The isolation of evolutionarily conserved Eag I end-clones from mouse chromosome 17 using cloned DNA

• Published in: DNA and cell biology Vol. 11; no. 8; p. 613
• Main Authors: Höglund, M, Sidén, T, Röhme, D
• Format: Journal Article
• Language: English
• Published: United States 01.10.1992
• Subjects: Animals; Bacteriophage lambda; Base Sequence; Chromosomes; Clone Cells; Cloning, Molecular; Conserved Sequence; Cricetinae; Crosses, Genetic; Deoxyribonucleases, Type II Site-Specific; DNA Probes; DNA, Recombinant; Genetic Markers; Genomic Library; Histocompatibility Antigens - genetics; Hybrid Cells; Mice - genetics; Repetitive Sequences, Nucleic Acid; Restriction Mapping
• ISSN: 1044-5498
• DOI: 10.1089/dna.1992.11.613

To isolate DNA markers from mouse chromosome 17, a genomic phage library was constructed from the mouse-hamster CMGT cell hybrid RcE-B52. This hybrid contains a chromosomal fragment from the distal end/flanking region of the t complex on mouse chromosome 17. Recombinants of mouse origin were identified by using a panel of mouse-specific repetitive sequences as a probe. A total of 1,500 mouse phage recombinants were isolated. These were found to represent 250-300 individual recombinants, comprising about 4 Mbp of cloned mouse DNA. The pooled mouse recombinant phages were used to construct an Eag I end-library. This was achieved by the specific insertion of a marker plasmid in Eag I recognition sites when present in the mouse inserts of the recombinant phages. The Eag I end-fragments were subsequently subcloned using a simple procedure taking advantage of the inserted plasmid. A total of 56 individual Eag I end-fragments were identified. These were found to contain recognition sites for rare cutting enzymes at high frequency. A large proportion (73%) were found to be evolutionarily conserved in human DNA. Furthermore, a significant fraction of these fragments, two of six tested, appears to detect specific cDNAs in a 8.5-day mouse embryo cDNA library.