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The chicken genome contains nucleotide sequences homologous to the transforming genes (oncogenes) of a number of avian retroviruses. We have isolated chicken DNA (c-myc) that is homologous to the oncogene (v-myc) of the avian myelocytomatosis virus MC29 and have compared the structures of the cellular and viral genes. Results from restriction endonuclease mapping of c-myc and from analysis of heteroduplexes between the DNAs of the cellular and viral genes show that c-myc is homologous to 1,500 nucleotides in v-myc DNA. This homologous region is interrupted in c-myc by an intron-like sequence of 1,100 nucleotides which is absent from v-myc. Nuclear RNA from normal chicken cells contains at least five species of transcripts from c-myc ranging from 2.5 to 6.5 kilobases in length. By contrast, cytoplasm contains only the 2.5-kilobase c-myc RNA. These features of the c-myc gene and its nuclear transcripts are characteristic of normal cellular genes and suggest that the myc gene is of cellular rather than viral origin. The exons in c-myc may define two functional domains in the gene and may therefore facilitate the dissection of the different oncogenic potentials of the MC29 virus.

U RNAs are highly abundant small nuclear RNAs involved in the processing of messenger RNA. Most U RNA genes are thought to be transcribed by RNA polymerase II (pol II). However, evidence has recently been presented that U6 RNA genes are transcribed by RNA polymerase III (pol III). In the light of these results it was surprising to find that the 5' flanking region of a mouse U6 RNA gene includes a perfect copy of the octamer sequence motif, ATTTGCAT, found in many RNA polymerase II transcription enhancer elements. In the present study we show that deletion of mouse U6 gene sequences upstream of nucleotide position -217, including the octanucleotide motif, reduces U6 transcription by 90% when assayed in Xenopus laevis oocytes, suggesting the presence of a distant control element. DNase I footprinting of the 5' flanking region of the U6 gene shows protection of the octanucleotide sequence. Moreover, the 5' flanking sequence from -217 to -315 can replace the enhancer of a human U2 RNA gene. We therefore conclude that although U6 RNA genes appear to be transcribed by pol III, they are preceeded by an enhancer-like element which can functionally substitute for the enhancer of a pol II-transcribed U RNA gene.

ABSIRACTEarly region 3 of the adenovirus type 2 genome encodes three proteins with molecular weights of 16,000,14,500, and 14,000 (E3/16, E3/14.5, and E3/14 (8,9). The glycoprotein is associated with the cell membrane where it is complexed with the cell transplantation antigen (8, 10). The structures of the ES mRNAs were recently deduced by RNA/DNA heteroduplex analysis (3,4). Several mRNAs with common 5' ends were found early after infection. These mRNAs differ in splicing patterns and several of them do not have coterminal 3' ends.In this paper, mRNAs encoded in early region 3 were purified and the proteins encoded by the different mRNAs were identified in a cell-free protein-synthesizing system. The primary structure of the ES/19 glycoprotein was also determined by aligning the protein NH2-terminal sequence with the known DNA sequence (11).MATERIALS AND METHODS Procedures for extraction of early viral mRNA, cell-free synthesis in mRNA-dependent reticulocyte lysate, preparation of rough microsomes from dog pancreas, tryptic peptide analysis, immunoprecipitations, and NaDodSO4/polyacrylamide gel electrophoresis have been described (8,12). Restriction enzyme fragments of Ad 2 DNA were prepared (13) and their purity was determined by agarose gel electrophoresis before use. Ad RNA was purified by hybridization to Ad 2 DNA bound to nitrocellulose filters (14). Amino Acid Sequence Analysis. Purified Ad mRNAs were translated in vitro in the presence of 200 ,tCi (1 Ci = 3.7 X I0W becquerels) of 3H-labeled amino acids. The translation products were purified by NaDodSO4/polyacrylamide gel electrophoresis with [s5S]methionine-labeled proteins as markers. The proteins were eluted from the gel (12), mixed with 1 mg of bovine serum albumin, and precipitated with trichloroacetic acid. Sequence analysis was performed in a Beckman 890C sequencer together with 1 mg of apomyoglobin. Degradations were performed for 20-25 cycles with a 0.1 M Quadrol program in the presence of Polybrene (15). Repetitive yields of both the labeled protein and the carrier ranged between 92% and 95%. RESULTSCell-Free Synthesis with Purified mRNAs. Restriction enzyme fragments from the E3 region of the viral genome were prepared and used for selection of viral mRNAs (Fig. 1). The selected mRNAs were translated in vitro and the [a5S]methionine-labeled products were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. The EcoRI D and HindIII H fragments selected mRNAs for two early viral polypeptides with sizes of 16,000 and 14,000 daltons (ES/16 and ES/14) (Fig. 2). The HindIII L fragment selected the mRNA for the ES/16 protein; the mRNA for the E3/14 protein was predominantly selected by the EcoRI E fragment. This fragment also selected the mRNA for a 14,500-dalton protein (E3/14.5) as well as small amounts of the E3/16 protein. The E3/16 protein is t1500 daltons larger than the unglycosylated E3/19o protein synthesized in the presence of tunicamycin (Fig. 2, lane b). Restriction enzyme fragments derived from early regions 2 and 4 (EcoRI B + C, F...

SummaryTwo so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild-type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling-circle structures with double-stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling-circle replication.

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Genes for the human small nuclear RNA U2 are present within 6.2-kilobase-pair-long tandem repeats. The haploid human genome contains approximately 20 such repeats, organized in one or a few very large clusters.The small nuclear RNAs (snRNAs) comprise a family of highly conserved RNA species, present in the nucleus of the eukaryotic cell (reviewed in ref. 1). Circumstantial evidence suggests that U1 RNA and possibly other snRNA species serve their function in connection with the maturation of heterogeneous nuclear RNA to spliced mRNA (2-4). Considerable effort has been devoted to studies of human snRNA genes (5-17). One main conclusion from these studies is that the mammalian cell contains numerous loci that are related to the snRNAs, many of which appear to represent pseudogenes. Because of the presence of numerous pseudogenes for snRNA, it has been difficult to isolate and determine the structure of functional mammalian snRNA genes. In the present report, we describe a human locus, designated U2/6, that contains multiple genes for U2 RNA. MATERIALS AND METHODSIsolation of Clones Containing U2 RNA Sequences. The U2/6 clone was obtained from the human genomic DNA library of Lawn (7,18). This library contains fragments generated by partial cleavage with endonucleases Alu I and Hae III inserted with EcoRI linkers. Fragments from the recombinant phage were subcloned using the pBR322 vector. Cloning experiments were carried out according to Swedish guidelines for work with recombinant DNA.DNA Sequence Analysis. The Taq I fragments shown in Fig. LA were cloned in the Cla I site of pBR322 and sequenced according to Maxam and Gilbert (19 Cloning of a Locus for Human U2 RNA. A locus designated U2/6 was isolated from the human DNA library of Lawn (7, 18) and a 4.4-kilobase-pair (kb)-long Pst I fragment was subcloned in the pBR322 vector. A restriction enzyme cleavage map was established for the resulting clone (pU2.6/1) (Fig. 1A) and relevant parts of the fragment were sequenced by the Maxam and Gilbert procedure (19). A comparison between the established sequence and the sequence of rat U2 RNA (22) reveals four differences, which occupy positions 108, 110, 111, and 116.A more detailed analysis of the established U2/6 sequence reveals some interesting features, and the results are presented in Fig. 2 and will be discussed below.Previous studies have shown that snRNA loci often are connected with repetitive sequences belonging to the socalled Alu family (5, 9). To test whether the U2/6 locus also contained such sequences, a DNA probe (Blur 8) (29) was hybridized to fragments of the U2/6 recombinant. The result revealed the presence of an Alu-like sequence and its position is shown in Fig. 1B.The U2/6 Recombinant Is Unstable When Propagated in Escherichia coli. When the U2/6-X recombinant was propagated on a large scale, two populations of phage particles were isolated after CsCl gradient fractionation. Electron microscopic heteroduplex analyses of 27 molecules showed that 6.8 ± 0.4 kb had been deleted from the gen...

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.