nucleosome - free origin region

A nucleosoroe-free region or gap containing the or igin of repl icat ion and the transcriptional promoter elenents 1s observed on 20 to 25% of the SV40 mini chromosomes Isolated at physiological 1on1c strength late In In fec t ion. We used the preferential sens i t iv i ty of the gapped minichromosomes to rest r ic t ion enzymes to obtain sucrose gradient fractions containing 50 to 80% of gapped molecules. The same fractions are also enriched 1n RNA polymerase B ( I I ) molecules engaged 1n transcr ipt ion. Using electron microscopy, we demonstrate here that the transcriptional complexes are preferent ia l ly sensitive to res t r ic t ion enzyme digestion, which Indicate that they represent a subpopulation of the gapped minichromosomes. INTRODUCTION Simian virus 40 (SV40) offers a model system to study the re lat ionship between chroroatin structure and gene expression. Late during the Infection cycle, the SV40 genome can be extracted and purif ied from nuclei of Infected cells 1n the form of a m1n1chromosome (1), free of cel lu lar chromatin (2-6). Electron microscopy has shown that 20 to 25% of the extracted minichromosomes contain a nucleosome-free region (gap) extending over approximately 400 bp (7, 8). The gap region 1s preferential ly cut in the nuclei of Infected cells by digestion with DNAse I and specific patterns of DKAse I hypersensitive sites within this region have been observed (9, 10). The region organised 1n an altered chromatin structure (the 0RI region on the physical map) on gapped minichromosomes contains the origin of replication (11) and the DMA sequences Involved in the regulation of both early and late transcription (1). As the gapped molecules and those which exhibit Increased ONAse I sensit iv i ty within the ORI region are well correlated (9, 12), 1t has been suggested that the generation of an altered chroroatin structure could lead to the formation of an "open window" within the nucleosoroal structure, thereby Increasing the accessibil i ty of DNA for binding of macromolecules Involved In transcription (9, 13-15). © IRL Pre» Limited, Oxford, England. 2045 Nucleic Acids Research We have previously reported (16) that gapped mini chromosomes represent a subpopulation of mini chromosomes, which are preferential ly accessible to res t r ic t ion enzymes and par t icu lar ly sensitive to simultaneous digestion with BglI and Mspl. This observation offered the possib i l i ty to prepare sucrose gradient fractions enriched 1n gapped mini chromosomes and thereby to further analyze the biological significance of the altered chromatin structure of the gapped molecules. We report here biochemical and electron microscopy studies which d i rect ly show that at least 60% of the v i ra l transcript ional complexes extracted from nuclei late 1n Infection at physiological ionic strength are mini chromosomes with a nucleosome-free region. MATERIALS AND METHODS Digestion of SV40 minichranosomes and sucrose gradient centr i fugat ion. SV40 m1n1chromosomes labeled in vivo with [C] thymidine (2) were extracted from Infected cells 40 hours post-Infection and puri f ied at physiological 1on1c strength (3). The 75S peak fractions were pooled, concentrated and incubated with single cleavage si te rest r ic t ion enzymes (see Results) as described (16). The digestion was stopped by addition of EDTA (5 mM f inal concentration) and the samples were dialysed for 2 hours at 4*C against 10 mM Tr1ethanolatn1ne (TEA) pH 7.5, 50 mM NaCl, 2 raM d i th io th re i to l (DTT), 1 mM ethylenediaminetetraacetate (EDTA) and 0.5 mM phenylmethylsulfonylf luoride (PMSF). At the end of d ia lys is , the material was loaded on a 5-3OT (w/v) sucrose gradient in 10 mM TEA pH 7.5, 0.13 M NaCl, 2 nfl DTT, 0.1 raM EDTA and 0.5 mM PMSF. After 3 hrs centrifugation 1n a SW41 rotor (Beckman) at 40.000 rpm and 4*C, the fractions (0.5 ml) were col lected and 10 i l l a l iquots, after addition of SDS (0.5% f inal concentration), were analyzed by electrophoresis on 1% agarose gel . Assay for RNA synthesis in v i t r o . Assays for RNA synthesis in v i t ro were carried out with fraction aliquots from the sucrose gradient (see above). The samples (120 j i l ) were adjusted to 50 raM TEA pH 8.00, 2.2 mM MnCl2, O. l t Sarkosyl, 300 mM ammonium sul fate, 1 raM DTT, 0.5 mM each of ATP, GTP, CTP and 0.05 mM [H] UTP (11.8 C1/ramole, Amersham) and incubated for 1 hour at 32°C. The reactions were stopped by addition of EDTA (5 mM, f ina l concentration) and the samples stored at 4*C. The assay mixtures were adsorbed onto f i l t e r s of DE-81 1on exchange paper (Whatman) and washed at least 10 times with 0.45 M NajHPO^, twice with water and once with ethanol. After a i r drying, the f i l t e r s were