Construction and characterization of a NotI-BsuE linking library from the human X chromosome.
We describe the construction and characterization of methylation-resistant sequence-tagged NotI linking clones specific for the X chromosome, referred to as NotI-BsuE linking clones. The approach consists of methylating the X-chromosome-specific cloned DNA with BsuE methylase (M. BsuE), an enzyme that methylates the first C residue in the CGCG sequence, followed by selection of the methylation-resistant NotI sites by insertion of a kanamycin-resistance gene in the clones cleavable by NotI. The frequent occurrence of NotI sites in CpG islands is expected to cause methylation of a large number of NotI sites with BsuE methylase, thereby rendering them resistant to NotI cleavage. Thus, the combination of M. BsuE and NotI yields less frequent cutting than the NotI alone. We have isolated, partially sequenced, and characterized 113 NotI-BsuE linking clones, and mapped 50 clones to various regions along the chromosome.
Arenstorf
H P
HP
Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Kandpal
R P
RP
Baskaran
N
N
Parimoo
S
S
Tanaka
Y
Y
Kitajima
S
S
Yasukochi
Y
Y
Weissman
S M
SM
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D015342:DNA Probes; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008040:Genetic Linkage; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid; D014960:X Chromosome
D015342:DNA Probes; D004247:DNA; C055352:DNA modification methylase BsuE; D015257:DNA-Cytosine Methylases; C039566:CGCG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases