Characterisation of amplified DNA in methotrexate-resistant mouse cells

The isolation and mapping of DNA sequences which are amplified in Methotrexate-resistant EL4/8 mouse cells is described. EL4/8 DNA, partially digested with BamHI or Hindill, was cloned in the lambda vector, EMBL3 and in the cosmid vector, pJB8, respectively. 22 different X clones, containing over 300kb of EL418 amplified sequences, were, identif led and isolated by using a differential screening method. These X clones were then used to select 98 cosmid clones potentially also containing EL4/8 amplified sequences. The representation of amplified sequences in the cosmid clones was analysed by comparing the band pattern produced after gel electrophoresis of a mixture of Hindill-digested cosmid DNAs with that of Hindill-digested total genomic EL4/8 DNA. The results suggested that approximately one half of the EL4/8 amplified sequences were represented in the cosmid clones isolated. The EL4/8 DNA present in each cosmid clone was analysed by gel electrophoresis following digestion with Hindill and with BamHI. The restriction fragments produced in each case were sized using a digital microdensitometer. Groups of cosmids containing common restriction fragments were identified. These groups of related clones were both edited and extended after analysis of the cloned sequences by hybridisation with a selection of single-copy sequence probes. These probes included a plasmid clone containing a cDNA to the dhfr gene (pdhfrll). MIF-homologous sequences were also identified. The results from the above analyses enabled 9 separate groups of cosmid and lambda clones to be formed. The DNA present in each group was mapped, to an extent which depended largely on the number of overlapping clones identified in that group. In total, approximately 600kb of EL4/8 amplified DNA sequences were mapped. The maps which were constructed showed that in several different regions of DNA, alternative sequence arrangements were present. Some of these structures must have been produced by intraor interchromatid recombination during, or after, DNA amplification in EL4/8 cells. Generally, the recombination events were found to have occurred in regions of the DNA which contain repeated sequences. In the largest group of clones, which spanned about 85kb of DNA and which included the dhfr gene, a minimum of 3 alternative sequence organisations was identified. One of the 3 arrangements was identical to that found by others in a mouse sarcoma cell line and thus probably represents the wild-type sequence arrangement. The other two arrangements must represent ones formed during DNA amplification in EL4/8 cells. Two types of alternative structures for a given region of DNA were found. In one type, the sequences characteristic of each. structure were amplified to an equal extent in EL4/8 cells. In the other type, the sequences of each variant were amplified to a different level. These results suggested that at least two-rounds of DNA amplification, followed by recombination, had occurred in EL4/8 cells. Limited evidence was found for a gradient of amplified sequences, such as might be expected if amplification occurs by disproportionate replication from a single origin.