Biomedical subjects
C Ambrose
Publications and source records attributed to C Ambrose.
Cardiac pacing: a new technique.
Explore the source record for details and available documents.
The flexibility and topology of simian virus 40 DNA in minichromosomes.
The linking number of DNA in minichromosomes increases by 2 turns during SV40 assembly. Changes in temperature also influence the average linking number of the total intracellular forms of SV40 DNA. When the isolated minichromosomes assembled in vivo are incubated with topoisomerase I at 33 degrees C in vitro, the linking number of SV40 DNA decreases. This decrease is about: -1.1 turns for minichromosomes with an average nucleosome spacing of 198 base pairs (bp), wt776; and -0.6 turns for minichromosomes containing a shorter average nucleosome repeat (177 bp), tsC219. The difference between the average linking number of naked SV40 DNA relaxed with topoI at 33 degrees C and minichromosomes relaxed with the enzyme at the same temperature indicates that SV40 chromatin contains on the average 26 nucleosomes. However, the results of studies obtained both on DNA flexibility in chromatin and in naked DNA, and on the shape of the topoisomer distribution curves, indicate that all of the minichromosomes, regardless of their overall structure, do not contain the same number of nucleosomes; this heterogeneity may be as large as 8 nucleosomes. We find no apparent correlation between the amount of minichromosomes containing unconstrained torsional stress and the abundance of the molecules with a structure characteristic of transcriptionally active chromatin.
Simian virus 40 protein VP1 is involved in spacing nucleosomes in minichromosomes.
We have investigated the average nucleosome spacing in the chromatin from several simian virus 40 virion assembly mutants temperature-sensitive in the major capsid protein VP1. Viral assembly intermediates that accumulate in cells infected with mutants that block virion assembly at the propagation step (tsB) have an average nucleosome repeat length similar to that of wild-type SV40 chromatin, approximately 198(+/- 4) base-pairs. This repeat length is longer than that of the host (BSC-40) cellular chromatin, which has a value of 187(+/- 4) base-pairs. In contrast, SV40 chromatin from cells infected with virus containing a mutation that blocks virion assembly at the initiation step (tsC) has a significantly shorter average repeat length of 177(+/- 4) base-pairs. At the permissive temperature (33 degrees C), tsC chromatin has a nucleosome spacing periodicity essentially the same as that of wild-type SV40 chromatin. In addition to possessing a chromatin structure with nucleosomes that are, on the average, closer together, tsC chromatin contains a nuclease-hypersensitive or open region in nearly all molecules, but apparently the same number of nucleosomes. These findings suggest that nucleosomes are deposited initially on newly replicated SV40 chromatin in such a way as to leave the DNA region containing the origin of replication and transcription enhancers uncovered. Subsequent interaction with capsid proteins appears to increase the average nucleosome spacing and consequently to cover the open region for encapsidation.
A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region.
The initiation of simian virus 40 assembly is blocked at the nonpermissive temperature in cells infected with the viral capsid protein VP1 mutant tsC219. Greater than 95% of the minichromosomes isolated from these cells are accessible to cleavage by Bgl I and Sph I, which recognize the sequences near the viral replication origin and in the transcription enhancer elements, respectively. The accessibility of the Ori region to Bgl I is considerably reduced when virion assembly is allowed to proceed in tsC219-infected cells at the permissive temperature. A reduced accessibility to Bgl I is also observed for chromatin isolated from cells infected with wt776, the wild-type parental strain of tsC219. For wt776 chromatin, variability to Bgl I sensitivity is observed and this can be correlated to the relative virion-to-chromatin yield. A similar correlation is not apparent for restriction endonucleases that recognize sequences within the coding region of simian virus 40 chromatin. These results, considered together, indicate that, when virion assembly initiation is blocked, nucleosomes are nonrandomly arranged with respect to the viral regulatory sequences. It appears that the open regulatory region in minichromosomes is established during replication and that a protected regulatory region is generated with the onset of virion assembly.