Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

Abstract

To define the DNA sequences required for the expression of the polyomavirus early transcription unit, we cloned part of the viral genome in a plasmid vector, isolated mutants bearing lesions introduced in vitro within DNA sequences upstream of the transcriptional start site, and measured the capacity of these various mutant genomes to transform cells and to function as templates for transcription in vitro by comparison with wild-type DNA. One set of mutants bore 5' unidirectional deletions beginning at position -810 and extending downstream to position +4. Another set of mutants bore 3' unidirectional deletions starting at position +4 and progressing upstream to position -311. The last set of mutants bore internal deletions between positions -810 and +4. Analyses of the properties of these mutant DNAs led us to conclude that the region between positions -403 and -311 includes an enhancer of gene expression. Deletion of this area from the viral genome reduced gene expression in vivo to 1 to 2% of wild-type levels, as measured by transformation assays. Moreover, this region increased the frequency of transformation of thymidine kinase-negative Rat-2 cells by the herpes simplex virus thymidine kinase (tk) gene from 5-to 20-fold. This occurred only if the polyomavirus sequences were covalently linked to the tk gene and then occurred independently of their orientation or position relative to the tk gene. A second transcriptional element is located downstream of the enhancer between positions -311 and -213. This element together with the enhancer was sufficient to bring about transformation of Rat-1 cells at nearly wild-type frequencies, and together these elements constitute the minimal sequences required for gene expression in vivo. The sequences making up the second element may be functionally duplicated downstream of position -165 (between positions -165 and -60). This was revealed by the characterization of mutant genomes with deletions between positions -349 and -60. The role of these redundant elements is not known; however, they may be analogous to the 21-base-pair repeats of simian virus 40. Finally, sequences between positions -57 and -1 were required for accurate and efficient transcription in vitro. However, this DNA stretch, which includes the TATA box and major transcriptional start sites, was not absolutely required for gene expression in vivo. We conclude that the polyomavirus promoter comprises multiple functional elements which are distributed across a DNA stretch of about 400 base pairs.

Sequence-dependent DNA replication in preimplantation mouse embryos.

“…These sequences have been cloned into pML-1 (62). Since none of these plasmids expressed PyV large T-antigen, they did not replicate when transfected into mouse 3T6 cells, but a-1-core, a-core, and 3-core did replicate when transfected into mouse MOP-8 cells that expressed PyV T-antigen from an integrated PyV genome (60,62). A plasmid carrying a deletion in the core sequence (a-1; Fig.…”

“…Only pBla DNA (16,24,67), strong and weak gene enhancer elements (21,22,37,48,60,79), host range mutations that allow replication in mouse EC cells (29,72,80), DNase I hypersensitive chromatin sites (38), SV40 ori (20; DePamphilis and Bradley, in press), and PyV transcription and translation initiation signals (17, 41, 60) are shown. PyV mutants that grow on EC PCC4 cells carried a duplication of a portion of the a-element which substituted for a deleted portion of the 13-element.…”

Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes.

“…DNA and nuclear factors for several reasons. (i) The regulatory region of Py has been studied in great detail and contains transcriptional enhancer elements (3, 18), upstream promoter elements (1), as well as elements essential for viral replication (32,33,44,47). In the case of Py, these regulatory elements are partially overlapping (44, 47) and functionally dependent on each other (4).…”

“…Some of these mutations alter the Py B enhancer (18,26), as well as the upstream promoter element (1). (iii) Functional domains of the Py F9-1 mutant that have been analyzed recently in vivo with the help of BAL 31 deletion mutants (1) can now be tested by appropriate in vitro assays.Toward this end, the Py control region determined in vivo (1,3,18,26,32,33,44,47) was examined by using the retention gel assay (10, 13) to define specific interactions between cellular factors and viral DNA fragments. The results of these experiments demonstrate a specific interaction with the Py B enhancer element as reported recently (37).…”

“…Toward this end, the Py control region determined in vivo (1,3,18,26,32,33,44,47) was examined by using the retention gel assay (10, 13) to define specific interactions between cellular factors and viral DNA fragments. The results of these experiments demonstrate a specific interaction with the Py B enhancer element as reported recently (37).…”