Mechanism of DHFR* amplification involving a ring chromosome intermediate. (a) Chromosome 3 copy number profile in untreated 1M-57 cells. Shown are the log2 ratios ordered according to position on the May 2004 freeze of the human genome sequence. The copy number gain extending from 3pter to RP11-233L3 reflects the presence of the two normal copies of chromosome 3 and the additional piece of chromosome 3 in HCT116+chr3 cells. The DHFR* insertion site is indicated by the arrowhead. (b) Chromosome 3 copy number profile in methotrexate resistant colony 1M-57_2. Two regions of amplification are evident at 3pter and near the distal end of 3q. Copy number losses include material from 3p and 3qter distal to DHFR*. (c) Proposed mechanism leading to amplification of the region around DHFR*. The ends of chromosome 3 fuse to create a ring chromosome with loss of material distal to DHFR* on 3q. Breakage at positions indicated by the arrowheads at anaphase results in the metacentric chromosome that now carries duplication of juxtaposed 3p and 3q sequences. The process can be repeated to generate additional copies of the 3p and 3q sequences. (d) FISH with RP11-107D22 (red), which contains sequences flanking the DHFR* integration site on 3q and RP11-28P14 (green), which maps to 3p. Shown are pseudocolor images showing hybridization signals on the chromosomes and the corresponding DAPI image (gray).