Mapping host range-specific oligonucleotides within genomes of the ecotropic and mink cell focus-inducing strains of Moloney murine leukemia virus.

The site of recombination of a mink cell focus-inducing strain (Mo-MuLV83) derived from an ecotropic Moloney murine leukemia virus (Mo-MuLV) was mapped by fingerprint analysis of the large RNase T1-resistant oligonucleotides, employing a two-dimensional gel electrophoresis method. Mo-MuLV83, in contrast to the ecotropic Mo-MuLV, demonstrated a broadened host range, i.e., growth not only on mouse cells but also on mink cells, and recombination involved the env gene function. The genomic RNA of these two viruses shared 42 out of a total of 51 to 53 large T1 oligonucleotides (81%) and possessed a similar subunit size of 36S. Most of these T1 oligonucleotides were mapped in their relative order to the 3' polyadenylic acid end of the viral RNA molecules. There were 10 common oligonucleotides immediately next to the 3' termini. A cluster of 7 (in Mo-MuLV83) or 10 (in Mo-MuLV) unique T1 oligonucleotides were mapped next to the common sequences at the 3' end, and they all appeared concomitantly in a polyadenylic acid-containing RNA fraction with a sedimentation coefficient slightly larger than 18S. Therefore, the env gene of Mo-MuLV was situated at a location approximately 2,000 to 4,000 nucleotides from the 3' end of the genomic RNA, and the gene order of Mo-MuLV appeared to be similar to that of the more rigorously determined avian oncornaviruses. cDNA(SFFV) specific for the xenotropic sequences in the spleen focus-forming virus RNA hybridized to the cluster of unique oligonucleotides of Mo-MuLV83 RNA. This suggests that the loci of recombination involve the homologous env gene region of a xenotropic virus.     

Sequence and location of large RNase T1 oligonucleotides in bacteriophage Qbeta RNA.

Twenty-nine oligonucleotides, 11 to 26 nucleotides in length, arising by complete RNase T1 digestion of bacteriophage Qbeta RNA and isolated by two-dimensional polyacrylamide gel electrophoresis, were sequenced. Their location within the genome was established with two methods. (a) In vitro synthesis of Qbeta RNA plus strands was started synchronously, using minus strands as template and nucleoside [alpha-32P]triphosphates as substrate; after various times, the reaction was stopped and the length of the products formed was correlated with their content of T1 oligonucleotides. (b) Qbeta [32P]RNA was elongated with poly(A) using terminal riboadenylate transferase; after mild treatment with alkali the fragments were fractionated by size and the poly(A)-containing molecules of each size class were isolated by chromatography on poly(U)-Sephadex and assayed for T1 oligonucleotides. The oligonucleotides in the 5' region were localized more precisely with method a, those near the 3' end with method b; in the middle region, the results of the two sets of analyses confirmed each other. The use of these oligonucleotides in the sequence determination of Qbeta RNA is discussed.     

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.     

Size analysis and relationship of murine leukemia virus-specific mRNA''s: evidence for transposition of sequences during synthesis and processing of subgenomic mRNA.

Virus-specific mRNA from purified polyribosomes of mouse cells infected with Moloney murine leukemia virus (M-MuLV) was analyzed by electrophoresis in agarose gels, followed by hybridization of gel slices with M-MuLV-specific complementary DNA (cDNA). The size resolution of the gels was better than that of sucrose gradients used in previous analyses, and two virus-specific mRNA's of 38S and 24S were detected. The 24S virus-specific mRNA is predominantly derived from the 3' half of the M-MuLV genome, since cDNAgag(pol) (complementary to the 5' half of the M-MuLV genome) could not efficiently anneal with this mRNA. However, sequences complementary to cDNA synthesized from the extreme 5' end of M-MuLV 38S RNA (cDNA 5') are present in the 24S virus-specific mRNA, since cDNA 5' (130 nucleotides) efficiently annealed with this mRNA. The annealing of cDNA 5' was not due to repetition of 5' terminal nucleotide sequences at the 3' end of M-MuLV 38S RNA, since smaller cDNA 5' molecules (60 to 70 nucleotides), which likely lack the terminal repetition, also efficiently annealed with the 24S mRNA. The sequences in 24S virus-specific mRNA recognized by cDNA 5' are not present in 3' fragments of virion RNA that are the same length. Therefore, it appears that RNA sequences from the extreme 5' end of the M-MuLV genome may be transposed to sequences from the 3' half of the M-MuLV 38S RNA during synthesis and processing of the 24S virus-specific mRNA. These results may indicate a phenomenon similar to the RNA splicing processes that occur during synthesis of adenovirus and papovavirus mRNA's.     

A hybridization procedure for the isolation of specific RNA segments applied to the analysis of bacteriophage Qbeta RNA.

A method for the isolation of RNA fragments originating from defined regions of bacteriophage Qbeta RNA minus strands is described. Large RNase T1 oligonucleotides were isolated on a preparative scale from Qbeta RNA. The nucleotide sequences (13 to 26 nucleotides) and map positions of these oligonucleotides were known from previous work (Billeter, M. A. (1978) J. Biol. Chem. 253, 8381-8389). After addition of AMP residues (50 in the average) using terminal adenylate transferase, these pure oligonucleotides were hybridized to 32P-labeled Qbeta RNA minus strands synthesized in vitro. Fragments in the size range of 100 to 500 nucleotides were then generated by partial digestion with RNase T1. Fragments hybridized to such oligonucleotides were recovered by chromatography on poly(U)-Sephadex and then resolved according to their size by polyacrylamide gel electrophoresis. The specificity and reproducibility of the method as well as its suitability for the sequence analysis of Qbeta RNA was verified by using in particular a linker oligonucleotide derived from a Qbeta RNA region near the 3' end. The sequence catalogues of the RNase T1 and RNase A oligonucleotides of two fragments isolated in this way, 202 and 310 nucleotides in length, were established and all fragments isolated were shown to contain a sequence complementary to the linker oligonucleotide.     

Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

The nucleotide sequence at the 5'' end of foot and mouth disease virus RNA.

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.     

Action of Ribonuclease T1 on 30S Ribosomes of Escherichia coli and Its Role in Sequence Studies on 16S Ribonucleic Acid

Two large ribonucleic acid (RNA) fragments have been obtained from T1-RNase-treated 30S ribosomes of Escherichia coli. One fragment, about 475 nucleotides long, contains all the unique oligonucleotides found by Fellner and associates in sections of 16S RNA designated P, E, E', and K, and one-half the large oligonucleotides of section A. The other large fragment is about 300 nucleotides long and contains the oligonucleotides found in sections C, C', C'. The isolation of these large fragments seems to confirm the arrangement of sections within 16S RNA. There are also recovered from nuclease-treated ribosomes three small fragments, one (120 nucleotides long) from the 5' end, one (26 nucleotides long) from the 3' OH end of the chain, and another section (66 nucleotides long) from the middle of the 16S RNA chain. Small molecular weight material is also generated by nuclease treatment, and about half this material is derived from a region close to the 3' OH end of the 16S RNA chain. This indicates that the most accessible part of the rRNA of E. coli 30S ribosomes is a region 100 to 150 nucleotides long near the 3' end of the chain. A general scheme is proposed to explain the generation of the various-sized RNA products from the rRNA of the 30S ribosome.     

Comparison of the genomic organization of Kirsten and Harvey sarcoma viruses.

Current studies were undertaken to compare the genomes of Kirsten murine sarcoma virus (Ki-MuSV), Harvey murine sarcoma virus (Ha-MuSV), and the replication-defective endogenous rat virus to understand the function of these viral RNAs. Genome organization and sequence homology were studied by fingerprinting large RNase T1-resistant oligonucleotides and by cross-protecting homologous oligonucleotides against RNase A and T1 digestion with complementary DNA prepared from each of the other viral RNA. Ki-MuSV and Ha-MuSV were found to share an extensive series of rat-derived oligonucleotides begining ca. 1 kilobase (kb) from the 3' end and extending to within 1.5 kb of the 5'end of Ki-MuSV RNA. The total map distance covered in ca. 5.5 kb. The eight oligonucleotides covering the 1.5 kb at the 5' end of Ki-MuSV RNA were not found in Ha-MuSV RNA. Five out of these eight oligonucleotides, however, could be designated with certainty to be of rat virus origin. Since Ha-MuSV is 6.5 kb in size and Ki-MuSV is 8 kb in size, the major difference between them is the 1.5 kb from the replication-defective endogenous rat virus sequences at the 5' end of Ki-MuSV not present in Ha-MuSV. Consistent with the difference in the genome structure, these two sarcoma viral RNA'S yielded distinct major translation products in cell-free systems, I.E., A 50,000-dalton polypeptide (P50) from Ki-MuSV and a 22,000-dalton polypeptide (p22) from Ha-MuSV. These polypeptides may provide the necessary protein makers for identifying in vivo virus-coded proteins.     

More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA.

The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.     

Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase.     

Nucleotide sequence of the 5'' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5'' splicing site for subgenomic mRNAs

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).     

Physical map of the Kirsten sarcoma virus genome as determined by fingerprinting RNase T1-resistant oligonucleotides.

From analysis of the large RNase T1-resistant oligonucleotides of Kirsten sarcoma virus (Ki-SV), a physical map of the virus genome was deduced. Kirsten murine leukemia virus (Ki-MuLV) sequences were detected in T1 oligonucleotides closest to the 3' end of the viral RNA and extended approximately 1,000 nucleotides into the genome. The rat genetic sequences started at this point and extended all the way to the very 5' end of the RNA molecules, where a small stretch of Ki-MuLV sequence was detected. By comparison of the fingerprints of Ki-SV RNA and the RNA of the endogenous rat src genetic sequences, it was found that more than 50% of the T1 oligonucleotides were similar between Ki-SV and the endogenous rat src RNA, suggesting an identical primary nucleotide sequence in over 50% of the viral genomes. The results indicate that Ki-SV arose by recombination between the 5' and 3' ends of Ki-MuLV and a large portion of the homologous sequences of the endogenous rat src RNA.