Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ exonuclease III digestion-random primer extension procedure.

Abstract

Fixed chromosomes from human amniotic fluid cells and peripheral blood lymphocytes were digested in situ with exonuclease III and the single stranded DNA obtained was used as template for an in situ random primer extension. Under these conditions an R banding pattern, more evident in lymphocytes than in amniocytes, was obtained. Nevertheless, constitutive heterochromatin of chromosomes 1, 16, Yq, and mainly the pericentromeric region of chromosome 9 was far more intensely labelled in amniocytes than in lymphocytes. Fluorescence in situ hybridisation with a specific classical satellite DNA probe, showed that this differential labelling was dependent on a greater sensitivity of chromosome 9 constitutive heterochromatin to exonuclease III digestion in amniocytes than in lymphocytes, thus indicating qualitative differences in this region between both human cellular materials. (J Med Genet 1995;32:32-35 In the present paper we present a modified in situ random primer extension procedure on exonuclease III treated chromosomes to show that there are exceptions to items (2) and (3) mentioned above. In fact, in the model we have tested, R bands coexist with labelled constitutive heterochromatin in chromosomes 1, 9, 16, and Yq. Moreover, this is shown to be specific to human amniocytes, human lymphocytes producing a different banding pattern. Materials and methods CULTURES AND METAPHASE PREPARATIONHuman amniotic fluid cells from four persons were obtained by amniocentesis, performed at 14 to 16 weeks' gestation, and cultured at 37°C in the dark with lOml Ham's F10 medium supplemented with 10% fetal calf serum, antibiotics, L-glutamine, hepes-buffer pH 7-2, and ultroser G. After a week of incubation, the amniocytes were subcultured, and harvested two days later by the trypsin suspension method or mechanically detached.Human peripheral blood lymphocytes were obtained from four normal persons, ranging from newborn to 35 years old. Cultures were set up for 72 hours at 37°C with 4 ml TC 199 medium supplemented with 10% fetal calf serum, antibiotics, and phytohaemagglutinin.Both amniocytes and lymphocytes were arrested at metaphase for three hours with 0 5 ztg/ ml colchicine, treated for 10 minutes with 0 075 mol/l KCI at 37°C, fixed three times in methanol:acetic acid (3:1), and spread on opposite sides of the same glass slide by the air dry method.

Phenomenon of selective staining of pericentromeric heterochromatin regions in chromosomes from spontaneously dividing cells by the acridine orange fluorochrome

“…On the other hand, the same staining pattern of these HRs is observed in the placenta and embry onic tissues, where they are condensed and methy lated [18,19,23]. Conversely, the hypomethylated and decondensed HRs of amniocytes [24] cannot be dif ferentiated by means of AO staining. Indirect data that the HRs of chorion CTB chromosomes contain sin gle stranded DNA have been obtained using nick translation with DNase I [18].…”

Evidence of a differential organization of chromatin containing terminal or interstitial (TTAGGG)_n repeats by in situ digestion with nucleases

“…Thus, the degree of enzymatic activity after ExoIII digestion is correlated with the amount of ssDNA generated, whereas that after Bal 31 digestion is correlated with the amount of dsDNA lost. If a (TTAGGG) n sequence area is highly digested by ExoIII, a large quantity of ssDNA is generated, which serves as a target for the hybridization of a large amount of probe and yields a strong FISH signal (Ferná ndez et al, 1995). Heat denaturation is not performed, since ExoIII activity is equivalent to enzymatic denaturation.…”