Comings 1973 - The Mechanism of C- And G-Banding of Chromosomes

Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserued

Experimental Cell Research 77 (1973) 469-493

THE MECHANISM OF C- AND G-BANDING OF CHROMOSOMES

D. E. COMINGS, EVANGELITA AVELTNO, T. A. OKADA and H. E. WYANDT

Department of Medical Genetics, City of Hope National Medical Center, Duarte, Calif. 91010, USA

SUMMARY

A series of biochemical, staining and electron microscopy techniques were utilized to investigate the mechanisms of C- and G-banding. These led to the following conclusions. I. The treatment of fixed chromosomes with 0.07 N NaOH for 30 to 180 set removes from 16

to 81% of the DNA from the chromosomes. 2. On average, the complete C-band technique removes 60% of the DNA. 3. This DNA is preferentially extracted from the non-C-band regions. 4. In marked contrast to this, all G-band techniques (except 1) removed less than 9 o,, of the

chromosomal DNA. 5. Most of the G-band techniques, including those using trypsin, remove very little protein from

the chromosomes. 6. Feulgen staining indicated that neither C- nor G-banding can be explained on the basis of

different amounts of DNA along the length of the chromatid. 7. Treatment of chromosomes witkalkali or prolonged treatment with trypsin tends to destroy

G-bands, while C-bands remain. 8. The combined use of acridine orange and Giemsa staining indicate that, (a) repetitious DNA

in situ renatures in seconds while non-repetitious DNA renatures in minutes; (6) Neither C- nor G-banding depends upon the differential renaturation of DNA for its effect.

9. G-banding is more delicate and relatively mild conditions allow staining of both C- and G-bands. To obtain only C-bands the chromosome must be treated more harshly to disrupt or destroy the G-bands.

10. DNA-non histone protein interactions probably play an important role in the production of both C- and G-banding.

in recent years the techniques of chromosome banding have provided cytogeneticists with some powerful new tools. These include the use of C-banding [31] to stain centromeric type heterochromatin, and Q- and G-banding [31] to produce differential staining in the chromosome arms. While it has often been suggested that these techniques are sensitive to variations in base composition of the DNA, or that they are causing dissociation and reassociation of repetitious DNA, the fundamental mechanism of their action is little understood. The present study is an investigation of the following questions: What do the various techniques inherent in

C- and G-banding do to fixed chromosomes? How much DNA, RNA and protein is removed? Do the treatments designed to denature DNA really do so? Under in situ conditions does repetitious DNA renature more rapidly than non-repetitious DNA? Is differential condensation of chromatin an important factor? And finally: What is the role of DNA-non histone protein interactions?

These questions have been investigated by biochemical and radioisotope analysis of extracts of fixed chromosomes and extracts of isolated, fixed, heterochromatin and euchromatin; by Feulgen, acridine-orange

Exptl Cell Res 77 (1973)

470 D. E. Comings et al.

and Giemsa staining; DNA ultracentrifugation of chromosome extracts, and whole mount electron microscopy.

MATERIALS AND METHODS

Preparation of pure metaphase cells Two cell lines were used, Chinese hamster V79, and Mouse L cells. They were grown in 250 ml plastic Falcon flasks in McCoy media with penicillin and streptomycin, 10 % fetal calf serum, and 1% non- essential amino acids. To obtain metaphase cells, 15 to 25 flasks were shaken to remove loose cells. Fresh media with 0.05 % Colcemid was added and incubation continued for 2 to 4 h. The flasks were shaken again and the metaphase cells collected by centrifugation. With the Chinese hamster cells this resulted in 98 to 100% metaphase cells and 95 to 98 % for mouse L cells.

The metaphase cells were resuspended in 0.075 % KCI, incubated at 37°C for 10 min, pelleted gently, and fixed in ice-cold 1 : 3 glacial acetic acid/methanol. The fix was replaced every 30 min x 3.

Biochemical treatments

The chromosome preparations were treated with one or more of the various chemical solutions that have been used in the process of C- and G-banding. Since all of the procedures begin with hypotonic treated, methanol-acetic acid fixed, and air dried chromosomes. this was taken as the baseline and the following’were then applied: (1) Distilled water for 2 min: (2) 0.07 N NaOH for 30. 60. 90 or 180 sec. The d-l&&ding techniques include an initial step which involves treatment of the chromosomes with alkali, usually in the form of 0.07 N NaOH 14. 111. (3) 0.07 N NaOH for 30 set and this extract saved: fhe slides were then washed in 70 and 90% ethanol and incubated in 6 x SSC overnight and this extract also saved. Technique of Arrighi & Hsu [4]. (4) 6 x SSC, 65°C overnight, technique of Arrighi & Hsu without alkali treatment. (5) 0.07 N NaOH, 90 set and extract saved. The slides were then washed in ethanol, air dried, and placed in Sorensen buffer, pH 6.8 at 60°C for 24 h and the extract saved. Tech- nique of Schnedl [51]. (6) in one experiment the slides were exposed to Sorensen buffer for only 30 min. (7) 0.07 N NaOH in 0.112 N NaCl, 30 set and extract saved. The slides were then washed 3 times in 12 x SSC and incubated in the same at 65°C for 60-72 h and extract saved. Techniaue of Drets & Shaw [26]. (8) 0.07 N NaOH, 30 set and extract saved. Then washed and incubated in 12 x SSC, 65°C 72 h. Technique of Drets & Shaw except that the NaOH was not in 0.112 N NaCl. (9) 2 x SSC, 60°C 1 h. Technique of Sumner, Evans & Buckland 1541. (10) 0.14 M Na,P04, pH 9.0, room temperature, 5 min. Technique of Patil, Merrick & Lubs [48]. They found that molarities of phosphate from 0.003 to 0.14 all worked and we chose the highest. (11) 20 mM phosphate, pH 6.5,87”C for 12 min. Technique

of Dutrillaux & Lejeune [28]. (12) 0.2 N HCl for 30 min. An HCI treatment has frequently been used to remove histones arior to additional treatments. (13) distilled water, ?OOC, 10 min. An alternative denaturation procedure. (14) 0.005 % trypsin (Worth- ington 3 x recrystallized) in Ca2+ and Mg2+ free buffered saline solution. for 5 to 120 sec. Many banding techniques using proteolytic enzymes have been reported. We have found that exposure of chromosomes to this solution for 10 set gives reproducible G-banding.

Biochemical analysis For biochemical analysis the number of fixed metaphase cells was counted in a Coulter Counter and from 5 x lo6 to 5 x 10’ cells were air dried onto the inner surface of small 5 mm ID x 40 mm long test tubes. Various chemical solutions were removed and the extracts analysed for DNA by the fluorometric technique of Hinegardner [36], for RNA by the orcinol technique [45], and for protein by the Lowry technique.

Radioisotope analysis For radioisotope studies the interphase cells were incubated in McCoy media with 3H-thymidine, 1.5 pCi/ml (17.5 Ci/mM, New England Nuclear), or SH-5-uridine. 1.5 r&i/ml (22 Ci/mM): or in leucine- free McCoy media ’ with SH-leuci&, 1.0 ,&i/ml (40 CijmM), for 16 h prior to harvest, to label DNA, RNA and protein respectively. The fixed metaphase cells were air dried onto the distal 20 mm portion of glass slides. These were dipped into small rectangular plastic containers (sawn-off 50 ml Falcon Flasks), containing 10 ml of the various chemical solutions. After appropriate periods the glass slides were removed and air dried. The portion containing the cells was cut off with a diamond pencil, placed into a scintillation vial and 10 ml of Aquasol (New England Nuclear) plus 1 ml of distilled water or 0.07 N NaOH added. Three 1.0 ml aliauots of the extract were placed in three scintillation vials, and 10 ml of Aquasol added. The vials were counted in a Beckman scintillation counter until the counts stabilized. Knowing the total counts extracted and counts remaining on the slides, it was possible to calculate the percentage of labeled DNA, RNA or protein extracted by the various procedures.

Isolation of euchromatin and heterochromatin To determine the amount of DNA extracted from ourified. fixed euchromatin and heterochromatin. ‘Chinese- hamster and mouse L cells were labeled with SH-thymidine as above. The cells were removed with a rubber policeman, washed twice in NMT (0.15 M NaCl. 5 mM M&l,. 0.01 M Tris, pH 8.0), once in 0.01 M Tris, 5 mM MgCl*, IO-; M NaSG,, 0.5 % Triton-X, pH 8.0), once in 0.01 M Tris, 5 mM MgCl,, 1O-3 M NaSO$, 0.5 % Triton-X, pH 8.0, and homo- genized in this buffer with a tight-fitting Teflon homogenizer. Homogenization and washing was continued until clean nuclei were obtained. They were


Mechanism of C- and G-banding 471

then washed twice in 0.25 M sucrose and allowed to swell. The heterochromatin + nucleoli, intermediate fraction and euchromatin were isolated as described previously 117, 41). The respective fractions were fixed in 1 : 3 glacial acetic acid/methanol, air dried on slides and examined in the same manner as the labeled, fixed, metaphase cells.

Giemsa, Feulgen and acridine orange (AO) staining

To determine the effects of different treatments on (a) the differential staining of heterochromatin and euchromatin; (6) the amount of DNA extracted; (c) the denaturation and renaturation of DNA; fixed, air dried, and treated mouse and Chinese hamster chromosomes were stained with Giemsa, Feulgen or AO.

The treatments consisted of the following: (1) Control. no treatment. (2) 0.02 N HCl for 30 min. rinsed in distilled water and air dried, then treated with pancreatic ribonuclease (Sigma) 100 ,ug/ml in 2 :: SSC at 37°C for 60 min, rinsed in 2 x SSC, 70 % ethanol, 90 % ethanol, and air dried [4]. (3) 0.07 N NaOH for 2 min, then l/lOth volume of 40% formaldehyde added to bring to 4% formaldehyde for 2 min. (4) treatment 2, then 0.07 N NaOH for 2 min, then transferred to 10% formaldehyde for 60 min. (5) same as treatment 4 except in NaOH for 5 min. (6) 0.07 N NaOH in 4 % formaldehyde for 2 min. (7) 0.2 N NaOH in 4% formaldehyde for 2 min. (8) treatment 2, then in distilled water at 100°C for 10 min, then placed in 10% formaldehyde for 60 min. (9) 9 parts l/10 SSC and 1 part formamide at 100°C for 2 min, then brought to 4 % formaldehyde [53]. (10) treatment 2, then 10% formaldehyde in 0.05 M Tris, pH 7.0 at 100” for 10 min. then fixed in 10 % formaldehyde at room temperature for 10 min. (11) same as 10 but heat for 60 min. (12) same as 10 but heat for 120 min. (13) treatment 2, then 0.07 N NaOH for 2 min, then washed in 6 x SSC and incubated in 6 x SSC at 65°C overnight, then transferred to 10% formaldehyde at room temperature for 60 min. (14) same as 13 but fixed by adding 40% formaldehyde to the 6 x SSC to make it 4% with formaldehyde at 65°C for 1 min. (15) 6 x SSC, 65°C overnight, then brought to 4% with formaldehyde for 1 min. (16) 2 x SSC, 65°C overnight, then brought to 4% with formaldehyde for 1 min; (17) 9 parts l/l0 SSC plus 1 part formamide at 100°C for 2 min, then in 2 x SSC at 65” for 20 set, then brought to 4% formaldehyde for 1 min [53]. (18) same as 17 but in 2 2: SSC for 1 min. (19) same as 17 but in 2 x SSC for 5 min. (20) 0.07 N NaOH for 2 min, then in Sorensen buffer at pH 6.8, 60°C for overnight, then brought to 4 % with formaldehyde at 60°C for 1 min. (21) 0.07 N NaOH 2 min, drain 10 set, distilled water 20 set, drain 10 set, 70 % ethanol 20 set, drain 10 set, absolute ethanol 60 set, air dry. (22) 0.005 % trypsin for 20 sec.

For Giemsa staining, Giemsa Blood Stain Stock (Matheson, Coleman & Bell, from Curtin) was used. 1.5 ml of 0.2 M Na,HPO,+50 ml distilled water was adjusted to pH 8.5, then 1.5 ml of absolute

methanol and 2.5 ml of stock stain was added. The slides were usually stained for 10 min.

For Feulgen staining Schiff reagent was prepared as described in the instructions provided with-basic fuschin from National Aniline Division of Allied Chemical Corp. The slides were hydrolysed for 6 min in N HCl at 6O”C, then placed directly in Schiff reagent for 1 h. They were then rinsed 3 ,: in a solution containing 10 ml 1 N HCl, 10 ml lo:,, sodium metabisulfate and 200 ml distilled water; then washed in distilled water and dried. The Feulgen- stained chromosomes were photographed in a Zeiss photoscope with 95 mm High Copy-Contrast Film. This was then projected onto 4 ,: 5 inch Kodak Commercial Film. A Joyce-Loebl microdensitometer was used to make tracings perpendicular to the long axis of the chromosome. These were taken at the region of the centromeric heterochromatin and along the arms of the chromosomes and averaged. For each treatment group five different chromosomes were so examined. Similar tracings were made of Giemsa stained, untreated and C-banded chrorno- somes.

For A0 staining the technique of Rigler et al. [49] was used. This consisted of placing the slide in 50”; acetic anhydride in pyridine for 15 min, hydration through successively decreasing concentrations of ethanol to distilled water, placing in Mcllvane buffer at pH 4.1 for 5 min, then in lo-” M A0 in the same buffer for 15 min, followed by successive washes in buffer alone. The preparations were mounted in buffer under a coverslip and sealed.

In some cases the chromosomes were simply stained for 5 min in 0.125 mg/ml A0 prepared by diluting 1 vol of 0.1 9, aqueous solution with 7 vol of 0.1 M phosphate buffer, pH 6, then washed in phosphate buffer alone and mounted in buffer [53]. The results with the two methods were the same.

DNA extraction and ultracentrifugation

To determine if satellite or non-satellite DNA was preferentially extracted by alkali, fixed metaphase mouse cells were air dried on slides and extracted with 0.07 N NaOH for 3 min. The pH was then immediately brought back to neutral by dipping the slides in SSC (0.15 N NaCI, 0.015 N Na citrate, pH 7.0) or by adding to the extract an equal-molar amount of HCI. The extract was made up to, and the cells were scraoed into. 0.25 % sodium dodecvl sulfate in 0.15 ^N NaCI,’ 0.01 M EDTA, pH 7.2. Pronase (autodigested at 37°C for 1 h) was added, 1 mg/ml, for 24 h at 37°C. Then a l/IO vol of NaCLO, was added [3] with shaking for 1 h at 37’C. The rest of the DNA extraction was as described previously 117, 411. The DNA was examined by analytical ultracentrifugation [41].

Whole mount electronmicroscopy

Logarithmically growing mouse L cells were exposed to 0.05 &ml of Colcemid for O-4 h and the

- I

metaphase cells shaken off. They were pelleted and washed once with buffered salt solution, pelleted again, and resuspended in the fluid from the sides

Exptl Cell RFS 77 (1973)


Table 1. Biochemical analysis of extracts of fixed metaphase chromosomes

Treatment’

pig DNA pug RNA pg Protein extracted % DNA extracted extracted per lo6 cells extracted per lo6 cells per lo6 cells

1 Distilled water 0.0 0.0 0.0 0.0 2 0.07 N NaOH 30 set 2.2 18.0 3.95 8.38 2 0.07 N NaOH 60 set 2.8 23.7 7.42 13.1 2 0.07 N NaOH 180 set 6.8 56.5 17.2 47.4

6a 0.07 N NaOH 90 set 2.06

46.7 17.2 37.3 66 Sore,. buff. 60°C 30 min

8:6 24.9 20.2 53.9

6c Total 71.6 37.4 91.2

8a 0.07 N NaOH 30 set 2 40.2 14.5 38.5 8 b 12 x SSC 65°C 72 h 8c Total 4:8

ii:; 16.9 - 31.4 -

9 2 x SSC 60°C 1 h 0.0 0.0 36.5 4.75 10 0.14 M NasHPOd pH 9.0 5 min 0.0 17.4 2.54 12 0.2 N HCl30 min 0.3

29: 15.0 2.0

a For details see comparable number under Biochemical Treatments, section of Materials and Methods, p. 470.

of the tube. This concentrated suspension was allowed to run down a glass trough onto a surface of distilled water in a small Petri dish. After spreading, the chromosomes were picked up on Formvar and carbon- coated copper grids, and exposed to the following: (a) no treatment; (b) 0.005 % trypsin in Cae+ and Mga+-free isotonic salt solution for l-10 set; (c) EDTA 0.01 M for 10 set; or (d) 0.07 N NaOH for l-10 sec. The grids were then dehydrated through 50, 75, and 95 % absolute ethanol, washed twice in amyl acetate, then air dried. The specimens were examined with an Hitachi HS-8 electronmicroscope at 50 kV.

RESULTS

Biochemical analysis of chromosome extracts

In our initial experiments the extracts of fixed metaphase chromosomes were examined by biochemical methods. Between 5 and 50 million fixed, Chinese hamster metaphase cells were dried onto the sides of small test tubes and then exposed to various chemical treatments. The results are shown in table 1. Taking the DNA content of metaphase cells as 12 picograms it was possible to estimate the percent of DNA extracted. This indicated that 0.07 N NaOH for 30, 60 and 180 set extracted 18.0, 23.7 and 56.5 % respectively of the chromosomal DNA. When the chromosomes were exposed first to 0.07 N NaOH


then to Sorensen buffer at 60°C for 30 min additional amounts of DNA were extracted. Treatment with 0.2 N HCl for 30 min extracted only 2.4% of the DNA, and either 2 x SSC at 60°C for 1 h or 0.14 M Na,HPO, at pH 9.0 for 5 min failed to extract any DNA as determined biochemically. Since we did not have figures for the RNA or protein content per metaphase cell it was not possible to calculate the percent lost for these extrac- tions. However, considerable amounts of RNA, ranging from 3.95 to 37.4 ,ug/106 cells were extracted by all treatments and the protein extracted ranged from 2 to 91 ,ug/106 cells. To obtain more precise figures on the percentage of DNA, RNA and protein lost we turned to the examination of radioisotope-labeled chromosomes.

Radioisotope analysis of chromosome extracts

The radioisotope labeled, fixed, Chinese hamster metaphase cells were air dried on glass slides and treated with the various chemical solutions. The counts from the extracted material and the counts from the material remaining on the slides, were determined to


Table 2. Radioisotope analysis of extracts of fixed Chinese hamster metaphase chromosomes

Treatmenta

% DNA extracted % RNA extracted :‘” Protein extracted -.---

x Range n x Range n x Range II -.--

1 Distilled water, 120 set 0.5 2 0.07 N NaOH, 30 set 21.4 2 0.07 N NaOH, 60 set 25.1 2 0.07 N NaOH, 90 set 30.9 2 0.07 N NaOH, 180 set 49.0 3 a 0.07 N NaOH, 30 set 21.8 3 b 6 ;x SSC. 65°C. 24 h 36.9 3c Total ’ 58.6

4 6 x SSC, 65°C 24 h 28.3

5 a 0.07 N NaOH, 90 set 20.1 5 b Sk. buff. pH 6.8,6o”C, 24 h 44.4 5c Total 64.5

6 a 0.07 N NaOH, 90 set 45.8 6 b Sk. buff. pH 6.8,6o”C, 30 set 8.6 hc Total 54.4

7 n 0.07 N NaOH, 0.1 N NaCI, 30 set 3.9

; f 1211a~SC, 65”C, 72 h 4.3 8.2

9 2 tSSC60”C,lh 3.7

10 0.14 M Na,PO,, pH 9.0,5 min 0.7

1 I 20 mM POI, pH 6.5, 87°C 12 min 2.8

12 0.2 N HCl, 30 min 0.3

13 Distilled water, lOO”C, 10 min 2.5

0.1-0.9 4 16.1-26.4 4 19.6-35.0 4 23.5-37.5 3 22.4-81.1 4 21.1-22.4 2 15.4-58.4 2 37.7-79.5 2

16.0-40.6 2

13.9-32.4 3 35.4-54.5 3 57.3-68.4 3

1 1 1

2.44.8 4 3.5-5.7 4 5.9-9.8 4

0.0-7.9 4

0.0-1.6 4

1.8-4.2 3

0.1-0.4 4

0.9-4.1 2

0.3 0.1-0.4 2 29.7 19.9-43.7 4 34.1 31 .o-40.9 4 41.3 35.7-44.6 4 52.3 46.8-62.7 4 55.6 46.1-65.1 2 34.2 30.9-37.4 2 89.8 83.5-96.0 2

76.6 69.1-90. I 2

29.7 26.4-32.9 2 57.3 53.9-60.6 2 87.0 86.8-87.2 2

21.2 17.1-25.3 2 55.4 55.2--55.6 2 76.6 72.3-80.8 2

35.7 356-35.8 2

2.5 2.2L2.8 2

22.1 15.8828.3 2

0.4 0.2X.6 3

2.3 1.3-3.2 2

0.3 0.2-0.4 19.2 14.3-24.3 22.1 20.7-24.6 21.4 18.2--24.7 29.9 27.6-32.1

3.6 3.9-3.2 24.0 18.7-29.3 27.5 32.5-32.5

7.1

21.2

6.5. 7.’

17.7L23.3 2.6-5.4

20.3 -28.7 4.1

25.2

5.3 4.1-6.5 6.9 4.1-9.0

12.6 8.2-16.3

4.6 3.775.0

3.0 2.5-3.5

3.3 3.2-4.6

6.0 5.2.-7.2

3.2 I .l-5.3

2 2 2

2

3 3

3

3

2 --

a For details see comparable number under Biochemical Treatments, section of Materials and Methods, p. 470.

allow calculation of the percentage of labeled DNA, RNA or protein lost (table 2).

The most striking results were obtained when the DNA was labeled. Distilled water, at room temperature for 2 min (treatment l), removed only 0.5 % of the DNA and some of this may merely represent incompletely extracted labeled nucleotides. Treatment with 0.07 N NaOH for 30, 60, 90 and 180 set (treatment 2) extracted 21.4, 25.1, 30.9, and 49.0 :b of the chromosomal DNA. The results were fairly uniform for the 30 to 90 set time intervals. A considerable degree of variability was found with the 180 set treatment, ranging from 22.4 to 81.1% DNA extracted. We suspect this is merely a bio-

chemical correlation of the fact that the results from the staining procedures themselves are frequently quite variable. When the procedure of Arrighi & Hsu [4] was used (treatment 3), consisting of 0.07 N NaOH for 30 set followd by 6 x SSC at 65°C for 24 h, the NaOH step removed 21.8 0:) and the 6 x SSC removed another 36.9 96, for a total of 58.6% extraction of the DNA. When 6 x SSC at 65°C for 24 h was used alone (treatment 4), 28.3% DNA was removed. Thus the C-banding technique removed a considerable amount of chromosomal DNA. When the procedure of Schnedl [Sl] was used, consisting of 0.07 N NaOH for 90 set, followed by Sorensen buffer, pH 6.8, at

Exptl Cell Re.s 77 (1973)


Table 3. Radioisotope analysis of 0.005% trypsin extracted, 3H-leucine labeled, fixed Chinese hamster metaphase cells

% Protein extracted Treatment % DNA time 1 2 3 x extracted

5 set 0.0 0.0 0.6 0.2 - 10 set 1.4 0.1 0.0 0.5 - 15 set 0.5 0.5 0.6 0.5 - 30 set 2.8 4.6 0.4 2.6 - 60 set 9.8 9.0 6.3 8.4 0.0 2 min 13.2 0.2 3 min 0.6 4 min 1.3

60°C for 24 h (treatment 5), the NaOH step removed 20.1% and the Sorensen buffer removed another 44.4 o/o for a total of 64.5 % DNA extracted. In a single experiment in which the Sorensen buffer was applied for only 30 min (treatment 6), it removed only an additional 8.6% DNA. This is the only G-banding technique reported that uses NaOH and the only one that removes large amounts of DNA, and in our ex- perience it gives more intense staining of the C-band regions and less in the arms than the other G-band techniques. All the other G-banding procedures removed relatively little DNA. For example, the technique of Drets & Shaw [26] (treatment 7), involves adding 0.112 M NaCl to the 0.07 N NaOH. After 30 set of this treatment only 3.9 % of the DNA was extracted. When this was followed by 12 x SSC at 65°C for 72 h, only another 4.3 % was removed, for a total of 8.2% DNA lost. The technique of Sumner, Evans & Buckland of 2 x SSC, at 60°C for lh (treatment 9), removed only 3.7% DNA; the technique of Pat& Merrick & Lubs of 0.14 M (or less) Na2P0,, at pH 9.0 for 5 min (treatment lo), removed 0.7% DNA; and the R-banding technique of Dutrillaux & Lejeune of 20 mM PO,, pH 6.5 at 87°C for 12 min (treatment ll), removed 2.8 % DNA. Ex-

ExptI Cell Res 77 (1973)

posure to 0.2 N HCl for 30 min (treatment 12), a preliminary step used in many banding techniques, removed negligible-amounts of DNA, 0.3 %; and boiling in distilled water for 10 min (treatment 13), also removed very little DNA, 2.5 %.

Since alkali readily hydrolyses RNA, the relatively large amounts of RNA extracted by all procedures utilizing NaOH was not unexpected. For example, with 0.07 N NaOH alone for 30, 60, 90 and 180 set, the percent of RNA extracted was 29.7, 34.1, 41.3 and 52.3 respectively. Many of the salt treatments, including those containing SSC, also removed considerable amounts of RNA. Distilled water, boiling water, the pH 9.0 phosphate, and the 0.2 N HCl removed relatively little RNA.

Exposure to 0.07 N NaOH, for various times, removed from 19.2 to 29.9 % of the metaphase protein, while all other treatments removed less than 8 %.

The DNA and protein extracted by the trypsin techniques are shown in table 3. Even though this technique presumably works

Table 4. Extraction of DNA from heterochromatin, intermediate fraction, and euchromatin (percent 3H-DNA extracted)

0.07 N NaOH

D.W. 1’ 3’ 5’

Chinese hamster, 1st expt Heterochromatin 2.2 Intermediate fraction 6.5 Euchromatin 5.3

Chinese hamster, 2nd expt Heterochromatin 1.55 Intermediate fraction 4.8 Euchromatin 5.6

Mouse Heterochromatin 0.0 Intermediate fraction 0.0 Euchromatin 0.0

9.5 17.2 16.3 23.2 26.3 24.2 32.1 31.1 34.3

9.2 16.7 16.5 20.9 25.2 30.3 26.5 23.4 41.1

0.0 4.8 0.0 0.0 13.6 11.3

21.8 34.4 38.5

Mechanism of’ C- and C-handing 415

Fig. 1. Giemsa staining of mouse chromosomes. (a, b) No treatment; (c) 0.2 N HCI 30 min, rinsed, air-dried, then treated with ribonuclease; (d) 0.07 N NaOH for 5 min; (e) complete C-banding technique with HCI, RNase, NaOH and 6 x SSC at 65°C overnight; (f) same as (e) but without the NaOH step; (8) distilled water, 100°C for 10 min; (h) 10% formaldehyde in SSC, 100°C for 10 min; (i) 10% formaldehyde in 0.05 M Tris, pH 7, 98°C for 10 min; (j) same as in (i) for 2 h. All figures at ,’ 2 000.

by removing certain proteins, there was sur- suggests that the results with this technique prisingly little protein extracted. We obtained may arise from protein alteration rather good banding with this solution of trypsin than protein extraction. after treating the cells for only 10 set, and yet this removed only 0.5 % of the labeyed pro- Radioisotope analysis of heterochromatin and

tein. Increasing the times of treatment to euchromatin extracts

30, 60 and 120 set resulted in the removal of The extraction of rather large amounts of 2.6, 8.4 and 13.206 protein respectively. This DNA with 0.07 N NaOH suggested that the

Exptl Cc,ll Rrs 77 c 1973)


Fig. 2. Feulgen staining of mouse chromosomes. (a) No treatment; (b) HCI-RNase; (c) 0.07 N NaOH for 2 min; (d) 0.07 N NaOH for 5 min; (e) complete C-band technique; (f) same as (e) but without NaOH step. All figures at x 2 000.

C-banding, in part, may be due to the differential extraction of euchromatic DNA and resistance to extraction by hetero- chromatic DNA. To test this, Chinese hamster and mouse L cells were labeled overnight with 3H-thymidine and the heterochromatin, intermediate and euchromatin fractions isolated, fixed, and air dried on slides (see Methods). These were then extracted with distilled water or with 0.07 N NaOH for 1, 3 and 5 min. The results are shown in table 4. The distilled water removed from 0.0 to 6.5 % of the label. The NaOH consistently extracted the most DNA from the euchromatin fraction, less from the intermediate fraction, and the least from the heterochromatin fraction. For example, in the second Chinese hamster experiment, 0.07 N NaOH for 5 min removed 16.5 % of


the DNA from the heterochromatin, 30.3 “/b from the intermediate fraction, and 41.1% from the euchromatin even though the number of counts on the slide was approximately the same for all three fractions. Thus, in the interphase cells, the DNA of the heterochromatin was much more resistant to extraction by alkali than that of the euchromatin. Since Chinese hamster heterochromatin is not unusually enriched in repetitious DNA [16, 171 this would appear to be a characteristic of the condensed heterochromatin per se rather than a characteristic of chromatin containing repetitious DNA.

Giemsa staining

If the C-banding techniques are the result of preferentially extracting euchromatic DNA with NaOH, then the centromeric hetero-

Mechanism of C- and G-handing 377

Table 5. Microdensitometry of the centromeric heterochromatin and the arms of mouse chromosomes treated and stained w,ith Feulgen and Gitmsa

Treatment

Centromeric heterochromatin Arms

Areaa % DNA lostb Areaa

Area cent. hct __.

‘lo DNA lostb Area arms

Feulgen stain Control, no treatment HCl-RNase 0.07 N NaOH, 1 min 0.07 N NaOH, 2 min 0.07 N NaOH, 5 min

Giemsa stain Control, no treatment 0.07 N NaOH, 2 min, 6 -:

SSC 65°C overnight

2135 4200 - 0.51 2334 0.0 3947 6.0 0.59 1414 33.7 1994 52.5 1.38 1751 17.9 2166 51.9 1.18 1073 49.1 1 275 69.6 1.53

232 849 0.27

769 443 1.74 -_-..

a Average of 5 different chromosomes, in nut?. ” Area control-Area treatment/Area control x 100.

chromatin would be expected to become visible after alkali treatment alone. To investigate this a series of slides were treated and stained with Giemsa (fig. 1). It was interesting that in chromosomes which were not treated the centromeric heterochromatin could be clearly seen as regions which stained lighter than the chromosome arms (fig. 1 a, b). The same result was obtained after treatment with HCl and RNase (fig. 1 c). A marker chromosome with numerous secondary constrictions was consistently seen (arrow). When the chromosomes were treated with HCI, RNase and 0.07 N NaOH for 1 to 5 min without the SSC overnight, the centro- merit heterochromatin was now darkly stained and the arms were pale (fig. Id). When the complete procedure was used, including 6 x SSC at 65°C overnight, the centromeric heterochromatin was darkly stained, the arms were light, and the marker chromosome showed heterochromatin at all the secondary constrictions (fig. le). The same results were obtained when they were treated with 6 x SSC, 65°C overnight without the NaOH step (fig. If). This is consistent

with the radioisotope findings that the 0.07 N NaOH alone and the 6 x SSC at 65°C overnight alone, both removed considerable amounts of DNA.

When the chromosomes were exposed to boiling water for 10 min the chromosome arms became diffuse while the centromeric heterochromatin remained condensed (fig. lg). When exposed to boiling 1090 formaldehyde in SSC for 10 min the arms were better preserved but the centromeres were still slightly more densely stained (fig. I h). Exposure to boiling 10% formaldehyde in 0.05 M Tris, pH 7.0, for 10 min resulted in paler chromosomes (fig. 1 i), and after 2 h the chromosomes were only ghosts (fig. 1 j).

Feulgen staining

As a further check of the effect of various treatments on the differential extraction of DNA from euchromatin and heterochromatin, the slides were stained with Feulgen and the amount of DNA remaining at the area of the centromeric heterochromatin and at the arms was determined by microdensitometry (see Methods). The results are shown



Giemsa - Trypa,,

B Feuigen - No Treatment

Feulgen - Trypsin

Fin. 3. Mouse chromosomes treated with 0.005 % trypsin for 10 set and stained with Feulgen or Giemsa. (A) Trynsin treated. stained with Giemsa: (B) no treatment, stained with Feulgen; (C) trypsin treated, stained with Feulgen. The insets are photographs of the original chromosomes. The line figures represent microdensitometry tracings of the photographs.

in fig. 2 and table 5. In the untreated slides there was uniform and dark staining all along the arms and the centromere regions (fig. 2a). By densitometry there was more DNA in the arms than in the centromere, the ratio of the two being 0.5 I. After treatment with HCl and RNase the chromosomes still stained uniformly although in some there was a slight suggestion of relatively greater staining at the centromere (fig. 2b). By densitometry there was no loss of DNA from the centromere regions and minor loss from the arms (6.0 %), with the ratio of DNA in the centromere region to that in the arms being 0.59. When the chromosomes were treated with 0.07 N NaOH for 1 to 5 mitt, the centromere regions were moderately well stained while the arms were pale (fig. 2c-e). By densitometry there was a 17.9 to 49.7 74 loss of DNA from the centromere regions, and 51.9 to 69.6% loss from the arms, with ratios of centromeric area to arm area of 1.18 to 1.53. Similar results were obtained if the height of the curve rather than the area was used. Treatment with ‘6x SSC at 65°C for 24 h alone -also produced chromosomes. that were stained more intensely at the


centromeres and the arms showed a ghost- like outline (fig. 2f). When the slides were treated with both NaOH and SSC overnight, the Feulgen staining was similar to that in fig. 2e but was too light for adequate densitometry studies.

Similar tracings were made on Giemsa- stained slides to determine if the change in intensity of the centromere regions was disproportionately greater with this stain (table 5). The ratio of centromere area to arms area on untreated Giemsa stained slides was 0.27, while the ratio for C-banded chromosomes was 1.74. This indicates that in the untreated chromosomes the centromere region stained disproportionately less than the amount of DNA present, and in the C-banded chromosomes it stained disproportionately greater than the amount of DNA present. Similar results were obtained with G-banded chromosomes.

Giemsa and Feulgen staining of G-banded chromosom.es

The above results indicate that the removal of significant amounts of non-centromeric DNA is an integral part of C-banding as done by the Arrighi-Hsu technique. This raises the question, Is interband DNA removed in the G-band techniques? To investigate this, mouse chromosomes were treated with 0.005 % trypsin for 10 set and stained with Giemsa and Feulgen. By Giemsa staining, striking bands were produced (fig. 3A) but no bands were produced by Feulgen staining of either the untreated (fig. 3B) or trypsin treated (fig. 3C) chromosomes. This is consistent with the radioisotope findings which indicate removal of chromosomal DNA is not necessary for G-banding, and indicates that’variations in the amount of DNA along the chromosomes is not the explanation for G-banding. Also, the observation that excellent staining of the C-band regions is produced


Table 6. A0 and Giemsa staining of mouse chromosomes following various treatments

Treatment

Acridine orange

Centro- mere Arms

Ciemsa Interpretations

Centro- C-band Arm mere Arms Fig. DNA DNA C-banding

Controls

1 No treatment 2 HR

Green Green

Green Green

Denaturing conditions

3 NaOH 2 min, +4% HCHO 2 min Red Red

4 HR, NaOH, 2 min, 10”: HCHO 60 min Green Orange

5 HR NaOH 5 min, IO”;, HCHO 60 min Green Orange

6. NaOH in 4% HCHO 2 min Orangwed Green

7 0.2 N NaOH in 4 ?o HCHO 2 min Orange-red Orange

8 HR. D.W. 100°C 10 min, 10 9: HCHO 60 min Green Red

9 l/l0 ssc tf 100°C 2 min, 4 “,b HCHO 1 min Red

10 HR, 10% HCHO 100°C 10 min Red

11 HR 10% HCHO 10&C 60 min Ghost

12 HR, 10% HCHO 100°C 120 min Ghost

Red

Red

Ghost

Ghost

Pale red +-t++ -I-

Denaturing and renaturing conditions

13 HR, NaOH 2 min, 6 x SSC 65°C ON, 10% HCHO 60 min Green

14 HR, NaOH 2 min, 6 x SSC 65°C ON, 4% HCHO fix Red

15 6 x SSC 65°C ON, -4% HCHO 1 min Red

16 2 x SSC 65°C ON.

Pale red

Pale red

’ -t 4 % HCHO I min Red

17 l/l0 SSC+f 100°C 2 min, 2 x SSC 65 0 20sec, -4” 10 HCHO 1 min Green

18 l/l0 SSC+f 100°C 2 min, 2 x SSC 65°C 1 min, --f 4 % HCHO 1 min Green

31 ~- 721817

Pale red

Red

Orange

la h -+1-i lc’

Native Native Reversed Native Native Reversed

like 1 II Denat.

like 1 d Renat.

Id Renat.

like 1 h P. denat.

like 1 /z Denat.

Renat.

like I h Denat.

Ii Denat.

like lj --

lj -

Denat. :

P. renat. -j :

P. renat. -t

Native. 0

P. denat. -t

Denat. -:

Denat. 0

Denat. 0

0

- 0

le Renat. Denat. -.- -! -c ,

like I e Denat. Denat. I- -1 ‘- -L-

If Denat. Denat. ./ I- ,.

like lf Denat. Denat. L- f

like lh Renat. Denat. +

like 1 h Renat. P. denat. +



Table 6 (continued)

Treatment

Acridine orange Giemsa Interpretations

Centro- Centro- C-band Arm mere Arms mere Arms Fig. DNA DNA C-banding

19 l/10 SSC+f 100” 2 min, 2 x SSC 65°C 5 min, -4% HCHO 1 min Green Green ++ ++ like lh Renat. Renat. t

20. NaOH 2 min, SGren buff. 60°C 0 N, -4% HCO 1 min Orange Green +++ +-1- - P. renat. Renat. i-+-l

21 0.07 N NaOH 2 min, wash. Green Green- +++ +c like Id Renat. Denat.- + +

orange- renat. red

Trypsin treatment 22 0.005% trypsin,

20 set Green Green +++-I- +?- 4a Native Native -!--k-t+

Abbreviations: P, partly. Denat., Denatured. Renat., renatured. HR, 0.2 N HCI 30 min, RNase 60 min. HCHO, formaldehyde. 10% HCHO 60 min, slide transferred to 10% formaldehyde for 60 min. +4% HCHO 1 min, 40% formaldehyde added to bring to 4% formaldehyde for 1 min. ON, overnight. f , 10% formamide.

with this technique (fig. 3A, arrows), indicates that DNA does not have to be preferentially extracted to produce C-banding and that the Giemsa staining, even in this region, is out of proportion to the amount of DNA present. Merrick & Lubs [43] have observed, and we have confirmed (fig. ll), that prolonged exposure to trypsin tends to destroy the G-bands while the C-bands persist. This suggests that the G-bands are more delicate than the C-bands, and if one wishes to have only C-banding it is necessary to destroy the G-bands. This can be done either by exposure of the chromosomes to NaOH and hot SSC overnight to preferentially remove the DNA of the arms, or by prolonged exposure to trypsin to preferentially disrupt the chromatin of the arms.

Acridine orange (AO) staining

The use of several techniques that pur- portedly denature and renature DNA in situ


raises several important questions. They include the following. (1) Is chromosomal DNA double stranded after fixation or does the use of glacial acetic acid and air drying itself denature the DNA? (2) Does alkali or heat denature all the DNA? (3) Do the renaturing conditions actually result in the preferential renaturation of C-band DNA leaving the DNA in the arms denatured? (4) Even if preferential renaturation does occur is this the cause of the differential staining or is it merely a coincidental associated phenomenon? Put in a different way, does chromatin containing double-stranded DNA stain more intensely than chromatin containing single stranded DNA? The use of AO, which stains double-stranded DNA green and single-stranded DNA red, can be used to answer these questions. Table 6 shows the results of the A0 staining of chromosomes after various treatments and com- pares them with the results of Giemsa stain-


ing of duplicate slides. The table also refers the reader to the appropriate figures illustrat- ing the appearance of these chromosomes.

Before getting into this data it should be pointed out that fixation of the chromosomes with formaldehyde, after they were treated, was done to prevent renaturation of DNA so that its actual status at the time of treatment could be assessed. It was found that the manner in which this fixation was carried out was very important. Initially we removed the slides from the treating solution and placed them in 10 % formaldehyde buffered to pH 7.0 in SSC, phosphate buffer, or 0.05 M Tris, and left them at room temperature for 60 min. In other experiments we added l/l0 vol of 40% formaldehyde directly to the treatment solution, at the same temperature as the treatment, and left it for 1 min [53]. The results were frequently different with the two procedures. When exposed to denaturing conditions the first frequently allowed C-band DNA to renature while this was prevented by the second procedure.

Does fixation and air drying denature chromosomal DNA? When the chromosomes were only fixed and air dried they stained uniformly green (no. 1, table 6). The same was true following treatment with 0.2 N HCl for 30 min and exposure to RNase at 37°C for 1 h (no. 2). By Giemsa staining the centromeric DNA was less intensely stained than the arms, and by A0 staining it was occasionally orange in color. Thus, fixation or treatment with HCl and RNase either does not denature the DNA or may slightly denature some of the centromeric DNA. This must be qualified by the observation that flame drying or sitting on the shelf for prolonged periods of time usually resulted in the red staining of untreated chromosomes. We have consistently fixed our chromosomes

in ice-cold fixative, a procedure which may help prevent denaturation. de la Chapelle et al. [25] came to a similar conclusion that air drying did not denature the DNA, whereas flame drying did.

Does NaOH and 100” heat denature centro- merit and arm DNA? When the chromosomes were exposed to 0.07 N NaOH for 2 min (no. 3), or to l/lOth SSC and 10% formamide at 100°C for 2 min (no. 9) [53] and then fixed by adding formaldehyde directly to the solutions, both the centromeres and the arms stained red. The same result was obtained when the chromosomes were placed in 10 “6 formaldehyde and heated at 100°C for 10 min (no. 10). Thus, NaOH and 100°C heat denatures both the centro- merit and the arm DNA. However, some interesting variations were observed. For example, when the chromosomes were exposed to NaOH for 2 or 5 min and then transferred to the formaldehyde (nos. 4, 5) the centromeres were now green and the arms orange, indicating some renaturation was occurring at the centromere before the DNA could be fixed. If the slides were placed in 0.07 N NaOH that was already brought to 4 ‘I,, with formaldehyde, and left for 2 min, the centromeres were now orange or red and the arms green (no. 6) suggesting the C-band DNA was more susceptible to alkali denaturation than the DNA in the arms. This could be due to its greater AT content [38]. When exposed to 0.2 N NaOH in 4 ~0 formaldehyde, for 2 min (no. 7), the centromeres were again more denatured (orange to red) than the arms (orange). If exposed to boiling distilled water for 10 min and then transferred to IO SC formaldehyde at room temperature (no. 8), the DNA of the centromeres was renatured while the DNA of the arms was still denatured. When exposed to IO”;, formaldehyde (in 0.04 M Tris, pH 7.0) at 100°C for 1 or



Native DNA

w Slide /,,,,A

911; ,

s; E Ki z (c gm /cm3 - - -- -

Fig. 4. Analytical ultracentrifugation of native mouse DNA, DNA left on the slide after extraction of fixed mouse metaphase chromosomes with 0.07 N NaOH for 3 min, and DNA extracted from the slides with NaOII.

2 h (nos. 11, 12), the chromosomes were disrupted and ghost-like.

Do renaturing conditions result in the preferential renaturation of centromeric DNA? Some of the above results have already provided an answer to this in that several treatments resulted in green centromeres with red arms. When the complete C-band procedure was carried out and the slides then transferred to the formaldehyde (no. 13) the centromeres were green and the arms red. It would seem logical to assume that this was because the 6 x SSC at 65°C overnight was acting as a renaturing condition. Closer in- spection, however, makes another interpreta- tion more likely. Thus, if instead of trans- ferring the slide to formaldehyde, the for-


maldehyde is added directly to the 6 x SSC and left for 1 min at 65°C (no. 14) then both the arms and the centromeres are red, and if 2 x SSC or 6 x SSC at 65°C overnight is used alone, without the NaOH step (nos. 15, 16), then transferred to formaldehyde, the centromeres are orange and the arms pale red, indicating this SSC treatment was actually denaturing rather than renaturing the DNA. Stockert & Lisanti [53] noted that when chromosomes were exposed to l/l0 SSC in 10 % formamide for 2 min and formaldehyde then added, all the DNA was red. When denatured and then renatured in 2 x SSC at 65°C for 20 set, the centromeres were green while the arms were red. When renatured for 3 to 5 min, the centromeres and the arms were green. We have repeated these experiments (nos. 9, 17-19) with the same results.

All of these findings indicate that either NaOH or heat at 100°C will denature all the DNA, but after these conditions are removed the centromeric DNA renatures almost instantly and the DNA of the arms very quickly follows. Thus only the briefest of time is required for the preferential renaturation of centromeric DNA. Much longer treatment and the DNA in the arms also renatures. The prolonged exposure to 2 or 6 xSSC overnight at 65°C in the usual C-banding technique is probably resulting in the re-denaturation of all the DNA, but when it is removed from this solution the centromeric DNA once again renatures more rapidly than the DNA in the arms.

Is differential renaturation of centromeric DNA responsible for C-banding? If it is, then chromatin containing denatured DNA should stain poorly, chromatin containing renatured DNA should stain intensely, and those conditions which result in green centromeres and red arms, by A0 staining,

Mechanism of C- avid G-handing 483

Figs 5-11. Whole mount electronmicroscopy of mouse metaphase chromosomes, untreated and treated with 0.005 % trypsin. Fig. 5. Untreated. Much of the chromatin is still adherent to the nuclear membrane. There is a suggestion of some coiling patterns which are symmetrically represented in both sister chromatids. x 11 000. Figs 6-11. Water spread, picked up on grids, treated with 0.005 % trypsin in Ca2+- and Mg2+-free balanced salt solution for 10 set, then dehydrated in ethanol washes. The centromeric heterochromatin is much more resistant to dispersion than the chromatin in the arms. In fig. 8 there is a suggestion of differential condensation in the arms (nrrows). In fig. 12 there has been some clumping of the non-centromeric chromatin in the distal portion of the arm (arrow). Fig. 7, x 5 0OO;fig. 8, x 13 SOO;&. 9, x 17 3OO;fig. IO, 2: 18OOO;fig. II. x 18000; fig. 12, x 8 100.

Exptl Ccl1 Res 77 (1973)


Figs 12-14. Whole mount electronmicroscopy of mouse metaphase chromosomes, untreated and treated with 0.07 N NaOH. Fig. 12. Untreated. The degree of coiling of the centromeric heterochromatin is similar to that of the arms. x 12 500. Fig. 13. Treatment with NaOH for 2 sec. The chromatin in the arms is dispersed while that in the centromere region remains intact. x 13 000. Fig. 14. Treatment with NaOH for 10 sec. The chromatin in the arms is mostly destroyed, while that in the centromere region is more resistant to destruction. x 10 500.

should show the best C-banding. Although at 65°C had denatured DNA in both their some results like nos. 4, 5 and 13 would centromeres and arms (no. 14). Finally, the seem to support these assumptions, other experiments indicate they clearly do not hold. Thus chromatin containing DNA that is denatured can stain either intensely (nos. 3, 9, 14, 15) or poorly (nos. 8, 10, 13-17), and chromatin containing DNA that is native or renatured can stain either intensely (nos. 1, 2, 4, 5, 8, 13, 18, 20-22) or poorly (nos. 1, 2, 20, 22). The best example of preferential renaturation of the centromeric DNA with denaturation of the DNA in the arms was

chromosomes treated with trypsin showed beautiful C-banding (no. 22) (plus G-banding) and yet all the DNA was still in its native DNA state. These experiments seem to indicate that even though denaturation and preferential renaturation of centro- merit DNA is occurring under the conditions of C-banding, it actually has little to do with the mechanism of the differential staining.

Differential acridine-orange staining in the observed in no. 17, yet these chromosomes chromosome arms. When the mouse chromo- showed poor C-banding, and intensely C- somes were placed in 0.07 N NaOH in 4 ‘x, banded chromosomes which were treated formaldehyde for 2 min, then washed in with formaldehyde while still in the 6 x SSC ethanol and stained with AO, some chromo-



somes showed striking differential staining in the arms, with some parts yellow-green and others orange-red. Similar studies with human chromosomes indicate that the red bands correlate well with the AT-rich G-banded regions [15a].

DNA ultracentrifugation

To further determine whether non-satellite DNA was preferentially extracted by the alkali, slides containing mouse metaphase cells were exposed to 0.07 N NaOH for 3 min. The slides and the extract were then neutralized to pH 7, the DNA extracted and

Fig. 15. Primary and secondary constrictions by normal staining, C- banding, and whole mount electronmicroscopy. LM, light microscopy of the untreated; Giemsa stained marker chromosome showing light staining of the centromeric heterochromatin and of the two secondary constrictions in the short arm and the four in the long arm. C-banding light microscopy of the same chromosome after C-banding. The primary and secondary constrictions are now intensely stained. EM, whole mount electronmicroscopy of the marker chromosome treated for 10 set with 0.005 % trypsin. It can be seen that the centromere and the secondary constrictions both contain the same type of heterochromatin that is resistant to decondensation by brief trypsin treatment.

examined by analytical ultracentrifugation (see Methods). Native mouse DNA extracted from unfixed liver nuclei contained 10% satellite DNA with a buoyant density of 1.690 g/cm3 and 90 % main band with a density of 1.701 g/cm3 (fig. 4). DNA extracted from mouse liver nuclei that had been fixed in 1 : 3 glacial acetic acid/methanol, and air dried on slides, showed the same charac- teristics as native DNA indicating the fixation itself had not altered the DNA and the procedure for extracting of DNA from fixed materials was not selectively removing satellite or non-satellite DNA. The DNA remaining



loo/ 10-q

, , /,,,, I/ , , , /,I,, / , / ,,,,,, , , ,,////, , , / ,,, / r , , ,,,,,, , , I,(,/, 10-s 10-p lo-’ I00 IO’ 102 id3 -724

Fig. 16. Abscissa: Cot (mole set/l); ordinate: % reassociation. Cot curve for mouse DNA in situ and in vitro. The in situ points represent renaturation of centromeric

DNA at 1 set and renaturation of all DNA at 3 to 5 min. (See text for calculations.) The in vitro results are based on data from Britten & Kohne [7] and Laird [40].

on the slides was enriched in satellite (40 “/b), which banded at a buoyant density of 1.694 g/cm3. Since this is the density of renatured satellite DNA [17], the NaOH presumably denatured the satellite and it then immediately renatured upon neutralization in a manner similar to that occurring in the A0 studies. The main band DNA had a buoyant density of 1.704 g/cm3. This is considerably less than the density of denatured main band DNA (1.719 g/cm3 [17]), but higher than native DNA. Thus, this main band DNA that was

and what main band DNA there was, banded over a broad range with a mean density of 1.721 g/cm3. We suspect that most of the extracted main band DNA did not renature and its single strandedness made it susceptible to being broken down into pieces that were too small to come to equilibrium in the cesium chloride and were thus lost. This would result in a relative enrichment of the satellite, even in the extract, despite the fact that NaOH preferentially extracts non-satellite DNA.

not extracted also underwent rapid renaturation following neutralization of the NaOH. Whole mount electron microscopy

This may be due to the fact that it is repeti- Brief treatment of the unfixed chromosomes tious main band DNA, or, being in situ may with dilute trypsin had the effect of producing have allowed the non-repetitious DNA to C-banding at the electron microscope level. rapidly renature in the same manner as seen Thus, the centromeric constitutive hetero- in the A0 experiments. Thus, the examina- chromatin in the mouse remained tightly con- tion of the DNA remaining on the slides tracted while the chromatin in the arms be- after NaOH extraction tends to confirm the came readily dispersed (figs 6-11). In un- radioisotope, Feulgen and electronmicro- treated specimens the degree of condensation scopy (see below) findings that non-satellite usually remained relatively uniform through- DNA is preferentially extracted. This inter- out the length of the chromosome (figs 5, 12) pretation, however, is complicated some- although in some cases it was possible to what by what was observed in the extract. The see differential condensation of the centro- extracted DNA (fig. 4) was even more merit chromatin even in untreated prepara- enriched in renatured satellite DNA (62%) tions [20]. The untreated chromosome in



Fig. 17. Variability in trypsin banding. Fixed mouse chromosomes were treated with 0.005 % trypsin for 10 sec. Although most chromosomes show good G-banding some were overtreated and the G-bands were partially (a) or completely (b) disrupted leaving only the centromeric C-bands. x 2 000.

fig. 5 still shows many sites of attachment to the nuclear membrane [18-211 and it is of interest that the pattern of condensation of chromatin onto the membrane is symmetrical for the two chromatids. Some suggestion of differential condensation of chromatin in the arms, like that in fig. 7 (arrows) was seen in a number of specimens. Whether these regions correspond to G-banding sites was not determined. In some chromosomes the trypsin produced dense banding in the arms as well as at the centromeres, fig. 11 (arrow). Exposure of the chromosome to EDTA also resulted in differential dispersion of the chromatin of the arms compared to that of the centromere.

The relative resistance of the centromeric heterochromatin to destruction by NaOH could also be demonstrated by electron microscopy. Thus, in fig. 12 the chromosome is untreated, in fig. 13 it is treated with 0.07 N NaOH for 2 set, and in fig. 14 it is treated for 10 sec. Marked differential dispersion is apparent at 2 set and at 10 set almost all of the chromatin is destroyed except for the two masses of tightly condensed centromeric heterochromatin.

The marker chromosome in the mouse cells was of particular interest. By light microscopy the untreated Giemsa stained chromosome shows pale staining primary and secondary constrictions (fig. 15). C-banding

32- 721817

indicates the presence of heterochromatin at each of the secondary constrictions. When treated briefly with dilute trypsin, electron microscopy shows the tightly condensesd bands of heterochromatin that are resistant to dispersion (fig. 15). This indicates that the tight condensation of this chromatin is not simply the result of its being compacted around the kinetochore.

DISCUSSION

Mechanism of C-banding

It is unlikely that RNA or histones are playing a role in C-banding since the treatment of the chromosomes with RNase or acid does not affect the results. There are 5 remaining major possibilities. C-banding could be due to: (1) A higher concentration of DNA in the C-band regions in untreated chromosomes; (2) the presence of DNA with a different base composition; (3) the presence of repetitious DNA wih the staining being due to differential renaturation; (4) The selective disruption of non-C-band DNA by alkali or trypsin; (5) DNA-non histone protein interactions.

(1) Higher concentration of DNA in C-band region. Even though constitutive heterochromatin seems almost synonymous with tightly condensed chromatin, this refers to



its status in the interphase cell. The situation is possibly different during mitosis. The densitometry tracings of mouse chromosomes stained with Feulgen suggested that, if anything, the concentration of DNA in the centromeric heterochromatin was actually less than the concentration in the arms (table 5). This is consistent with the observation that following the treatment of chromosomes with cold [IO, 24, 341 or colchicine [ 131, the lesser degree of condensation of the heterochromatin becomes even more apparent. This implies that the major feature of C-band heterochromatin is that its degree of condensation is fixed and is thus relatively more condensed during interphase and relatively less condensed during metaphase [13]. During mitosis the centromeric heterochromatin also stains less intensely with Giemsa (fig. la, b). Thus, this first possi- bility would seem to be an unlikely explanation of C-banding.

(2) Base composition. This can be effectively ruled out by the fact that the satellite DNA localized to constitutive heterochromatin may be AT-rich, GC-rich or neutral [17] and still produce C-banding.

(3) Preferential renaturation of repetitious DNA. The results with the combination A0 and Giemsa staining of chromosomes treated in various ways (table 6) indicated the following: (a) the fixation of the chromosomes or their treatment with 0.02 N HCl and RNase does not denature the DNA; (b) treatment with 0.07 N NaOH for 2 min, or 100°C heat, denatures the DNA of both the centromeres and the arms; (c) once the denaturing conditions are removed the C- band DNA renatures almost instantly, and the DNA of the arms soon follows; (d) treatment with 2 or 6 x SSC at 65°C overnight actually re-denatures the DNA; (e) depend-


ing on the conditions, chromatin containing single stranded DNA can stain with Giemsa either intensely or poorly, and chromatin containing native or renatured DNA can stain intensely or poorly; (f) intense C- banding can be seen in chromosomes in which (i) both the centromeric and arm DNA is double-stranded; (ii) in which it is single- stranded in both; (iii) in which the centromeric DNA is double-stranded and the arm DNA is single-stranded; (iv) in which the centro- merit DNA is single-stranded and the arm DNA is double-stranded.

Thus, although selective renaturation of C-band DNA can be obtained under a number of conditions, this seems to be unrelated to the ability of the chromosomes to show C- banding. This is consistent with previous studies in Chinese hamster cells which suggest that C-banding may be seen in heterochromatin that is not excessively enriched in repetitious DNA [16, 171 and the observation that post-fixation of mouse chromosomes with 10 1;, formaldehyde in 0.4 :’ SSC,

followed by careful washing, does not affect the ability of the DNA to denature and renature, but does prevent the differential staining of the C-band regions [53].

(4) Selective disruption of non-C-band DNA. The principal feature of the C-banding technique is the exposure of fixed chromosomes to alkali for 1 to 2 min, followed by incubation in 2-6 x SSC at 65°C overnight. Alkali alone and the SSC treatment alone also produce C-banding (fig. lrl, f), but the results are better when both are used. Bio- chemical and radioisotope analysis of extracts of fixed chromosomes indicate that both the alkali and the SSC remove significant amounts of DNA from the chromosomes. Radioisotope analysis of extracts of isolated and fixed euchromatin and heterochromatin, densitometry studies of Feulgen stained chro-


mosomes, analytical ultracentrifugation of extracted and unextracted DNA, and electron microscopy of alkali-treated whole mount preparations, all indicate that DNA is selectively removed from the chromosome arms and selectively retained by the centromeric heterochromatin. Although this could be the entire explanation of C-banding, several observations suggest it is not. For example, relatively selective C-banding can also be obtained by treating chromosomes with trypsin for longer than necessary to produce G-banding [43] (fig. ll), and yet the radioisotope experiments indicate this is removing relatively little DNA. This suggests that the selective C-banding is produced whenever the more delicate G-bands in the arms are disrupted either by removal of their DNA or by disruption of their organization. This is probably the explanation for the observation that following the C-band procedure, chromosomes which are stained with quinacrine show no evidence of Q-banding in the arms but do stain intensely at the centromere regions [25, 321.

(5) DNA-protein interactions. The comparative densitometry of chromosomes stained with Feulgen or Giemsa (table 4) also indicated that the selective removal of DNA in the arms does not completely explain C-banding. The Giemsa stains the C-band region of untreated chromosomes disproportionately lighter, and stains the same regions of treated chromosomes disproportionately greater than the amount of DNA present. The most likely explanation for this behaviour is that certain DNA-protein interactions are playing an important role in C-banding, as well as G-banding.

little effect1 [13], RNA and histones appar- ently play no role.

(1) Higher concentrations of DNA in G-band regions. Although whole mount electronmicroscopy did provide some evidence of differential coiling of chromatin into bands in the chromosome arms (fig. 7) the comparative Giemsa and Feulgen staining (fig. 3) clearly indicated that this alone cannot explain the G-banding.

(2) Base composition. Most of the studies of the relationship between base composition and DNA banding have been done in rela- tion to quinacrine or Q-banding. However, since Q- and G-banding are identical, with only a few exceptions [31], these studies are pertinent. Some suggest that Q-banding is related to GC content [I]. Weisblum & Haseth [56] however, have shown that AT containing polymers enhance quinacrine fluorescence while CC containing polymers quench it; and Ellison & Barr [29] have demonstrated that the intensely staining regions in Samoaia leonensis, a drosophilid fly, are AT-rich.

In addition, through the use of fluorescent- labeled antibodies to guanosine, cytidine and adenosine, Schreck et al. [51a] have shown that AT content plays a role in Q-banding. This conforms with the finding that Q-banding in mammals correlates well with late replicating regions [33], and late replicating DNA tends to be AT-rich [8, 14, 551. It correlates poorly with the fact that the centromeric heterochromatin in the mouse, containing AT-rich satellite, stains poorly with quinacrine. One thing seems clear, if base composition is important as a primary

Mechanism of G-banding * Since treatment of chromosomes with 0.2 N HCI

The same questions can be asked of G- for 30 min causes some decrease in the intensity of

banding. Again, because RNase and acid have G-banding some zcid-soluble proteins may be in- valved.



factor in G-banding, it is not a linear relationship since the differential staining obtained with Giemsa is far greater than that allowed by the relatively narrow range of base composition in mammalian DNA.

(3) Preferential renaturation of repetitious DNA. It has frequently been suggested that G-banding may also be picking up highly or moderately repetitious DNA [51, 54, 571. There are a number of reasons, however, to suspect this is not the case. (a) A much stronger case can be made for repetitious DNA and preferential renaturation being involved in C-banding and yet the results with comparative A0 and Giemsa suggest it is not the primary factor; (b) A0 staining provided no evidence for preferential denaturation or renaturation in the arms of G- banded chromosomes; (c) radioisotope and nuclear subfraction studies suggest that late replicating, intercalary heterochromatin [ 13, 151 in the chromosome arms is probably composed of DNA which is not unusually enriched in repetitious DNA [16, 171; (d) very gentle methods, such as treatment with 0.005 % trypsin for 10 set, produced excellent G-banding without denaturing DNA.

(4) Selective disruption of non-G-band DNA. It is possible that the various treatments inherent in producing G-banding might either remove or disperse interband DNA. Several observations speak against this being any more than a modifying factor. (a) The radioisotope experiments indicate that very little DNA is extracted by the different G- banding techniques; (b) Feulgen stained G-banded chromosomes show no significant variations in the density of DNA along the arms; (c) Q-banding, which produces basi- cally the same results as G-banding, is ex- tremely mild and when chromosomes are post- stained with Giemsa no banding is present,

‘Exptl Cell Res 77 (1973)

indicating there is something about these regions, other than a differential density of chromatin, that is responsible for the banding.

(5) DNA-protein interactions. The many observations that proteolytic agents produce excellent G-banding [9, 27, 30, 521 provide evidence that DNA-protein interactions are an important factor in this type of banding (although the Ca2+ and Mg2+, free buffers by themselves, can also produce banding [25a]). Since the removal of histones does not affect the banding, the proteins involved are presumably non-histone proteins (or arginine-rich histones since they may not be completely removed by the fix [23]). While there is much evidence that there are quanti- tative differences in theamount of non-histone proteins present in euchromatin compared to heterochromatin [13, 151 there may also be qualitative differences in the form of S-S banding, phosphorylation, acetylation, or allosteric changes [22]. We have examined the non-histone proteins of isolated euchromatin and heterochromatin by SDS gel electrophoresis, and some differences were observed. These are described in a later paper [23a]. Other evidence that DNA-protein interactions are important in the banding reactions is discussed in more detail elsewhere [15].

In summary, the above evidence suggests that DNA-non histone protein interactions are the most important factor in both C- and G-banding. G-banding seems to be more ‘delicate’ and many relatively mild treatments are capable of altering these interactions in such a way that subsequent staining with Giemsa produces both C- and G-banding. For example, one feature that all of the G- banding techniques have in common is exposure to solutions capable of removing Ca2+ or Mg2+. This may affect the proteins in heterochromatin differently than those in


euchromatin. To obtain only C-banding it is necessary to treat the chromosome more harshly to disrupt or destroy the G-banded regions.

Relevance to in situ hybridization

The observation that the treatment of fixed chromosomal preparations with NaOH removes up to 80% of the DNA, that this DNA preferentially comes from the chromosome arms rather than the centromeric heterochromatin, and that both repetitious and non-repetitious DNA rapidly renature in situ, suggests the technique of in situ hybridization as usually performed is not functioning at its maximum capacity. Obviously, if most of the DNA of the arms is extracted there will be little chance for it to be involved in hybridization. The same holds if most of the DNA is renatured as soon as it is removed from denaturing conditions. From the present studies it would seem that a far better technique would be to denature in salt t formamide at 100°C for 2 min, then bring the solution to 60 or 65°C for hybridization.

Reassociation of DNA in situ versus in vitro

The observation that the C-band DNA is reassociated in 1 to 20 set while the DNA in the arms is reassociated in 3 to 5 min, allows a useful comparison of the Cot [7] of in situ versus in vitro DNA. The following calculations will use the human chromosome number 13 since the necessary data are avail- able and it is comparable in size to the acro- centric chromosomes of the mouse. Taking the diploid DNA content of mammalian cells as 6 x IO-l”, and knowing the relative size of the 13 chromosome, it is possible to calculate that its chromatid contains 1.07 x 10m13 g of DNA [5]. Using 325 D as the average molecular weight of a nucleotide in DNA, this represents 3.29 x IO-l6 moles of nucleotides. The approximate volume of

the chromatid can be calculated from light microscope measurements [46] as 5.8 r 0.9 x 0.9 pm or 4.70 pm3, or 4.7 x 10-l” I. This gives an approximate concentration of 7.0 v~ 1O-2 moles/l of DNA. Since some of the A0 studies suggest the satellite DNA can re- associate in as little as 1 set, this has occurred at a Cot of 7.0 x lo-‘. If the arm DNA is 9011, reassociated at 5 min this has occurred at a Cot of 2.1 x 10’. This crude Cot curve is compared to that obtained with DNA in vitro in fig. 16, and suggests that the reassociation of non-repetitious DNA in situ

may take place 100 times more rapidly than in vitro. These values, however, should not be accepted uncritically, since we do not really know what the effective concentration of DNA in the chromatid is. It could conceivably be 100 times greater in which case the renaturation rate would be the same as that in vitro. What this really means is that there are two potential explanations for the rapid in situ renaturation of non-repetitious IDNA. (1) Fixation in situ may hold the DNA half- helices close together so that they do not have to search for each other and can rapidly renature, or (2) the effectike concentration of DNA in situ is so great that non-repetitious DNA can renature within minutes. It should be apparent that in reality there is very little difference between these two explanations. Given the high effective concentration of DNA in situ they are both stating the same thing in different ways. It is also apparent that the manipulation of heat and alkali followed by fixation and A0 staining is probably incapable of meas- uring the true rate of renaturation of satellite DNA in situ. If we assume that satellite DNA is largely renatured by a Cot of 10e3 171, and the in situ concentration is somewhere between 7.0 x lO-‘L and 7.0 M/l, the actual rate of in situ renaturation of satellite DNA would be somewhere between 1 I IO and

E.uptl Cell Rex 77 (1973)


l/l 000th of a second. It would obviously be impossible to alter the in situ denaturing conditions fast enough to detect this. These observations are consistent with the experi- ence of others who have also found that DNA in situ renatures very rapidly [29, 44, 531.

Secondary constrictions

The early studies of Heitz [35] and McClin- tack [42] and later biochemical investigations of the bobbed locus in Drosophila [50] and the 0-nu mutant in Xenopus Zuevis [6], have clearly indicated that the nucleolus organizer, the secondary constriction associated with the nucleolus, and ribosomal cistrons are essentially synonymous entities. But what about the numerous other secondary constrictions that are not usually associated with the nucleolus? These may be seen in normal chromosome preparations, but they are enhanced by cold [lo] or chemical [12,37,47] treatments, and are frequently associated with C-banding heterochromatin and repetitious DNA (the secondary constriction on human chromosome number 9 being a good example [2, 391). The high frequency with which they occur, compared to the relative paucity of nucleolus organizers, suggests some secondary constrictions do not contain rDNA but simply represent non-centromeric sites of C-banding heterochromatin. For example, in the Mexican axolotl, Callan [IO] noted that under normal conditions there was a single secondary constriction associated with the nucleolus, while after cold treatment, a process which accentuates constitutive heterochromatin, one or more secondary constrictions were observed in almost every chromosome. Do these also contain ribosomal cistrons or are they a different type of secondary constriction?

The marker chromosome in the present studies is pertinent to this question. By

Giemsa staining of normally fixed chromo-


somes, the centromeric heterochromatin and the five secondary constrictions (figs 1 a, 6, 1.5) stain less intensely than the remaining portions of the chromosome. After C-banding these same regions that were light staining, now stain intensely and the remaining portions of the chromosome are lightly stained (figs 1 e, 15). By electron microscopy it is clearly seen that both the primary and the secondary constrictions contain C-band heterochromatin. This redistribution of centromeric heterochromatin into several regions along the arms probably took place by a series of inversions and translocations, or unequal sister strand crossovers. We feel this is consistent with the presence of two types of secondary constriction, one containing ribosomal RNA and forming the nucleolus organizer (which may or may not be associated with heterochromatin), and the other consisting purely of the centromeric type of heterochromatin.

This work was supported by NlH grant GM 15886 (D. E. C.). H. E. W. was supported by grants AM 13173, HD 05082, and Children’s Bureau Grant from Division of Genetics, University of Oregon Medical School.

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Received June 26, 1972 Revised version received August 14. 1973

Exptl Cdl Res 77 (1973)

Comings 1973 - The Mechanism of C- And G-Banding of Chromosomes

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Transcript of Comings 1973 - The Mechanism of C- And G-Banding of Chromosomes