Relationship of mRNA from productively infected cells to the complementary strands of adenovirus type 2 DNA (original) (raw)
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Journal of Molecular Biology, 1974
ABSTRACT Separation of the complementary strands of adenovirus type 2 DNA by poly(U,G)-CsCl density gradient centrifugation permitted studies of Ad2‡ DNA renaturation with independently variable concentrations of each complementary strand. Single-stranded DNA was isolated by hydroxylapatite chromatography following exhaustive incubation under such conditions, and was found to selectively represent sequences of the complement present in excess during the incubation. This result was exploited in a general method for isolation of complementary strand-specific sequences of radioactively labeled Ad2 DNA or restriction enzyme fragments of Ad2 DNA. Liquid phase saturation-hybridization experiments were carried out with labeled DNA representing each complementary strand of the six endo R.EcoRI cleavage fragments of Ad2 DNA and unlabeled messenger RNA prepared from HeLa cells late after productive infections with Ad2. The results were combined with the known endo R.EcoRI cleavage map of Ad2 DNA to construct a low-resolution map showing physically separated regions, on both complementary strands of Ad2 DNA, represented in mRNA late after infection.
An mRNA fraction coding for hexon polypep-tide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonu-clease cleavage fragments of viral DNA were visualized in the electron microscope, branched forms were observed in which 160 nucleotides of RNA from the 5! terminus were not hydrogen bonded to the single-stranded DNA. DNA se uences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17,20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of vira RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA. Most eukaryotic mRNAs bear modifications at both termini; their 3' termini have a tract of poly(A) that ranges in length from 30 to 200 bases (1-4), while their 5' termini are typically capped with a methylated guanine joined through a 5'-5' py-rophosphate linkage to a second nucleotide methylated at its 2' position (5, 6). Both types of modifications of eukaryotic mRNA are known to occur after transcription. All adenovirus mRNAs are thought to contain poly(A) tracts at their 3' termini (7) and be capped with a methylated guanine (8, 9). Specific restriction endonuclease cleavage fragments of adenovirus 2 (Ad2) DNA have permitted the mapping of regions of the genome expressed as mRNA and viral proteins during different stages of the lytic cycle (10-12). Little is known about the molecular mechanisms of viral mRNA synthesis. An important aspect of late mRNA synthesis is thought to be the processing and selection of viral mRNAs from the nucleus (18, 14). We have purified a late Ad2 hexon mRNA and found evidence providing some insight into the mechanism of synthesis of this mRNA. MATERIALS AND METHODS Isolation of Ad2 DNA and RNA. Polyribosomal RNA was prepared from Ad2-infected cells 32 hr after infection as described by Flint and Sharp (14, 15) and selected by chromatography on poly(U)-Sephadex (16). R-Loop Mapping. The R-loop hybridization mixture was essentially that of Thomas et al. (17) and contained 70% (vol/ vol) formamide [Matheson, Coleman, and Bell, 99%, further purified as described by Duesberg and Vogt (18)]; 0.20 M Tris-HCl, pH 7.91; 0.50 M NaCI; 0.01 M EDTA; Ad2 DNA at 10 ,g/ml; and purified hexon mRNA at 1-10 Ag/ml. This mixture was incubated at 52.50 for 2-3 hr and spread on a hy-The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3171 pophase of water with internal length standards of DNA from bacteriophage qX174, 5375 bases (19). Hybridization to Single-Stranded Ad2 DNA. Hybridiza-tions of either polyribosomal poly(A) or purified hexon mRNA with restriction endonuclease fragments of Ad2 DNA were carried out in reaction mixtures of-80% formamide; 0.40 M NaCl; 0.04 M 2-(N-morpholino)ethanesulfonic acid (Mes), pH 6.2; 0.01 M EDTA; DNA at 10 lg/ml; and hexon mRNA at 1.0-10 ,ug/ml (20). The sample was incubated at 57-60° for 2-3 hr. RESULTS Adenovirus late mRNAs begin to appear on polyribosomes about 13 hr after infection and continue to accumulate in the cell throughout the lytic cycle (21). Thus, to fractionate the most abundant late mRNAs, polyribosomes were prepared from cells 32 hr after infection with Ad2 and poly(A)-containing mRNA was selected by chromatography on poly(U)Sephadex columns. These mRNAs were then resolved into different molecular weight fractions by electrophoresis in 2.4-4.0% linear gradient polyacrylamide gels containing a uniform concentration of 7 M urea. After staining with ethidium bromide, distinct fluorescent bands were present in gels containing mRNA from virus-infected cells that were not found in gels containing identically prepared HeLa cell mRNA (Fig. 1A). These virus-specific RNAs were selectively labeled when [32P]phosphate was added to infected cells 24 hr after infection and the same mRNA fractions were prepared (Fig. 1B). RNA from the predominant ethidium bromide-staining band migrating 1.5 times faster than 28S rRNA in Fig. 1A and marked with a large arrow has been shown to code for the hexon polypeptide by in vitro translation (S. M. Berget, B. E. Roberts, and P. A. Sharp, data not shown). Furthermore, this RNA has been mapped by the R-loop technique (see below) to a region of the genome known to code for hexon (12) and is complementary to the r strand of the viral DNA (11). This mRNA species, therefore, will be referred to in the following sections as hexon mRNA. R-Loop Mapping of Hexon RNA. The R-loop technique developed by White and Hogness (22) and Thomas et al. (17) was used to position purified hexon mRNA on the viral genome. RNA eluted from a gel similar to that shown in Fig. IA was incubated, as described in Materials and Methods, with either total Ad2 DNA or restriction endonuclease fragments. Of the 43 total Ad2 DNA molecules scored as containing R-loops, 41 were observed to have a single region of hybrid, while two molecules contain a second R-loop, apparently in the region of the genome coding for the 1O(K polypeptide (12). Fig. 2 shows two examples of R-loops resulting from hy-bridization of hexon mRNA to fragments generated by the cleavage of Ad2 DNA with the EcoRI restriction endonuclease Abbreviation: Ad2, adenovirus 2. * We dedicate this work to the memory ofJerome Vinograd, a nan who loved science.
Journal of virology, 1977
Complementary strand-specific adenovirus DNA, either full length or from restriction enzyme cleavage fragments, was used to estimate the fractional representation and abundance of viral sequences in two adenovirus type 2 (Ad2)-transformed rat cell lines, A2F19 and A2T2C4. The reassociation method introduced is based on the linear relationship, after exhaustive hybridization, between the inverted fraction of hybrid DNA and the molar ratio of probe to cellular DNA in the reaction mixture. The amount of viral DNA in A2F19 cells represents 12 to 14% of the viral genome at a level of around seven copies per diploid cell equivalent. For the cell line A2T2C4, the pattern of integrated viral DNA sequences is more complex. With full-length Ad2 DNA strands as a probe, about 56% of the probe was represented in cellular DNA. When each of the four BamHI fragment strands of Ad2 DNA was used as a probe, the fraction of the viral DNA present also amounted to around 56% with one to five copies from ...
Studies on the nature of the linkage between the terminal protein and the adenovirus DNA
Biochemical and Biophysical Research Communications, 1980
The termini of human adenovirus types 7 and 2 DNA extracted from the virions using pronase and protease K are covalently linked to peptides that contain tyrosine residue(s). The peptide attached to each terminus can be labeled with [1251] i __n_ vitro. The linkage between the peptide moiety and the terminal nucleotide is sensitive to snake venom phosphodiesterase, Aspergillus nuelease S I and microeoccal nuelease. The available data suggest that the peptide is linked to the terminal nucleotide of these DNA molecules through a phosphodiester bond. Human Ad DNA extracted from the virion using guanidine hydrochloride is a linear, duplex molecule containing a protein of 55,000 daltons tightly associated with its termini (I-8). Treatment of the Ad virion or the DNA-protein complex with a protease results in a linear DNA whose 5' ends were blocked to digestion with 5' exonucleases and phosphorylation by phosphatase, polynucleotide kinase and [y_32p] ATP (9). However, the 3'-specific E. coli exonuclease III acts on such viral DNA molecules (10,11,12). These observations suggested that the 55K protein bound (presumably covalently) to the 5' termini leaves a residual amino acid or peptide attached to the 5' terminal nucleotide upon treatment with protease K or pronase. The 5' terminal nueleotide has been shown to be a dC residue (9,13-15). In this communication, we wish to report that the viral DNA isolated from Ad 2 or Ad 7 serotype by treatment with protease K and/or pronase can be specifically labeled at the 5' termini with [125I] using chloramine-T as the oxidizing agent (16). Although under some circumstances iodine may react with sulf
Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2
Journal of virology, 1978
Complementary strand-specific adenovirus DNA of full length or from endonuclease BamHI fragments was used as a probe to estimate the fractional representation and abundance of viral sequences in five hamster cell lines (Ad2HE1-5) transformed with UV-inactivated adenovirus type 2. The fraction of the viral genome present in the five transformed cell lines varied from 44% in the Ad2HE5 cell line to 84% in the Ad2HE3 cell line. The number of viral DNA copies per diploid cell equivalent ranged from 1.8 in the Ad2HE1 line to 7.1 in the Ad2HE4 line. In vivo labeling with [35S]methionine followed by immunoprecipitation with an antiserum against adenovirus type 2 early proteins revealed virus-specific polypeptides with molecular weights of 42,000 to 58,000 in extracts from all five hamster cell lines. Several other early viral polypeptides were detected in some of the adenovirus type 2-transformed hamster cell lines.
Genome expression and mRNA maturation at late stages of productive adenovirus type 2 infection
Journal of Virology, 1976
RNA from adenovirus 2-infected KB cells was annealed in liquid with RNA in vast excess to viral heavy (l) and light (r) 32P-labeled DNA strands. Hybridization kinetics were analyzed by computer to estimate the number of viral RNA abundance classes, their relative concentrations, and the fraction of each DNA strand from which they originated. Early whole cell RNA extracted 5 h postinfection annealed rapidly to 10 to 15% of l and r strands and then slowly to final values of 60 and 40% of l and r strands. By 9 h postinfection the expression of late genes was apparent and whole cell RNA annealed to 20 and 75% of l and r strands. Whole cell RNA extracted between 12 and 36 h postinfection annealed to 7 to 15% and 75 to 90% of l and r strands. Late nuclear RNA hybridized to 10 and 90% of l and r strands, and late polyribosomal RNA hybridized to 20 and 75% of l and r strands. Based upon kinetic analyses, we estimate that mRNA synthesized exclusively during late stages arises from about 6 to...
The EMBO journal, 1984
The adenovirus type 2 (Ad2)-transformed hamster cell line HE5 contains one or very few integrated copies of Ad2 DNA. At the site of insertion of Ad2 DNA, the cellular DNA sequence has been completely preserved and has homologies to small unpolyadenylated, cytoplasmic RNAs of 300 nucleotides in length and to minority populations of smaller RNAs present in HE5 cells and in normal hamster cells. The 300-nucleotide RNA is present on average in approximately 20 copies per cell. This RNA, and shorter RNAs, reveal homologies to the hamster DNA sequence of approximately 400 nucleotides to the right of the site of insertion of Ad2 DNA, which is present in one or very few copies per genome. The nucleotide sequence of the DNA segment homologous to this RNA does not contain open reading frames in excess of a sequence encoding 18 amino acids. Thus, it is unlikely that the small RNAs are actually translated and their function is unknown. The nucleotide sequence does not exhibit similarities to kn...
Journal of virology, 1978
Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein synthesized early after injection of human cells with adenovirus type 2 (Ad2) showed that polypeptides of 11,000 (11K), 12K, 14K, 15K, 19K, 21K, 24K, and 72k molecular weight were present in infected but not in mock-infected cells. These polypeptides corresponded in electrophoretic mobility to the following polypeptides synthesized in vitro by using mRNA complementary to specific regions of the Ad2 genome: 11K, 19K, 21K (91.5 to 96.8 map units), 14K (78.2 to 83.4 map units), 72K (62.4 to 67.9 map units), and 15K (4.9 to 11.0 map units). Polypeptides of 25K, 17K, 15.5K, and 13K were also synthesized in vitro, but have not yet been detected in infected cells. In addition, six adeno-specific polypeptides of 38 to 50K molecular weight could be discerned in infected cells if two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis was used to compare extracts from infected and mock-infected ce...
Biological activity of the intact and cleaved DNA of the simian adenovirus 7
Nucleic Acids Research, 1979
Only the deproteinized DXA preparations of the simian adenoviras of the type 7 (BA 7) exhibited transforming and tumorigenic activity. The complex of the SA7 DXA with terminal protein (TP) did not exhibit either transforming or tumorigenic activity in cell cultures. In contrast to the transforming potential the infectious titers of the DNK -TP complex for the monkey kidney cells were 30-50 times higher than those of pure DNA. Cleavage of the SA7 DNA by specific endonucleases enhanced the tumorigenic potential of pure DNA, suppressed its infectivity and did not affect the lack of transformation capacity of the DNA -TP complex. The onc-gene was localizpd in the left terrminal fragment with the minimal size 4,3x10FD in the case of R.Sal I. The tumorigenic activity was found to decrease with an increase in the size of the DNA fragment containin the onc-gene. fectivity of the isolated DNA preparations4 but its properties are yet unknown. This paper describes the simultaneous studies of the in-C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England Volume 6 Number 9 1979 Nucleic Acids Research 3119 Nucleic Acids Research fectivity and the transforming and oncogenic activities of the DNA and the DNl -TP complex both in the intact form and after cleavage with various restrictases. The preliminary results of these studies were reported at th.e VI-the USSR-USA Symposium on the oncogenic viruses7.