distal short arm of chromosome 1 to which the U1 genes have been previously mapped (Lund et al., Mol. Cell. Biol. 3:2211-2220, 1983; Naylor et al., Somat. Cell Mol. Genet. 10:307-313, 1984). We confirmed our in situ hybridization results by genomic blotting experiments with somatic cell hybrid lines with translocation products of human chromosome 1. These experiments provide further evidence that class I U1 pseudogenes and the true U1 genes are not interspersed. The results, along with those published elsewhere (Bernstein et al., Mol. Cell. Biol. 5:2159-2171, 1985), suggest that gene amplification may be responsible for the sequence homogeneity of the human U1 gene family.

The DNA immediately flanking the 164-base-pair Ul RNA coding region is highly conserved among the approximately 30 human Ul genes. The Ul multigene family also contains many Ul pseudogenes (designated class I) with striking although imperfect flanking homology to the true Ul genes. Using cosmid vectors, we now have cloned, characterized, and partially sequenced three 35-kilobase (kb)