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Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope
Author(s) -
ArndtJovin Donna J.,
RobertNicoud Michel,
Jovin Thomas M.
Publication year - 1990
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1990.tb02947.x
Subject(s) - biology , dna , polytene chromosome , chromatin , biophysics , confocal , heterochromatin , fluorescence microscope , nucleus , cell nucleus , confocal microscopy , interphase , microbiology and biotechnology , fluorescence , chromosome , optics , genetics , gene , physics
SUMMARY Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu‐ and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi‐parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co‐localization of nuclear components. DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi‐stranded). We have assessed by laser scanning microscopy the presence of left‐handed Z‐DNA in polytene chromosomes of Diptera as well as the spatio‐temporal distribution of Z‐DNA binding proteins in whole‐mount Drosophila embryos and ovaries. We have determined the 3‐D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti‐BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.