21 The Combined Use of Nuclease in Situ Digestion and FISH (NU-FISH)
277
Checking differences in satellite DNAs
Differences in chromatin organisation have been inferred by using NUFISH in human lymphocytes. Extended or condensed chromatin in interphase nuclei and chromosomes were "in situ"-digested by the restriction
endonuclease Alu I; the remaining DNA was hybridised with alphoid
probes specific for chromosome 1 and chromosome X (OIZ5 and
DXZl loci) and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). With this experiment we analysed the differential
DNA removal produced by Alu I at specific repetitive DNA sequences
with known restriction site frequency and distribution. Fluorescent hybridisation signals were quantified by high-resolution digital image analysis.
The results showed that the specific repetitive DNA at D9Z1 locus (classical) remained resistant to digestion (compare Fig. 2a, a'). On the other
hand, although restriction sites at the DIZ510cus were at least as frequent
as at the DXZllocus, they showed different hybridisation intensities after
digestion; while DNA at the DXZl locus was extensively removed (compare Fig. 2b, b'), that at the D1Z510cus was only partially removed (compare Fig. 2c, c'). These results indicate that chromatin organisation within
constitutive heterochromatin partially prevents DNA extraction and, specifically, that alphoid DNA sequences seem to be differentially organised
in different chromosomes. The same results were obtained from condensed and extended chromatin, suggesting that higher-order chromatin
organisation does not influence "in situ" DNA cleavage and removal by
Alu I (compare Fig. 2a-c with d-g) (Fernandez et al. 1997).
Checking differences between tissue-specific cells
When fixed chromosomes obtained from human amniotic fluid cells and
peripheral blood lymphocytes were "in situ"-digested with ExoIII followed
by "in situ" random priming extension, an R-banding pattern was obtained in both cases. However, this pattern was more evident in lymphocytes than in amniocytes. In addition, constitutive heterochromatin of
chromosomes 1, 16, Yq, and mostly the pericentromeric region of chromosome 9 were far more intensely labelled in amniocytes than in lymphocytes. By using a specific classical satellite DNA probe, FISH showed that
this differential labelling was due to the greater sensitivity to ExoIII digestion of the constitutive heterochromatin of chromosome 9 in amniocytes.
This experiment shows cryptic and qualitative differences in constitutive
heterochromatic regions between different cells (Fernandez et al. 1995).
277
Checking differences in satellite DNAs
Differences in chromatin organisation have been inferred by using NUFISH in human lymphocytes. Extended or condensed chromatin in interphase nuclei and chromosomes were "in situ"-digested by the restriction
endonuclease Alu I; the remaining DNA was hybridised with alphoid
probes specific for chromosome 1 and chromosome X (OIZ5 and
DXZl loci) and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). With this experiment we analysed the differential
DNA removal produced by Alu I at specific repetitive DNA sequences
with known restriction site frequency and distribution. Fluorescent hybridisation signals were quantified by high-resolution digital image analysis.
The results showed that the specific repetitive DNA at D9Z1 locus (classical) remained resistant to digestion (compare Fig. 2a, a'). On the other
hand, although restriction sites at the DIZ510cus were at least as frequent
as at the DXZllocus, they showed different hybridisation intensities after
digestion; while DNA at the DXZl locus was extensively removed (compare Fig. 2b, b'), that at the D1Z510cus was only partially removed (compare Fig. 2c, c'). These results indicate that chromatin organisation within
constitutive heterochromatin partially prevents DNA extraction and, specifically, that alphoid DNA sequences seem to be differentially organised
in different chromosomes. The same results were obtained from condensed and extended chromatin, suggesting that higher-order chromatin
organisation does not influence "in situ" DNA cleavage and removal by
Alu I (compare Fig. 2a-c with d-g) (Fernandez et al. 1997).
Checking differences between tissue-specific cells
When fixed chromosomes obtained from human amniotic fluid cells and
peripheral blood lymphocytes were "in situ"-digested with ExoIII followed
by "in situ" random priming extension, an R-banding pattern was obtained in both cases. However, this pattern was more evident in lymphocytes than in amniocytes. In addition, constitutive heterochromatin of
chromosomes 1, 16, Yq, and mostly the pericentromeric region of chromosome 9 were far more intensely labelled in amniocytes than in lymphocytes. By using a specific classical satellite DNA probe, FISH showed that
this differential labelling was due to the greater sensitivity to ExoIII digestion of the constitutive heterochromatin of chromosome 9 in amniocytes.
This experiment shows cryptic and qualitative differences in constitutive
heterochromatic regions between different cells (Fernandez et al. 1995).