Electron microscopic demonstration of the presence of amplified sequences at the 5''-ends of the polyoma virus late mRNAs.

Electron microscopic techniques were used to examine the structure of the leader sequences at the 5'-ends of the late polyoma virus mRNAs. The three late mRNA's were partially purified and hybridized to an E. coli plasmid containing two polyoma virus genomes inserted in tandem. The hybrids were spread by the cytochrome c-formamide technique and visualized in the electron microscope. These studies revealed that whereas the body of a given mRNA molecule can hybridize with only one of the two corresponding body sequences in the two adjacent viral genomes, the leader of the same mRNA molecule can hybridize with both copies of the leader sequence-specific DNA. The mVP1 and mVP3 RNA species thus generated hybrids containing two loops, while mVP2 molecules formed hybrids containing one loop. Hence, the leaders of the three polyoma virus late mRNA species must contain two or more repeats of a sequence transcribed from a unique DNA segment. Length measurements showed that most leaders in the late mRNA's consist of at least 200 nucleotides and some contain up to 500 nucleotides, whereas the basic repeat sequence contains about 60 nucleotides.     

Complementary strands of CELO virus DNA.

When alkali-denatured DNA from CELO virus (an avian adenovirus) was annealed for 15 min at 37 C in 0.1 M NaCl, 70% of the molecules formed single-stranded circles. This is probably due to base pairing of complementary sequences not more than 110 nucleotides long at the ends of the single strands and implies an inverted terminal repetition in the duplex DNA similar to that reported for the DNA from human adenoviruses. The circular molecules had a uniform length that was approximately the same as that of linear single-stranded molecules. The complementary strands of CELO virus DNA were separated on a preparative scale, and at least 40% of the heavy strands and 56% of the light strands were found to be intact as judged by the formation of single-stranded circles.     

The 30S Moloney sarcoma virus RNA contains leukemia virus nucleotide sequences.

The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.     

Transcription of DNA from the 70S RNA of Rous Sarcoma Virus II. Structure of a 4S RNA Primer

The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus. We purified these primers in three different ways-by isolation of the covalent complex between primer and nascent DNA, by differential melting of the 70S RNA, and by two-dimensional electrophoresis in polyacrylamide gels. The 4S RNAs purified by these procedures were homogeneous and possessed very similar if not identical nucleotide compositions and sequences. The RNAs were approximately 75 nucleotides long, had pG at the 5' terminus and CpCpA(OH) at the 3' terminus, and contained a number of minor nucleotides characteristic of tRNA. In contrast to most tRNA's, the primer lacked rTp and contained Gp (Psip, Psip, Cp) Gp (possibly in place of the characteristic sequence GprTpPsipCpGp). At least 50% of the 4S primers available on 70S RNA were utilized in a standard polymerase reaction in vitro.     

Capped and polyadenylated low-molecular-weight RNA synthesized by vaccinia virus in vitro.

In the presence of ATP plus two other ribonucleoside triphosphates or in reactions containing all four ribonucleoside triphosphates and actinomycin D, vaccinia virus synthesizes in vitro discrete low-molecular-weight RNA molecules ranging in size from about 20 to several hundred bases. A novel feature of these small RNA molecules is that they are capped and methylated at the 5' terminus, containing both mGpppGm and mGpppAm type cap structures, and in addition these molecules are polyadenylated at the 3' terminus. Hybridization of these RNAs to restriction fragments derived from vaccinia virus DNA indicates a considerable degree of complexity, suggesting the presence of a large number of promoters throughout the genome. However, measurable sensitivity to pancreatic RNase of the 5' capped end of these RNAs while in hybrid form to the DNA suggests other possible roles for these small RNAs in vaccinia virus mRNA biogenesis.     

Characterization of DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis

In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3' → 5' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.     

Processing enzyme ribonuclease E specifically cleaves RNA I. An inhibitor of primer formation in plasmid DNA synthesis

When the RNA processing enzyme RNAase E is inactivated in an Escherichia coli strain carrying derivatives of the colicin E1 plasmid, a small RNA, about 100 nucleotides long, accumulates. Structural analysis of this RNA showed that it is RNA I, the RNA that inhibits plasmid DNA synthesis. RNA I is a specific substrate for RNAase E and the cleavage takes place between the fifth and sixth nucleotides from the 5' end of the molecule. This is only the second natural RNA substrate that has been found, so far, for the RNA processing enzyme ribonuclease E, the other being a precursor for 5 S ribosomal RNA. It is remarkable that nine nucleotides around the cleavage sites are identical in both substrates: (Formula: see text). Therefore, we suggest that at least part of the interaction between RNAase E and its substrate is controlled by these nine nucleotides.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.