Genetica 78: 57-62, 1989 © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands 57

Evolution of digestibility by Hind III: an analysis by light and electron microscopy

J. M6ndez l, A. M. Gonz~tlez 1, A. M. Insua ~ & V. J. Goyanes 2 1Departamento de Gendtica, Colegio Universitario de La Corufla, Universidad de Santiago, 15071 La Corufla, Spain 2Secci6n de Gen~tica, Hospital "T. Herrera" (INSALUD), 15006 La Corufia, Spain Reprint requests to be addressed to J. M~ndez

Received 8.2.1988 Accepted in revised form 16.9.1988

Abstract

Digestion of Chinese hamster metaphase chromosomes from the Don ceil line by Hind III restriction en- donuclease followed by Giemsa staining were analysed by light and electron microscopy. The evolution of di- gestibility was studied and four digestion stages were characterized by different levels of chromosome structure. Three different condensation stages were established according to morphological criteria of length, width and separation among chromatids. It was observed that there are statistically significant differences in the diges- tion progress at the three condensation stages previously defined.

Introduction

Restriction endonucleases have been used to test the differential organization of chromatin at different chromosomal regions on mammal metaphase preparations. These enzymes have been described as able to induce different banding patterns on fixed chromosomes (Sahasrabuddhe et al., 1978; Mez- zanotte et al., 1983; Miller et al., 1983). Such patterns include C-, g- and NOR banding.

Chromosomes treated with restriction en- donucleases show the same conventional chro- mosomal pattern obtained by other G-bands and NOR methods, but it has been established that mechanisms are different in each case (Comings, 1978; Sumner, 1982; Lica & Hamkalo, 1983). C- banding patterns obtained by endonuclease diges- tion are not coincident with those previously described (Bianchi et al., 1984).

The effect of digestion with restriction en- donucleases followed by Giemsa staining suggests that structural organization of chromatin plays a

primary role in determining enzymatic activity on different chromosomal DNA regions (Sahas- rabuddhe et al., 1978; Lica & Hamkalo, 1983; Mez- zanotte et al., 1983).

In this work, fixed Chinese hamster metaphase chromosomes have been treated by Hind III en- donuclease in an attempt to determine a relationship between chromosomal digestion and chromatin fiber packing.

Metaphase preparations have been analysed by light and electron microscopy. We have established three different condensation stages: low condensa- tion (LC), medium condensation (MC) and high condensation (HC), and evaluated the different digestion degree at each stage.

Material and methods

Chromosome preparation and digestion by restriction endonuclease

Metaphase chromosomes were obtained from Chi-

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nese hamster Don cell line cultures by standard tech- niques. Digestion was performed with Hind III (New England Biolabs). The enzymatic solution was pre- pared by dissolving 40 units of enzyme in 1 ml of in- cubation buffer (50 mM NaCI; 50 mM Tris HC1, pH 8.0; 10 mM MgCI 2 and 100 #g/ml bovine se- rum albumin).

Metaphase spreads, aged 1-4 days at 4 °C, were incubated in Coplin capsules containing this en- zymatic solution for 1-24 hours. The incubation was performed at 37 °C. After restriction treatment the preparations were washed in 100 mM Na 2 EDTA, pH 7.5, at room temperature and stained by 4°70 Giemsa solution in phosphate buffer salt, pH 6.88. Control chromosomes were treated as described but without the enzyme. Standard chro- mosomes were stained without post-fixation treat- ment.

Chromosomes were examined with a Nikon microscope (rood. Optiphot) equipped with a 100 × Planachromatic objective (NA 1.35) and were photo-recorded on Agfa Pan 100 film.

Metaphase transference and preparation for electron microscopy

Slides were immersed in a 1% solution of photo- graphic film in chloroform and allowed to dry verti- cally. Then, chromosomal metaphases were spread onto these film-coated slides, air-dried and incubat- ed by the same method employed by light microsco- py.

Under observation by light microscopy, metaphases on these film-coated slides were selected making circles of 2 mm diameter employing a mark- er objective (Nikon).

The film was cut employing a blade and floated in a waterbath. EM grids were placed on the circles, collected with a piece of filter paper and heat-dried at 60°C for 24 h, as reported by Goyanes & M6ndez (1982). Grids were examinated with a Zeiss 109 Turbo electron microscope operating at a voltage of 50 kW. Metaphases were photo-recorded on Agfa Ortho 25 film.

Statistical analysis

The contingency Chi-squared statistic was used to test (at 95°70 and 990/o levels of significance) whether there were significant differences in the numbers of metaphases at each degree of digestion (I, II, III, IV) between groups of metaphases defined by the stage of chromosomal condensation (LC, MC, HC) at different incubation times.

R e s u l t a n d d i s c u s s i o n

Control and standard metaphase chromosomes re- vealed a uniform Giemsa staining, while enzyme- treated chromosomes showed different digestion degrees.

Morphologically, the differences between metaphase chromosome condensation allowed us to establish three condensation stages: - low condensation chromosomes (LC): low and

narrow chromosomes which show non- differentiated chromatids;

- m e d i u m condensation chromosomes (MC): short chromosomes with wide and slightly sepa- rated chromatids;

- high condensation chromosomes (HC): chromo- somes shortest and widest with totally sepa- rated chromatids.

Hind-III endonuclease activity on metaphase chromosomes from our Don cell line show four characteristic digestion stages: - Stage I. Chromosomes treated with Hind III re-

vealed a uniform Giemsa staining. We did not ob- serve differences between control and standard chromosomes. Stage-I chromosomes observed under electron microscopy showed well-defined chromatids and centromeres. Chromatin fiber was tightly packed (Figs. la and lb).

- Stage II. Chromosomes revealed a differential staining. Chromatids were observed well in- dividualized. Strongly stained structures, similar to beads-on-a-string, were visualized (chromo- meres). Under EM these chromomeric electron- dense structures were placed parallel along chro- mosome arms. Centromere and telomere areas conserved their usual structures and chromatin

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Fig. 1. Chinese hamster: metaphase at stage I observed (a) by LM; (b) by EM. No digestion effect is observed; (c) Stage II, LM. Dark regions show structures similar to beads-on-a-string; (d) Under EM, no linear chromosomal differentiation is observed. Dark structures (chromomeres) are located at areas where, later, chromosome bands appear. Bar in a and c represents l0 / tm and in h and d 1 /.tm.

fiber appeared slightly collapsed (Figs. lc and ld). Stage III. Differential staining revealed a G- banding pattern. Chromosomes observed under EM appeared collapsed with fused chromatids and G-bands as electron-dense areas. Centromere and telomeres were unaffected (Figs. 2a and 2b). Stage IV. Staining capacity decreased notably. Chromosomes appeared as 'ghost-like' structures and any type of lineal differentiation could not be observed, neither chromomeres nor bands. EM observation revealed 'swollen' chromosomes and only contour delineation could be appreciated. This was produced without chromosome distor- sion (Figs. 2c and 2d).

These results corroborate the G-banding pattern obtained by Mezzanotte et al. (1983) and Mezzanotte and Ferrucci (1984). It also suggests that the process of digestion by this endonuclease is gradual and con- tinuous and that, previous to the appearance of the banding pattern, there exists a characteristic stage which shows strongly stained chromomeric struc- tures.

Table 1 summarizes the percentages of metaphases at each digestion stage and the total numbers of metaphases analyzed at the three con- densation stages defined. It can be observed that in LC chromosomes the stage I of digestion disap- peared after 1 h. More than 50% of metaphases reached stage III (G-banding pattern) after 3 h of

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Fig. 2. Chinese hamster: (a) Metaphase at stage III by LM; a clear differential staining can be observed (G-banding pattern); (b) EM, banding pattern characterized by very electrondense areas, chromatids appear more fused than in stage II, centromere and teiomeres con- serve their structures; (c) 'Ghost' metaphase, LM, chromosomes 'swollen' and well-delineated; (d) High resolution power of EM, chromo- some morphology suggests a remarkable loss of chromosomal components. Bar in a and c represents 10 #m and in b and d 1 #m.

treatment. In MC and HC chromosomes stage I of digestion only disappears after 3 h. An incubation time of 6 h is needed to obtain more than 5007o of metaphases at stage III. Each condensation group showed a characteristic behaviour that depends on the time required to reach the different digestion stages; the evolution of digestibility was faster in LC chromosomes and it slowed down as chromosomal condensation increased.

Results from Table 1 were analysed by a contin- gency chi-squared statistic test (Table 2). We com- pared the number of metaphases at each digestion stage between pairs of condensation groups at

different incubation times. The test revealed signifi- cant differences between groups for incubation times up to 6 h. However, statistically significant differences were not observed after 24 h of treat- ment, and we suggest that the homogeneity of the response between the different condensation stages is produced by a prolonged incubation time.

Actually it is not clear whether restriction activity depends on the presence or absence of specific tar- gets along chromosomal DNA or on the structural organization in specific chromosomal regions (Mez- zanotte et al., 1983). Lica and Hamkalo (1983) and Mezzanotte and Ferrucci (1984), employing type-II

Table 1. Evolution of digestibility according to condensation stage/digestion stage relation (expressed in percentages).

Condensation stages

IT MN LC MN MC MN HC

I II II1 IV I II III IV I II 1II IV

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1 h 142 0.70 61.27 38.03 0.00 332 1.81 89.46 8.73 0.00 461 9.98 85.25 4.77 0.00 2 h 91 0.00 60.44 39.56 0.00 211 4.74 71.56 23.70 0.00 254 22.05 61.42 16.53 0.00 3 h 85 0.00 15.29 84.71 0.00 204 0.49 56.86 42.65 0.00 210 1.90 78.10 20.00 0.00 4 h 106 0.00 29.25 70.75 0.00 242 0.00 72.73 27.27 0.00 197 0.00 89.34 10.66 0.00 5 h 44 0.00 36.36 63.64 0.00 77 0.00 71.43 28.57 0.00 112 0.00 85.71 14.29 0.00 6 h 95 0.00 12.63 87.37 0.00 217 0.00 27.19 72.81 0.00 293 0.00 34.47 64.85 0.68

24 h 35 0.00 0.00 85.71 14.29 134 0.00 2.24 85.82 11.94 219 0.00 3.20 9.41 6.39

IT = Incubation times; I, II, III, IV = Digestion stages; MN = Number of metaphases analyzed.

Table 2. X 2 values.

Comparation Digestion times between condensation groups 1 h 2 h 3 h 4 h 5 h 6 h 24h

LC-MC 60.34*** 11.09"** 42.94*** 57.85*** 14.19"** 7.95** 0.94 ns (2) (2) (2) (l) (1) (1) (2)

MC-HC 16.57"** 29.10"** 25.65*** 18.87"** 5.79* 4.76 ns 3.48 ns (2) (2) (2) (1) (1) (2) (2)

HC-LC 115.13"** 35.79*** 106.97"** 114.99"** 37.98*** 17.55"** 3.72 ns (2) (2) (2) (1) (1) (2) (2)

* --- p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant, p>0.05. X 2 Values obtained by contingency chi-squared statistic test comparing pairs of condensation groups at different incubation times ( ) = degrees of freedom.

restriction endonucleases suggest that highly con- densed chromatin structure plays a primary role in determining the restriction enzyme accessibility to mouse chromosomal DNA. The specific restriction sites in specific chromosomal regions would not be sufficient for restriction endonuclease activity (Mez- zanotte et al., 1983).

The relation between packing ratio and chromatin digestibility could be the cause of the evolution of the digestion process as suggested by Saharasbuddhe et al. (1978). The disappearance of the banding pat- tern after 24 h of treatment that we observed, sug- gests that the unaffected heterochromatic regions are digested too. A prolonged incubation with the

endonuclease could facilitate the access to recogniz- able sequences which could be 'protected' by the structural complexity of specific chromosomal regions. Maybe, as the digestion advances, the pres- ence of a recognizable sequence would acquire great- er significance.

References

Bianchi, N. O., Bianchi, M. S. & Cleaver, J. E., 1984. The action of ultraviolet light on the patterns of banding induced by re- striction endonucleases in human chromosomes. Chromoso- ma 90: 133-138.

Comings, D. E., 1978. Mechanisms of chromosome banding and

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implications for chromosome structure. Ann. Rev. Genet. 12: 25 -46.

Goyanes, V. J. & Mendez, J., 1982. Karyotyping chromosomes by electron microscopy. II. A method for the sequential examina- tion of spread and banded metaphases by light and electron microscopy. Hum. Genet. 62: 355-357.

Lica, L. & Hamkalo, B., 1983. Preparation of centromeric heter- ochromatin by restriction digestion of mouse L 929 cells. Chro- mosoma 88: 42-49.

Mezzanotte, R., Bianchi, U., Vanni, R. & Ferrucci, L., 1983. Chro- matin organization and restriction endonuclease activity on human metaphase chromosomes. Cytogenet. Cell Genet. 36: 562- 566.

Mezzanotte, R. & Ferrucci, L., 1984. Alterations induced in mouse chromosomes by restriction endonucleases. Genetica 64: 123-128.

Miller, D. A., Choi, J. C. & Miller, O. J., 1983. Chromosome localization of higher repetitive human DNAs and amplified ribosomal DNA with restriction enzymes. Science 219: 395-397.

Sahasrabuddhe, C. G., Pathak, S. & Hsu, T. C., 1978. Responses of mammalian chromosomes to endonueleases digestion. Chromosoma 69: 331- 337.

Sumner, A. T., 1982. The nature and mechanisms of chromosome banding. Cancer Genet. Cytogenet. 61: 59-87.