A. T. Sumner

Cytological mapping of human chromosomes: results obtained with Quinacrine fluorescence and the Acetic-Saline-Giemsa techniques

The similarities and differences between the banding patterns obtained in human chromosomes with the Quinacrine fluorescence and the Acetic-Saline-Giemsa (ASG) techniques are described. The use of these techniques to identify each chromosome pair in the human karyotype is discussed, as also is the use of the methods to identify aberrant chromosomes and to map points of exchange in translocations and inversions. A number of examples are used to illustrate the resolution permitted by these new methods. Seven polymorphic regions on normal chromosomes are described, which include four identified by fluorescence on chromosomes 3,4, 13, and 22. The secondary constrictions on chromosomes 1, 9, and 16, which had previously been observed in conventionally stained preparations from favourable material, are particularly clear in all cells treated with the Giemsa techniques. The new methods make it possible to detect small differences in size between the heterochromatic blocks at these regions in homologous chromosomes. The benefit to human genetics of studying the familial segregation of both structurally rearranged and normal, but polymorphic chromosomes, where the chromosomes or parts of chromosomes can be unambiguously identified is stressed.

Chromosome homology and heterochromatin in goat, sheep and ox studied by banding techniques

Peripheral blood lymphocyte metaphase chromosomes of three Bovoidean species have been studied using Quinacrine fluorescence and Giemsa banding techniques to give Q-, G-, and C-banding patterns. Q- and G-banding characteristics, coupled with chromosome length, enabled all of the chromosomes in each of the chromosome complements to be clearly distinguished, although some difficulties were encountered with the very smallest chromosomes. A comparison of G-banding patterns between the species revealed a remarkable degree of homology of banding patterns. Each of the 23 different acrocentric autosomes of the domestic sheep (2n=54) was represented by an identical chromosome in the goat (2n=60) and the arms of the 3 pairs of sheep metacentric autosomes were identical matches with the remaining 6 goat acrocentrics. A similar interspecies homology was evident for all but two of the autosomes in the ox (2n=60). This homology between sheep metacentric and goat acrocentric elements confirms a previously suggested Robertsonian variation. The close homology in G-banding patterns between these related species indicates that the banding patterns are evolutionarily conservative and may be a useful guide in assessing interspecific relationships. —The centromeric heterochromatin in the autosomes of the three species was found to show little or no Q-or G-staining, in contrast to the sex chromosomes. This lack of centromeric staining with the G-technique (ASG) contrasts markedly with results obtained with other mammalian species. However, with the C-banding technique these regions show a normal intense Giemsa stain and the C-bands in the sex chromosomes are inconspicuous. The amount of centromeric heterochromatin in the sheep metacentric chromosomes is considerable less than in the acrocentric autosomes or in a newly derived metacentric element discovered in a goat. It is suggested that the pale G-staining of the centromeric heterochromatin in these species might be related to the presence of G-Crich satellite DNA.

Mechanisms involved in the banding of chromosomes with quinacrine and Giemsa: I. The effects of fixation in methanol-acetic acid

Abstract A study has been made of the mechanism whereby specific banding patterns can be produced on chromosomes when stained with quinacrine or Giemsa. This paper describes the effects of methanol/acetic acid fixation on chromosomes. Fixation removes much material, the resultant fixed chromosome consisting of 2 to 3 parts of DNA to 1 part of non-histone protein. Both these components are uniformly distributed along the chromosomes. Although purified DNA is much denatured by methanol/acetic acid, such fixation only causes very slight denaturation of chromosomal DNA. Incubation in warm saline anneals this denatured DNA, but causes no redistribution or extraction of DNA or protein.

Mechanisms involved in the banding of chromosomes with quinacrine and Giemsa: II. The interaction of the dyes with the chromosomal components

Abstract It is shown that the dyes used to produce banding patterns on chromosomes, quinacrine and Giemsa, are bound to DNA, and not to non-histone protein, the other chromosomal component remaining after acetic acid fixation. Studies on fixed nuclei and on extracted DNA in gelatine films show that the amount of dye bound is not affected by whether the DNA is native or denatured, and is not directly related to the amount of DNA present. Quinacrine is bound to the DNA ionically. With Giemsa, a new magenta compound is formed in situ, consisting of two molecules of methylene blue and one of eosin; this compound is attached to the chromosome by hydrogen bonds. Both quinacrine and the magenta compound formed from Giemsa appear to be attached to DNA molecules at two separate points, and the available evidence suggests that the amount of dye bound is related to the concentration of the DNA. It is suggested that the dye molecules bridge longitudinally separated sites brought into close proximity by folding of the DNA, and that the spatial arrangement of sites in the chromosome is influenced by non-histone proteins. It is concluded that chromosome banding is thus a consequence of the reduction of dye binding in those regions where the DNA chains become sufficiently dispersed to prevent bridging by the dye molecules. Possible indirect effects of base composition and repetition on dye binding at certain chromosomal sites are discussed.

Protein-depleted chromosomes

Protein-depleted isolated Chinese hamster chromosomes have been obtained by different protein extraction procedures and examined by electron microscopy and SDS-polyacrylamide gel electrophoresis. Salt-resistant centromeric and telomeric structures are visible in protein-depleted chromosomes and the protein-depleted chromosomes appear to have a regular, longitudinal pattern in critical point dried preparations. The scaffold-like structure of protein-depleted chromosomes is highly affected by the ionic strength and composition of the extraction medium and by the spreading conditions. Nucleosomal histones of isolated chromosomes proved to be more sensitive to the sodium chloride treatment than histones of isolated chromatin. A small, but constant quantity of core histones was detected in 2 M salt extracted chromosomes and H3 and H4 histones of isolated chromosomes appeared to be resistant to the sodium deoxycholate treatment.

Some factors affecting the action of restriction endonucleases on human metaphase chromosomes

We have investigated whether restriction endonucleases produce bands on human chromosomes by extracting DNA, using staining methods which are stoichiometric for DNA. Restriction enzymes that produce C-band patterns appear to remove DNA extensively from chromosome arms. In general, however, those restriction enzymes that produce G-bands do not extract DNA from chromosomes, and their effects are believed to be due to conformational change in the chromosomal DNA; in these cases, the chromosomal regions affected appear to be determined by the chromosome structure and not by the specificity of the enzyme. DNA loss from chromosomes due to digestion by restriction enzymes may in some cases be uniform, although a G-banding pattern is visible after Giemsa staining.

Relative DNA contents of somatic nuclei of ox, sheep and goat

Diploid ox nuclei contain about 14% more DNA than nuclei from sheep of the same sex. Goat nuclei have a similar DNA content to those of sheep. In view of the similar chromosome banding patterns in these species, it appears that chromosome evolution must have involved numerous minute interstitial deletions or additions of DNA. Although chromosomes which have similar banding patterns in these three species may be regarded as homologous in this respect, and can be regarded as having a common evolutionary origin, they are not homologous for the quantity of their DNA.

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis

The distribution of quinacrine in relation to Q-banding on CHO chromosomes has been investigated using X-ray microanalysis. Technical problems involved in this type of experiment were studied in detail. It was necessary to use a solution of quinacrine acetate in acetic acid to ensure that the only chlorine detectable in quinacrine-stained chromosomes was in the quinacrine molecule. Electron irradiation during analysis rapidly destroys quinacrine fluorescence, but the chlorine is not lost from the chromosomes, and there are several reasons for supposing that a reliable distribution of quinacrine on the chromosome can be obtained by the method. — Small variations along the chromosome in the amounts of chlorine (representing quinacrine) and of phosphorus (mainly DNA) occur. The distribution patterns for chlorine and phosphorus show a good resemblance to each other for each homologous chromosome; quinacrine fluorescence patterns (Q-bands) do not resemble chlorine distribution patterns, however. The results of this study therefore support the view that Q-bands result from the differential quenching of fluorescence along chromosomes to which the quinacrine is essentially uniformly bound, and do not reflect differential binding of quinacrine along the chromosome.

Immunocytochemical demonstration of kinetochores in human sperm heads.

CREST sera have been used to identify kinetochores in mature mammalian sperm heads. It is necessary to decondense the sperm heads artificially to permit access of the reagents before the kinetochores can be demonstrated immunocytochemically. The distribution of kinetochores in the sperm heads appears to be random. These results show that the kinetochore antigen recognized by the CREST sera used here is retained during spermiogenesis and is passed on to the zygote at fertilization.

Suppression of quinacrine banding of human chromosomes by mounting in organic media

When Q-banded human chromosomes mounted in water are transferred to an organic mounting medium, the chromosomes show uniform bright quinacrine fluorescence. This change is reversible. It is inferred that quinacrine is bound uniformly along the chromosomes, and that Q-banding is a consequence of a non-uniform distribution along the chromosomes of chemical groups, probably proteinaceous, which affect the fluorescence efficiency of the bound quinacrine.