The telomeres of Tetrahymena ribosomal DNA are not sufficient for stabilizing linear DNA in Xenopus oocytes

Restriction fragments that include the telomeres of ribosomal DNA from Tetrahymena thermophila (TtrDNA) were ligated to the ends of linearized simian virus 40 (SV40) DNA. The linear SV40 DNA with TtrDNA ends, circular SV40 DNA, linear SV40 DNA, and intact TtrDNA were injected into the nuclei of Xenopus laevis oocytes and assayed for stability. The intact linear 21-kb TtrDNA and circular SV40 DNA were maintained stably for at least 72 h after injection while the linearized SV40 DNA, either with or without telomeric ends, was degraded rapidly. Limited digestion with micrococcal nuclease revealed that neither the intact TtrDNA nor the SV40 DNA with telomeric ends reconstituted into chromatin containing regularly spaced nucleosomes. Another linearized plasmid DNA (pBamC), 14 kb in length, also was not stable in Xenopus oocytes with or without the addition of TtrDNA telomeres. Therefore, TtrDNA telomeres by themselves are not sufficient for stabilization of linear DNA in Xenopus oocytes. Rather, linear TtrDNA is maintained stably because of additional sequence or structural information encoded within the molecule.     

DNA methylation diminishes bleomycin-mediated strand scission

Three DNA duplexes differing substantially in sequence were derived from pBR322 plasmid DNA and supercoiled SV40 DNA by digestion with appropriate restriction endonucleases. Following treatment with the restriction methylase HhaI (recognition sequence: GCGC) or HhaI and HpaII (CCGG), the unmethylated and methylated DNAs were compared as substrates for the antitumor agent bleomycin. Bleomycin-mediated strand scission was shown to diminish substantially at a number of sites in proximity to the methylated cytidine moieties, especially where multiple sites had been methylated within a DNA segment of limited size. Detailed analysis of the DNA substrates revealed that both strands of DNA within a methylated region became more refractory to cleavage by bleomycin and that the protective effect could extend as many as 14 base pairs in proximity to the 5-methylcytidine moieties. Among the methylated DNA segments that became more resistant to bleomycin cleavage was a HpaII site of SV40 DNA, methylation of which has previously been shown to diminish the synthesis of the major late viral capsid protein following microinjection into Xenopus laevis oocytes. Study of the cleavage reaction at varying salt levels suggested that diminished bleomycin strand scission may be due, at least in part, to local conformational changes of the DNA to Z form (or other non-B-form structures). The results are generally consistent with the hypothesis that one mechanism for the expression of selective therapeutic action by certain DNA damaging agents could involve the recognition of specific methylation patterns.     

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.     

A specific DNA unwinding activity associated with SV40 large T antigen

The incubation of highly purified large T antigen with relaxed, circular SV40 DNA in the presence of topoisomerase I (nicking closing enzyme) resulted in the introduction of negative superhelical turns in the DNA. ATP was not required for this reaction. A similar introduction of superhelical turns could also be obtained when a recombinant plasmid DNA (Y182), which contains sequences from both SV40 DNA and pBR322, was used. However, no effect was observed when relaxed pBR322 DNA, which does not contain SV40 DNA sequences, was incubated with T antigen in the presence of topoisomerase. These results are consistent with the hypothesis that large T antigen can recognize and unwind specific sequences on SV40 DNA.     

Preservation of Xenopus laevis rDNA-containing plasmid, pXlr101A, injected into the fertilized egg of Xenopus laevis

A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pXlr101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pXlr101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.     

Characterization and biological activity of cloned simian virus 40 DNA fragments

The biological activity of fragments of the SV40 genome was determined by manual microinjection of the fragments into the nuclei of mammalian cells. Fragments of the SV40 A gene (that codes for the T antigens) were obtained either directly by digestion with restriction endonucleases or after cloning into plasmid pBR322. Three different biological activities were studied: expression of T antigen, induction of cell DNA synthesis, and, in a few cases, reactivation of repressed ribosomal RNA genes. By using a number of fragments with deletions in the various portions of the SV40 A gene, we have been able to conclude that: 1) the sequences from 0.65 to 0.51 map units are not needed for the induction of cell DNA synthesis; 2) the sequences from 0.42 to 0.17 map units are not needed for the induction of cell DNA synthesis; and 3) the critical sequences for the induction of cell DNA synthesis, 0.51 to 0.42 map units, are different from those necessary for the reactivation of repressed ribosomal RNA genes (0.39-0.33 map units). These results indicate that the information for these two fundamental processes of cell proliferation resides in two separate and distinct domains of the SV40 A gene.     

Production of a T-antigen-related protein in mammalian cells after stable transformation with a cloned SV40 gene fragment

A recombinant plasmid based on pBR322 has been constructed which carries the replicator proximal early region of SV40 DNA, including the viral origin of replication (ORI). It lacks a major part of the tumour antigen 3'-coding region, the large T-antigen termination codon and the polyadenylation site. The recombinant plasmid was transferred together with the herpes simplex virus thymidine kinase gene, as a selectable marker into mouse LTK- cells. Integration and expression of the cloned SV40 gene fragment in TK+ transformants could be demonstrated by DNA restriction and blot hybridization and by immunofluorescence techniques.