Organization of RNA transcripts from a vaccinia virus early gene cluster

The detailed organization of the RNAs transcribed from an early gene cluster encoded by vaccinia virus has been determined from the information derived from several complementary techniques. These include hybrid selection coupled with cell-free translation to locate DNA sequences complementary to mRNAs encoding specific polypeptides; RNA filter hybridization to size and locate on the DNA mature RNAs as well as higher-molecular-weight RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; and fractionation of hybrid-selected mRNAs in an agarose gel containing methyl mercury hydroxide followed by the cell-free translation of these mRNAs to definitively ascertain the size of the mRNA encoding each polypeptide. The early gene cluster is located between 21 and 26 kilobases from the left end of the vaccinia virus genome and is encoded by a 5.0-kilobase EcoRI fragment which spans the HindIII-N, -M, and -K fragments. Transcribed towards the left terminus are four mature mRNAs, 1,450, 950, 780, and 400 nucleotides in size, encoding polypeptides of 55, 30, 20, and 10 kilodaltons, respectively. These mRNAs are colinear with the DNA template and are closely spaced such that the 5' terminus of one mRNA is within 50 base pairs of the 3' terminus of the adjacent RNA. In addition to the mature size mRNAs, there are higher-molecular-weight RNAs, 5,000, 3,300, 2,350, 2,300, 1,800, 1,700, and 1,350 nucleotides in size. The 5' and 3' termini of the high-molecular-weight RNAs are coterminal with the 5' and 3' termini of the mature size mRNA. The implications of this arrangement and the biogenesis of these early mRNAs are discussed.     

Early-replicating DNA from mosquito cells is associated with a distinct EcoRI fragment

In an effort to define an origin of bi-directional DNA replication (OBR) in mosquito genomic DNA, we applied methods that take advantage of characteristic features of single-stranded DNA to methotrexate-resistant Aedes albopictus cells. The Mtx-5011-256 cells contained approximately 1000 copies of a 200 kb amplicon containing the dihydrofolate reductase locus, which likely contained one or more replication origins. When Mtx-5011-256 cells were synchronized by treatment with hydroxyurea, released into the S phase of the cell cycle, and labeled in vivo with tritiated DNA precursors, a 1.9 kb EcoRI fragment was preferentially labeled in EcoRI-digested genomic DNA. Similarly, we detected a 1.9 kb EcoRI fragment in DNA from wild type cells after cell cycle synchronization and in vivo labeling. In a complementary method, unlabeled single-stranded DNA was isolated from Mtx-5011-256 cells, labeled in vitro, and hybridized to EcoRI-digested genomic DNA from mosquito cells. The labeled probe hybridized preferentially to a 1.9 kb fragment. Finally, a 1.9 kb EcoRI fragment was detected when nascent DNA was recovered from unsynchronized cells, made double-stranded by in vitro labeling, and digested with EcoRI. Taken together, these results suggest that in Aedes albopictus mosquito cells, many replication origins used at different times during S are flanked by EcoRI sites that define a 1.9 kb fragment, which has become more abundant in Mtx-5011-256 cells because it occurs in the dhfr amplicon. Tentative mapping of this origin to amplicon DNA remains ambiguous, further suggesting that a repeated sequence element occurs at or near the origin of replication.     

Enzymatic multiplication of a chemically synthesized DNA fragment.

A synthetic DNA fragment of 19 residues was enlarged by the enzymatic addition of deoxyadenylate residues to its 3'-end with calf thymus terminal deoxynucleotidyl transferase. The 3'-terminus of this elongated DNA strand was blocked with 2', 3'-dideoxyadenylate to prevent hydrolysis by the 3'-exonuclease function of E. coli DNA polymerase I. This elongated and 3'-blocked fragment was annealed to an oligomeric primer and used as a template for the synthesis of a complementary copy of the synthetic 19-mer. The product of such a repair synthesis was separated by gel filtration and analyzed by nearest neighbor techniques. All template strands were copied with complete repair in over 90% of the chains. Facile recovery of the elongated template by virtue of its size permitted repetition of the copy process, thus allowing accumulation of the desired strand.     

Characterization of polyoma virus early proteins expressed from vaccinia virus recombinants

We previously reported that live recombinant vaccinia viruses (VV) encoding either the large T (LT) or middle T (MT) antigens of polyoma virus (PyV) were able to induce rejection of tumors caused by PyV-transformed cells [Lathe et al., Nature 326 (1987) 878-880]. Here we present evidence that PyV early proteins expressed by the recombinants retain the biochemical characteristics of their authentic counterparts despite the cytopathic effect of VV infection. VV-encoded LT is a nuclear phosphoprotein, with specific DNA binding, ATPase and nucleotide-binding activities. VV-expressed MT associates with cellular kinases, particularly with pp60c-src, by which it is phosphorylated in vitro. Expression levels of LT and MT reached 10(6) molecules per infected cell. The use of VV as a vector is encouraged by the high expression level obtained and because VV infection does not seem to prevent appropriate post-translational processing of proteins encoded by VV recombinants.     

Herpes simplex virus mRNA species mapping in EcoRI fragment I.

We described the detailed characterization and high-resolution mapping of nine herpes simplex virus type 1 mRNAs encoded in EcoRI fragment I. Four of these mRNAs are partially colinear and encode the same sized polypeptide in vitro. Nucleotide sequence analysis of the DNA around the 5' ends of these mRNAs suggested that the larger may encode a small (ca. 100-dalton) polypeptide not resolvable by in vitro translation.     

Procedure for purification of intact DNA from vaccinia virus.

A procedure for the isolation of intact vaccinia DNA molecules by chromatography on hydroxyapatite in the presence of 6 M urea is described. When lysates of virions containing 0.5 to 10 microgram of DNA were employed, over 95% of the viral DNA could be recovered free of poteins. Vaccinia DNA molecules isolated in this manner sedimented at 68S in neutral sucrose gradients and had an average contour length of 62.3 micrometer when examined in an electron microscope, and the DNA could be cleaved with the restriction endonuclease EcoRI and BamHI. The results of these analyses showed that intact vaccinia DNA molecules of 120 X 10(6) to 130 X 10(6) molecular weight could be obtained by the procedures described.     

Identification and mapping of a 60 bp EcoRI fragment in the Dictyostelium discoideum ribosomal DNA

We have re-examined the organization of the transcribed sequences in the ribosomal DNA repeat unit of Dictyostelium discoideum. In addition to the four EcoRI fragments previously reported, we have identified and cloned a fifth fragment which defines a small portion of the 25S ribosomal RNA. The fragment is 60 bp long and is located between the 1.5 kbp and the 3 kbp EcoRI fragments of the ribosomal DNA repeat unit.     

Enzymatic sequence-specific spin labeling of a DNA fragment containing the recognition sequence of EcoRI endonuclease

Deoxyuridine analogs spin labeled in position 5 have been enzymatically incorporated sequence specifically into an oligodeoxyribonucleotide to form a spin-labeled 26-mer. The 26-mer contains the EcoRI-binding site and two labels which are located symmetrically close to the binding site. The labels are separated from one another far beyond the Heisenberg spin-exchange distance. The local base motion as determined by ESR spectroscopy is of the order of 4 ns in the oligonucleotide duplex. This is the same value as reported earlier for local T motions in polynucleotide duplexes, thereby providing direct experimental evidence that the ESR line shape of spin levels covalently attached to nucleic acids depends primarily on the local dynamics of the nucleic acid building blocks.     

Evidence of a repetitive sequence in vaccinia virus DNA.

Analysis of vaccinia DNA by reassociation kinetics revealed that 7% of the genome contains a sequence repeated 10 times. This sequence does not contain any host cell DNA, is viral specific, and is found in virions passed at either high or low multiplicities of infection.