The densitometric study of G bands on human metaphase chromosomes

Summary The method of densitometric measurement of G bands on human metaphase chromosomes is described, and some factors influencing the densitometric patterns are discussed. The densitometric patterns are reproducible and typical for a given chromosomal pair, although they display some degree of variability. Typical patterns of all human metaphase chromosomes are presented, with the patterns obtained from the measurement of some structurally abnormal chromosomes (t13q14q, t14q21q, Xp-).

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Zusammenfassung Wir beschreiben die Methode der photometrischen Darstellung von G-Banden an menschlichen Chromosomen und diskutieren über einigen Faktoren, die das Resultat beeinflussen. Die photometrischen Kurven sind reproduzierbar und für jedes Chromosomenpaar typisch. Selbstverständlich muß man mit geringen Abstufungen ihrer Variabilität rechnen. Wir zeigen unsere typischen Kurven aller menschlichen Chromosomen, die mit Hilfe der ASG-Technik gefärbt wurden. Gleichzeitig geben wir einige Kurven abnormaler Chromosomen wieder, die auch in unserem Labordargestellt wurden (t(13q14q), t(14q21q), Xp-).

     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

Quantitative analysis of high-resolution trypsin-Giemsa bands on human prometaphase chromosomes

Summary We have constructed ideograms of human prometaphase chromosomes from synchronized and from standard 72-h lymphocyte cultures. G banding was achieved by a trypsin-Giemsa (or Wright's stain) method.In addition to light (white) and dark (black) bands, we have distinguished three different shades of grey. This distinction is essential for proper identification of the increasing number of bands displayed by high-resolution chromosomes. The relative amount of chromatin in each category of staining intensity has been calculated and expressed as light value.The ideograms represent the maximal number of bands discernible with some consistency on prometaphase chromosomes, i.e., 721 euchromatic and 62 variable heterochromatic or heteromorphic bands.The ideograms are based on measurements. On selected printed copies of each chromosome derived from different cells and different individuals, the relative width of each band was measured in relation to the length of the respective chromosome arm. The measurements per chromosome were averaged and used for construction of the ideograms. The distance of each border between bands or sub-bands from the centromere has been calculated on a relative scale, with positions 0 at the centromere and 1.0 at the p terminus or q terminus.The numbering system for bands and sub-bands follows the Paris Conference (1971) recommendations.     

Fluorescent C bands of human chromosomes with 33 258 Hoechst stain

Summary Air-dried preparations of human metaphase chromosomes normally exhibit a Q band fluorescence pattern with 33 258 Hoechst stain while the C band regions of 1, 9, 16, and most acrocentric short arms appear dull. If these stained and mounted slides are stored at room temperature in the dark for several days, a spontaneous change from C-negative to C-positive bands sometimes occurs. We postulate that the pH of the buffered saline mounting medium during storage of the slides causes the C band shift since the results can be duplicated experimentally by lowering the pH of the mounting buffer from 5.5 to 4.0.     

Production of C and T bands in human mitotic chromosomes after heat treatment

Summary A modified thermal denaturation of human chromosomes in Hanks' solution at 91°C produces highly contrasted C and T bands. This improved double-banding system is a good tool in the analysis of ring chromosomes, for which R banding is often inadequate, as it is exemplified.Service Prof. J. Battin     

The synthesis of compound bands in Drosophila melanogaster salivary gland chromosomes

Small chromosome aberrations were utilized to construct compound bands in the 3C region of Drosophila melanogaster salivary gland chromosomes. The results imply that one should expect to find multiple functions associated with single bands, especially heavy bands. It is suggested that the natural occurrence of compound bands needs to be recognized and that exceptions to the one gene: one band correspondence are expected to occur.Journal Paper No. J-9459 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 1985     

The location of four human satellite DNAs on human chromosomes.

In situ hybridisation was carried out with ³H-cRNAs transcribed from four human satellite DNAs. The human metaphase chromosomes used were stained with quinacrine and photographed prior to hybridisation. This allowed accurate karyotyping of the autoradiographs. A method of quantitative analysis of grain distribution permitted identification of minor sites of hybridisation which could be distinguished from purely random grains. The hybridisation patterns for each of the transcribed satellite cRNAs were similar, with the C-band of chromosome 9 and the Y chromosome being the most heavily labelled sites. Other detectable sites of hybridisation were the centromeric regions of chromosomes 1, 5, 7, 10, 12, 13, 14, 15, 17, 20, 21 and 22. The cRNA transcribed from DNA satellite II, however, was the only one to hybridise to the centromere region of chromosome 16. The evolution of the human satellite DNAs and the validity of current models of satellite DNA function are discussed in the light of the present results.     

A possible correlation between DA-DAPI counterstaining and alu I-induced bands in fixed human chromosomes

DA-DAPI counterstaining is a technique which reveals the heterochromatic areas of chromosomes 1, 9, 15, 16 and Y in humans; a result which may be related to the peculiar characteristics of these chromosomal regions, particularly regarding their DNA content. The present report describes a different DA-DAPI stain reaction. Obtained under standard conditions, it shows positive, bright staining in a large number of chromosomes. Such a result is discussed in relation to the banding pattern produced by Alu I in human metaphase chromosomes, in view of the high degree of overlapping of the two patterns.     

Cd bands and centromeric function in dicentric chromosomes

Summary The Cd technique was applied to two cases of dicentric attached X chromosomes (XpXp and XqXq) and to cells from an established cell line of tumor origin (MaNo9) in which dicentrics with two active centromeres were present and dicentrics with one active and one inactive centromere. It was confirmed that the Cd technique discriminates between active and latent centromeres, and it was demonstrated that true dicentrics and dicentrics with one latent centromere can co-exist in the same cells. This indicates that the mechanism of centromere inactivation is a phenomenon that is specific to each chromosome and not generalized at the level of the individual cell.     

Detection of cryptic bands by AluI in eukaryotic chromosomes

Selective digestion of fixed chromatin with the restriction endonuclease AluI (which cuts the sequence AG CT) uncovers a specific and repeatable pattern of bands within the euchromatin of two species of grasshoppers and of the L929 mouse cell line, which are not detectable by means of other banding techniques such as C-bands, specific fluorochromes, or other restriction endonucleases. It is tentatively suggested that this chromatin represents a special class of repetitive DNA embedded in the euchromatin, not containing the AluI restriction site to the same extent as in euchromatin and not associated with C-banded heterochromatic material.     

The steroids of 2000-year-old human coprolites.

Six samples of human coprolites, some more than 2,000 years old, were analyzed for fecal steroid composition. Despite this very lengthy period of storage, the fecal steroids of coprolites were remarkably similar to those of stool samples collected today. The sterol nucleus was clearly rather stable under the dry environmental conditions of the Nevada Caves. The steroid content (microgram/g dried weight) of coprolite was low in comparison to that of modern man. The bile acid/cholesterol and plant sterol/cholesterol ratios of the coprolite, however, were similar to these ratios of the stools of modern man. In the six coprolites, an average 73% of the neutral steroids was digitonin-precipitable. This precipitate was composed of cholesterol and three plant sterols (campesterol, stigmasterol, and beta-sitosterol) and their bacteria-modified products. A portion of the neutral steroids had been converted to products tentatively identified as epimers of these steroids. Individual bile acids were identified in the coprolite. The bile acid composition of the coprolite was similar to that of the stool of modern man.     

A rapid technique for producing silver-stained nucleolus organizer regions and trypsin-Giemsa bands on human chromosomes

Summary A simple and rapid technique is described whereby the nucleolus organizer regions (NORs) of human chromosomes can be differentially stained with silver. This staining is followed by trypsin-Giemsa banding on the same metaphase chromosomes. The metaphases simultaneously exhibit silverstained NORs and G bands, allowing for the unequivocal identification of all chromosomes and greatly facilitating studies involving the NOR-bearing acrocentrics.