Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.     

An MSV-specific subgenomic mRNA in MSV-transformed G8-124 cells

An intracellular subgenomic RNA species from MSV-transformed G8-124 cells was characterized by electron microscopy of RNA:cDNA heteroduplexes using long cDNAs both MSV and MuLV. This subgenomic RNA, 3.1 kb long, consisted of 5'-derived sequences of about 0.4 kb joined to 2.7 kb of RNA derived from the 3' end of the RNA genome. The 3'-derived sequences included the residual sequences from the MuLV pol region and the acquired cellular sequences of MSV. The genome of MSV was shown to retain approximately 0.13 kb from the 5' end of the MuLV env region, including sequences which span the point in the MuLV env mRNA. No subgenomic MSV RNA could be detected, however, which consisted of a 5'-derived leader sequence spliced to the retained env region sequences. Nor could a subgenomic MSV RNA be detected in which a 5'-derived leader sequence was joined directly to the acquired cellular sequences. Although its translation products are unknown, the subgenomic MSV RNA was present in preparations of poly(A)+ polysomal RNA, consistent with this RNA functioning as a messenger. The structure of this 3.1 kb MSV subgenomic RNA suggests a possible role in the expression of 3'-encoded MSV information, possibly including transformation-specific sequences.     

Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA''s of B77 Avian Sarcoma Virus

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.     

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.     

RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA.

In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end. Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.     

Primary organization of nucleosomes. Interaction of non-histone high mobility group proteins 14 and 17 with nucleosomes, as revealed by DNA-protein crosslinking and immunoaffinity isolation

The binding sites for histones and high mobility group proteins (HMG) 14 and 17 have been located on DNA in the nucleosomal cores and H1/H5-containing nucleosomes. The nucleosomes were specifically associated with two molecules of the non-histone proteins HMG 14 and/or HMG 17 when followed by DNA-protein crosslinking and immunoaffinity isolation of the crosslinked HMG-DNA complexes. HMGs 14 and 17 were shown to be crosslinked in a similar manner to each core DNA strand at four sites: to both 3' and 5' DNA ends and also at distances of about 25 and 125 nucleotides from the 5' termini of the DNA. These sites are designated as HMG(143), (0), (25) and (125). The site HMG(125) is located at the place where no significant histone-DNA crosslinking was observed. The HMG(125) and HMG(25) sites lie opposite one another on the complementary DNA strands across the minor DNA groove and are placed, similarly to histones, on the inner side of the DNA superhelix in the nucleosome. The crosslinking of HMG 17 to the 3' ends of the DNA is much weaker than that of HMG 14. These data indicate that each of two molecules of HMG 14 and/or HMG 17 is bound to the double-stranded core DNA at two discrete sites: to the 3' and 5' ends of the DNA and at a distance of 20 to 25 base-pairs from each DNA terminus inside the nucleosome on a histone-free DNA region. Binding of HMG 14 or 17 does not induce any detectable rearrangement of histones on DNA and both HMGs seem to choose the same sites for attachment in nucleosomal cores and H1/H5-containing nucleosomes.     

Maize streak virus genes essential for systemic spread and symptom development

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The `+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary `-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.     

Macronuclear gene-sized molecules of hypotrichs.

The macronuclear genome of hypotrichous ciliates consists of DNA molecules of gene-sized length. A macronuclear DNA molecule contains a single coding region. We have analyzed the many hypotrich macronuclear DNA sequences sequenced by us and others. No highly conserved promoter sequences nor replication initiation sequences have been identified in the 5' nor in the 3' non-translated regions, suggesting that promoter function in hypotrichs may differ from other eukaryotes. The macronuclear genes are intron-poor; approximately 19% of the genes sequenced to date have one to three introns. Not all macronuclear DNA molecules may be transcribed; some macronuclear molecules may not have any coding function. Codon bias in hypotrichs is different in many respects from other ciliates and from other eukaryotes.     

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.     

At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.