A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids

"Splicing" of the precursor to an adenovirus mRNA was accomplished in isolated cell-free extracts. Nuclei were prepared from hypotonically swollen cells that had been labeled with [3H]uridine for 10 min prior to nuclear isolation.Addition of a "cytoplasmic" fraction was required for the splicing to occur. The nuclear precursor, a Foly(A)terminated RNA molecule approximately 5 kilobases fong, contained sequences complementary to the 58.5-75.9 region of the adenovirus 2 genome, including those sequences spliced out of the mature mRNA molecule. The in vitro spliced product was a poly(A)terminated RNA molecule identical in size to the cytoplasmic 72,000 Mr protein mRNA (2 kilobases long) in which the sequences encoded in the 70.7-75.9 region of the viral genome were spliced to those encoded at 58.7-65.6, with the sequences encoded at 66.1-70.7 deleted.Both the mRNAs that are produced from viral DNA in the cell nucleus (1-7) and many cellular mRNAs (8-11) contain sequences from noncontiguous regions of DNA. The only detectable nuclear primary RNA transcripts from adenovirus 2 (Ad-2) DNA (12, 13), as well as the genes for hemoglobin (14-16) and immunoglobulin light chains (17), also contain mRNA-specific sequences in a noncontiguous form. Therefore, it has been suggested that RNA-RNA "splicing" is necessary during mRNA biogenesis (1, 3). Recently, a precursor to yeast tRNA was described that contains 15 nucleotides intervening between two --40-nucleotide regions that constitute the mature tRNA (18)(19)(20). Cell-free extracts are capable of removing these intervening sequences and splicing the remaining segments together (20). In this report we describe the in vitro conversion of an Ad-2 nuclear mRNA precursor to a molecule with the size and sequence composition of the mature mRNA. We conclude that RNA-RNA splicing of parts of a nuclear precursor RNA molecule is probably a general mechanism in mRNA manufacture in higher cells. MATERIALS AND METHODSThe growth of HeLa cells, procedures for Ad-2 infection, and virus and virus DNA purification have all been described (21,22), as have techniques for extraction of RNA from isolated nuclei, sucrose sedimentation analysis (22), and gel analysis of the Ad-2-specific RNA (23).Preparation of DNA fragments by restriction endonuclease digestion has been described (24). Enzymes used for this work were purchased from New England BioLabs.The preparation of isolated nuclei basically followed the technique described earlier for in vitro RNA studies (22). De- RESULTSEarly in Ad-2 infection at least five individual transcription units, scattered on both strands of the Ad-2 DNA, are active in producing mRNA (12,(26)(27)(28). The average size of these transcription units is approximately 2-8 kilobases (kb), considerably smaller than the major late Ad-2 transcription unit (12, 13). However, like the late Ad-2 mRNAs, virtually all of the early Ad-2 mRNAs are composed of noncontiguously encoded sequences, some of which are at least 5 kb apart in the Ad-2 genome (29, 30). Previously,...

Section: Resultsmentioning

confidence: 87%

“…Early in Ad-2 infection at least five individual transcription units, scattered on both strands of the Ad-2 DNA, are active in producing mRNA (12,(26)(27)(28). The average size of these transcription units is approximately 2-8 kilobases (kb), considerably smaller than the major late Ad-2 transcription unit (12, 13).…”

Section: Resultsmentioning

confidence: 99%

In vitro RNA-RNA splicing in adenovirus 2 mRNA formation.

Blanchard¹,

Weber²,

Jelinek³

et al. 1978

“…The Rloop procedure was taken from published methods (12,17). The hybridization solution contained 4 ,g of rDNA per ml (0.6 nM with respect to half molecules) and 5-15 ,g of RNA per ml (7 nM in each species).…”

Section: Introductionmentioning

confidence: 99%

Localization of transcribed regions on extrachromosomal ribosomal RNA genes of Tetrahymena thermophila by R-loop mapping.

Cech¹,

Rio²

1979

R-loop hybridization and electron microscopy were used to map the RNA transcription products of the extrachromosomal rRNA genes of Tetrahymena thermophila. The mature 17S and 26S rRNAs and the nuclear 35S pre-rRNA and pre-26S rRNA were located with a precision of approximately 100 base pairs. A 370-base pair intervening sequence was found in the 26S coding region. It has the same size and relative location as that found in Tetrahymena pigmentosa [Wild, M. A. & Gall, J. G. (1979) Cell 16, 565-5731. One class of R-loop structures formed by nuclear p&-rRNA provided preliminary evidence for a primary transcript that contains the intervening sequence. The results suggested a processing scheme in which splicing of the intervening sequence is followed by a series of strand cleavages to give the mature 17S and 26S rRNAs. Analysis of the data also showed that RNADNA and DNADNA duplexes, when mounted for electron microscopy by the R-loop procedure, have the same length per base pair within 4%. The rRNA genes (rDNA) in the macronucleus of Tetrahymena are extrachromosomal molecules containing about 20,000 base pairs (bp) of DNA (1, 2). The linear molecules are palindromic, each half containing a set of rRNA coding sequences and adjacent spacer sequences (3, 4). The 10,000 palindromic rDNA molecules in the nucleoli of the macronucleus are probably formed by amplification of a single, nonpalindromic copy integrated in a micronuclear chromosome (5).As one of the few eukaryotic genes that can be isolated in a transcriptionally active chromatin state (6, 7), the Tetrahymena rDNA is of particular interest. For studies of chromatin structure and gene regulation it is necessary to have a detailed transcription map of the rDNA. Previous studies have revealed some general features. Gall et al. (8) showed that transcription proceeds bidirectionally from the central region toward the ends of the molecule. Engberg et al. (4) and Karrer (9) found that the 26S rRNA sequences are distal to the 17S rRNA sequences. Transcription of the smaller rRNA therefore precedes that of the larger rRNA in Tetrahymena as in other organisms (reviewed in ref. 10).The formation of R-loops, regions of RNA-DNA hybridization within a double-stranded DNA molecule, was first described by White and Hogness (11) and Thomas et al. (12). In the present study, we have used R-loop mapping to locate precisely the sequences coding for the mature cytoplasmic rRNAs and the nuclear precursor rRNAs on the rDNA of Tetrahymena thermophila.MATERIALS AND METHODS Growth of Cells. Tetrahymena thermophila strain B VII, obtained from M. Gorovsky, was grown in a medium containing 10 g of proteose peptone (Difco), 30 mg of SequestrineThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. 8 M urea (16) and were run at room temperature. RNA in the 8 M urea sample buffer was heated for 5 min at 65°C before being applied to denaturing gels.Isolation of rDNA. Ex...

“…The Ad5 ts mutant designated H5ts125 has been best characterized. The mutant gene product is coded by DNA between 62.4 and 67.1% from the left-hand end of the genome (19). The protein is a 72,000-dalton polypeptide which binds strongly to singlestranded Ad2 DNA (20).…”

mentioning

confidence: 99%

Temperature-sensitive replication of H5ts125 adenovirus DNA in vitro.

Horwitz¹

1978

A soluble adenovirus DNA replication system has been prepared in extracts of HeLa cells infected with the temrperature-sensitive mutant HMts125. These nuclear extracts synthesize full-sized viral DNA at 300 but are temperature sensitive in this function at 380. A 72,000-dalton DNA-binding protein purified from cells infected with wild-type virus complements the temperature-sensitive defect and restores viral DNA replication to 74% of normal values. We have recently described the preparation of a nuclear extract from adenovirus (Ad)infected cells that catalyzed the synthesis of viral DNA in a manner analogous to that used in whole cells (1). These extracts, which were free of chromosomal DNA, contained replicating Ad DNA and synthesized full-size viral DNA products. Similar approaches developed in prokaryotes and their infecting bacteriophages have allowed the analysis of various gene products required for DNA initiation and elongation (2)(3)(4)(5). The general approach has been to prepare extracts from bacterial cell mutants that are temperature sensitive (ts) in DNA synthesis and to use these extracts as a receptor to isolate biochemically complementing activity from wild-type