인간염색체의 DNA복제에 관한 세포유전학적 연구

초록

Replication mapping of chromosomes has been developed very recently, employing 5-bromodeoxyuridine (BrdU), a base analogue of thymidine incorporated during the DNA-synthesis (S) phase of the cell cycle. Chromosomal regions with BrdU-substituted DNA possess the property of reducing the fluorescence of Hoechst 33258 fluorescent dyes or decreasing the staining intensity of Giemsa. These different cytological properties of BrdU-substituted DNA thus allow the precise localization of DNA replicating regions of chromosomes. The BrdU-Hoechst 33258-Giemsa technique was employed to study patterns of chromosomal replication in human lymphocytes synchronized by Methotrexate (MTX). PHA-stimulated human peripheral lymphocytes from chromosomally normal subjects were cultured synchronized, and pulsed with BrdU or thymidine. The results obtained are as follows: 1. Synchronization of cultured lymphocytes with MTX and minimal exposure to colcemid produces sharp band definition and increased resolution in late prophase and prometaphase. 2. Human chromosomes synthesize DNA in a segmental but highly coordinated fashion. 3. The replication of the human genome is clearly biphasic. Euchromatin and intercalary and facultative heterochromatin replicate in a non-overlapping fashion. 4. The late replicating elements, such as G-bands, the Yh, C-bands and the entire LX, initiate replication after it has been completed in the autosomal R-bands (euchromatin). 5. Every prometaphase band is composed of a set of replication units or sub-band. This study confirms not only that chromosomal replication is tightly linked to chromosomal banding but also shows that this methodology is a potentially valuable clinical cytogenetic technique, because it: 1) produces sharp band definition and increased resolution in late prophase and prometaphase subbands, 2) differentiates not only the inactivated X chromosome from the rest of chromosomes but also segments of the Y Chromosome.