Transcriptionally active minichromosomes are attached transiently in nuclei through transcription units

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 Iysed and analysed subsequently in a 'physiological' buffer. Non-transcribed minichromosomes eluted from nuclei but transcriptionally active ones did not. Cutting attached minichromosomes with Haem enabled most of the resulting ~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.