Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.     

Mouse primase initiation sites in the origin region of simian virus 40.

The sites of initiation of DNA synthesis by purified mouse DNA primase in the origin-of-replication region of simian virus 40 (SV40) were examined. Using as template the separated strands of a cloned fragment of SV40 approximately equal to 300 base pairs (bp) long that includes the origin, we observed specific sites of initiation on the two strands. On the early strand that is the template for early mRNA synthesis, the primary starts are at four positions within 10 nucleotides of each other around nucleotide 5215 and an additional site around nucleotide 5147 that is used at one-sixth the frequency of the major sites. The major start sites on the early strand are within the 65-bp minimal origin of replication and lie between tumor antigen binding sites I and II. On the late strand that is the template for late mRNA synthesis, six major initiation sites were observed, each within the 3' C-C-C-G-C-C 5' sequence in the template that is repeated twice within each of the three 21-bp repeats that lie adjacent to the minimal origin, on its late side. A 6-bp deletion in the 65-bp minimal origin that eliminates its function as an origin reduced the major initiations around nucleotide 5215 on the early strand by 90% but did not affect initiations at the minor start site on the early strand or initiations on the late strand. Mouse DNA primase is able to recognize specific regions on the SV40 DNA. Those on the early strand are within the minimal origin of replication and those on the late strand are within the 21-bp repeat region necessary for maximum replication.     

Territorial limits and functional anatomy of the simian virus 40 replication origin.

The region at and near the simian virus 40 (SV40) DNA replication origin contains a series of palindromes, a 17-base pair (bp) A + T-rich sequence, three copies of a 21-bp repeat, and two copies of a 72-bp repeat. We have constructed a series of recombinant plasmids containing sequential deletions at the region of SV40 DNA replication origin starting from the end near the repeats. These deletions were introduced by using in vitro and in vivo techniques. The relative replication efficiency of these recombinant plasmids were directly assayed in COS-1 monkey kidney cells capable of providing the tumor antigen necessary for the replication of these molecules. Recombinants lacking both copies of the 72-bp repeat did not exhibit any reduction in replication efficiency. Recombinants lacking the 21-bp repeats showed decreased replication efficiency; the reduction in replication efficiency was proportional to the number of copies of the 21-bp repeat deleted in these recombinants. A recombinant retaining the palindromes at the region of SV40 DNA replication but lacking the A + T-rich sequence and the repeats failed to replicate. Based on these results, the SV40 DNA replication origin is subdivided into two regions, and their boundaries are defined. One of these two regions is a core region containing the 17-bp, 15-bp, and 27-bp palindromes and, quite likely, the 17-bp A + T-rich sequence which are necessary for replication. The other is an auxiliary region that consists of the 21-bp repeats and has a dose-dependent enhancement effect on replication efficiency.     

The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen.

We have studied the temporal regulation of simian virus 40 (SV40) late gene expression by construction and transient expression analysis of plasmids containing the transposon Tn9 chloramphenicol acetyltransferase gene placed downstream from the late control region. The SV40 origin region in the early (but not the late) orientation promotes chloramphenicol acetyltransferase gene expression efficiently in monkey cells lacking large tumor (T) antigen. In monkey cells producing T antigen, the promoter activity of the late control region is induced by approximately 1,000-fold above the basal level. By deletion and point mutagenesis, we define two domains of the late control region required for efficient induction with T antigen. Domain I is the minimal replication origin containing T-antigen binding site II. Domain II consists of the 72-base-pair (bp) repeat and a 19-bp downstream sequence up to nucleotide 270. Domains I and II should act synergistically because the absence of either one or the other decreases induction efficiency by 2 orders of magnitude. Though a complete copy of domain II is optimal, the origin-proximal 22-bp portion of this domain is sufficient. The 21-bp repeat, located between domains I and II, is dispensable for this induction, as are sequences located downstream from nucleotide 270 in the late orientation.     

DNA-transformed murine teratocarcinoma cells: regulation of expression of simian virus 40 tumor antigen in stem versus differentiated cells.

Thymidine kinase-deficient (TK-; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21)F9 teratocarcinoma stem cells have been transformed with a recombinant plasmid genome consisting of the pBR322 genome linked to a herpes simplex virus type 1 thymidine kinase gene (HSV-1 tk) and a simian virus 40 (SV40) genome. A clonal line of stem cells was obtained that contains only one copy of plasmid DNA, which is integrated into murine chromosomal DNA through a site on the pBRR322 genome. The HSV-1 tk gene, which is adjacent to the SV40 genome, is expressed in stem cells, whereas SV40 gene expression is not detectable. If differentiation of these stem cells is induced, the differentiated cells express SV40 early gene products. Thus, we have constructed a stem cell which contains a set of genes (SV40), the expression of which is regulated differently in stem and differentiated cells. This cell line could be used to determine the mechanism of suppression of expression of these genes in stem cells.     

Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.

The recombinant plasmid p102 based on pBR322 carrying approximately equal to 50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK- cells. TK+ cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA . DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed.     

Deoxyribonuclease I sensitivity of plasmid genomes in teratocarcinoma-derived stem and differentiated cells.

The DNase I (EC 3.1.21.1) sensitivities of the simian virus 40 (SV40) genome, the pBR322 genome, and the herpes simplex virus type 1 thymidine kinase (HSV-1 tk) gene have been compared in teratocarcinoma-derived stem (12-1) and differentiated (12-1a) cell lines established by transfection of thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21)-deficient F9 cells with DNA from a tripartite plasmid genome consisting of the pBR322 genome, the SV40 genome, and the HSV-1 tk gene. HSV-1 tk is present in both stem and differentiated cells; SV40 early proteins are present in differentiated cells but not in stem cells; the pBR322 genome is not expressed in either cell type. The SV40 and pBR322 genomes are more sensitive to DNase I digestion in stem cells than in differentiated cells, reflecting the DNase I-hypersensitivity of total stem-cell chromatin. The HSV-1 tk gene is the least sensitive to DNase I digestion in both cell types.     

Expression of herpes simplex virus glycoprotein C from a DNA fragment inserted into the thymidine kinase gene of this virus.

Previous reports have described mutants of herpes simplex virus type 1 that fail to produce or accumulate one of the major glycoproteins, glycoprotein C (gC). This defect is not lethal in cell culture, has been associated with the syncytial plaque morphology of some mutants, and may result from mutations that map to a region on the genome noncontiguous with the structural gene for gC. To investigate the conditions required for, and consequences of, gC expression in a specific genetic background, we have inserted a wild-type allele of the gC gene into the thymidine kinase gene (tk) of a gC- fusion-inducing viral mutant, strain MP. This was accomplished by identifying cloned viral DNA fragments homologous to gC mRNA, inserting the appropriate fragments into the viral tk cloned in pBR322, and then cotransfecting cells with the recombinant plasmids and DNA from strain MP, for selection of insertional TK- mutants. All TK- mutants containing insertions of appropriate sequences (in either orientation) into tk were found to express gC while maintaining the syncytial plaque morphology of strain MP. Elimination of the insertion from one of the TK- mutants was accompanied by loss of ability to produce gC. Our results permit more precise mapping of the DNA sequence encoding gC, to a subfragment of Sal I fragment R (map coordinates 0.620-0.640) and indicate also that promoter sequences for the gC gene may be located in this fragment. Moreover, we can conclude that the previously described regulatory mutation of strain MP does not prevent expression of gC from the DNA inserted into its gene tk and that the syncytial phenotype of MP cannot be due solely to absence of gC.     

DNA sequences similar to those around the simian virus 40 origin of replication are present in the monkey genome.

We report the molecular cloning of African green monkey genomic DNA segments that include regions of homology to the origin of replication of simian virus 40 (SV40). Three clearly different cloned segments 14 to 17 kilobase pairs (kb) long were isolated from a genomic library in lambda phage. We estimate that each of the three is repeated fewer than four times in the monkey genome. The SV40-like regions represent a small portion of the cloned segments, and these regions cross hybridize only weakly with one another. One of the three segments is described here in detail. Although the entire segment occurs only once or twice in the monkey genome, it contains DNA sequences (other than the SV40-like sequences) that are repeated elsewhere in the genome including in the other two cloned segments. The homology to SV40 is contained within about 300 base pairs of monkey DNA and is limited to the region around the viral replication origin. The nucleotide sequence of the SV40-like region was determined. It contains a large number of short stretches homologous to three specific noncoding domains around the SV40 origin of replication: the 27-base-pair region of dyad symmetry, the first set of (short) repeats that occur just on the late side of the origin, and, further in the late direction, the two 72-base-pair-long repeats. Although these components are grouped in the monkey DNA, as they are in SV40 DNA, their relative juxtaposition is scrambled.     

Mapping of transcription sites of simian virus 40-specific late 16S and 19S mRNA by electron microscopy.

Simian virus 40 (SV40) late 19S and 16S mRNAs were annealed to complementary regions of partially melted viral double-stranded SV40(LHpa II) DNA or SV40(LBam HI) DNA. The RNA-DNA hybrid regions within the DNA molecules were visualized as loops [with SV40(LBam HI) DNA] in the electron microscope. The data confirm the previous localizations of the 3' and 5' ends of 16S SV40 mRNA and of the 3' end of late 19S SV40 mRNA. The 5' end of the major stable SV40 late 19S mRNA has been positioned at 0.755 map unit. Thus, the sequences of viral DNA from 0.655 to 0.755 map unit, including the replication origin, are not converted into major stable species of late viral mRNA.     

Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro.

Simian virus 40 (SV40) large tumor antigen (T antigen) unwinds DNA containing the SV40 origin of replication. The origin requirement for unwinding can be satisfied by the 64-base-pair SV40 core origin that supports T-antigen-dependent DNA replication both in vivo and in vitro. The core origin contains three domains with specific DNA sequence features. These include an inverted repeat, a central T-antigen binding domain, and an adenine- and thymine-rich domain containing a DNA bending focus. The domain and spacer requirements of the core origin for DNA unwinding and replication in vitro are strikingly similar to the origin requirements for DNA replication in vivo. Thus, each of the three functional domains of the core origin contributes directly to the initiation of duplex DNA unwinding by T antigen.     

Mapping of sequences with 2-fold symmetry on the simian virus 40 genome: a photochemical crosslinking approach.

Sequences with 2-fold axes of symmetry have been detected and mapped on the simian virus 40 (SV40) genome by their ability to form hairpin turns in single-stranded SV40 DNA. Supercoiled SV40 DNA (SV40 I) was digested with restriction enzymes EcoRI and HpaII. The resulting linear DNA molecules with lengths of the complete SV40 genome were then denatured and photochemically reacted with 4,5',8-trimethylpsoralen (trioxsalen) at 16.0 +/- 0.5 degrees and different ionic strengths. Secondary structures on the single-stranded SV40 DNA were crosslinked and their positions analyzed by electron microscopy. There were no observable hairpin turns on the denatured SV40 DNA when it was photoreacted in 1 mM Tris-HCl/0.1 mM EDTA at pH 7.0. In 20 mM NaCl, one specific hairpin turn was detected at 0.17 +/- 0.02 map units on the map of EcoRI-digested SV40 DNA, where the 3' ends of early 19S mRNA, late 19S mRNA, and 16S mRNA of SV40 have been mapped. In 30 mM NaCl there are five more major hairpin turns besides the most stable one. The centers of four of these specific hairpin turns were mapped at 0.26 +/- 0.02, 0.68 +/- 0.03, 0.84 +/- 0.02, and 0.94 +/- 0.01 units on the map of EcoRI-digested SV40. The fifth one is at or near the unique EcoRI cleavage site on SV40 DNA. The possible functions of these sequences are discussed in terms of the nature of the promoter sites, the replication origin, the processing of RNA precursors, and regulation at the translational level.     

Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication.

We have inserted a 311-base pair DNA fragment containing the simian virus 40 (SV40) origin of DNA replication, the early promoter, and the tumor (T) antigen binding sites into a bacterial plasmid and cloned it. This recombinant plasmid, pSV01, binds to a purified T antigen in vitro and replicates in monkey cells when supplied with large T antigen. A series of deletion mutations was generated in the origin sequences of pSV01 DNA by mutagenesis in vitro. The replication of these mutant DNAs in monkey cells was compared with their ability to bind to purified D2 protein. Mutant DNAs deficient in binding to D2 protein also exhibit reduced levels of replication in monkey cells. These findings provide biochemical evidence that the initiation of SV40 DNA synthesis may involve a direct interaction of T antigen with sequences at the origin of replication.