3232'Med Genet 1995;32:32-35 Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ J Med Genet: first published as 10.1136/jmg.32.1.32 on 1 January 1995. Downloaded from exonuclease III digestion-random primer extension procedure J L Fernandez, A Campos, C L6pez-Fernandez, J Gosalvez, V Goyanes Abstract chromatin banding for certain human Fixed chromosomes from human amniotic chromosomes in not easily obtained. (3) The fluid cells and peripheral blood lympho- banding pattern resulting from a specific pro- cytes were digested in situ with exo- cedure is quite consistent across all human nuclease III and the single stranded DNA cellular types. obtained was used as template for an in In the present paper we present a modified situ random primer extension. Under in situ random primer extension procedure on these conditions an R banding pattern, exonuclease III treated chromosomes to show more evident in lymphocytes than in that there are exceptions to items (2) and (3) amniocytes, was obtained. Nevertheless, mentioned above. In fact, in the model we have constitutive heterochromatin of chro- tested, R bands coexist with labelled con- mosomes 1, 16, Yq, and mainly the peri- stitutive heterochromatin in chromosomes 1, centromeric region of chromosome 9 was 9, 16, and Yq. Moreover, this is shown to be far more intensely labelled in amniocytes specific to human amniocytes, human lympho- than in lymphocytes. Fluorescence in situ cytes producing a different banding pattern. hybridisation with a specific classical sat- ellite DNA probe, showed that this differ- ential labelling was dependent on a greater Materials and methods sensitivity of chromosome 9 constitutive CULTURES AND METAPHASE PREPARATION heterochromatin to exonuclease III di- Human amniotic fluid cells from four persons gestion in amniocytes than in lymph- were obtained by amniocentesis, performed at ocytes, thus indicating qualitative 14 to 16 weeks' gestation, and cultured at 37°C differences in this region between both in the dark with lOml Ham's F10 medium http://jmg.bmj.com/ human cellular materials. supplemented with 10% fetal calf serum, an- tibiotics, L-glutamine, hepes-buffer pH 7-2, (J Med Genet 1995;32:32-35) and ultroser G. After a week of incubation, the amniocytes were subcultured, and harvested two days later by the trypsin suspension method Chromosome banding procedures are powerful or mechanically detached. tools in clinical cytogenetics since they re- Human peripheral blood lymphocytes were on October 1, 2021 by guest. Protected copyright. produce specific longitudinal differentiation in obtained from four normal persons, ranging each single human chromosome. Additionally, from newborn to 35 years old. Cultures were de Centro Oncol6gico they can show interesting data on chromosome set up for 72 hours at 37°C with 4 ml TC 199 Galicia, Avda de medium supplemented with 10% fetal calf Montserrat sin, 15006 structure and function.' Recently, molecular La Corufia, Spain approaches, such as digestion with restriction serum, antibiotics, and phytohaemagglutinin. J L Femindez endonucleases or nick translation, have been Both amniocytes and lymphocytes were ar- rested at metaphase for three hours with 0 5 ztg/ Unidad de Genetica, successfully applied in situ on fixed chro- Hospital Teresa mosomes, resulting in several specific banding ml colchicine, treated for 10 minutes with Herrera, 15006 0 075 mol/l KCI at 37°C, fixed three times La Corufna, Spain patterns.23 on There are three general rules that apply to in methanol:acetic acid (3:1), and spread J L Fernandez opposite sides of the same glass slide by the air A Campos most of the banding procedures. (1) All band- V Goyanes ing techniques applied to human chromosomes dry method. Unidad de Genetica, result in a G banding (intercalary het- Departamento de erochromatin), an R banding (euchromatin), Biologia, Universidad or a C or C-like banding (constitutive het- EXONUCLEASE III (EXO III) DIGESTION-RANDOM Aut6noma de Madrid, selective PRIMER EXTENSION PROCEDURE 28049 Madrid, Spain erochromatin) pattern. (2) C banding C Lopez-Fernandez for certain human chromosomes, or an overlap Recently spread chromosomes were incubated J Gosalvez of G with several C banding patterns, can at 37°C for 20 minutes in a moist chamber simultaneously be obtained. However, con- with 150 units of E coli Exo III (Stratagene) in Correspondence to: ventional C banded preparations using the bar- 50,1 of incubation buffer. Slides were thor- Dr Fernandez. ium hydroxide technique sometimes show a oughly washed in 2 x SSC, pH 7, dehydrated Received 3 June 1994 Simultaneous euchro- in cold 70,90, 100% ethanol baths, and air Revised version accepted for residual R banding.4 publication 10 August 1994 matin and specific constitutive hetero- dried. Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes 33 In situ random primer extension was per- Control slides were denatured in 70% forma- formed using a multiprime DNA labelling kit mide/2 x SSC at 70°C for three minutes, de- (Amersham) in which dTTP had been replaced hydrated in an increasing ethanol series, and with biotinylated-1 6-dUTP (Boehringer incubated with the same probe. All slides were Mannheim). The reaction was carried out with washed in 50% formamide/2 x SCC, pH7, at J Med Genet: first published as 10.1136/jmg.32.1.32 on 1 January 1995. Downloaded from 30 pil ofthe labelling mixture in a moist chamber 43°C for 15 minutes and in 2 x SSC, pH7, at 37°C for 75 minutes. The slides were washed at 37°C for eight minutes. The bound probe twice with 2 x SSC, pH 7, for three minutes was detected by treatment with two layers of and once in PBD buffer. After a five minute fluorescein labelled avidin, intercalating a layer incubation with a blocking solution from a of biotinylated goat anti-avidin. Finally, chro- chromosome in situ hybridisation kit (Oncor, mosomes were counterstained with propidium Inc), the slides were incubated for 20 minutes iodide, mounted in an antifade solution, and in a moist chamber at 37°C with 60 gl of fluor- visualised with a Zeiss photomicroscope. escein labelled avidin solution (Oncor, Inc). Finally, they were washed in PBD buffer three times for two minutes each, mounted in an Results and discussion antifade solution, and visualised under epi- Exo III catalyses the removal of nucleotides fluorescence. from double stranded DNA, beginning with a Three control experiments were performed: free 3' hydroxyl end or nick.5 When used in (1) the chromosomes were not digested with situ on methanol-acetic acid fixed and spread Exo III; (2) the Klenow fragment of DNA air dried chromosomes, this enzyme removes polymerase I from E coli was not added to the nucleotides at spontaneously occurring nicks reaction mix; and (3) primers were not added and breaks in the DNA. This results in ex- to the reaction mixture. tensive single stranded DNA motifs that are capable of acting as targets for in situ hy- bridisation of biotinylated DNA probes.6 Con- FLUORESCENCE IN SITU HYBRIDISATION (FISH) sequently, these single stranded DNA areas can After Exo III digestion, some dehydrated slides also hybridise with oligonucleotides of random were incubated at 37°C with 30 gil of a classical sequences, which can then be extended by the satellite DNA probe specific for chromosome Klenow fragment of E coli DNA polymerase 1.7 9, D9Z1 locus, biotin labelled (Oncor, Inc). Incorporation of biotinylated nucleotides by This probe had been previously denatured in the polymerase could be finally detected under the hybridisation mix at 70°C for 10 minutes. fluorescence microscopy using a fluorescein tagged streptavidin. When using this method, all recently spread mitoses from amniotic fluid cells showed a faint R banding pattern (fig 1 A,B). Surprisingly, this euchromatin banding coexisted with highly labelled pericentromeric constitutive heterochromatin of chromosome 9. Constitutive heterochromatin of chro- http://jmg.bmj.com/ mosomes 1, 16, and Yq was also highlighted, though less intensely than in chromosome 9 (fig 1 A,B). Nevertheless, when chromosomes from human peripheral blood lymphocytes were similarly processed on the same slide, only a highly contrasted R banding pattern was obtained (fig IC). These R bands are similar to on October 1, 2021 by guest. Protected copyright. those obtained on thermally denatured human lymphocyte chromosomes when they are in situ random primed and extended.8 I Our control experiments with lymphocyte chromosomes showed that both the Exo III digestion and the Klenow polymerisation were necessary for the differential R labelling, al- though it was very faint. Initially, it appears that random primers were not essential but this differential labelling was greatly strengthened when they were added. Possibly the euchro- matic bands would be more sensitive to Exo III digestion, giving more single stranded DNA areas which are then filled by the Klenow poly- merase, mainly after the hybridisation of ran- dom primers. The differential sensitivity between R and G bands to Exo III could be explained in terms of a distinct chromosome organisation in both regions. For example, Figure 1 Metaphase spreads from human amniotic fluid cells (A,B) and from a human DNase I sensitivity studies show different lymphocyte (C) processed by Exo III digestion-random primer extension. Amniocyte be- chromosomes show a faint R banding pattern coexisting with highly labelled constitutive haviour ofboth chromosome domains9 and the heterochromatin, mainly of chromosome 9 (arrows) and of chromosomes 1, 16, and Yq. nick translation experiments using DNase I and However, lymphocyte chromosomes show only a sharp R banding pattern. restriction endonucleases sensitive to methyl- 34 Fernandez, Campos, LJpez-Ferndndez, Gosdlvez, Goyanes ation of CpG doublets'" also support this point toses examined, while it was as intense as ther- of view.