Scanning ion analytical microscopy for high-resolution detection of 5-bromo-2'-deoxyuridine incorporation in metaphase chromosomes.
We investigated the possibilities of using scanning ion analytical microscopy (SIAM) to detect bromine in human metaphase chromosomes. The experiments were performed after incorporation of the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), into the DNA or by in situ hybridization of a BrdU-labelled probe for the subcentromeric repeated DNA sequences. The possibilities offered by this microanalytical method were compared with immunofluorescent staining techniques. Well-defined maps of bands containing bromide were obtained with metaphase chromosomes that had incorporated BrdU during the late S-phase. Their patterns were similar to the labelling obtained by immunofluorescence. In addition, SIAM reveals the presence of bromine within constitutive heterochromatic regions in which BrdU is poorly detected by immunofluorescence. The comparison of the 12C14N, 31P and 81Br maps of controls and fluorescence plus Giemsa (FPG) metaphase chromosomes shows the loss of bromide from DNA during this treatment. SIAM emerges as a new powerful microanalytical technology for investigating chromosome structure further.
Bourgeois
C A
CA
Institut Curie-CNRS UMR 147, Paris, France. claire.bourgeois@curie.fr
Dennebouy
R
R
Gibaud
A
A
Gerbault-Seureau
M
M
Malfoy
B
B
Slodzian
G
G
Galle
P
P
Dutrillaux
B
B
D001966:Bromine / Q000032:analysis; D001973:Bromodeoxyuridine / Q000032:analysis*; D002478:Cells, Cultured; D002875:Chromosomes / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008214:Lymphocytes; D013058:Mass Spectrometry / Q000379:methods; D008677:Metaphase; D008853:Microscopy / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004247:DNA; D001973:Bromodeoxyuridine; D001966:Bromine