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.
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).
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.
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.
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).
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
These observations are summarized in table I.
- 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.
|Technique||Bands||Centromeres||Secondary constrictions||Satellites||Y chromosome
|Giemsa after heating||+||-
+||-||-||variable||short arm + long arm
|Q.M. staining||-||+ -
(1)||2||-||variable||long 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.
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.
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
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:
TURPIN, R. and LEJEUNE, J.: Les chromosomes humains (Gauthier-pillars,
Paris 1965). Manuscript received 12 November 1971; accepted for publication 14