Adrian T. Sumner

Scanning electron microscopy of mammalian chromosomes from prophase to telophase.

Changes in the morphology of human and murine chromosomes during the different stages of mitosis have been examined by scanning electron microscopy. Two important findings have emerged from this study. The first is that prophase chromosomes do not become split into pairs of chromatids until late prophase or early metaphase. This entails two distinct processes of condensation, the earlier one starting as condensations of chromosomes into chromomeres which then fuse to form a cylindrical body. After this cylindrical body has split in two longitudinally, further condensation occurs by mechanisms that probably include coiling of the chromatids as well as other processes. The second finding is that the centromeric heterochromatin does not split in two at the same time as the rest of the chromosome, but remains undivided until anaphase. It is proposed that the function of centromeric heterochromatin is to hold the chromatids together until anaphase, when they are separated by the concerted action of topoisomerase II acting on numerous similar sites provided by the repetitive nature of the satellite DNA in the heterochromatin. A lower limit to the size of blocks of centromeric heterochromatin is placed by the need for adequate mechanical strength to hold the chromatids together, and a higher limit by the necessity for rapid splitting of the heterochromatin at anaphase. Beyond these limits malsegregation will occur, leading to aneuploidy. Because the centromere remains undivided until anaphase, it cannot undergo the later stage of condensation found in the chromosome arms after separation into chromatids, and therefore the centromere remains as a constriction.

Mammalian chromosome banding — an expression of genome organization

Banding of metaphase chromosomes is an invaluable aid to analysing the complex genomes of vertebrates, but the biochemical basis for this phenomenon is poorly understood. Advances in molecular biology are beginning to point to features of genome organization that may play roles in chromosome banding.

ICF syndrome (immunodeficiency, centromeric instability and facial anomalies): investigation of heterochromatin abnormalities and review of clinical outcome

A further patient with the ICF syndrome (immunodeficiency, centromeric heterochromatin instability of chromosomes 1, 9 and 16 and facial anomalies) is described. This case is the second to be reported with consanguinity of the parents. This lends support to the theory of autosomal recessive inheritance. The features of the 15 published cases are reviewed. The clinical and cytogenetic characteristics of the syndrome are discussed, and new evidence provided as to the role of centromeres and centric heterochromatin in the production of chromosome aberrations. Correspondence with other authors has made possible a review of the clinical outcome in this condition.

Correlation of pachytene chromomeres and metaphase bands of human chromosomes, and distinctive properties of telomeric regions

By means of double staining with DAPI and chromomycin A3, we show that the chromomeres of human pachytene chromosomes are generally DAPI positive and chromomycin negative, like the G- or Q-bands of mitotic chromosomes. Thus we have demonstrated, using an objective technique not based on morphological comparisons, that chromomeres and G-bands are equivalent. However, terminal chromomeres and the ends of mitotic chromosomes, as well as a few other sites, are chromomycin positive and DAPI negative. The ends of human chromosomes appear, therefore, to contain a distinctive class of GC-rich DNA.

Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation.

SummaryA restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.

The distribution of genes on chromosomes: A cytological approach

Studies during the last 20 years have shown that the chromosomes of many organisms, especially those of higher vertebrates, consist of a series of segments having different properties. These can be recognized as, for example, G- and R-bands. Recent studies have indicated that genes tend to lie in the R-bands rather than in the G-bands, although the number of genes that has been mapped with high precision is, as yet, only a very small proportion of the total, probably much less than 1%. We have therefore sought to study the distribution of genes on chromosomes using a cytological approach in conjunction with “universal” markers for genes. Such markers include mRNA and the gene-rich, G + C-rich H3 fraction of DNA, both of which can be localized using in situ hybridization, and DNase I hypersensitivity, and digestion by restriction enzymes known to show selectivity for the CpG islands associated with active genes, both of which can be detected using in situ nick translation. We have chosen to use the approaches involving in situ nick translation and have shown that the patterns of DNase I hypersensitivity and of CpG islands on human chromosomes show a strict correspondence to R-banding patterns: Deviations from R-banding patterns reported by previous investigators who have made similar studies appear to be attributable to excessive digestion. On the other hand, we have not found the expected differentiation between the active and inactive X chromosomes; this may perhaps be attributable to such factors as the demethylation of some non-island CpGs in the inactive X and the possible alterations of chromatin structure caused by methanol-acetic-acid fixation affecting DNase I hypersensitivity.

Restriction endonuclease/nick translation of fixed mouse chromosomes: A study of factors affecting digestion of chromosomal DNA in situ

We used a restriction endonuclease/nick translation procedure to study the ability of certain enzymes, known to cleave mouse satellite DNA in solution, to attack satellite DNA in fixed mouse chromosomes. Although AvaII and Sau96I readily attack the mouse major satellite in fixed chromosomes, BstNI and EcoRII do not normally do so, although if the heterochromatin is uncondensed as a result of culture in the presence of 5-azacytidine, BstNI can attack it. No clear evidence was obtained for digestion in situ of the minor satellite of mouse chromosomes by MspI, the only enzyme reported to cleave this satellite. Our results show that the DNA of mouse heterochromatin is not merely not extracted by certain restriction enzymes, but is actually not cleaved by them. Chromatin conformation is therefore shown to be an important factor in determining patterns of digestion of chromosomes by restriction endonucleases.

Ageing of fixed cytological preparations produces degradation of chromosomal DNA

When fixed chromosome preparations were allowed to age for 1-72 h, they became progressively more susceptible to digestion by exonuclease III and by S1 nuclease. Analysis of DNA from these aged preparations on agarose gels showed that the molecular weight of the DNA decreased as ageing progressed. We conclude that DNA in fixed chromosome preparations becomes progressively degraded as the preparations age.

Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae)

The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.

Heterochromatin heterogeneity in Triturus marmoratus (Urodela: Salamandridae) demonstrated with specific DNA-binding fluorochromes and «in situ» restriction endonuclease/nick translation

SUMMARYThe chromosomes of Triturus marmoratus have been studied using base-specific fluorochromes, and restriction enzyme digestion followed by nick translation to reveal the sites of specific DNA sequences. Much of the heterochromatin of this newt contains sufficient concentrations of AT- and GC-rich DNAs to be detected by specific fluorochromes. Our application of the restriction enzyme/nick translation to newt chromosomes for the first time shows that this procedure is particularly valuable for studying the organisation of heterochromatin in these organisms. The centromeres of all chromosomes except those of pairs 10, 11 and 12 had similar properties; a positive reaction with DA/DAPI, chromomycin/DA, Hae III/nick translation and Hinf I/nick translation, but a negative reaction with Eco RI/nick translation. The reactions of the centromeres with the restriction enzyme/nick translation procedures can be correlated with the known properties of the satellite DNAs contained in these regions. On the other han...