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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.

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.

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...