Comparison of banding patterns of human chromosomes obtained with heating, fluorescence, and proteolytic digestion

B. DUTRILLAUX, CATHERINE FINAZ, J. DE GROUCHY, and J. LEJEUNE

Cytogenetics 11:113-116 (1972)


Résumé :

Abstract : Banding patterns of human chromosomes obtained with various new techniques - quinacrine mustard staining, staining after heating, and staining after proteolytic digestion - are compared: Depending on the technique, the same band may be stained or not, but the sequence of the bands is identical with all techniques.

Sommaire

Recent techniques have shown the existence of discrete chromosomal structures. A comparison of chromosome patterns observed alfer controlled heating, quinacrine mustard (Q.M.) staining, and proteolytic digestion indicates that these structures show a comparable sequence of bands.

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Methods

Blood cultures were obtained and hypotonic treatment (15 min in 15 vol. 1/6 horse serum in H2O, 1 vol. 3.39 % MgCl2, and 2.5 IU/ml hyaluronidase), fixation, spreading and drying were performed by the usual techniques (TURPIN and LEJEUNE, 1965; DE GROUCHY et al.,1970).

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Controlled heating.

The slides were treated with 20 mm phosphate buffer (pH 6.5) for 10 min at 87°C, rinsed with tap water, and stained with Giemsa (DUTRILLAUX and LEJEUNE, 1971). More than 100 patients were studied, meaning that several hundred cells were karyotyped.

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Quinacrine mustardfluorescence.

The preparations were stained with quinacrine mustard dihydrochloride (Q. M.) (Polysciences, Inc.) according to CASPERSSON et al. (1970). Slides were dipped in a solution of 5 mg Q.M, in 100 ml distilled water and rinsed three times in pH 7 Macllvaine buffer. Some 20 patients, i.e., more than 50 cells, were thus studied.

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Proteolytic digestion.

The slides were treated with a solution of 5 mg pronase (Calbiochem) in 100 ml distilled water for 3 to 6 rein at 37°C, rinsed, and stained with Giemsa (DUTRILLAUX et al., 1971). Some 21 individuals, i.e., more than 60 cells, were studied by this procedure. Similar results can be obtained with a-chymotrypsin instead of pronase (FINAZ and DE GROUCHY, 1971).

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Results

The strongly stained bands obtained after heating correspond to the weakly fluorescing bands of the Q. M, method as well as to the unstained but swollen bands revealed by proteolytic digestion. Conversely, the lightly stained bands of the heating procedure correspond to the brightly fluorescent bands and to the deeply stained bands of the prateolytic digestion method: particularly clear banding patterns are shown in fig. 1. Telomeric zones are always stained after heating.

Some exceptions, however, must be pointed out: (1) Centromeric regions are stained only after enzymatic digestion. (2) Secondary constrictions are never stained by any of the methods. (3) The short arms and satellites of the acrocentric chromosomes generally appear clear and swollen after proteolytic digestion, but their appearence varies with the other two techniques. (4) The pattern of the Y chromosome is variable and can not be systemized; the short arm is usually stained after heating; the long arm is brightly fluorescent with Q. M.

These observations are summarized in table I.


Fig. 1. - Chromosomes 1, 3, 7, 21, and 22 from three different individuals, showing banding patterns obtained with Giemsa staining after heating (left), with quinacrine mustard fluorescence (center), and with Giemsa staining after pronase digestion (right).

Table I. Comparison of staining patterns of human chromosomes obtained with Giemsa after controlled heating, with quinacrine mustard (Q.M.), and with Giemsa after proteolytique digestion.
TechniqueBandsCentromeresSecondary constrictionsSatellitesY chromosome
DistalIntermediate
Giemsa after heating+- +--variableshort arm + long arm variable
Q.M. staining-+ - (1)2-variablelong arm +
Giemsa after proteolytic digestion+ - (1)+more information needed
(1) pattern apposite that obtained with Giemsa staining after controlled heating. (2) Except for the usual polymorphism.

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Discussion

The various structures shown by the new techniques may depend, a priori, on different mechanisms. Banding, however, always occurs according to the same sequence. It therefore seems probable that the various treatments, namely, Q. M. staining, controlled heating, and proteolytic digestion, reveal a biochemical heterogeneity of the chromosome. On the one hand, it has been suggested that Q.M. is specific for G-C rich DNA segments. On the other hand, prateolytic digestion must obviously operate mostly on proteins. Controlled heating could act on both DNA and protein structure. The congruence of the structures revealed by these three techniques demonstrates that banding depends not only on the local base composition of DNA but also, and maybe mostly, an the local protein constitution.


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References

CASPERSSON T.; ZECH, L.; JOHANSSON, C. and MODEST, E. J.: Identification of human chromosomes by DNA reacting fluorescing agents. Chromosoma, Berl. 30: 215-227 (1970).

DUTRILLAUX, B.; GROUCHY, J. DE; FINAZ, C. and LEJEUNE, J.: Mise en évidence de la structure fine des chromosomes humains par digestion enzymologique (pronase en particulier). C.R. Acad. Sci. 273: 587-588 (1971).

DUTRILLAUX, B. and LEJEUNE, J,: Sur une nouvelle technique d'analyse du caryotype humain. C.R. Acad. Sci. 272: 2638-2640 (1971).

FINAZ, C. and GROUCHY, J. DE: Le caryotype humain après traitement par l'alpha-chymotrypsine. Ann. Génét. 14: 309-311 (1971).

GROUCHY, J. DE; ROUBIN, M. and BILLARDON, C.: Etudes chromosomiques a partir de cultures cellulaires. Modifications techniques. Ann. Génét. 13: 141-143 (1970).

TURPIN, R. and LEJEUNE, J.: Les chromosomes humains (Gauthier-pillars, Paris 1965). Manuscript received 12 November 1971; accepted for publication 14 December 1971.