A standardized method for the three-way differential staining of plant chromosomes and the scoring of SCEs per cell cycle.
We have made use of 2 alternative methodologies to obtain 3-way differential staining (TWD) in third-mitosis (M3) chromosomes of Allium cepa, which involve different uptakes of bromodeoxyuridine (BrdU) through 3 cell divisions, in order to evaluate the sister-chromatid exchanges (SCE) frequency on a per-cell-cycle basis. The main innovation introduced by us to the original protocols previously reported has been the use of 5-fluorodeoxyuridine (FdU) to inhibit endogenous thymidine (dT) synthesis. By using different [BrdU]:[dT] ratios in the presence of FdU the relative incorporation of BrdU into replicating DNA can be controlled. The use of 2 different approaches to obtain 3-way differentiation of sister chromatids allowed us to evaluate the role of BrdU in the induction of SCEs in our system. Both procedures rendered nearly 100% of M3 chromosomes showing TWD. An additional advantage of our methodologies is their high degree of reproducibility.
Escalza
P
P
Departamento de Biología Celular, Facultad de Biología, Sevilla, Spain.
Piñero
J
J
Cortés
F
F
D000490:Allium / Q000235:genetics*; D001973:Bromodeoxyuridine; D002453:Cell Cycle; D002875:Chromosomes; D004247:DNA / Q000187:drug effects / Q000235:genetics; D005467:Floxuridine / Q000494:pharmacology; D005821:Genetic Techniques; D012854:Sister Chromatid Exchange; D013194:Staining and Labeling
D005467:Floxuridine; D004247:DNA; D001973:Bromodeoxyuridine