Article

Expression of the autonomous parvovirus H1 genome: Evidence for a single transcriptional unit and multiple spliced polyadenylated transcripts

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Abstract

We identified viral transcripts in parvovirus H1-infected rodent cells using the S1 nuclease mapping technique of Berk and Sharp (1977, 1978). The most abundant viral transcript, present in both nucleus and cytoplasm, is approximately 2.8 kb long and represents about 56% of the viral genome. Less abundant viral transcripts of 3.0, 1.45 and 1.30 kb, and possibly other minor viral transcripts, are also detected in nuclear and cytoplasmic fractions. In contrast, a prominent 4.7 kb viral transcript which corresponds to 95% of the viral DNA is found only in the nucleus; this finding suggests that the parvovirus genome may function as a single transcription unit. Virus-infected cells pretreated with cycloheximide accumulate all these viral transcripts. Analyses of RNA-DNA hybrids (isolated from neutral agarose gels) by electrophoresis on alkaline agarose gels indicate that the 4.7, 3.0 and 2.8 kb viral transcripts are "spliced" RNAs. The nuclear-specific 4.7 kb transcript appears to be encoded by two noncontiguous DNA segments of 2.2 and 2.6 kb. The 3.0 and 2.8 kb transcripts are apparently encoded by.a 2.6 kb segment of DNA and one or more much smaller noncontiguous DNA segments, one of which is approximately 170 nucleotides long.

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Biochemical methods are presented for determining the structure of spliced RNAs present in cells at low concentrations. Two cytoplasmic spliced viral RNAs were detected in CV-1 cells during the early phase of simian virus 40 (SV40) infection. One is 2200 nucleotides in length and is composed of two parts, 330 and 1900 nucleotides, mapping from approximately 0.67 to approximately 0.60 and from approximately 0.54 to approximately 0.14, respectively, on the standard viral map. The other is 2500 nucleotides long and also is composed of two parts, 630 and 1900 nucleotides mapping from approximately 0.67 to approximately 0.54 and from approximately 0.54 to approximately 0.14, respectively. Correlation of the structure of these mRNAs with the structure of the early SV40 proteins, small T antigen (17,000 daltons) and large T antigen (90,000 daltons), determined by others suggests that: (i) translation of the 2500-nucleotide mRNA yields small T antigen; (ii) translation of the 2200-nucleotide mRNA proceeds through the splice point in the RNA to produce large T antigen (and thus large T antigen is encoded in two separate regions of the viral genome); and (iii) the DNA sequences between approximately 0.67 and approximately 0.60 present in both mRNAs are translated in the same reading frame in both mRNAs to yield two separate gene products that have the same NH(2)-terminal sequence. Therefore, expression of the early SV40 genes is partially controlled at the level of splicing of RNAs.
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In previous work, linear duplex molecules of adeno-associated virus, type 2 (AAV2), DNA were cleaved with the restriction endonucleases R-EcoRI, R-HindII, and R-HindIII. The physical order of the specific fragments obtained was deduced and oriented with respect to the DNA strand polarity and the direction of transcription. Stable AAV RNA is transcribed only from 70% of the minus DNA strand. We report here RNA-DNA hybridization experiments using these restriction fragments to obtain a more accurate map of the portion of the AAV genome represented in stable RNA. The data obtained with several sets of restriction fragments annealed to either whole-cell RNA or poly(A)-containing RNA were internally consistent. The AAV RNA annealed with a continuous region of the AAV DNA, beginning at 0.18 map units (18%) from the left end of the molecule and ending at 0.88 map units. In addition, the restriction endonuclease BamHI was found to make one specific cleavage in AAV2 DNA at 0.22 map units, which is 0.04 map units (i.e., 160 nucleotides) to the right ("down stream'') of the point corresponding to the 5' end of the viral mRNA.
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S1 nuclease (EC 3.1.4.X), a single-strand-specific nuclease, can be used to accurately map the location of mutational alterations in simian virus 40 (SV40) DNA. Deletions of between 32 and 190 base pairs, which are at or below the limit of detectability by conventional electron microscopic analysis of heteroduplex DNAs, have been located in this way. To map a deletion, a mixture of unit length, linear DNA, prepared from the SV40 deletion mutant and its wild-type parent, are denatured and reannealed to form heteroduplexes. S1 nuclease can cut such heteroduplexes at the nonbase-paired region to produce fragments whose lengths correspond to the position of the deletion. Similarly, specific fragments are produced when S1 nuclease cleaves a heteroduplex formed from the DNAs of SV40 temperature-sensitive mutants and either their revertants or wild-type parents. Thus, the positions of the nonhomology between these DNAs can be determined.
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Viral complementary DNA (cDNA) sequences corresponding to the gag, pol, env, src, and c regions of the Rous sarcoma virus genome were selected by hybridizing viral cDNA to RNA from viruses that lack the env or src gene or to polyadenylic acid [poly(A)]-containing RNA fragments of different lengths and isolating either hybridized or unhybridized DNA. The specificities, genetic complexities, and map locations of the selected cDNA's were shown to be in good agreement with the size and map locations of the corresponding viral genes. Analyses of virus-specific RNA, using the specific cDNA's as molecular probes, demonstrated that oncovirus-infected cells contained genome-length (30-40S) RNA plus either one or two species of subgenome-length viral RNA. The size and genetic content of these RNAs varied, depending on the genetic makeup of the infecting virus, but in each case the smaller RNAs contained only sequences located near the 3' end of the viral genome. Three RNA species were detected in Schmidt-Ruppin Rous sarcoma virus-infected cells: 39S (genome-length) RNA; 28S RNA, with an apparent sequence of env-src-c-poly(A); and 21S RNA, with an apparent sequence of src-c-poly(A). Cells infected with the Bryan high-titer strain of Rous sarcoma virus, which lacks the env gene, contained genome-length (35S) RNA and 21S src-specific RNA, but not the 28S RNA species. Leukosis virus-infected cells contained two detectable RNA species: 35S (genome-length) RNA and 21S RNA, with apparent sequence env-c-poly(A). Since gag and pol sequences were detected only in genome-length RNAs, it seems likely that the full-length transcripts function as mRNA for these two genes. The 28S and 21S RNAs could be the active messengers for the env and src genes. Analyses of sequence homologies among nucleic acids of different avian oncoviruses demonstrated substantial similarities within most of the genetic regions of these viruses. However, the "common" region of Rous-associated virus-0, an endogenous virus, was found to differ significantly from that of the other viruses tested.
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We have analyzed an autointerference phenomenon exhibited by adeno-associated virus type 2 (AAV) when grown in KB cells coinfected with adenovirus type 2 as the helper. Infectious AAV particles that banded at 1.41 g/cm3 in CsCl were purified by three cycles of centrifuging in CsCl equilibrium gradients. When cells were infected with an increasing multiplicity of these AAV particles there was a corresponding decrease in production of infectious progeny AAV. There was also an AAV multiplicity-dependent inhibition of production of infectious adenovirus and inhibition of Ad DNA replication. The viral DNA in the Hirt supernatant fraction extracted from cells infected with different multiplicities of AAV was analyzed in neutral sucrose gradients. At low multiplicities of infection with AAV, the main AAV DNA species synthesized was the mature 14.5 S (standard) viral genome. In higher multiplicity infections with AAV increasing amounts of aberrant 10 S AAV DNA molecules accumulated and the proportion of 14.5 S AAV DNA decreased. Restriction endonuclease cleavage showed that the 10 S DNA was enriched for the left- or right-hand terminal regions of the AAV genome. These molecules may be analogous to the previously characterized aberrant DNA molecules found in light-density AAV particles. Thus, the AAV autointerference is correlated with production of the aberrant deleted AAV genomes.
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In solutions containing 70% formamide hybridization of RNA to denatured DNA proceeded in the absence of DNA reannealing at temperatures permitting the optimum rate of formation of RNA-DNA hybrids. At low complementary DNA to RNA ratios the percentage of ribosomal RNA hybridized was substantially higher in 70% formamide than in solutions lacking formamide. The ability to perform RNA-DNA hybridization without competing DNA reannealing facilitates the description of the kinetics and stoichiometry of the reaction.
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A simple microscale procedure is described for the synthesis and purification of high specific activity α-[32P]deoxyribonucleoside 5′-triphosphates (300-1300 Ci/ mmol) and their utilization in labeling as little as 56 ng of adenovirus DNA or DNA restriction fragments to specific activities up to 4 × 108 cpm/μg by in vitro repair synthesis ("nick translation"). [3H]DNA of specific activity 1.5 × 107 cpm/μg has also been prepared by nick translation using commercial [3H]thymidine 5′-triphosphate (50 Ci/mmol). These products have been extensively characterized as to reassociation properties. Adenovirus 12 DNA and the EcoRI restriction fragments labeled by this procedure were shown to be identical with in vivo labeled viral DNA by three types of molecular hybridization measurements including (i) copy number determination by reassociation kinetics, of viral DNA in viral transformed cells, (ii) renaturation rate of viral DNA and restriction fragments and agreement with genetic complexity, and (iii) saturation-hybridization of labeled viral DNA with unlabeled restriction fragments. Finally, the distribution of radioactivity in restriction fragments prepared from in vitro labeled DNA showed that viral DNA is uniformly labeled in vitro. These studies represent the first direct demonstration that the in vitro labeling procedure produces sensitive, accurate, and representative DNA probes. Most important, our microscale procedure conserves on expensive nucleoside triphosphates, and valuable (perhaps irreplaceable) viral or tissue nucleic acids. Therefore, this is a versatile method for preparing labeled DNA probes for detecting homologous nucleic acid sequences in small quantities of viral, cellular, or tissue nucleic acids.
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The differences in the biochemistry of messenger RNA formation in eukaryotes compared to prokaryotes are so profound as to suggest that sequential prokaryotic to eukaryotic cell evolution seems unlikely. The recently discovered noncontiguous sequences in eukaryotic DNA that encode messenger RNA may reflect an ancient, rather than a new, distribution of information in DNA and that eukaryotes evolved independently of prokaryotes.
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We describe a technique for transferring electrophoretically separated bands of RNA from an agarose gel to paper strips. The RNA is coupled covalently to diazobenzyloxymethyl groups on the paper. After transfer and appropriate treatment of the paper to destroy remaining diazo groups, specific RNA bands can be detected by hybridization with 32P-labeled DNA probes followed by autoradiography. This procedure allows detection of specific RNA bands with high sensitivity and low background.
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Transcription of the genomes of the defective parvovirus, adenovirus-associated virus (AAV) type 2, and a helper virus, adenovirus (Ad) type 2, was studied in nuclei isolated from infected KB cells. As measured by the incorporation of [3H]UTP into acid-insoluble product, nuclei prepared 15.5 hr after coinfection with AAV and Ad (AAV-Ad nuclei) synthesized RNA for 10 min. Total RNA synthesis in AAV-Ad nuclei was greater than in uninfected nuclei but was only about that observed in nuclei from cells infected with Ad alone (Ad nuclei). DNA-RNA hybridization with RNA samples from the AAV-Ad nuclei revealed that the in vitro synthesis of AAV-RNA (14% of total) was twice that of Ad-RNA (6% of total). When Ad or AAV-Ad nuclei were assayed at various times after infection, total RNA production was comparable until AAV transcription was well established (12 hr); thereafter, a relative depression of total RNA synthesis was observed in the AAV-Ad nuclei. However, from 12 hr on, more AAV-RNA than Ad-RNA was synthesized in AAV-Ad nuclei, and a comparison with Ad nuclei suggested a preferential inhibition of Ad-RNA transcription in the AAV-Ad nuclei. Finally, it was found that AAV, Ad, and total RNA synthesis shared the same optimum salt concentration [0.02 M (NH4)2SO4], and that 95% of Ad-RNA synthesis and greater than 99% of AAV-RNA synthesis were inhibited by levels of α-amanitin which blocked cellular RNA polymerase II activity. These data suggest that the AAV genome is also transcribed by the same polymerase that transcribes the bulk of Ad-RNA and cellular heterogeneous nuclear RNA.
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The DNA of bacteriophage T7 is cut into seven unique fragments by the restriction endonuclease DpnII (or the equivalent MboI), 19 fragments by HpaI, and eight additional fragments by the combination of the two enzymes. The relative location of each fragment in the T7 DNA has been determined by a combination of techniques. If it is assumed that the length of any DNA molecule equals the sum of the lengths of the fragments produced from it by cleavage, and that electrophoretic mobility through agarose gels is a smooth function of the length of the DNA, then the known relationships between fragments provide enough conditions to define accurately the relative molecular weight of each fragment in the set. Absolute molecular weights are based on that of full-length T7 DNA. The fragments provide a convenient set of length standards covering the entire range from about 100 to 40,000 base-pairs (the length of T7 DNA). A horizontal slab gel system for electrophoresis on agarose gels is described. In this system, gels of very low concentrations do not distort during electrophoresis and accurate relative mobilities of large DNAs are obtained. Excellent resolution can be obtained for DNAs of molecular weights up to at least 26·5×106, a difference of less than 10% being readily resolved even for molecules of this size. Agarose and polyacrylamide gels can be prepared in alkaline solvents that denature native DNA and completely unfold the single strands. The fragments of T7 DNA have the same relative mobilities whether subjected to electrophoresis as single strands in alkaline gels or as double-stranded DNA in neutral gels, and resolution is comparable in the two states. Thus, electrophoresis in alkaline gels can provide accurate molecular weights for linear, single-stranded DNAs, and should be useful in analyzing DNA for single-strand breaks, depurinations or topological differences such as ring forms. In both neutral and alkaline gels, the relative mobilities of DNAs shorter than about 1000 base-pairs (or bases) are essentially insensitive to changes in voltage gradient, at least over the range of voltage gradients commonly employed. However, relative mobilities become increasingly sensitive to voltage gradient the larger the DNA, with DNAs longer than about 20,000 base-pairs (or bases) being severely affected. This effect is probably due to the ease with which large DNA molecules can be deformed from their equilibrium conformations, thus permitting them to penetrate channels in the gel that would exclude them in their unperturbed conformations. As a practical matter, this means that low voltage gradients must be used for separations of large DNAs by gel electrophoresis.
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The cleavage map of H-1 replicative-form DNA to the bacterial restriction endonuclease EcoRI, HaeII, HaeIII, HindII, HindIII, and HpaII has been determined. The 5'-phosphoryl end of the viral strand is on the right end of the molecule at or near the replication origin. Evidence is presented for the presence of inverted self-complementary sequences at the right end that differ from those at the left end. These sequences allow a foldback of the DNA after denaturation, and a minority of the native replicative-form DNA has the foldback configuration. The possible role of these structures in H-1 DNA synthesis is discussed.
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The DNA-containing (full) particles of minute virus of mice contain three polypeptide species, designated A (Mr=83,000), B (Mr=64,000) and C (Mr=61,000). These three proteins are compared here by tryptic and chymotryptic fingerprinting after radio-iodination of their tyrosyl residues in vitro. Polypeptide B and C digests are almost identical, thus confirming the precursor-product relationship between them suggested by previous kinetic studies. Both types of fingerprint show one peptide which occurs in the C polypeptide but not in B, indicating that the cleavage in vivo may not occur at either a tryptic or chymotryptic site. In addition to the relationship between B and C, all of the sequence of B is present in the largest polypeptide A, which constitutes≈16% of the total virion protein. The A polypeptide contains additional tyrosyl peptides, comprising about 20% of the total, which do not occur in either B or C.Proteolytic digestion of intact full particles in vitro shows that the cleavage of B in vivo can be closely mimicked by trypsin and to a lesser extent by chymotrypsin. However, the B polypeptide in the empty virion is resistant to cleavage by either enzyme in vitro, indicating that it adopts a different conformation in each particle type. This correlates well with the in vivo observation that empty particles contain polypeptide B, in addition to A, and do not contain any polypeptide C. The A polypeptide is completely resistant to cleavage by either enzyme in either particle, suggesting that the conformation of the common sequence in polypeptide A may be similar to that adopted by polypeptide B in empty virions. A scheme for the maturation of infectious parvovirus virions is described.
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We have developed a simple and sensitive method for detecting, sizing and mapping RNA transcripts from viral or cloned DNAs. This technique has been used to examine the cytoplasmic transcripts produced during the early phase of adenovirus 2 (Ad2) infection of HeLa cells. Unlabeled total cytoplasmic or oligo (dT)-selected cytoplasmic RNA is hybridized to restriction fragments of 32P-labeled viral DNA in 80% formamide under conditions above the Tm of the DNA duplex, but below the Tm of the RNA-DNA hybrid duplex (Casey and Davidson, 1977). DNA complements precisely the length of the hybridized RNA are generated by treating with single-strand-specific S1 endonuclease under conditions which do not introduce strand breaks into hybrid duplex. The sizes of the S1-resistant single-stranded DNAs are then determined by alkaline agarose gel electrophoresis (McDonnell, Simon and Studier, 1977). A restriction fragment which terminates within a region coding for an mRNA yields a band equal in size to the portion of the mRNA transcribed from that restriction fragment. This allows unique mapping of coding regions relative to restriction endonuclease cleavage sites.
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The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the "nuclear skeleton." SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.
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The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [3H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.
Article
We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.
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An important objective in studying adenovirus associated virus (AAV) multiplication is to determine the number of functions supplied by helper viruses and to define the biochemical role of each. A detailed analysis is presented of the kinetics of synthesis of viral DNA and RNA in DB cells coinfected with AAV type 2 (AAV2) and adenovirus type 2 (Ad2) as helper. Data obtained suggest that adenovirus provides separate helper functions for AAV DNA and RNA synthesis, and that these functions appear sequentially after adenovirus infection.
Article
Nucleic acid hybridization procedures were used to measure the extent of transcription of adenovirus-associated virus (AAV) deoxyribonucleic acid (DNA) in KB cells in the presence of either adenovirus or herpes simplex virus as the helper. Annealing of AAV ribonucleic acid to AAV DNA was monitored by a hybridization inhibition assay on nitrocellulose filters or by hydroxyapatite chromatography. These experiments confirmed the previous observation that, in the presence of either type of helper virus, only one strand of AAV DNA (the thymidine-rich or "minus" strand) is transcribed in vivo. However, it was found that only 70 to 80% of this strand appears to be transcribed in vivo. Furthermore, studies with minus strands employing hydroxyapatite chromatography and nuclease S(1), which specifically degrades single-stranded DNA, indicated that up to 20% of the minus strand is self-complementary. It seems likely that these self-complementary sequences account for the bulk of that portion of the minus strand (20 to 30%) which is not transcribed in vivo.
Article
We have studied the viral-specific RNA synthesized after infection of a permissive cell line with Kilham rat virus (KRV). The RNA was shown to be virus specific by analysis of its nucleotide base ratios and by hybridization with KRV and cellular DNA. Viral RNA is synthesized as early as 2 h after infection. This viral RNA synthesis occurs before viral progeny DNA synthesis which is initiated at 7 to 8 h after infection. The predominant viral RNA synthesized before and after viral progeny DNA synthesis has a sedimentation coefficient of approximately 18S in dimethylsulfoxide-sucrose gradients and a calculated molecular weight of 6.5 x 10(5) to 7.5 x 10(5). KRV contains a molecule of single-stranded DNA with a molecular weight of approximately 1.6 x 10(6). If the viral-specific 18S RNA is a homogenous species, it would account for 40 to 50% of the viral genome. A small amount of 26S viral RNA with a molecular weight of 1.6 x 10(6) to 1.7 x 10(6) can also be detected. If this 26S RNA is a single viral-specific entity, it could represent a transcription of the entire KRV genome.
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The synthesis of two virus-induced proteins, VP1 (mol wt 92,000) and VP2' (mol wt 72,000), was detected in the cell and in purified virions from H-1 parvovirus propagated in synchronous cell cultures.
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We have studied viral RNA synthesized in KB cells coinfected with adenovirus-associated virus type 2 (AAV-2) and adenovirus type 2 (Ad2) as the helper. RNA was analysed in nonaqueous, denaturing solvents either by sucrose density gradient sedimentation in 99% dimethyl sulfoxide or by acrylamide gel electrophoresis in 98% formamide. The analyses revealed two populations of AAV RNA, (i) a single, discrete 20 S species and (ii) a very heterogenous population ranging in size from 4 S to 18 S. In the nucleus both populations of AAV RNA were observed but in the cytoplasm the 20 S RNA was mainly present in the polysome fraction and the heterogenous RNA was mainly in the nonpolysomal fraction. The 20 S RNA therefore appears to be the only functional AAV message species. The heterogenous AAV RNA may arise from incomplete transcription or degradation of the 20 S RNA in the nucleus or cytoplasm. The molecular weight of the 20 S AAV RNA was approximately 0.9 × 106–1.0 × 106 which accounts for the entire region of the AAV genome that is stably transcribed. This 20 S RNA may be monocistronic and thus AAV DNA may contain only one gene.The 20 S AAV RNA did not appear to be formed by posttranscriptional cleavage of a larger nuclear precursor. In the same experiments the posttranscriptional cleavage of Ad RNA was clearly observed. At least nine species of Ad RNA were present in the cytoplasm late after infection and at least six of these were associated with polysomes. Nonpolysomal, cytoplasmic Ad RNA was heterogenous but contained one discrete, 5.5 S species (VA RNA) which was transported to the cytoplasm more rapidly than either Ad or AAV message RNA or cellular rRNA.
Article
A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.
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Several strains of Escherichia coli 15 harbor a prophage. Viral growth can be induced by exposing the host to mitomycin C or to ultraviolet irradiation. The coliphage 15 particles from E. coli 15 and E. coli 15 T− appear as normal phage with head and tail structure; the particles from E. coli 15 TAU are tailless. The complete particles exert a colicinogenic activity on E. coli 15 and 15 T−; the tailless particles do not. No host for a productive viral infection has been found and the phage may be defective. The properties of the DNA of the virus have been studied, mainly by electron microscopy. After induction but before lysis, a closed circular DNA with a contour length of about 11.9 μ is found in the bacterium; the mature phage DNA is a linear duplex and 7.5% longer than the intracellular circular form. This suggests the hypothesis that the mature phage DNA is terminally repetitious and circularly permuted. The hypothesis was confirmed by observing that denaturation and renaturation of the mature phage DNA produce circular duplexes with two single-stranded branches corresponding to the terminal repetition.
Article
The two complementary strand species of 5-bromodeoxyuridine-substituted, adenovirus-associated virus type 2 (AAV-2) deoxyribonucleic acid were preparatively separated in CsCl density gradients and further purified by sedimentation through 5 to 20% sucrose. The base composition of each strand species was determined, and it was found that the species banding at a greater density in CsCl (heavy strands) had an expected higher thymidine content (26.5%) than that 21.7%) of the less dense species (light strands). Furthermore, the base composition of in vivo-synthesized, AAV-specific ribonucleic acid was similar to that of light-strand deoxyribonucleic acid, and this ribonucleic acid apparently hybridized only with heavy strands. These observations indicate that the heavy-strand species alone serves as the transcriptional template in vivo. This study represents the first instance in which the base composition and specificity of in vivo transcription have been determined for each of the complementary strands of an animal virus deoxyribonucleic acid.
  • Carter B.J.
  • Khoury G.
  • Rose J.A.
The Replication of Mammalian Parvoviruses.
  • Ward D.C.
  • Tattersall P.