Host-Cell Repair of Vaccinia Virus and of Double Stranded RNA of Encephalomyocarditis Virus

IN normal human cells DNA which has been damaged by ultraviolet radiation is repaired by excision of thymidine dimers and by repair replication. Patients suffering from xeroderma pigmentosum have a hereditary defect of the excision step and therefore their cells repair ultraviolet-induced lesions in their DNA less efficiently than do normal cells^1–4. An analogous situation has been well characterized in bacteria⁵.     

Evidence for RNA-DNA Complexes in Haemolytic Plaque-forming Cells containing Ribonuclease Resistant RNA

INCORPORATION of ³H-thymidine into antibody-producing haemolytic plaque-forming cells (p.f.c.) has been interpreted as evidence of DNA synthesis in a proliferating population of antibody-synthesizing cells^1–6. Although ³H-5-uridine has been used as the most specific label available for RNA^{7, 8}, there are cells in which its specificity as an RNA precursor is not absolute; because of incorporation into DNA^{9, 10} through ³H-5-deoxycytidine. The autoradiographic modification² of the Jerne-Nordin plaque technique¹¹ provides a powerful tool for studying nucleic acid synthesis in a small population of antibody-producing cells amidst large populations of non-antibody-producing cells. Tritiated thymidine and ³H-5-uridine are incorporated specifically into DNA and RNA respectively in this system, though not all RNA in p.f.c. is degradable by ribonuclease. The work described here indicates that resistant RNA is associated with DNA and is more abundant in antibody-producing cells than in non-antibody-producing cells. These findings may offer a clue to mechanisms of antibody formation.     

Rat C-Type Virus induced in Rat Sarcoma Cells by 5-Bromodeoxyuridine

HALOGENATED derivatives of uridine are highly effective inducers of latent C-type RNA viruses^1,2 and have been successfully used to induce viruses identical to, or similar to, the C-type RNA tumour viruses in mouse, rat and human cells^3–6. In previous experiments we used 5-bromodeoxyuridine (BrUdR) for induction of focus-forming virus in non-productive rat cells that have been transformed by mouse sarcoma virus². We describe here the induction of a C-type RNA virus in the cells of the rat tumour cell line XC, which contains the Rous sarcoma virus genome⁷. The induced virus possesses the group specific (gs) antigens of rat C-type viruses but not those of chicken C-type viruses.     

New Method of Studying the Precursor-product Relationship between High Molecular Weight RNA and Messenger RNA

THREE principal methods have been used to test whether giant heterogeneous RNA is a precursor to cytoplasmic messenger RNA in animal cells. First, competition-hybridization to DNA has shown that there is certainly a degree of similarity between the two types of molecule^1–5. The conditions used in most of these experiments allowed only the hybridization of the reiterated fraction of DNA or RNA. It is possible therefore that the competing sequences are similar, but not identical and in any case may represent only a small fraction of the total sequences. Scherrer et al.⁶, using conditions which might allow the hybridization of some of the slow sequences of DNA and which would give meaningful competition, conclude that there is a precursor-product relationship between giant heterogeneous RNA and cytoplasmic messenger RNA. Darnell, following the uptake of labelled nucleotides into the two fractions⁷, has shown that a precursor product relationship is possible although not proven. Third, the differential inhibition of synthesis of the two fractions by cordycepin⁸ has been taken to show that the giant heterogeneous RNA cannot be a precursor to the cytoplasmic messenger RNAs. Although these data would confirm previous observations on the rate of uptake and decay of label in nucleoplasmic and cytoplasmic RNA in the presence of actinomycin⁹, this type of experiment is rather indirect and open to other interpretations because the inhibitors might not act exclusively and directly on one step of the overall metabolic pathway. A relationship has been clearly established between high molecular weight precursors and the final RNA produced only for SV40 transformed cells¹⁰. In this case the hybridization experiments were much simplified because SV40 DNA, which is very small in comparison with animal DNA, is readily available. In this communication we describe a direct and general way of approaching the problem which makes novel use of molecular hybridization and can be applied to messenger RNAs which are not easily labelled. The method also overcomes the difficulty of hybridizing to the extremely complex DNA of higher organisms.     

7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

Effect of Interferon on Some Aspects of Transformation by Polyoma Virus

WHEN BHK 21 hamster cells are infected with polyoma virus¹, there is no vegetative growth of virus, but stably transformed cells appear. These transformed cells are more easily transplanted than BHK 21 cells; they initiate their growth cycle in otherwise restrictive cultural conditions such as the absence of serum, high density and suspension; they grow with random orientation and have exposed on their surfaces receptor sites for certain glycoprotein agglutinins^2–5. The proportion of stably transformed cells is low, even after high doses of virus. But a much higher proportion (sometimes all cells) shows abortive transformation—changes characteristic of transformation, but which last only a few days. In suspension cultures, for example, most of the infected cells grow into small colonies of four to thirty-two cells⁶. In surface cultures deprived of serum DNA synthesis is initiated and the cells may then divide at least once⁷: they also temporarily lose the normal parallel orientation and develop the typical random appearance of transformed cells. Moreover, the polyoma nuclear T-antigen and also a surface antigen detected by immunofluorescence, appear temporarily in most polyoma infected BHK 21 cells⁸, while 3T3 cells exposed to SV40 virus show transient exposure of cell surface sites reacting with conconavalin A (ref. 9).     

Single RNA Polymerase Binding Site Isolated

One approach to study the structure of promoter regions^1–3 is to isolate RNA polymerase binding sites. Several attempts have been made to isolate such sites as protected DNA fragments^4–10, but so far the DNA isolated has not definitely shown to be only one or a few specific sites. We report here the isolation of a single RNA polymerase binding site from the replicative form (RF) DNA of bacteriophage fd. The site as isolated is a short double-stranded DNA with a unique nucleotide sequence.     

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression ^{1,2,3,4,5,6,7}. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion ^8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation ^10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide ^3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex ¹⁴; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex ¹³.Prior studies mapping RNA occupancy at chromatin have revealed substantial insights ^15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution ¹⁷. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA''s structure or functional domains.     

RNA-dependent DNA Polymerase in Human Lymphocytes during Gene Activation by Phytohaemagglutinin

RNA-DEPENDENT. DNA polymerase an activity initially thought to be unique to RNA tumour viruses^1–3, is now known to be present in normal human lymphocytes⁴, which are differentiated and seldom divide in vivo or in culture. The addition of phytohaemagglutinin (PHA) or other mitogens (including appropriate antigens) transforms lymphocytes into actively proliferating cells⁵. Several enzymes which function in gene activation and DNA replication specifically increase in activity during this transformation^6,7. We now report that RNA-dependent DNA polymerase also behaves in this way.     

Translation of Globin Messenger RNA in a Heterologous Cell-free System

MESSENGER-SPECIFIC initiation factors, capable of discriminating between classes of messenger RNAs (mRNAs) or different cistrons in viral RNA, have been implicated in the regulation of protein synthesis in bacteria^1–5. Comparable but less detailed observations have also been made in eukaryotic systems^6–10. For example, RNA extracted from a mammalian virus (encephalomyocarditis virus, EMC) cannot be translated in a reticulocyte cell-free system unless the system is fortified with an extract from responsive cells—in this case, Krebs II ascites cells⁶. Such results imply the existence of tissue-specific factors and lead to questions whether this incompatibility is reciprocated by an inability of the Krebs II ascites cell system to respond to the mRNA for globin.