• Fine-structural aspects of bromodeoxyuridine incorporation in sister chromatid differentiation and replication banding.

      Jack, Elspeth M; Harrison, Christine J; White, Gavin R M; Ockey, Charles H; Allen, Terence D; Department of Ultrastructure, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. (1989-10)
      The structure of harlequin-stained chromosomes following substitution with low levels of 5-bromodeoxyuridine (BrdUrd) over two cell cycles and high levels over the last part of one cycle (replication banding) was studied in Chinese hamster ovary (CHO) cells. By using correlative light (LM) and scanning electron microscopy (SEM), it was shown that the effects of both the ultraviolet light (u.v.) and hot SSC treatment steps of the harlequin staining procedure were necessary to obtain sister-chromatid differentiation (SCD) or replication banding. u.v. treatment alone resulted in dark Giemsa staining of both chromatids with SEM morphology of short compact protuberances and an overall flattened smooth appearance in both the unsubstituted and BrdUrd-substituted chromatids, a morphology essentially similar to that of untreated chromosomes. SSC alone on the other hand resulted in dark-staining chromatids with an SEM morphology of raised, loosely packed loops of fibres in both types of chromatids. u.v. and SSC treatment together resulted in differentiation, with dark-staining unifilarly (TB) chromatids in the LM corresponding to raised loosely packed loops in the SEM and pale bifilarly (BB) chromatids corresponding to the smooth compact flattened SEM appearance. Where the BrdUrd-substituted strand became the template (BT), or when the nascent strand TB contained high levels of BrdUrd substitution in replication banding, the chromatid stained pale and showed the compact smooth appearance in the SEM. The Giemsa staining ability and ultrastructural morphology of harlequin staining is discussed with respect to putative DNA loss and also in terms of preferential protein-protein, protein-DNA cross-linkage in BrdUrd-containing DNA. These changes are also compared with the ultrastructural morphology observed after other banding methods, where deterioration of protein and DNA-protein interaction resulting in aggregation of chromatin fibres appears to be the major mechanism.
    • Scanning electron microscopy of human metaphase chromosomes.

      Allen, Terence D; Jack, Elspeth M; Harrison, Christine J; Claugher, D (1986)
      Preparative methods for scanning electron microscopy of chromosomes are dependent on the original source of material. Chromosomes extracted from unfixed metaphase cells via isolation buffers tend to show topography and surface morphology which may have been induced by the choice of isolation buffer itself. Furthermore, this type of preparation often precludes any chromosome identification, as many metaphases have been pooled, and also the chromosomes from these preparations are not suitable for the banding techniques regularly used in clinical cytogenetics. Our own approach has been to use the standard cytogenetic approach, starting with methanol-acetic acid fixed, air dried metaphase spreads, allowing both identification of individual chromosomes, and also the facility for various banding procedures such as G and C banding to be performed. Chromosomes are subsequently "reprepared" for SEM, using rehydration, glutaraldehyde fixation, and osmium impregnation using Thiocarbohydrazide (TCH). This method produces chromosomes which can be examined at high resolution, without metallic coating, for their topography, surface morphology and chromatin organisation, and the changes produced by banding techniques which give rise to a structural alterations resulting in differential staining in the light microscope.
    • The structural basis for C-banding. A scanning electron microscopy study.

      Jack, Elspeth M; Harrison, Christine J; Allen, Terence D; Harris, R; Department of Ultrastructure and Department of Cell Biology, Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester M20 9BX, UK (1985)
      The same C-banded human polymorphic chromosomes were observed in the light microscope (LM) and then in the scanning electron microscope (SEM) to investigate the structural changes produced by the C-banding technique. C-banded regions, which stained positively in LM, were highly condensed with tightly packed chromatin fibres, resembling non-banded chromosomes. In striking contrast, adjacent non-C-banded regions were represented by loosely arranged fibres, resembling G-banded chromosomes. The significance of these observations in relation to current theories on the effects of C-banding on chromosome structure is discussed.
    • A structural basis for R- and T-banding: a scanning electron microscopy study.

      Jack, Elspeth M; Harrison, Christine J; Allen, Terence D; Harris, R; Department of Ultrastructure Paterson Laboratories, Christie Hospital, Manchester M20 9BX UK (1986)
      The structure of reverse (R)-banded and telomeric (T)-banded chromosomes was studied by examination of the same chromosomes first in the light microscope (LM) followed by the scanning electron microscope (SEM). This procedure demonstrated a structural basis to both the R- and T-banding techniques. A direct correlation was shown between the LM staining patterns and the structural patterns observed in the SEM. In the R-banded chromosomes the positively stained R-bands, viewed by LM, corresponded to highly fibrous three-dimensional regions in the SEM. The negatively stained R-interbands corresponded to flatter regions from which material appeared to have been extracted. These structural observations strongly support the suggestion that chromosomal material is preferentially lost from the R-interbands with aggregation of fibres in the R-bands. T-banded chromosomes showed a similar structure to the R-banded chromosomes. The positively stained T-bands located at the telomeres corresponded to regions of highly aggregated fibres. The remainder of the chromosome, corresponding to the negatively stained area, had a flattened and extracted appearance. These similarities in morphology between the T- and R-banded chromosomes support the view that T-bands result from a progressive breakdown of the R-banded chromosome structure.