William R. Folk

5' termini of polyoma virus early region transcripts synthesized in vivo by wild-type virus and viable deletion mutants

Abstract The 5′ termini of polyoma virus early region transcripts synthesized during the productive infection of permissive mouse cells by wild-type or tsa virus, and those expressed in a variety of transformed rodent cell lines, have been mapped on the viral genome. Results were obtained using the S 1 nuclease and primer extension gel mapping procedures. Principal 5′ ends of cytoplasmic polyadenylated mRNAs in every instance mapped between nucleotides 145 and 156 (numbering according to Soeda et al. , 1980) in the DNA sequence, 17 to 28 base-pairs before the translational initiation codon for early proteins and 26 to 37 base-pairs after a sequence agreeing with the TATA box consensus. Our data implied a minimum of two different termini with this region of the genome, at nucleotide 147 ± 2 and at 152 ± 2. The 5′ ends at 147 ± 2 were particularly common in the mRNA overproduced after thermal inactivation of the large T protein at late times during infection. The sequences determining the principal 5′ termini, unlike the analogous sequences in the closely related simian virus 40 (SV40), are distinct from those involved in the viral origin of DNA replication. A number of minor alternative 5′ termini of cytoplasmie mRNAs. located both before and after the principal 5′ ends, were also detected. Of those downstream from the principal termini, one at nucleotide 300 ± 2 was prominent. Although this apparent 5′ end is well within the early protein coding sequence, it occurs at a position 31 ± 2 base-pairs after a second TATA box. The several minor apparent 5′ termini mapping upstream of the principal termini occurred primarily in the vicinity of the highly conserved papovavirus DNA replication origin sequence. This sequence includes a third TATA box. Two of the minor 5′ termini, at 14 ± 2 and at 20 ± 2. were near the consensus distance from this TATA box, but the others mapped within or before it. Early region mRNA extracted at late times from the cytoplasm of cells infected with wild-type virus, or with tsa virus at the permissive temperature, was usually (but not invariably) enriched for RNA species with apparent 5′ termini mapping in the replication origin region, as well as for even longer RNAs. Such RNAs were correctly spliced and had the normal polyadenylated 3′ ends. They were very minor in the cytoplasmic mRNA overproduced after thermal inactivation of the large T protein at late times during infection, but the nuclear RNA from these cells comprised giant species with highly heterogeneous apparent 5′ ends, including predominantly those in the origin region and others further upstream. Nuclear viral RNA from most transformed cell lines lacked the giant species and had principal 5′ termini in the nt145 to 156 region. We consider two models to account for the presence of the longer mRNAs in the cytoplasm of infected cells at late times during infection. The first postulates a shift in transcriptional initiation sites to upstream positions because of the repressor action of the large T protein. The second, which we favour, proposes that the longer species occur because of inefficient transcriptional termination, which leads to transcription around the entire circular genome and consequently to the eventual accumulation of long cleavage products in the cytoplasm. We further studied the mRNAs expressed by three viable deletion mutants (Bendig et al. , 1980) that lack all or part of the sequence determining the principal 5′ termini and the TATA box that precedes it. These efficiently synthesized early region mRNA with slightly or highly heterogeneous 5′ ends. Two of the mutants (dl-75 and dl-17) produced mRNAs with principal alternative 5′ ends located slightly before or after the deletions. The 5′ ends of these mutant mRNAs corresponded to those of very minor transcripts of wild-type templates. These results suggest that sequence information other than the TATA box has an important role in specifying the approximate position of mRNA 5′ ends.

Closed circular DNAs with tandem repeats of a sequence from polyoma virus

The R, restriction endonuclease from bacteria containing the RTF-1 plasmid cleaves polyoma virus DNA at a single site. Virus stocks which have been passaged through cells several times at moderate or high multiplicities contain closed circular DNAs which are not cleaved by the R, endonuclease. One such population of DNAs, is not infectious, and when sheared and denatured, reassociates at a rate approximately four times faster than polyoma DNA. The predominant DNA in the population is 10% smaller than polyoma DNA and contains a fraction of the polyoma genome tandemly repeated three to four times. There is no significant contamination of the covalently closed circular DNAs by repetitive host sequences. A model explaining how such trandemly repeated molecules could arise by abortive rounds of polyoma DNA replication is discussed.

Regulatory mutants of polyoma virus defective in DNA replication and the synthesis of early proteins

Abstract In polyoma virus the origin of replication, the 5′ ends of early mRNAs, and the initiation codon for early protein synthesis map within an approximately 200 bp region of the genome. We have previously reported the isolation and partial characterization of viable mutants of polyoma virus with deletions in this important regulatory region of the genome. Three of the mutants with large deletions, one of which had significantly altered growth properties, have been further characterized with respect to their nucleotide sequence alterations and their levels of viral DNA replication and of early protein synthesis. The nearly coincident deletions in mutants 17 and 2–19 reduce the capacity of these viruses to replicate, even in the presence of a coinfecting virus; thus they help define one boundary of the origin of DNA replication. The deletion in mutant 75 appears to remove sequences that are essential for efficient expression of early genes, but has little or no effect upon DNA replication. Its defect is complemented in trans by wild-type virus. All three mutants eliminate sequences which are candidates for RNA polymerase and ribosome binding sites near the initiation codon for early proteins.

Sites in the polyoma genome cleaved by restriction endonuclease HindII

Abstract The Hemophilus influenzae HindII endonuclease cleaves polyoma DNA into two fragments, approximately 90 and 10% the size of polyoma linear DNA. One of the HindII sites is located in the H. parainfluenzae HpaII-F fragment, and the other in the HpaII-A fragment. Both sites have the sequence

Use of [1 or 3-3H, U-14c]glucose to estimate the synthesis of glycerolipids via acyl dihydroxyacetone phosphate

Summary BHK-21-cl3 fibroblasts were incubated with [1-3H, U-14c]glucose or [3-3H, U-14c]glucose to produce intracellular [3H]NADPH via the phosphogluconate pathway. 3H and 14C were then determined at the three positions of glycerol in glycerol phosphate, saponifiable glycerolipids, alkyl ether glycerolipids and plasmalogens. The 3H/14C ratio at C-2 in glycerol of saponifiable glycerolipids is 2–10 fold greater than in glycerol phosphate and approaches the ratio found in ether-containing glycerolipids. This suggests that a significant fraction of the glycerolipids in BHK-21-cl3 fibroblasts is synthesized via acyl dihydroxyacetone phosphate.

An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII.

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.

Polyoma virus replication in BHK-21 cells: semi-permissiveness is due to cellular heterogeneity.

Abstract Normal and transformed BHK-21 cells are heterogeneous in their capacity to support replication of polyoma virus. In situ hybridization has been used to demonstrate rare cells in which large numbers of polyoma genomes accumulate following infection by wild-type virus or induction of polyoma A gene mutants. Different transformed cell lines vary in the frequency of occurrence of these rare cells. Such cells may be an important determinant of the frequency of transformation of BHK-21 cells by polyoma virus.

Tandem Repetition of the Origin of DNA Replication in Defective Polyoma Virus DNA's

The sequences derived from the polyoma virus genome which are tandemly repeated in a class of noninfectious covalently closed circular DNA’s (Folk, W. R., and Wang, H. E., Virology 61, 140-155 (1974) have been identified. Specific fragments of polyoma DNA produced by cleavage with the HpaII endonuclease have been purified, and the homology between each fragment and the noninfectious DNA’s containing tandem repeats has been measured by DNA reassociation kinetics. The majority of the repeated sequences in the noninfectious DNA’s are derived from the region of the genome containing the origin of DNA replication. We have previously characterized a population of noninfectious covalently closed circular DNA’s derived from polyoma virus (Folk and Wang, 1974). The predominant DNA in the population was shown to contain three to four tandem repeats of a sequence comprising approximately 20% of the polyoma genome. Although the noninfectious DNA’s are only lo-15% smaller than infectious polyoma DNA, major portions of the polyoma genome (including the site cleaved by the EcoR, endonuclease) have been deleted in the defective DNA’s. A model indicating how DNA’s containing tandem repetitions of a unique sequence might arise as a result of abortive replication of the polyoma genome was suggested; the model predicted that the repeated sequence would contain the site for the initiation of DNA replication. In this report we extend the characterization of the sequences in the noninfectious DNA’s by demonstrating that the predominant repeated sequence is derived from the region of the genome that includes the origin of DNA replication.

A map of the sites in the polyoma genome cleaved by endonuclease AluI

Abstract The 29 sites in the polyoma genome cleaved by endonuclease (endo) A lu l and the site cleaved by endo Xba I have been identified. The A lu I fragments range in size from 0.9 to 37 × 10 4 daltons; 17 sites are located in the early region, and 12 are in the late region. The Xba I site is located 17% of the genome away from the Eco RI site, towards the terminus of DNA replication.

Isolation of polyoma viruses lacking endonuclease hindlll sites

Spontaneous mutants of polyoma virus have been isolated which lack either one or both of the two endo HiI however, one member of this group grows poorly in comparison with wild-type virus. Other mutants contain large deletions, have lost either of the Hi&III sites, and appear to require complementation by helper viruses in order to complete their growth cycle.