Minichromosome from BK virus as a template for transcription in vitro.

Abstract

BK virus DNA can be extracted from virions as a nucleoprotein complex containing about 20 nucleosomes. Transcription of this "minichromosome" with Escherichia coli RNA polymerase indicates that both initiation and elongation of RNA chains are reduced by the presence of nucleosomes. Hybridization analysis of RNA made on the complex shows preferential transcription of one region of BK virus genome. No increase in strand selection is observed with respect to transcription of purified superhelical BK virus DNA. In recent years, a number of studies have been reported on the structural and biological properties of human papovavirus BK (1-5). Originally isolated from the urine of an immunosuppressed patient (6), this virus has been shown recently to induce malignant transformation in hamster cells (7-9). Interest in this infectious agent has further increased since it was shown that antibodies to BK virus (BKV) are widespread in the human population (10-12) and since BKV DNA was found in human tumors (13 DNA Purification. BKV DNA was extracted from purified virions as follows. Five milliliters of virus suspension containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.5% sodium dodecyl sulfate (NaDodSO4), and 50 jg of proteinase K per ml (Merck) was heated at 500 for 30 min and then incubated at 370 for 3 hr. CsCl (5.3 g) and ethidium bromide (2 mg) were then added and the density was adjusted to 1.600 g/cm3. After centrifugation in a Spinco Ti5O rotor at 43,000 rpm and 100 for [50][51][52][53][54][55][56][57][58][59][60] hr, fractions containing the superhelical form (F I) of DNA were pooled, extracted with isopropanol, and dialyzed against 10 mM Tris-HCl (pH 7.5)/1 mM EDTA.Transcription Assay. Fractions from the sucrose gradient used to isolate the nucleoprotein complex (see above) were assayed for template activity in an in vitro transcription system with E. coli RNA polymerase. Assays were performed by adding 50 ,ul of each fraction to 50 ,ul containing 80 mM Tris-HC1 (pH 7.9), 20 mM Mg9l2, 200 mM NaCl, 0.8 mM EDTA, 8 mM dithiothreitol, 0.8 mM each ATP, GTP, and CTP, and 0.4 mM [a-32P]UTP (specific activity 8 X 104 cpm/nmol). Final NaCl concentration was 150 mM. Reactions were started by addition of 5 ,ul of E. coli RNA polymerase (0.5 unit, purification procedure to be described elsewhere). After a 30-min incubation at 370, reactions were precipitated with 5% trichloroacetic acid/1% Na pyrophosphate and filtered on GF/C filters. Radioactivity was then determined. In all experiments, Abbreviations: BKV, BK virus; SV40, simian virus 40; NaDodSO4, sodium dodecyl sulfate. 1126The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Transcription of nucleosomal DNA in SV40 minichromosomes by eukaryotic and prokaryotic RNA polymerases

“…Presence of cellular contaminants was also a problem (Chambon, personal communication). The same minichromosomes are present inside the papovavirions, and large quantities of them can be isolated intact and free of cellular contaminants by mild alkaline treatment (6,7). We think that minichromosomes isolated in this way are very convenient templates for model studies of viral and cellular chromatin transcription by exogenous RNA polymerases.…”

“…Superhelical SV40 DNA is transcribed asymmetricaly by E. coli RNA polymerase, the majority of transcripts being complementary to the early (E) strand (14,15). The relevance of this observation to SV40 transcription in infected cells was made doubtful by the finding that DNAs of two other members of the papovavirus group (polyoma virus and BKV), closely related to SV40, are transcribed symmetrically under the same conditions (16,12). However, the ability of E. coli RNA polymerase to preferentially recognize promoters for one of the two strands on SV40 DNA allows one to ask whether the constraints imposed on viral DNA by nucleosomes result in an alteration of this selectivity.…”

“…The adsorbent was washed with 500 ml of the above buffer and transferred to a glass column, from which total RNA polymerase activity was eluted with DEAE buffer containing 0. (12). the presence of nucleosomes, both eukaryotic and prokaryotic RNA polymerases can transcribe portions of minichromosomal DNA that include a few nucleosomes.…”

“…We have recently shown that BK-virus minichromosomes can act as transcription templates in an in vitro system using E.coli RNA polymerase (7). In the present study, SV40 minichromosomes extracted from virions were transcribed with eukaryotic RNA polymerase B and with E.coli RNA polymerase.…”

Integrated and free viral DNA in hamster tumors induced by BK virus.

“…32P-Labeled BKV DNA Probe. Viral DNA was extracted from CsCl-purified virions by disrupting the virions in NaDodSO4 at 50°C and digesting with proteinase K. Superhelical BKV DNA was then purified in a CsCl/ethidium bromide density gradient (15). Labeling with deoxyribonucleoside [32P]triphosphates by nick-translation was catalyzed by Escherichia coli DNA polymerase I (Boehringer) and will be described in detail elsewhere.…”