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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A052
For the analysis of the 3D-genome nano-structure the precise determination
of 3D distances between small specifically labelled chromatin regions in the
interior of 3D-conserved cell nuclei is necessary eventually in the living
cell. This necessitates the use of far field microscopical methods. A new
light mircroscopical approach, Spectral Precision Distance Microscopy (SPDM)
was developed which allows the determination of 3D distances far below the
resolution limit. Spectral Precision Distance Microscopy (SPDM) is based on:
a) the use of point-like objects labelled with different spectral signatures
and b), spectrally differential registration of images and c) three-
dimensional image analysis. In situ measurements revealed, that 3D distances
in intact cell nuclei can be measured with an resolution equivalent of about
50 nm. This analysis tool was applied to study the 3D-positioning of the
adenine nucleotide translocase genes ANT2 and ANT3 on both X-chromosome
territories (Xa active and Xi inactive) in human female amniotic cell nuclei.
ANT2 is transcriptionally active on Xa, but inactive on Xi. ANT3 however
escapes X-inactivation. In this first SPDM analysis multicolour fluorescence
in situ hybridisation (FISH), confocal laser-scanning microscopy and
three-dimensional image analysis was combined and revealed that the relative
positions of the ANT2/3 genes correlated with the activation state. On the
active X-chromosome (Xa), the active genes ANT2 and ANT3 were located in a
peripheral position in the chromosome territory. In the inactive X-chromosome
territory (Xi) the active ANT3 gene was also located at a peripheral position,
whereas the inactive ANT2 gene was shifted significantly (appr. 200 nm)
towards a more interior position in the chromosome territory.
SPECTRAL PRECISION DISTANCE MICROSCOPY (SPDM) IN HUMAN GENOME RESEARCH:
3D-NANOSTRUCTURE ANALYSIS OF X-CHROMOSOMAL GENES.
Edelmann P +,*, Esa A +, Dietzel S #, Cremer T #,*, Cremer C +,*
+ Institute of Applied Physics, Research Division Applied Optics and
Information Processing, * Interdisciplinary Centre for Scientific Computing
University of Heidelberg, Heidelberg,
# Institute of Anthropology and Human Genetics, Ludwig-Maximilians-University,
Munich, Germany