Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide.

We have constructed a cDNA library from the cytoplasmic RNAs of Raji cells, a Burkitt's lymphoma cell line latently infected with Epstein-Barr virus. We report here the characterization of a cDNA representing a spliced RNA transcribed from the IR1-U2 region of the viral genome. The cDNA is 1007 bp long. The 5' region contains three tandem repeats of two exons, 66 and 132 bp, which are transcribed from the IR1 repeats. The 3' region is formed from four exons transcribed from U2. An open reading frame extends from the 5' end to position 784, and includes the repeats. This reading frame presumably corresponds to the carboxy-terminal 261 amino acids of a polypeptide containing several repeats of a 66 amino acid sequence. Since it would be encoded by the IR1-U2 region of the viral genome, the putative polypeptide might be involved in the process of growth-transformation of B-lymphocytes.     

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.     

Simple repeat sequence in Epstein-Barr virus DNA is transcribed in latent and productive infections.

The BamHI K region of Epstein-Barr virus DNA is transcribed in latently infected cells from Burkitt tumors and in growth-transformed B-lymphocytes latently infected with Epstein-Barr virus. We determined the nucleotide sequence of a 1,153-base pair HinfI fragment in BamHI fragment K from the B95-8 Epstein-Barr virus isolate. The fragment contains a remarkable 708-base pair simple sequence repeat array, designated IR3, which is composed of only three nucleotide triplet elements: GGG, GCA, and GGA. The triplets are organized into three repeat units: GCAGGA, GCAGGAGGA, and GGGGCAGGA. Immediately 3' of IR3 are tandem nearly perfect direct repeats of two different 24-base pair sequences. IR3 is conserved at a colinear position in the DNAs of other Epstein-Barr virus isolates, and a homologous sequence maps at the same location in the genome of a genetically related baboon herpesvirus, herpesvirus papio. IR3 is transcribed from left to right in latently infected, growth-transformed IB4 cells. It encodes part of a 2.0-kilobase exon of the 3.7-kilobase cytoplasmic polyadenylated RNA previously detected in IB4 cells (van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1981). IR3 also encodes parts of 2.4- and 1.0-kilobase RNAs in productively infected B95-8 cells.     

Bovine papilloma virus transcription: polyadenylated RNA species and assessment of the direction of transcription

The bovine papilloma virus type 1 (BPV-1)-specific RNA species were identified in virus-induced bovine warts, hamster tumors, and transformed hamster and mouse cells. In each case two major species were present (1.1 and 1.3 kilobases [kb]). Also two species of 1.6 and 1.8 kb appearing in variable amounts were found. Only in the keratinized periphery of the warts, where virus replication takes place, was it possible to reveal an additional 2-kb RNA species. In this tissue, however, the 1.6-kb species was not detected. The basal part of a bovine wart contained an additional minor, 2.9-kb, BPV-1-specific RNA sequence. By hybridization with purified defined BPV-1 DNA fragments it was shown that most of the coding sequences of the 2-kb species were transcribed from a region between 0.02 and 0.19 map units. The majority of the coding sequences of the smaller species in transformed cells were located in the region between 0.31 and 0.61 map units. The putative 5' ends mapped between 0.72 and 0.96 map units. Oligodeoxythymidylic acid-primed [(32)P]cDNA was synthesized from various RNA preparations to generate probes for the detection of 3' termini of the polyadenylated BPV-1 RNAs. By hybridization across the BPV-1 genome only one signal between the map positions 0.30 and 0.40 was obtained when RNA from transformed cells and from a tumor was used as a template. In contrast, RNA from the periphery of a wart led to the detection of an additional signal which was confined to the region between 0.96 and 1.00 map units. From the arrangement of both the 3' termini and the coding areas along the viral genome it appears that several RNA species are transcribed from one DNA strand.     

Molecular cloning of unintegrated visna viral DNA and characterization of frequent deletions in the 3' terminus

Visna viral DNA, like other retroviral DNA, exists in two circular forms in infected cells. The larger probably contains two copies of the LTR, the smaller, one copy. Recombinant DNA techniques were used to clone unintegrated circular visna viral DNA in the lambda WES . lambda B vector. Circular visna viral DNA was digested with the restriction enzyme SstI, which yields a 9.2-kb viral DNA fragment containing 90% of the viral genome colinear with the restriction map of linear viral DNA. This fragment extends from a site about 900 bp from the left (5') end of the viral DNA molecule, through the 3' region, including U3 and R sequences at its right (3') end. The recombinant clones isolated contain visna viral DNA inserts which range in size from 3.1 kb to 9.2 kb. All the clones contain the 5' region intact, but most had sustained deletions of varying lengths in the 3' terminal region of the cloned fragment.     

Fimbriation genes of Salmonella enteritidis.

From a cosmid clone, a 5.3-kilobase (kb) HindIII fragment of Salmonella enteritidis DNA containing the fimA gene was subcloned into bacteriophage T7 promoter vectors in both orientations. Predominantly mature fimbrin (14,000 Mr) was produced by clones containing the 5.3-kb insert, whereas the original cosmid clone predominantly accumulated a prefimbrin precursor (16,500 Mr). T7 RNA polymerase-dependent expression of the 5.3-kb insert and of a series of nested deletions revealed several membrane-localized polypeptides (80,000, 40,000, 29,000, 25,000, and 16,500 Mrs) transcribed in the same direction as fimA as well as a single polypeptide (9,000 Mr) transcribed in the opposite direction. Mini-Mu and TnphoA (Tn5 IS50L::phoA) transposon mutagenesis was used to identify a 2- to 3.5-kb region downstream of fimA that affected fimbrin production and processing. A more distant region (greater than 7 kb), revealed by Tn10 and Mu dI mutagenesis, was also required for fimbriation but did not hybridize with the 5.3-kb fragment. Yet another distant region did hybridize to the 5.3-kb fragment, suggesting the existence of other fimbriation-related genes.     

Identification and nucleotide sequences of two similar tandem direct repeats in Epstein-Barr virus DNA

Epstein-Barr virus DNA is known to have partially homologous segments, designated DL and DR, near the left and right ends of the long unique region (Raab-Traub et al., Cell 22:257-267, 1980). DL and DR are each partially composed of tandem direct repeat sequences. DL contains 11 to 14 repeats of a 124-base-pair sequence designated IR2. DR contains approximately 30 direct repeats of a 103-base-pair sequence designated IR4. The DL and DR sequences have colinear partial homology for approximately 2.4 and 1.5 kilobase pairs to the right of IR2 and IR4, respectively. IR2 and IR4 are similar sequences and evolved in part from a common ancestor. Both sequences are 84% guanine and cytosine and have limited homology to Epstein-Barr virus IR1 and to the herpes simplex virus type 1 inverted terminal repeat "a" sequence. IR2 encodes part of an abundant 2.5-kilobase persistent early EBV RNA expressed in productively infected cells, but does not encode part of the 3-kilobase Epstein-Barr virus RNA which is transcribed from the adjacent IR1-U2 region of the Epstein-Barr virus genome in latently infected cells.     

Structural analysis of a 1.7-kilobase mouse mammary tumor virus-specific RNA

We have detected a mouse mammary tumor virus (MMTV)-specific 1.7-kilobase (kb) polyadenylated RNA in mammary glands of several mouse strains. In BALB/c mice, it is the only MMTV-specific RNA species present. C3H and GR mammary glands and tumors contain, in addition, 3.8- and 7.8-kb MMTV RNAs. Nuclease S1 analysis was performed to map 1.7-kb polyadenylated RNA. It contains predominantly long terminal repeat (LTR) sequences. The 5' end maps approximately 134 nucleotides upstream from the 3' end of the LTR. Colinearity with complete proviral DNA continues to a site about 153 nucleotides downstream from the left (5') LTR. No sequences from the middle part of proviral DNA were found. Colinearity with proviral DNA is resumed 72 nucleotides upstream from the right (3') LTR. The nucleotide sequence in this area is TTCCAGT, which is a splice acceptor consensus sequence. The anatomy of 1.7-kb RNA indicates that it may serve as a messenger for the 36,700-dalton protein encoded by the LTRs of MMTV.     

Characterization of the bovine herpesvirus 4 major immediate-early transcript.

The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain.     

Sensitivity to UV radiation of small nuclear RNA synthesis in mammalian cells.

It was demonstrated previously that the synthesis of small nuclear RNA (snRNA) species U1 and U2 in human cells is very sensitive to UV radiation. In the present work, the UV sensitivity of U3, U4, and U5 snRNA synthesis is shown to be also high. The synthesis of U1, U2, U3, U4, and U5 snRNAs progressively decreased during the first 2 h after UV irradiation (this was not observed in polyadenylated RNA) and had not returned to normal rates 6 h after UV exposure. In contrast, the restoration of 5.8S rRNA synthesis began immediately after UV irradiation and was essentially complete 6 h later. A small fraction of U1 and U5 (and possibly U2 and U3) snRNA synthesis remained unaffected by high UV doses, when cell radiolabeling began 10 min after UV irradiation. The present data suggest that a factor other than the level of pyrimidine dimers in DNA (possibly, steps in the post-irradiation DNA repair process) plays an important role in the mechanism of UV-induced inhibition of U1-U5 snRNA synthesis.     

Nuclease S1-sensitive sites in multigene families: human U2 small nuclear RNA genes.

We show here that human U2 small nuclear RNA genes contain a 'strong nuclease S1 cleavage site' (SNS1 site), a sequence that is very sensitive to digestion by nuclease S1. This site is located 0.50-0.65 kb downstream of the U2 RNA coding region. It comprises a 0.15-kb region in which (dC-dT)n:(dA-dG)n co-polymeric stretches represent greater than 90% of the sequence. Nuclease S1 is able to excise unit length repeats of the human U2 RNA genes both from cloned fragments and total human genomic DNA. The precise locations of the cleavage sites are dependent on the superhelicity of the substrate DNA. In negatively supercoiled substrates, cleavages are distributed over the entire 0.15-kb region, but in linearized substrates, they occur within a more limited region, mainly at the boundary of the SNS1 site closest to the human U2 RNA coding region. Nuclease S1 cleavage of negatively supercoiled substrates occurs at pHs as high as 7.0; in contrast, cleavage of linearized substrates requires a pH less than 5.0, indicating that supercoiling contributes to the sensitivity of this site. Mung bean nuclease gives results similar to that observed with nuclease S1.