DNA-RNA Hybrid Molecules Research Articles

Novel Determinants of Intestinal Colonization of Salmonella enterica Serotype Typhimurium Identified in Bovine Enteric Infection

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle.

Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt

The genetic elements of herpesvirus origins of lytic replication have been characterized in detail; however, much remains to be elucidated concerning their functional role in replication initiation. In the case of the Epstein-Barr virus (EBV), we have found that in addition to the two well-defined critical elements required for lytic replication (the upstream and downstream essential elements, UEE and DEE), the origin of lytic replication (OriLyt) also requires the presence of a GC-rich RNA in cis. The BHLF1 transcript is similar to the essential K5 transcript identified at the Kaposi's sarcoma-associated herpesvirus OriLyt. We have found that truncation of the BHLF1 transcript or deletion of the TATA box, but not the putative ATG initiation codon, reduce OriLyt function to background levels. By using an antibody specific for RNA-DNA hybrid molecules, we found the BHLF1 RNA stably annealed to its DNA template during the early steps of lytic reactivation. Furthermore, expression of human RNase H1, which degrades RNA in RNA-DNA hybrids, drastically reduces OriLyt-dependent DNA replication as well as recruitment of the viral single-stranded DNA binding protein BALF2 to OriLyt. These studies suggest that a GC-rich OriLyt transcript is an important component of gammaherpesvirus lytic origins and is required for initial strand separation and loading of core replication proteins.

Response to: The Schizosaccharomyces pombe imprint — nick or ribonucleotide(s)?

Vengrova and Dalgaard believe our observations are better explained by their ribonucleotide(s) replacement model [1xRNase-sensitive DNA modification(s) initiates S. pombe mating-type switching. Vengrova, S. and Dalgaard, J.Z. Genes Dev. 2004; 18: 794–804Crossref | PubMed | Scopus (66)See all References1] rather than by the dephosphorylated nick model we proposed [2xA programmed strand-specific and modified nick in S. pombe constitutes a novel type of chromosomal imprint. Kaykov, A. and Arcangioli, B. Curr. Biol. 2004; 14: R915–R917Abstract | Full Text | Full Text PDF | PubMed | Scopus (30)See all References2]. Here we wish to review the two models in the context of previously published work and discuss the observations made in the accompanying letter.Recently, Vengrova and Dalgaard stated that the imprint can be purified intact, or cleaved in some conditions. We believe it may be purified as cleaved in all conditions, since we were never able to isolate intact imprinted mat1 upper strand. Furthermore, they propose that the imprint is composed of either one or two ribonucleotides, and that starting from an intact or nicked molecule, treatments with RNase T2 or NaOH remove one ribonucleotide from the 5′-end mat1-distal upper strand, converting the imprint into a gapped molecule [1xRNase-sensitive DNA modification(s) initiates S. pombe mating-type switching. Vengrova, S. and Dalgaard, J.Z. Genes Dev. 2004; 18: 794–804Crossref | PubMed | Scopus (66)See all References1].Our recent work [2xA programmed strand-specific and modified nick in S. pombe constitutes a novel type of chromosomal imprint. Kaykov, A. and Arcangioli, B. Curr. Biol. 2004; 14: R915–R917Abstract | Full Text | Full Text PDF | PubMed | Scopus (30)See all References2] was designed to further analyze the molecular nature of the imprint and to directly challenge the nick and RNA models. A PstI site was inserted at mat1, and shows that a nick is located at a precise and fixed position, as observed in the wild-type strain, and is independent of RNase T2 treatment (yielding no gaps), which is incompatible with the presence of a 5′-end ribonucleotide(s). In addition, the potential caveat that our engineered strain containing PstI affects the number of nucleotides modified in a sequence-specific manner is not supported by the PstI series of mutant strains, which allows us to position the nick next to any of the four bases (stated as data not shown in [2xA programmed strand-specific and modified nick in S. pombe constitutes a novel type of chromosomal imprint. Kaykov, A. and Arcangioli, B. Curr. Biol. 2004; 14: R915–R917Abstract | Full Text | Full Text PDF | PubMed | Scopus (30)See all References2]). Altogether, our data support the simplest and most economical model, that the imprint at mat1 is a nick containing 3′OH and 5′OH termini and is resistant to RNase T2 treatments.Here, Vengrova and Dalgaard attempt a unifying explanation. They suggest that two populations of imprint exist, containing either one or two ribonucleotides, and propose that hydrolysis of a putative DNA–RNA–DNA hybrid molecule containing a single ribonucleotide would yield a nicked molecule in which the ribonucleotide will remain attached to the 3’ end of the nick. In principle, this new interpretation could allow our set of data [2xA programmed strand-specific and modified nick in S. pombe constitutes a novel type of chromosomal imprint. Kaykov, A. and Arcangioli, B. Curr. Biol. 2004; 14: R915–R917Abstract | Full Text | Full Text PDF | PubMed | Scopus (30)See all References2] to become compatible with a ribonucleotide on the 3’ end but not the 5’ end of the nick [1xRNase-sensitive DNA modification(s) initiates S. pombe mating-type switching. Vengrova, S. and Dalgaard, J.Z. Genes Dev. 2004; 18: 794–804Crossref | PubMed | Scopus (66)See all References1].We agree that a unifying explanation would be ideal. To achieve this, we should not make more assumptions before answering the following questions. What are the methods used by Dalgaard and colleagues to prepare intact/nicked DNA? How is their PCR approach able to synthesize across the intact heat-labile imprinted strand? What is the direct evidence for a mixed population of one and two ribonucleotides? Ultimately, identification of the machinery responsible for the imprint remains the major issue.

The Schizosaccharomyces pombe imprint — nick or ribonucleotide(s)?

The Schizosaccharomyces pombe imprint — nick or ribonucleotide(s)?

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides.

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5-10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.

Mapping Initiation Sites for Simian Virus 40 DNA Synthesis Events In Vitro

Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.

Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.

Evaluation of an Oligonucleotide Probe for Identification ofCampylobacterSpecies

A total of 209 well-defined Campylobacter, Helicobacter , and Wolinella strains were tested with a Gen-Probe oligodeoxyribonucleotide probe for Campylobacter jejuni , Campylobacter coli , and Campylobacter lari . Released ribosomal RNA was hybridized with the acridinium-ester-labeled probe. The amount of light proportional to the number of DNA-RNA hybrid molecules was captured by a photo-multiplier and converted to a digital signal. A reading of more than 50,000 relative light units (RLU) was considered positive. After isolates were cultivated, lysis and hybridization procedures were completed in 30 minutes. All 104 strains within the target species were positive, as well as two Campylobacter hyointestinalis strains. The remaining 103 strains, including 15 C hyointestinalis strains, were negative. Our data indicate a sensitivity of 1.0 and specificity of 0.98 (P <.001).

Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

A DNA fragment encoding Ribonuclease H (EC 3. 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant. The primary amino acid sequence showed 56% similarity to that of E. coli RNase HI but little or no homology to E. coli RNase HII. Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts. However, T. thermophilus RNase H has one more cysteine than its E. coli homologue. Stability of the RNase H in extracts of T. thermophilus to elevated temperatures was the same for the protein expressed in E. coli. T. thermophilus RNase H should, therefore, be a useful tool for editing RNA-DNA hybrid molecules at higher temperatures and may also be stable enough to be used in a cyclical process. It was suggested that regulation of expression of the RNase H may be different from that of E. coli. RNase HI.

In Situ Hybridization

Twenty years ago Gall and Pardue1 reported a method to detect RNA-DNA hybrid molecules in cytologic specimens. Since then diagnostic in situ hybridization has become a standard method to detect DNA and RNA.The method has also been modified for use in electron microscopy2-4 but it is not easy to preserve both the hybridization properties and an acceptable morphology. Most postembedding techniques are useful for DNA hybridization but not for RNA analyses. Some of our problems may be solved with microwave procedures for EM fixation,5 but at present, ultrathin cryosectioning is the method of choice to preserve both DNA and RNA molecules. This sophisticated and time-consuming technique is not, however, in widespread use in EM laboratories, and preembedding nuclear acid hybridization will probably be preferred for quite some time.Colloidal gold rather than radioactive probes seems to be the marker to favor. Protein A-gold complexes have been used to demonstrate cellular RNA sequences,3 and preembedding in situ hybridization with colloidal gold has been used in order to demonstrate cytomegalovirus DNA in human fibroblasts.4The in situ hybridization electron microscopy method demonstrates which genes are present, their rearrangements, viral genomes, and so on, and even shows their exact subcellular localization. Detection of viral DNA as well as oncogenes may once more focus the interest of the ultrastructural pathologist on the nucleus. In combination with immunoelectron microscopy, we have a powerful tool to follow the dynamic patterns of oncogenes, the resulting mRNA, and finally, the protein product within the tumor cells.Ultrastructural Pathology welcomes papers on the techniques and applications of in situ hybridization electron microscopy.

Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.

Hepatitis B viral DNA in infected human liver and in hepatocellular carcinoma.

Blot-hybridization analysis of DNA forms from hepatitis B virus (HBV) in the liver of chronic carriers of hepatitis B surface antigen (HBsAg) has revealed the presence of both relaxed and closed circular viral DNA and of novel viral DNA-RNA hybrid molecules in patients with complete virions, with the hepatitis B e antigen (HBeAg), or with both, in serum. One carrier of HBsAg with no detectable virus or HBeAg in his serum had small amounts of free viral DNA in his liver sample, a finding suggesting the potential for production of complete virus; another such carrier had only HBV DNA integrated in cellular DNA and, thus, may have lost the ability to replicate virus. The liver sample of one of eight patients with antibodies to HBsAg in his serum, but no HBsAg, contained small amounts of free viral DNA. Analysis of tissue from hepatocellular carcinoma revealed evidence for integrated viral DNA sequences in multiple-cellular DNA sites, and stoichiometric analysis suggested that the tumors were monoclonal in origin. These results demonstrate the presence of a new form of HBV in infected human liver and reveal that serological profiles are not always a reliable guide in determining the presence of potentially infectious forms of HBV in the liver.

Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous dna polymerase reaction

Particles purified from the liver of hepatitis B virus-infected patients were previously shown by us to incorporate 32P-deoxynucleotides into viral DNA and DNA-RNA hybrid molecules when incubated in a DNA polymerase reaction mixture. In this investigation, similar particles from duck and ground squirrel livers infected with viruses closely related to HBV were also shown to incorporate 32P-deoxynucleotides into viral-specific DNA and DNA-RNA hybrid molecules when incubated in vitro in a DNA polymerase reaction mixture. The particles from duck hepatitis B virus-infected liver contained a heterogeneous population of hybrid molecules, while those from ground squirrel hepatitis virus-infected liver contained hybrid molecules with densities similar to those found in HBV particles including a distinct population of molecules with an average density of 1.57 g/cm 3. Brief endogenous DNA polymerase reactions with particles from all three livers, resulted in incorporation of 32P-deoxynucleotides into viral DNA of DNA-RNA hybrid as well as viral DNA molecules. When the reaction was continued in the presence of a 1000-fold molar excess of unlabeled deoxynucleotides, a decrease in [ 32P]DNA in the DNA-RNA hybrid region of the Cs 2SO 4 density gradient and a proportional increase in [ 32P]DNA in the DNA region of the gradient was observed. This effect was seen most dramatically with particles isolated from freshly obtained ground squirrel hepatitis virus-infected livers in which 90% of the pulse labeled DNA in the hybrid species at the buoyant density of 1.57 g/cm 3 appeared to be converted to a form with the buoyant density of pure DNA (1.42 g/cm 3). Storage of virus particles at 4°, or prior freezing of infected ground squirrel liver almost completely abolished the ability of the endogenous DNA polymerase activity to incorporate 32P-deoxynucleotides into hybrid molecules, while incorporation into DNA molecules was apparently unaffected. These results suggest that different enzymatic activities catalyze synthesis of the viral DNA in DNA-RNA hybrids and in molecules with buoyant density of pure DNA. Thus particles from infected liver synthesize DNA of DNA-RNA hybrid molecules which can be converted in the particles into molecules with the buoyant density of pure DNA. This indicates that DNA-RNA hybrids may be intermediates in viral DNA replication and that the mechanism of hepatitis B virus (and closely related viruses of ground squirrels and ducks) DNA replication differs from that known for other DNA viruses.

Hepatitis B virus particles of plasma and liver contain viral DNA-RNA hybrid molecules

Hepatitis B virions in plasma (Dane particles) are known to contain small circular DNA molecules. The experiments described here indicate that virions in plasma, as well as particles from hepatitis B virus-infected human liver, also contain viral DNA-RNA hybrid molecules, and deoxynucleotides can be incorporated into the DNA of these hybrids by DNA polymerase activities in the virions. Thus, two viral DNA synthetic reactions appear to take place in virions: repair of the single-stranded region of circular DNA molecules and synthesis or elongation of the DNA strand of DNA-RNA hybrid molecules. Centrifugation of virion nucleic acid to equilibrium in Cs 2SO 4 density gradients revealed the presence of viral DNA-RNA hybrid molecules over a density range of 1.45 to 1.60 g/cm 3. Distinct species of hybrid molecules were found with an average density of 1.57 g/cm 3 in Dane particles and 1.52 and 1.57 g/cm 3 in particles from liver. Fractionation of nucleic acid from Cs 2SO 4 density gradients by gel electrophoresis demonstrated that the majority of hybrid molecules migrated faster than molecules with the density of pure DNA (1.42 g/cm 3). One notable exception was the finding of DNA-RNA hybrid molecules migrating slower than open circular viral DNA. Characterization of viral DNA-RNA hybrids by heat denaturation Cs 2SO 4 density gradient fractionation, and recombinant ] single-stranded probe hybridization revealed that the hybrid molecules consisted of viral plus-strand RNA hydrogen bonded to viral minus-strand DNA sequences. Data obtained by pancreatic ribonuclease digestion revealed that the hybrid molecules at density 1.45 to 1.52 g/cm 3 contained HBV RNA strands base paired over only part of their length in contrast to the hybrid species at density 1.57 g/cm 3 which contained RNA strands apparently base paired over most of their length. Further characterization showed that the hyrid at 1.57 g/cm 3 contained genome-length minus-strand viral DNA. The experiments rule out the possibility that the hybrid molecules are transcriptional complexes. Data presented in a companion manuscript indicate that the hybrid molecules may represent intermediates in the synthesis of viral DNA in the endogenous DNA polymerase reaction.

Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver

Human liver tissues obtained at autopsy from two patients chronically infected with hepatitis B virus (HBV) were found to contain several distinct species of HBV DNA. Southern blot analysis using a nick-translated HBV [ 32P]DNA probe identified specific DNA bands migrating at the positions expected for linear double-stranded DNA of 3.6 and 2.0 kb. These DNA bands were shown to represent relaxed circular and closed circular (supercoiled) HBV DNA, respectively. In addition to these distinct bands several minor bands as well as a heterogeneous population of HBV DNA molecules were present. When infected cell nuclei were isolated, and the nuclear and cytoplasmic nucleic acid separately analyzed, the nuclear fraction contained the 2.0-kb DNA species. This species was shown to be supercoiled 3.2-kb HBV DNA by electron microscopy, restriction endonuclease digestion, and thermal denaturation. The cytoplasmic fraction contained DNA forms similar to those found in virions isolated from plasma (i.e., migration in the position of linear double-stranded molecules of 3.6 and 3.2 kb) and no supercoiled DNA was detected. Particles isolated from the cytoplasmic fraction were able to incorporate dNTPs into viral DNA sequences. Southern blot analysis of the nucleic acid isolated from the particles revealed the presence of HBV DNA forms migrating in positions expected for 3.6- and 3.2-kb linear double-stranded molecules as well as a heterogeneous population of HBV molecules. The 3.6- and 3.2-kb species were identified as relaxed circular and double-stranded linear genome-length HBV DNA. Digestion of the viral nucleic acid with pancreatic ribonuclease increased the electrophoretic mobility of a portion of the heterogeneous HBV molecules and resulted in the appearance of a distinct 1.9-kb DNA band suggesting the same viral DNA was complexed with RNA. Experiments to be reported elsewhere showed this DNA species to be genome-length minus-strand HBV DNA which was released from DNA-RNA hybrid molecules by RNase digestion. Thus, supercoiled HBV DNA exists free in the nucleus of infected liver cells and cytoplasmic particles contain relaxed circular and linear HBV DNA as well as a heterogeneous population of HBV DNA and DNA-RNA hybrid molecules, and a DNA polymerase reaction in the particles results in incorporation of dNTP into DNA strands of these molecules.

In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.

Mechanism of release of the avian retrovirus tRNA Trp primer molecule from viral DNA by ribonuclease H during reverse transcription

Employing enzymatic reactions containing reverse transcriptase and appropriately defined substrates, we have demonstrated that the tRNA Trp primer molecule required for the initiation of DNA synthesis is cleaved from viral DNA by an enzymatic activity associated with the reverse transcriptase molecule. Since the α subunit of reverse transcriptase facilitates release of the tRNA Trp primer from viral DNA and this activity is inhibited by a known inhibitor of reverse-transcriptase-associated RNAase H, it appears that the RNAase H activity, rather than the DNA endonuclease activity, is involved in this reaction. The cleavage site for RNAase H—mediated removal of the tRNA Trp primer from viral DNA is located at or near the tRNA Trp—viral DNA junction, and transcription of most, if not all, of the tRNA Trp-binding site into (+) polarity DNA occurs before RNAase-H—mediated cleavage takes place. These studies indicate that an additional function can be ascribed to the reverse-transcriptase-associated RNAase H activity, which in this instance acts like an endonuclease, not requiring the unblocked termini of an RNADNA hybrid molecule for its activity.

Characterization of simian virus 40 transcriptional intermediates in infected CV 1 cell nuclei

Simian Virus (SV40) transcriptional intermediates (T.I.) were isolated from infected cell nuclei incubated in vitro in the presence of the four ribonucleoside triphosphates. The nascent mRNA strands in the viral DNA-RNA hybrid molecules were hydrogen bonded to their template by 200–250 nucleotides on the average, as judged from the extent of their RNase resistance and the aspect of T.I. under electron microscope after treatment with 50 per cent formamide. The RNA polymerase involved (RNA polymerase II) synthesized up to full length transcripts at a rate of approximately 150 nucleotides/min. at 25°C. Each SV40 infected cell was found to contain about 200 active T.I. molecules at the peak of late transcription. The DNA in the T.I. molecules was exclusively form I DNA only in cells infected with the tsA30 mutant of SV40 that had been transferred to non-permissive temperature in order to arrest DNA replication, but both form I DNA and molecules behaving as replicative intermediates (R.I.) in wild type infected cells.

The Kinetics of Synthesis of Infectious Unintegrated Virus DNA in Rous Sarcoma Virus Infected Chicken Cells

Infectious virus nucleic acids were extracted from the Hirt supernatant of chicken embryo fibroblasts infected for different times with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV). Infectious DNA and DNA-RNA hybrid molecules could be recovered from 6 h after infection in experiments using 1.5 X 10(9) infected cells. Only small amounts of infectious virus DNA could be purified 6 h after infection whereas at 24 h approximately one infectious DNA molecule could be recovered for each input virus infectious unit. At 24 h, both infectious supercoiled and non-supercoiled molecules were found. The specific infectivity of the supercoiled fraction was less than that of the non-supercoiled fraction. Infectious supercoiled DNA could be recovered from 16 h after infection. Evidence is presented that both forms of unintegrated virus DNA may rest unintegrated for at least 8 days in the cell, though chronically infected cells were shown to contain less than one unintegrated molecule per 10(2) to 10(3) cells.

DNA structure: evidence from electron microscopy.

The contour lengths of phiX174 DNA duplex and RNA-DNA hybrid molecules were measured by several commonly used electron microscopic techniques. The countour length of the hybrid molecules corresponds to a rise of 2.5 to 2.6 angstroms per base pair, as expected for the A conformation, while the length of phiX174 duplex DNA similarly measured corresponds to a 2.9-angstrom rise, very different from 3.4 angstroms of the classic B form. Thus any chromatin structure parameter based on electron microscopy and a rise of 3.4 angstroms must be reappraised. The possibility that DNA in dilute solution also has a rise of 2.9 angstroms and a screw of 10.5 base pairs per turn is discussed.