R-banding of human chromosomes by heat denaturation and Giemsa staining after

amethopterin-synchronization
C. L. RICHER
Departement d'Anatomie, Universite de Montreal, MontrPal (QuP.), Canada H3C 357
and
Dkpartement de Pathologie et Centre de Recherche pediatrique, Hbpital Ste-Justine, Montreal (Que.),Canada H3T lC.5
M. MURER-ORLANDO AND R. DROUIN'
Departement d'Anatomie, Universite de Montreal, Montreal, (Quk.),Canada H3C 357
Can. J. Genet. Cytol. Downloaded from [Link] by [Link] on 01/10/15

Received August 17, 1982

RICHER,C. L., M. MURER-ORLANDO, and R. DROUIN.1983. R-banding of human chromosomes by heat denaturation and
Giemsa staining after amethopterin-synchronization. Can. J. Genet. Cytol . 25: 26 1 - 269.
Human late prophase to late metaphase chromosomes were prepared after amethopterin cell synchronization. R-banding was
produced by heat denaturation followed by Giemsa staining (RHG). Haploid sets of prophase chromosomes contain approx-
imately 850 bands. Sequences of chromosomes of different degrees of condensation are presented; their analysis provides
helpful information to identify the elongated chromosomes and to follow band subdivision. The heat denaturation technique
is free from most of the disadvantages encountered with R-banding by 5-bromodeoxyuridine incorporation. Giemsa stained
R-bands produced by heat denaturation on prophase and prometaphase chromosomes are useful to analyse the numerous
chromosome anomalies involving R-bands. In conjunction with G-banding, it is also important to compare adequately the
positive and negative regions of each chromosome to define the anomalies with precision.

RICHER,C. L., M. MURER-ORLANDO et R. DROUIN. 1983. R-banding of human chromosomes by heat denaturation and Giemsa
staining after amethopterin-synchronization. Can. J. Genet. Cytol. 25: 26 1 - 269.
For personal use only.

Des chromosomes humains aux stades allant de la prophase tardive a la metaphase tardive ont ete obtenus apres syn-
chronisation cellulaire par l'amethopterine. Un marquage R a eti produit par denaturation thermique et coloration au Giemsa
(RHG). Un lot haploide de chromosomes prophasiques contient environ 850 bandes. Des series de chromosomes presentant
differents degres de condensation sont illustries; leur analyse fournit des renseignements utiles pour identifier les chromosomes
allonges et suivre la subdivision de leurs bandes. La technique de denaturation thermique permet d'eviter la plupart des
inconvknients rencontres lors du marquage R par incorporation de bromodesoxyuridine. Les bandes R produites par dena-
turation thermique et coloration au Giemsa sur les chromosomes prophasiques et prometaphasiques sont utiles a I'analyse des
nombreuses anomalies chromosomiques portant sur des bandes R. En parallele avec le marquage G. les bandes R sont
importantes aussi pour localiser adequatement les regions positives et negatives de chaque chromosome afin de definir les
anomalies avec precision.

Introduction This paper describes R-bands produced by heat dena-

The various banding techniques have proven their turation, and Giemsa staining (RHG) of the prophase
usefulness even on short contracted chromosomes. and prometaphase chromosomes obtained after cell
Obviously they take on added precision when applied to synchronization with amethopterin.
elongated late prophase and prometaphase chromo-
somes; localization of breakpoints and analysis of ab- Materials and methods
normal chromosomes are thus made much easier. The Heparinized peripheral blood (0.3 mL) is cultured in 5 mL
technique of chromosome synchronization proposed of medium containing 75% Eagle minimum essential medium
by Yunis (Yunis 1976; Yunis et al. 1978) uses (Flow Laboratories), 25% fetal calf serum (Grand Island Bio-
amethopterin, low colcemid exposure, and direct logical Co. (GIBCO)), 0.1 % garamycine (Shering), 1 % glu-
Wright staining for G-banding. Viegas-Pequignot and tamine (Flow Laboratories), and 1 % phytohaemagglutinin
Dutrillaux ( 1978) have proposed synchronization with (Wellcome). Following a period of incubation of 66 to 72 h
thymidine followed by 5-bromodeoxyuridine (BrdU) at 37"C, amethopterin (methotrexate, Lederle Laboratories) is
incorporation; R-banding is observed in fluorescence added at the final concentration of lop7 M. After 17 h the
cultures are washed twice with unsupplemented media and
after staining with acridine orange. However, R-
suspended in supplemented media containing l o p 5 M thy-
banding without BrdU incorporation has not yet been midine (Baker Chemical Co.) and incubated again at 37°C
reported for amethopterin-synchronized lymphocytes. for 2 to 5 h. No colcemid is added to the medium before
harvesting.
'Stagiaire de recherche, Ministere des Affaires sociales and At the end of the culture period, the cells are centrifuged at
Compagnie Poulenc LtCe. 2000 rpm for 10 min, resuspended in 0.075 M KC1 and
 262 CAN. J . GENET. CYTOL. VOL. 25. 1983

incubated at 37°C for either 12 or 24 min. The cells are then four R-subbands separated by three G-subbands (R-
centrifuged for 5 min and resuspended in 3: 1 methanol - negative subbands). The karyotype shown in Fig. 6.
acetic acid fixative for 20 min. This fixation is repeated twice. was chosen from the 100 prepared in the range of 850
Finally, spreads are prepared by letting one drop of the sus- bands per haploid set.
pension fall on slides which have just been removed from the
freezer, and thus are cold and covered with condensation.
This spreading is usually done right after the third fixation; Discussion
however, a few slides were prepared much later ( 1 -12
months) from a cell suspension kept in fixative at 4OC; these The technique used to obtain elongated chromo-
presented a sufficient number of well-spread metaphases for somes can be relatively simple. The amethopterin block
analysis. is effective when used with culture media which are
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On the day following spreading and any day during the devoid of at least one nucleotide and its immediate
following month, heat denaturation (Dutrillaux and Lejeune precursors. The time elapsed between the release of the
197 1 ; Dutrillaux 1975) will produce well-marked R-bands on amethopterin block and harvesting need not be ex-
these chromosomes. For this, the slides were immersed in tremely precise. We adopted 3 h as suitable for good
Earle's salts solution (prepared in the laboratory or prepared results and convenient for the laboratory schedule. It
commercially from GIBCO) with the pH adjusted to 5.2 by
was found unnecessary to add colcemid with this tech-
adding 0.067 M KH,PO,, and heated at 86-87OC. After 2 to
30 min in the hot solution (depending on the number of days nique; furthermore this eliminates the chromosome
after spreading), the slides were rinsed under tap water and condensation possibly induced by this substance. On
then stained for 10 min in 4% Giemsa (Harleco) containing the other hand, the hypotonic treatment is very im-
4% of Sorensen's phosphate buffer at pH 6.7. The Giemsa portant. Its length was doubled compared with the one
stained R-bands were photographed in phase contrast using an used in standard techniques; this made the chromosome
orange filter; this permitted analysis of small sharp R-bands spreading so much better that no sophisticated method
not clearly visualized under ordinary lighting. had to be used. Three changes of fixative were found to
be sufficient.
For personal use only.

Results Concerning R-banding by the heat denaturation tech-

Preparations containing an adequate number of elon- nique, it should be noted that the time of denaturation
gated chromosomes were obtained when 2 to 4 h had must be slightly shortened to band adequately prophase
elapsed between the release of the amethopterin block and prometaphase chromosc~mes (Viegas-Pequignot
and harvesting. Too many contracted chromosomes and Dutrillaux 1978). Furthermore, we consider that
were present after 5 h. Chromosomes at all stages are the type of Giemsa used is important for this technique.
present on our slides, from late prophase to late meta- Of all the kinds of Giemsa previously tried in our labo-
phase. But while there are very few late metaphase ratory, Harleco gave the best results.
chromosomes, the earlier stages are much better repre- Most important, is the fact that the length of dena-
sented than on standard preparations. In different blood turation must be adapted not to the time elapsed since
samples there is not always a correlation between the fixation, but rather to the time elapsed after spreading.
shorter time and the number of earlier stages present. This can be very handy; the banding need not be done
The longest hypotonic treatment (24 min) was found immediately, but, if convenient, the chromosomes may
necessary to decrease the overlapping of decondensed be kept in the fixative and slides prepared 3-8 days
chromosomes. prior to denaturation.
Unlike G-bands, R-bands appear more quickly on We think it is very useful that R-banding can be
elongated chromosomes. The time of denaturation is applied to prometaphase and prophase chromosomes by
approximately 30 min for 1-day-old slides and de- the heat denaturation technique. Indeed, heat dena-
creases to approximately 2 min for 1 -month-old slides. turation is not as simple as incorporation of BrdU and
Three- to eight-day-old slides seem to give the best one needs to get accustomed to it, but it lacks the
results. The optimum time for denaturation depends on disadvantages found after BrdU treatment. Yunis
the number of days after spreading as shown by the ( 198 1) reports that bromodeoxyuridine tends to de-
banding times adequate for slides prepared from cell crease the mitotic index and can produce differentially
suspensions kept over a period of 1 year. elongated chromosomes with excessive breaks. Anoth-
The number of R-bands varies according to the er drawback of BrdU incorporation is the asymmetry
length of the chromosome as displayed in Figs. 1 to 4. sometimes found between the two homologous chro-
In Fig. 5 it is possible to see in chromosomes 2, 8, 12, mosomes, especially those which make their DNA syn-
and 14, the change in the number of bands from late thesis near the middle of the S-phase (Dutrillaux et aI.
prophase to late metaphase. For example, the telomeric 1976); this may hinder band to band comparison of the
band of the long arm of chromosome 2 opens up into two homologues. Furthermore, BrdU incorporation de-
 RICHER ET AL.
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FIG. 1. Various degrees of condensation in R-banded (RHG) human chromosomes from different cells: from late metaphase
(left) to late prophase (right). Chromosomes 1 to 4.
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 RICHER ET AL
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FIG.3. Various degrees of condensation in R-banded (RHG) human chromosomes from different cells: from late metaphase
(left) to late prophase (right). Chromosomes 10 to 16.

times even the centromeric index (Schollmayer et al. In conclusion, we suggest that Giemsa stained R-
1981). bands produced by heat denaturation of prophase and
 CAN. J . GENET. CYTOL. VOL. 2 5 , 1983
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FIG.4. Various degrees of condensation in R-banded (RHG) human chromosomes from different cells: from late metaphase
(left) to late prophase (right). Chromosomes 17 to 22, X and Y.

prometaphase chromosomes are useful to analyse the indicates positive R-bands may represent regions richer
numerous chromosome anomalies involving R-bands . in transcribed genes (Sumner 1981). Indeed, pheno-
This is particularly important because of evidence that typic malformations are frequently associated with R-
 RICHER ET AL.
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For personal use only.

RG.5. Chromosomes 2, 8, 12, and 14 are shown here with guidelines to illustrate the increasing number of bands when
chromosomes are observed at earlier stages.

band anomalies (Ganner and Evans 1971; Hoehn 1975), each chromosome to define the anomalies with pre-
and DNA sequences that are transcribed into messenger cision. Indeed, for chromosome studies it is always
RNA are localized by in situ hybridization in the preferable to use two different techniques to obtain
negative G-bands (Yunis et al. 1977). Furthermore, complementary banding patterns, thus defining clearly
RHG-banding is used in conjunction with G-banding to every band of the chromosome and helping to pinpoint
compare adequately the positive and negative regions of the regions involved in chromosomal rearrangements.
 268 CAN. J. GENET. CYTOL. VOL. 25, 1983
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FIG. 6. Late prophase karyotype , R-banded (RHG).

We hope that this work, by presenting the sequences bands and subbands during mitosis, will facilitate the
of chromosomes which show the evolution of the R- recognition of the R-banded prophase chromosomes for
 RICHER ET AL. 269

diagnostic use. With this aim in view, we are presently mosome banding. Am. J . Hum. Genet. 27: 676-686.
making a detailed study of the R-bands obtained on SCHOLLMAYER, E., D. SCHAFER,B. FRISCH,and E.
prophase chromosomes. SCHLEIERMACHER. 1981. High resolution analysis and dif-
ferential condensation in RBA-banded human chromo-
somes. Hum. Genet. 59: 187- 193.
Acknowledgments SUMNER, A. T. 1981. The nature of chromosome bands and
This study was supported by grants from the Medical their significance for cancer research. Anticancer Res. 1:
Research Council of Canada and from the Fondation 205-216.
Justine-Lacoste-Beaubien. W e thank Elise Menard and VIEGAS-PEQUIGNOT, E., and B. DUTRILLAUX. 1978. Une
mCthode simple pour obtenir des prophases et des prome-
Knne Guenette for skillful technical assistance.
taphases. Ann. GenCt. 21: 122-125.
Can. J. Genet. Cytol. Downloaded from [Link] by [Link] on 01/10/15

YUNIS,J . J . 1976. High resolution of human chromosomes.

Science (Washington, D.C.), 191: 1268- 1270.
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somes humains. Monographie des Annales de GCnCtique. human neoplasia. Hum. Pathol. 12: 540-549.
Expansion Scientifique Fran~aise,Paris. YUNIS,J . J . , M. T. KUO,and G. F. SAUNDERS. 1977. Local-
DUTRILLAUX, B., J . COUTURIER, C.-L. RICHER,and E. ization of sequences specifying messenger RNA to light-
VIEGAS-PEQUIGNOT. 1976. Sequence of DNA replication staining G-bands of human chromosomes. Chromosoma,
in 277 R- and Q-bands of human chromosomes using a 61: 335-344.
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GANNER,E., and H. J . EVANS.1971. 'The relationship V. A. BENJUSCH,V. S. DEMINTSEVA, and N. G.
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