The isolation of evolutionarily conserved Eag I end-clones from mouse chromosome 17 using cloned DNA.
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.
Höglund
M
M
Department of Molecular Genetics, Wallenberg Laboratory, University of Lund, Sweden.
Sidén
T
T
Röhme
D
D
D000818:Animals; D010582:Bacteriophage lambda; D001483:Base Sequence; D002875:Chromosomes; D002999:Clone Cells; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D003433:Crosses, Genetic; D015342:DNA Probes; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific; D005819:Genetic Markers; D015698:Genomic Library; D006649:Histocompatibility Antigens / Q000235:genetics; D006822:Hybrid Cells; D051379:Mice / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping
D015342:DNA Probes; D004274:DNA, Recombinant; D005819:Genetic Markers; D006649:Histocompatibility Antigens; C046751:endodeoxyribonuclease XmaIII; D015252:Deoxyribonucleases, Type II Site-Specific