Organization of shared and unshared sequences in the genomes of chicken endogenous and sarcoma viruses.

The genome of the genetically transmitted endogenous C type virus of chickens, RAV-O, is closely related to that of Rous sarcoma virus (RSV). Nevertheless, these viruses differ widely in oncogenicity and regulation by the host cell. Competitive hybridization analysis of ¹²⁵I-labeled genomic RNA demonstrated that the genome of RAV-O lacks about 35% of the sequences of nondefective RSV which formed hybrids with proviral DNA from RSV-infected cells, and that the genome of transformation-defective deletion mutants of RSV (td RSV) lacks about 15% of these sequences. Conversely, about 12% of the RAV-O sequences forming hybrids with normal chicken cell DNA were not detected in the sarcoma virus. A technique was developed to map the location of these unshared sequences by competitive hybridization. The deletion in the genome of td RSV was seen to begin at about 0.2 and to end at about 0.05 of the genome length from the 3′ end of sarcoma virus RNA, confirming the results of other laboratories using the method of mapping RNAase TI resistance of oligonucleotides. The 35% of RSV sequences missing and/or diverged in the genome of RAV-O were concentrated within 40% of the sarcoma virus genome from the 3′ end, and most of this large section did not appear to form hybrids with chicken DNA under the conditions of these experiments. A low level of hybrid formation was, however, detected between uninfected chicken cellular DNA and a small fraction of the nucleotides in the region of the td deletion. Analysis of RAV-O 3′ end fragments demonstrated that the genomic sequences of RAV-O missing in RSV were concentrated at the 3′ end of the endogenous viral genome. We conclude that the sequence differences between endogenous and sarcoma viruses are largely concentrated in specific regions of the viral genome.     

Transformation by subgenomic fragments of Rous sarcoma virus DNA

Subgenomic fragments of Rous sarcoma virus (RSV) DNA, generated by Eco RI digestion of DNA of RSV-infected chicken cells, induced transformation of NIH/3T3 mouse cells with efficiencies that were 100–1000 fold lower than the efficiency of transformation by intact RSV DNA. Analysis of the DNAs of NIH cells transformed by Eco RI-digested RSV DNA indicated that these cells contained no more than 2 × 10⁶ daltons of RSV DNA, and did not contain sequences from the 5′ terminus of RSV RNA which are included in the leader sequence of subgenomic src mRNA of RSV-infected cells. The product of the RSV src gene (pp60^src), however, was produced in apparently similar quantities by NIH cells transformed by Eco RI fragments of RSV DNA and by intact RSV DNA. Thus expression of the src gene of RSV in NIH cells transformed by subgenomic fragments of RSV DNA did not require the terminal sequences of the RSV genome, which appear to be involved in synthesis and processing of src mRNA in RSV-infected cells. DNAs of NIH cells transformed by Eco RI-digested RSV DNA were found to induce transformation in secondary transfection assays with efficiencies that were similar to the efficiency of transformation by intact RSV DNA. These results suggest that transformation by subgenomic fragments of RSV DNA may be a consequence of integration of src gene-containing DNA fragments in the vicinity of a promoter site in the recipient cell genome, leading to efficient expression of the RSV src gene.     

Total nucleotide sequence of a nearly full-size DNA copy of satellite tobacco necrosis virus RNA

pSTNV-1 is a chimera plasmid that contains a nearly full-size double-stranded DNA copy of the satellite tobacco necrosis virus RNA genome (see preceding paper by van Emmelo et al., 1980) and we report here the complete nucleotide sequence of this STNV² DNA insert. The results show that except for 23 nucleotide pairs corresponding to the 5′ end of STNV RNA, a full-size STNV DNA copy is present in pSTNV-1. The total nucleotide sequence of the STNV genome contains 1239 residues. The amino acid sequence of the coat protein can be deduced from the 5′ half of the DNA message strand and shows a rather hydrophobic carboxyl-terminal region and a basic amino-terminal region. The 3′ untranslated part of the viral RNA is 622 nucleotides long. A secondary structure model for the 5′ end showing an interaction with a segment in the 3′ half is proposed. The 3′ end region can be folded into a transfer RNA cloverleaf-like structure with an anticodon for AUG.     

Molecular Mapping of Rice Blast Resistance Gene Pi-k h in the Rice Variety Tetep

We have used rice line Tetep as a resistant donor with the aim of mapping a durable blast resistance gene Pi-k ^h using RAPD and AFLP techniques in conjunction with bulk segregant analysis. An F₂ mapping population consisting of 205 plants was generated by crossing Tetep with HP2216, a highly susceptible cultivar. Inoculation with specific isolate (PLP-1) of Magnaporthe grisea at seeding stage showed that the Pi-k ^h gene inherited as a single dominant gene in F2 population. RAPD analysis was performed with 240 primers to detect polymorphism between resistant and susceptible parents. Of these, 48 primers produced polymorphic banding pattern between resistant and susceptible parents. Bulk segregant analysis was performed with 48 primers of which 5 showed polymorphism between resistant and susceptible bulks. A 700 bp DNA band was obtained in resistant F2 plants with primer 5-129 indicating its linkage to the resistance gene. Out of 64 AFLP primer combinations used for polymorphism survey between HP 2216 and Tetep, 11 AFLP primer combinations were able to distinguish the resistant and susceptible bulks. An AFLP band of 75 bp obtained with primer combination, E-TAlM-CTC co-segregated with the resistance gene. The RAPD marker 5-129₇₀₀ and AFLP₇₅ were placed on the linkage map at a distance of 2.1 eM and 15.1 eM flanking to Pi-k ^hgene, respectively. The RAPD band closely linked to Pi-k ^h gene was sequenced and used for the development of CAPs markers which also co-segregated with resistant phenotype in the mapping population. On sequence analysis and homology search of RAPD fragment with whole rice genome sequence database and the information available on physical, genetic and sequence maps of rice, the co-segregating CAPs marker was placed at long arm of rice chromosome 11. CAPs marker developed in this study showed polymorphism in different rice cultivars grown in North-Western Himalayan region and is being used for the pyramiding of Pi-k ^h gene along with other blast resistance genes using marker-assisted selection.     

Analysis of FBJ-MuSV provirus and c-fos (mouse) gene reveals that viral and cellular fos gene products have different carboxy termini

The complete nucleotide sequence of the FBJ-MuSV proviral DNA and the cellular homolog (c-fos) of its oncogene (v-fos) have been determined. The 4026 nucleotide long FBJ-MuSV proviral DNA contains two long terminal repeats, a substitution of 1639 nucleotides of mouse cellular DNA (v-fos) and the 3′ end of the env gene derived from FBJ-MuLV. The sequences of the parental FBJ-MuLV and the cellular c-fos (mouse) gene share five of five nucleotides at the 5′ end and ten of 11 nucleotides at the 3′ end of the v-fos substitution. When compared with the v-fos sequences, the c-fos gene contains four discontinuous regions, three of which are flanked by sequences characteristic of introns. Direct sequence analysis of c-fos (mouse) RNA by primer extension demonstrates that the fourth discontinuity is due to a 104 bp deletion in the v-fos gene. As a consequence of the deletion, the predicted v-fos and c-fos gene products differ at their C termini.     

RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.     

Retroviruses as mutagens: Insertion and excision of a nontransforming provirus alter expression of a resident transforming provirus

Integration of retroviral DNA appears to occur randomly in host genomes, suggesting that retroviruses can act as insertion mutagens. We have confirmed this prediction by showing that the nontransforming retrovirus, Moloney murine leukemia virus (M-MuLV), can insert its provirus within the selectable target provided by a single provirus in a clonal rat cell line (B31) transformed by Rous sarcoma virus (RSV). Analysis of over 60 morphological revertants of M-MuLV-superinfected B31 cells revealed two lines with inserts of M-MuLV proviruses within the RSV provirus but outside the transforming gene of RSV (src), at sites 0.6 and 4.0 kb from the 5′ end. The inserts did not inactivate initiation of RSV RNA synthesis but did affect elongation or processing, or both, generating species with the 5′ end of RSV RNA linked to sequences that presumably derive from the inserted M-MuLV DNA. In one mutant line, most of the insert was excised at low frequency, apparently by homologous recombination between repeated sequences at the ends of M-MuLV DNA. After excision, RSV src mRNA was present in normal amounts, and the cells resumed a transformed appearance. In at least four independent lines, large portions of the left end of the RSV provirus (from 1 to 6 kb) and variable amounts of leftward flanking cellular DNA (from 0.5 to 10–15 kb or more) were deleted, without nearby insertions of M-MuLV DNA. The deletions removed the putative promoter for synthesis of RSV RNA; in the two cases examined, no RSV RNA was detected. These deletions may represent a second mutational effect of the superinfection by M-MuLV.     

Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA.

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T₁ and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1³ (see following paper).     

Sequences from the beginning of the fiber messenger RNA of adenovirus-2.

Small restriction fragments, from around co-ordinate 86.6 on the adenovirus-2 genome, have been used as primers for direct DNA sequence analysis by Sanger's (Sanger et al., 1977) chain termination method with Ad2² DNA as template. The genomic sequences obtained have been compared with sequences deduced using fiber messenger RNA from Ad2 or Ad2⁺ ND5-infected cells as template. With one primer, Hha 54, the sequences complementary to mRNA match those of the genome for 10 nucleotides but then differ from those found on the genome because this primer hybridizes near the point at which the leader sequence becomes joined to the main body of fiber mRNA. Using Ad2⁺ ND5 fiber mRNA as template, the sequence beyond the point of divergence matches the known sequence of the third leader component of one of the late Ad2 mRNAs, that encoding the hexon polypeptide. With Ad2 fiber mRNA, a heterogeneous sequence continuation is found, in accordance with earlier findings that two major species of fiber mRNA are present, which differ in the nature of the leader component joined to the main body of fiber mRNA (Chow &; Broker, 1978). Nevertheless, the data suggest that both leader components appear to become joined to the same nucleotide at the start of the main body of fiber mRNA.The AUG codon, which probably encodes the N-terminus of the fiber polypeptide, occurs just two nucleotides beyond the point at which these leader segments become spliced to the main body of fiber mRNA.     

Two adenovirus mRNAs have a common 5′ terminal leader sequence encoded at least 10 kb upstream from their main coding regions

The messenger RNAs encoding two late adenovirus serotype 2 (Ad2) proteins, fiber and 100K, were purified by hybridization to restriction endonuclease fragments of Ad2 DNA followed by electrophoresis on polyacrylamide gels containing 98% formamide. The 5′ terminal oligonucleotides generated by RNAase T1 digestion of the messengers were selected by dihydroxyboryl-cellulose chromatography. Both mRNAs gave an identical 5′-undecanucleotide with the general structure 7mG^5′ppp^5′A^mC^(m)U(C₄,U₃)G. This undecanucleotide could be removed by mild RNAase treatment from the mRNA after hybridization to DNA fragments containing the main coding sequence of the messenger. In contrast, a small region defined by Bal I-E (14.7–21) protects this undecanucleotide from RNase. A second region contained within both Hind III-B (17–31.5) and Hpa I-F (25.5–27.9), although unable to protect the undecanucleotide, hybridizes to both fiber and 100K mRNAs and protects a similar sequence of 100–150 nucleotides. These observations suggest that both mRNAs contain a long common sequence, complementary to at least two different sites on the Ad2 genome remote from the start of these two genes. The implications of these findings are discussed, and a general mechanism is presented for the biosynthesis of mRNAs from larger precursor molecules, based on intramolecular ligation.     

Evidence for the presence of ribonucleotides at the 5' termini of some DNA molecules isolated from Escherichia coli polAex2.

We have purified a set of small DNA molecules from various strains of exponentially growing Escherichia coli, including E. coli polAex2. This material included very short molecules (2 S), the nascent DNA (“Okazaki fragments”) and some longer molecules. Most of the [³H]thymidine incorporated during a brief period of labeling was found in the 5 S to 15 S Okazaki fragments. There was a large number of the 2 S molecules in the cell. The properties of the 5′ ends of these molecules were investigated using three procedures. (1) The DNA preparation, pulse-labeled with [³H]thymidine, was reacted with polynucleotide kinase and ATP to insure that all 5′ ends were phosphorylated. After subjection of the DNA to alkaline hydrolysis, the proportion of incorporated ³H pulse-label that became susceptible to digestion by spleen exonuclease was determined. In different experiments there was an increment of up to 20% in the amount of pulse-labeled E. coli polAex2 DNA that could be hydrolyzed by the exonuclease after treatment with alkali. (2) As in the preceding protocol, phosphorylation of the 5′ ends was assured by reaction with kinase and ATP; the preparation was then treated with alkali and the number of 5′-OH ends generated that could be labeled with ³²P using [γ-³²P]ATP and kinase in a second reaction was determined. The data indicated that 3 to 30% of the molecules could be labeled after alkali digestion, but not before. (3) The DNA molecules were reacted with kinase and [γ-³²P]ATP after having been exposed previously to alkaline phosphatase. The end-labeled molecules were then subjected to an alkaline hydrolysis and the resulting hydrolysate chromatographed on a polyethyleneimine-cellulose thinlayer plate. Alkali treatment was found to release 2′(3′),5′-ribonucleoside diphosphates from 1 to 30% of the molecules; pAp and pGp predominated. Control experiments showed that these ribonucleotides were covalently linked to the 5′ ends of polydeoxyribonucleotides. Curiously, the smaller the DNA molecule the less likely it was to possess a 5′-terminal ribonucleotide. Very few apparent RNA/DNA molecules were observed in the non-polAex2 strains tested. These observations are in part in agreement with previous reports, and we infer that at least some of the nascent E. coli polAex2 DNA molecules are initiated in vivo with a ribonucleotide primer. The relatively smaller proportion of molecules with apparent 5′-terminal ribonucleotides among the smaller DNA molecules and in strains other than E. coli polAex2 suggests to us that there may exist a mechanism for initiating DNA molecules that does not require an RNA primer.     

Initiator RNA of nascent DNA from animal cells.

Nascent DNA synthesized by intact cells has been examined for the presence of RNA that may function as a primer in the discontinuous synthesis of DNA. A low molecular weight fraction that contains nascent DNA was isolated from a human lymphoblastoid cell line in logarithmic growth. After labeling the 5′ ends with bacteriophage T4 polynucleotide kinase and [γ-³²P]ATP, and digestion of the DNA with DNAase, a DNAase-resistant oligonucleotide was isolated. This fragment consisted of approximately 9 ribonucleotide residues, with 5′ terminal purines ($\text{A}\text{G}\text{=}\text{3·5}\text{1}$), plus one to three 3′ terminal deoxynucleotides resulting from incomplete removal by DNAase. Approximately 10% of short nascent DNA chains contained the nonanucleotide molecule. An additional 20% of the nascent DNA contained ribooligomers shorter than 9 residues, with 5′ termini substantially increased in pyrimidines, which may result from degradation of the nonanucleotide. These results extend previous studies that demonstrated a similar ribooligonucleotide present at the 5′ end of most or all short nascent DNA chains synthesized in broken cell systems. Together with the results obtained by Reichard and co-workers (Reichard et al., 1974) with polyoma virus, the data support a mechanism by which a short initiator RNA serves as primer for discontinuously synthesized DNA in animal cells.