Assignment of the Human CDC21 (MCM4) Gene to Chromosome 8q11.2

The MCM/P1 family genes encode proteins that are reminiscent of the replication licensing factor first hypothesized by Blow and Laskey to explain the cellular mechanism that ensures the replication of DNA only once per cell cycle in eukaryotic cells. We previously identified MCM/P1 proteins as a component of a complex containing licensing factor activity. So far, six kinds of MCM/P1 genes have been identified in yeast, frog, and human, and this genomic organization has been shown to be conserved widely among eukaryotes. The chromosomal locations for human MCM/P1 genes (this paper, HsMCMs) have been determined by the fluorescence in situ hybridization (FISH) technique. These locations are 3q21 for HsMCM2, 6p12 for HsMCM3, 22q13 for HsMCM5, 2q14 for HsMCM6, and 7q21 for HsMCM7. Only the chromosomal localization of the HsMCM4 gene remains undetermined. To investigate the chromosomal localization of HsMCM4 (symbol, MCM4; name, minichromosome maintenance deficient 4), we obtained a 3.3-kb cDNA fragment encoding the HsMcm4 protein by PCR using appropriate oligonucleotides primers designed from the reported sequence (X74794) and DNA from a human cDNA library (HeLa cell) as substrate. The identity of the cDNA clone as HsMCM4 was confirmed by DNA sequencing. Using this plasmid DNA harboring HsMCM4 cDNA as a probe, we performed chromosome mapping utilizing the FISH technique. FISH to 5-bromodeoxyuridine (BrdU)-incorporated human metaphase chromosomes was performed as described previously. Briefly, a human peripheral blood lymphocyte culture was synchronized by the thymidine block method, and differential replication staining was carried out using BrdU. A biotinylated 3.3-kb cDNA probe (200 ng) was hybridized with metaphase spreads in 10 mu l of 50% formamide containing 2x SSC and 10% dextran sulfate at 37 degree C overnight. After hybridization, the slides were washed for 5 min in 2x SSC at 72 degree C and in 1x PBD for 5 min at room temperature. The fluorescence was detected using fluorescein (FITC)-labeled avidin, followed by the association of biotinylated anti-avidin antibody and the second cycle binding of FITC-labeled avidin (Biotin-FITC detection kit; Oncor) as described before. The preparations were counterstained with propidium iodide and examined using an Olympus AX80 fluorescence microscope. Specific FISH signals generated from the biotinylated HsMCM4 cDNA were observed on the pericentromeric region of the long arm of chromosome 8. Of the 100 metaphase sp