Construction and characterization of a yeast artificial chromosome library for Xpter-Xq27.3: a systematic determination of cocloning rate and X-chromosome representation.
We describe the construction and characterization of a human X-chromosome-specific yeast artificial chromosome (YAC) library. Starting with 60 micrograms of hybrid cell line genomic DNA, we generated over 150,000 recombinants, over 90% of which range from 150 to 500 kb. From these recombinants, 3300 human-positive YACs (representing coverage of 4.5 X chromosomes) were identified by genomic human DNA hybridization. Mapping of random clones revealed that they are derived from the X chromosome in a regionally unbiased fashion, and screening with single-copy X-chromosome probes has repeatedly produced YACs from the library. By determining the frequency of YAC clones containing both hamster and human repetitive sequences, we estimated that approximately 11% of clones contain discontiguous sequences. Taken together, the low cocloning rate, the unbiased coverage, and a consistent recovery of YACs using specific X-chromosome markers indicate that YAC technology can be used for extensive cloning and mapping purposes. Because a certain amount of genomic rearrangement is present in YAC libraries, chromosome walking must be undertaken with a degree of caution.
Lee
J T
JT
Howard Hughes Medical Institute, Department of Human Genetics, University of Pennsylvania, School of Medicine, Philadelphia 19104.
Murgia
A
A
Sosnoski
D M
DM
Olivos
I M
IM
Nussbaum
R L
RL
D000818:Animals; D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014960:X Chromosome
D004247:DNA