Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

Abstract

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei of Xenopus laevis oocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 minichromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.Several types of systems have been developed for studying transcription of cloned eucaryotic genes: microinjection of DNAs into nuclei of Xenopus oocytes (see reference 11 for review); transcription in monkey cells of genes linked to simian virus 40 (SV40) vectors (12, 28); expression of DNAs stably introduced into mammalian cells in culture (see reference 29 for review); and in vitro transcription with cell-free extracts (1,22,38,40). Together with the availability of DNA sequencing and in vitro mutagenesis techniques, these systems have enabled much progress to be made in the identification of elements within eucaryotic DNAs that are involved in regulating promotion and initiation of RNA synthesis. However, except for the elegant studies of Brown, Roeder, and their colleagues on control of transcription Xenopus 5S RNA genes (see reference 18 for review), only limited progress has been made to date concerning the roles played by either specific cellular chromosomal proteins or chromatin structure in regulating eucaryotic gene expression.

Sequence-dependent DNA replication in preimplantation mouse embryos.

“…Similar observations were made with SV40 DNA injected into mouse oocytes (manuscript in preparation) and two-cell embryos (data not shown), demonstrating that DNA replication is not required for this transition to be observed. Similar changes also have been observed with DNA injected into Xenopus oocytes (32,59,84) and eggs (47) and have been shown to result from the organization of injected DNA into chromatin. In contrast to the relative stability of DNA injected into nuclei, at least 90% of form I DNA injected into the cytoplasm of one-or two-cell embryos was degraded completely within 24 h (data not shown).…”

In vivo catenation and decatenation of DNA.

“…1 Lastly, the most interesting question raised by these studies is whether catenation and decatenation of DNA play a crucial role in nucleosome formation as well as in DNA replication and recombination. Findings consistent with an affirmative answer to this question include: (i) topoisomerases are needed to relieve the supertwists that are generated when DNA wraps around cores of histone proteins (7); (ii) a correlation exists between the appearance in oocytes of injected DNA molecules that are fully reconstituted into chromatin and the disappearance of DNA catenanes (14); and (iii) linear DNA neither catenates nor forms chromatin containing regularly spaced nucleosomes in vivo (13).…”

“…While performing experiments concerned with the reconstitution into chromatin of circular simian virus 40 (SV40) DNA injected into the nuclei of Xenopus laevis oocytes, we noted that much of the DNA went through an intermediate form in which it did not readily enter a 1.4% agarose gel even though it had been deproteinized by treatment with both proteinase K and phenol (13,14); after a few hours, it reappeared as monomer supercoiled DNA (see Fig. 5 of reference 14 for an example).…”

“…Supercoiled (form I) 3H-labeled SV40 DNA (>991% monomers; specific activity, ca. 1.5 x 105 cpm/,Lg) was isolated from virus-infected monkey cells and purified as described previously (14). Although initially containing less than 5% nicked circular molecules (form II), the preparation of DNA used in the experiments described here contained approximately 60% nicked molecules because of radiation damage and nonspecific endonucleolytic cleavages that occurred during prolonged storage at 4°C.…”

Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes

“…It has been demonstrated that protein-free plasmid DNA injected into oocyte nuclei becomes organized in a nucleosomal chromatin structure, but only a very small fraction of these 'minichromosomes' is transcriptionally active whereas the majority of the molecules persists as inactive chromatin [4][5][6]. Recently, attention has been focussed on the interdependence of chromatin assembly with formation of stable transcription complexes using this type of in vivo gene expression assay [7,8]. Here, we used an S-150 extract prepared from X. laevis oocytes [9] for controlled in vitro chromatin assembly.…”