H.J. Evans

Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

Abstract3H-rRNA obtained from Xenopus laevis tissue cultured cells, or a 3H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the 3H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus3H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived 3H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much 3H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of 3H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Downs patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of 3H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.

Forty four probands with an additional “marker” chromosome

SummaryInformation is presented which has been obtained from an exhaustive examination of 44 probands with a supernumerary marker chromosome (mar) and their families. The data include the derivation of the mar, frequency in various populations, inheritance and possible effect on fertility, congenital abnormality, and mental ability. The practical problems in assessing the risk of abnormality in a foetus discovered during prenatal diagnosis to be carrying a mar, are discussed.

Radiation-induced chromosome aberrations in nuclear-dockyard workers.

The incidence of chromosome aberrations in peripheral blood lymphocytes of 197 dockyard workers has been followed over a 10-yr period. These workers were exposed to mixed neutronγ radiation during the refuelling of nuclear reactors, but most exposures were below the internationally accepted maximum permissible level of 5 rem per yr. There was a significant increase in chromosome damage with increasing exposure, aberration frequency was a linear function of dose and was influenced by age and time of blood sampling after exposure.

Sister chromatid exchange in human chromosomes from normal individuals and patients with ataxia telangiectasia

A new fluorescence plus Giemsa staining technique now makes the detection of sister-chromatid exchange (SCE) a relatively easy matter in cells containing 5-BrdU-substituted DNA. The technique has been applied to human cells to examine the distribution of SCE between different people and within different chromosomes. The results show: (1) That there were no large differences in the incidence of SCE between blood leukocyte chromosomes from male and female adults and newborn, and that similar frequencies were found in cells from two patients with ataxia telangiectasia which, nevertheless, showed the typical increases in chromosomal aberrations. (2) The distribution of SCE between chromosomes in the complement was found to be proportional to chromosome length, although the smaller chromosomes were under-represented, but not significantly so. (3) The distribution of SCE within chromosomes was nonrandom, with a deficiency in the centromeric and an excess in the mid-arm regions. There was no evidence for an excess of SCE in chromosome regions rich in AT DNA sequences. (4) The frequency of SCE is to some extent dependent of 5-BrdU concentration, but the influence of concentration is minimal within the range of from 1 to 160 muM. Human cells exposed over two cell cycles at these higher BrdU levels have around 14 SCE per cell-a frequency virtually identical with that observed in cultured cells from the Chinese hamster, wallaby, and rat kangaroo.

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.

The relationship between patterns of DNA replication and of Quinacrine fluorescence in the human chromosome complement

Cultured human peripheral blood lymphocytes were labelled with 3H-thymidine in the early or late S phase prior to mitosis. Quinacrine fluorescence patterns in metaphase chromosomes were then recorded photographically and the slides reprocessed for autoradiography so that the same metaphase cells were examined with the two techniques. The intensity and distribution of 3H-thymidine labelling was compared with the intensity and distribution of Q fluorescence with particular reference to chromosomes 1, 13, 14, 15, 17, 18, 19, 20, 21 and 22. It was found that chromosome regions showing bright fluorescence were also late replicating and that, in general, patterns of late replications reflected the patterns of fluorescence. Exceptions to this generalisation included the late labelling X chromosome in cells of female origin and areas near the centromeres on chromosomes 1, 9, 16 and 22. These centromeric regions show a dull fluorescence but, with exception of chromosome 9, are strongly Giemsa-positive in the ASG staining technique. On the basis of staining reaction, late replicating heterochromatic regions fall into five categories, the relationships and functional significance of these categories is discussed.

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.

Uptake of 3H-thymidine and patterns of DNA replication in nuclei and chromosomes of Vicia faba

Abstract 1. 1. A significant incorporation of 3 H-thymidine into the DNA of root tip cells of Vicia faba is shown to occur within 5 min of exposure to the labelled precursor. 2. 2. When roots are exposed to water after a 30 min 3 H-thymidine treatment, all the labelled compound is used up or washed out within 10 min of removal from the active solution. However, post-treatment of roots with a high concentration of inactive thymidine, following a water wash, results in a slow depletion of the “hot” pool, labelled thymidine being available for incorporation into DNA for up to 40 min after the removal of the roots from the “hot” solution. 3. 3. The distribution of grain counts between nuclei exposed for short periods to 3 H-thymidine does not conform to a Poisson distribution, the data suggesting a basic exponential rate of incorporation by the cell nuclei. 4. 4. Observations on interphase nuclei and metaphase chromosomes of cells exposed to 3 H-thymidine at various periods during the S phase show ( a ) that DNA replication is initiated at a large number of sites over the chromosome complement, there being no sequential progression from the distal to proximal chromosome regions, and ( b ) that replication in the heterochromatic chromosome zones is confined to late S . 5. 5. The available information on the time of DNA replication in eu- and heterochromatin in a variety of species is reviewed, the evidence indicating that positive heteropycnotic regions, whether in sex chromosomes or in autosomes, always undergo a late replication. The possibility that this delayed replication of heterochromatin is associated with genetic inactivity and the presence of arginine-rich histone is discussed.