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Viral minichromosomes provide simple models for chromatin domains. The sequences attaching them to larger nuclear structures were mapped; attachments were defined operationally by their ability to prevent chromatin fragments electroeluting from nuclei. Cells encapsulated in agarose microbeads were lysed and analysed subsequently in a 'physiological' buffer. Non-transcribed minichromosomes eluted from nuclei but transcriptionally active ones did not. Cutting attached minichromosomes with HaeIII enabled most of the resulting approximately 400 bp fragments to elute. Analysis of residual fragments showed that no single sequence was responsible for attachment; rather, minichromosomes were attached at only one or two points through a promoter or part of a transcription unit. This suggests that RNA polymerases mediate attachments that change dynamically as the template slides past the attached enzyme. As, under optimal conditions, polymerases continue to elongate roughly at the rate found in vivo, these attachments are unlikely to be generated artifactually.

Type

Journal article

Journal

J Cell Sci

Publication Date

08/1993

Volume

105 ( Pt 4)

Pages

1143 - 1150

Keywords

Animals, Binding Sites, Cell Line, Cell Nucleus, Chromatin, Chromosomes, DNA Replication, DNA, Viral, DNA-Directed RNA Polymerases, Humans, RNA, Viral, Simian virus 40, Transcription, Genetic, Transfection