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Analysis of cloned human genomic loci homologous to the small nuclear RNA Ut established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of Ut RNA. Although all of the Ul genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of Ul pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the Ut gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the Ut sequence in class II and III Ul pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the Ut gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III Ut pseudogenes were generated by an RNA-mediated mechanism involving the insertion of Ul sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of Ul pseudogenes described here and may have been created by an RNAmediated insertion event.The extensive molecular analysis of eucaryotic genes accomplished in recent years has drastically altered our classical notions of gene structure derived from the study of bacteria. For example, earlier paradigms concerning the strict colinearity between a gene and its product have been undermined by the discovery that intervening sequences interrupt many eucaryotic genes, and the characterization of RNA polymerase III control regions that lie within the transcription unit itself (for a review, see reference 14) has confounded previous ideas about the exclusively upstream location of promoter elements. Similarly, the discovery of extraordinarily varied patterns of gene organization in eucaryotic multigene families and the widespread occurrence in these families of defective gene copies known as pseudogenes (see, for example, references 4, 8, 11, 16, 18, 21, 28, 34, 36, 37, 40, 46, 48, 50, 56) required a major reorientation in our thinking about the structure and evolution of eucaryotic genomes.We have been investigating the structure and organization of the multigene families for the t Present address: Department of Microbiology and Immunology, University of California, San Francisco, CA 94143. human small nuclear RNAs (snRNAs) Ut, U2, and U3. Our previous work established that many or most of the members of each of these families are actually pseudogenes which are dispersed in the human genome (11). DNA sequence analysis of several cloned loci demonstrated that t...

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)....

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Analysis of cloned human genomic loci homologous to the small nuclear RNA U1 established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of U1 RNA. Although all of the U1 genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of U1 pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the U1 gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the U1 sequence in class II and III U1 pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the U1 gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III U1 pseudogenes were generated by an RNA-mediated mechanism involving the insertion of U1 sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of U1 pseudogenes described here and may have been created by an RNA-mediated insertion event.