Role of specific simian virus 40 sequences in the nuclease-sensitive structure in viral chromatin.

A nuclease-sensitive region forms in chromatin containing a 273-base-pair (bp) segment of simian virus 40 DNA encompassing the viral origin of replication and early and late promoters. We have saturated this region with short deletion mutations and compared the nuclease sensitivity of each mutated segment to that of an unaltered segment elsewhere in the partially duplicated mutant. Although no single DNA segment is required for the formation of a nuclease-sensitive region, a deletion mutation (dl45) which disrupted both exact copies of the 21-bp repeats substantially reduced nuclease sensitivity. Deletion mutations limited to only one copy of the 21-bp repeats had little, if any, effect. A mutant (dl135) lacking all copies of the 21- and 72-bp repeats, while retaining the origin of replication and the TATA box, did not exhibit a nuclease-sensitive region. Mutants which showed reduced nuclease sensitivity had this effect throughout the nuclease-sensitive region, not just at the site of the deletion, indicating that although multiple determinants must be responsible for the nuclease-sensitive chromatin structure they do not function with complete independence. Mutant dl9, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the BglI site is 146 bp away from the site of the deletion.     

Relationship among location of T-antigen-induced DNA distortion,auxiliary sequences,and DNA replication efficiency

T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork.     

Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV

We have isolated three types of pBR322-AAV recombinant plasmids that contain deletions within the 145 bp AAV terminal repeats. When the plasmids were transfected into human cells, mutants that contained deletions within the left (type I) or right (type II) terminal repeat were viable. Of four mutants examined that contained deletions in both termini (type III), only one was viable. All of the viable mutants produced AAV virions that contained wild-type AAV DNA. Furthermore, the viable type III deletion could be converted to a nonviable mutant by deleting all copies of an 11 bp sequence from its termini. We conclude that there is an efficient mechanism for correcting deletions within the AAV termini. A model that could account for these observations is also discussed.     

Roles of the TGACT repeat sequence in the yeast TRP5 promoter

Yeast genes under general amino acid control contain multiple copies of a sequence known as the TGACT repeat in the 5'-flanking DNA. The yeast TRP5 gene contains two copies of the TGACT repeat sequence in its 5'-flanking region. The upstream TGACT repeat of TRP5 is required for normal basal expression as well as derepression by general control. Synthetic oligonucleotides containing a TGACT sequence were inserted into previously constructed TRP5 control region deletion mutants. A synthetic 17-base pairs (bp) oligonucleotide containing a TGACT copy along with flanking nucleotides from HIS3 was able to restore derepression in all deletion mutants tested. The 17-bp oligonucleotide also functioned bidirectionally. Replacements in the upstream control region by synthetic oligonucleotides indicated that sequences other than the TGACT repeat are required for high basal expression. Replacements of the downstream repeat sequence by the 17-bp oligonucleotide suggest its main role in this position is for derepressed expression. High level derepressed expression was found to correlate with the presence of two repeats.     

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.     

Activity of simian virus 40 late promoter elements in the absence of large T antigen: evidence for repression of late gene expression.

We used chloramphenicol acetyltransferase transient expression to examine the activity of the promoter elements of the simian virus 40 late promoter in the absence of large T antigen. Since the experiments were done in permissive CV-1 cells, these conditions mimic the state which exists early in the viral lytic cycle before the onset of replication and T-antigen-mediated trans activation. Our data, using deletion analysis, indicate that removal of the 21-base-pair (bp) repeat region causes as much as a 10-fold increase in activity of the late promoter elements. This result suggests that the 21-bp repeat sequences may be involved in repression of the late promoter elements during the early phase of the lytic infection. This is supported by competition analysis which indicates that increasing amounts of competitor containing only the 21-bp repeat region results in increased activity of the intact promoter. A model for the activity of the late promoter through the course of lytic infection is presented.     

Nonviable mutants of simian virus 40 with deletions near the 3'' end of gene A define a function for large T antigen required after onset of viral DNA replication

Deletion mutants of simian virus 40 (SV40) with lesions at the three DdeI sites near the 3' end of the early region were constructed. Mutants with deletions at 0.203 and 0.219 map units (mu) which did not change the large T antigen reading frame were viable. This extends slightly the upstream boundary for the location of viable mutants with deletions in the 3' end of the A gene. Mutants with frameshift deletions at 0.193 and 0.219 mu were nonviable. These are the first nonviable mutants with deletions in this portion of the A gene. None of the three nonviable mutants with deletions at 0.219 mu produced progeny viral DNA. These three mutants all used the alternate reading frame located in this portion of the SV40 early region. The mutant with a deletion at 0.193 mu, dlA2459, was positive for viral DNA replication and was defective for adenovirus helper function. All of these mutations were located in the portion of the SV40 large T antigen which has no homology to the polyoma T antigens. These results indicate that this portion of large T antigen is required for some late step in the viral growth cycle and suggest that adenovirus helper function is required for productive infection by SV40.     

Physical and Genetic Characterization of Deletion Mutants of Simian Virus 40 Constructed In Vitro

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.     

Cellular mobile genetic elements in the regulatory region of the pneumotropic mouse polyomavirus genome: structure and function in viral gene expression and DNA replication

DNA from the murine pneumotropic virus was extracted from virus in lung tissue of infected mice, and the regulatory region of the genome was amplified by PCR. The regulatory region of individual plasmid cloned DNA molecules appeared to have heterogeneous enhancer segments, whereas the protein-coding part of the genome had a uniform length. Nucleotide sequence analysis revealed that the majority of the DNA molecules had a structure differing from the standard type. A 220-bp insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was prominent. There were two variants of the 220-bp insertion, differing at two nucleotide positions at one of the termini. Other DNA molecules had complete or partial deletions of these structures and surrounding sequences in the viral enhancer. However, the end of the insertion at nucleotide 142 was frequently preserved. The viral early and late promoter activity of the variant regulatory regions was tested in a luciferase reporter assay by using transfected NIH 3T3 cells. In relation to the standard-type DNA, all variants, including a G272T mutant, had much stronger late promoters. In contrast, the early promoter activity was influenced in a positive or negative direction by individual mutations. Also, the activity of the viral origin of DNA replication was affected by the sequence variation of the regulatory region, although the effects were smaller than for the late promoter. Analysis by Southern blotting and quantification using dot blots showed that approximately 10(3) copies of material related to the 220-bp insert in murine pneumotropic virus DNA was present in mouse and human DNA but not in Escherichia coli DNA. Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220-bp viral DNA segment. These data together with the nucleotide sequence analysis suggest that the 220-bp insertion is related to a transposable element of a novel type.