21 The Combined Use of Nuclease in Situ Digestion and FISH (NU-FISH)
279
Checking differences in telomere DNA sequences
Telomere-like Chinese hamster interstitial pericentromeric DNA sequences and human telomeres are differentially affected by "in situ" digestion with ExoIII or nuclease Bal 31 (Fig. 3a-f). Quantification of the
FISH signal produced by a telomeric probe (poly-TTAGGG) by digital image analysis, showed that ExoIII was 2.6 times more active in interstitial
(Chinese hamster) than in terminal telomeric (human) chromosome regions. On the other hand, nuclease Bal 31 was 2.3 times more effective in
terminal than in interstitial telomeric sequences. Both results support the
hypothesis that the same DNA sequences can be differentially organised,
depending on the chromosome region where they are located and probably on their functional role (Fernandez et al. 1998).
Checking DNA methylation
Tandem REs that recognise the same restriction site but differentially
cleave DNA according to the pattern of DNA methylation can be employed
for the «in situ" digestion of fixed chromatin. For example, isoschizomers
such as Msp I or Hpa II, which recognise CCGG sites, act differentially on
DNA depending on whether the internal cytosine is methylated or not.
While Msp I cleave CCGG sites independently of methylation, Hpa II
does not cleave this site if the internal cytosine is methylated. RE-FISH
using this pair ofisoschizomers and post-hybridisation with rDNA probes,
permits the visualisation of DNA methylation at CCGG sites.
For example, the grasshopper Eyprepocnemis plorans has a family of
extra B-chromosomes containing long repeats of silent rDNA. When these
chromosomes are digested with Mspl and Hpall and the amount of fluorescence recovered after FISH using rDNA probes is measured, higher values of fluorescence are detected in B-chromosomes digested with Hpall
than in Mspl-digested Bs (Fig. 3g-i). This result indicates less severe DNA
removal with Hpall because of internal cytosine methylation. In fact,
when the same experiment is performed on Southern-blotted DNAs, differences in DNA methylation are observed at these sites (Gosalvez et al.
1997b).
Acknowledgements. This work was supported by the Consejo de Seguridad Nuclear, grants
from FIS and SAF (Spain) and European Community.
279
Checking differences in telomere DNA sequences
Telomere-like Chinese hamster interstitial pericentromeric DNA sequences and human telomeres are differentially affected by "in situ" digestion with ExoIII or nuclease Bal 31 (Fig. 3a-f). Quantification of the
FISH signal produced by a telomeric probe (poly-TTAGGG) by digital image analysis, showed that ExoIII was 2.6 times more active in interstitial
(Chinese hamster) than in terminal telomeric (human) chromosome regions. On the other hand, nuclease Bal 31 was 2.3 times more effective in
terminal than in interstitial telomeric sequences. Both results support the
hypothesis that the same DNA sequences can be differentially organised,
depending on the chromosome region where they are located and probably on their functional role (Fernandez et al. 1998).
Checking DNA methylation
Tandem REs that recognise the same restriction site but differentially
cleave DNA according to the pattern of DNA methylation can be employed
for the «in situ" digestion of fixed chromatin. For example, isoschizomers
such as Msp I or Hpa II, which recognise CCGG sites, act differentially on
DNA depending on whether the internal cytosine is methylated or not.
While Msp I cleave CCGG sites independently of methylation, Hpa II
does not cleave this site if the internal cytosine is methylated. RE-FISH
using this pair ofisoschizomers and post-hybridisation with rDNA probes,
permits the visualisation of DNA methylation at CCGG sites.
For example, the grasshopper Eyprepocnemis plorans has a family of
extra B-chromosomes containing long repeats of silent rDNA. When these
chromosomes are digested with Mspl and Hpall and the amount of fluorescence recovered after FISH using rDNA probes is measured, higher values of fluorescence are detected in B-chromosomes digested with Hpall
than in Mspl-digested Bs (Fig. 3g-i). This result indicates less severe DNA
removal with Hpall because of internal cytosine methylation. In fact,
when the same experiment is performed on Southern-blotted DNAs, differences in DNA methylation are observed at these sites (Gosalvez et al.
1997b).
Acknowledgements. This work was supported by the Consejo de Seguridad Nuclear, grants
from FIS and SAF (Spain) and European Community.