Micromanipulation and physical mapping of the chicken Z chromosome.

摘要

An integrated approach has been developed for physical mapping of the chicken Z chromosome via the construction of a large-clone genomic bacterial artificial chromosome (BAC) library and strategies for obtaining Z-specific sublibraries, primarily to obtain Z-chromosome DNA sequences. A large-insert BAC library has been constructed from male chicken genomic DNA. Fragments of increased average size were cloned by coupling a second size strategy using pulsed-field gel electrophoresis (PFGE) with optimized electroporation that favoured transformation of Escherichia coli DH10B cells with very large plasmids. The overall library consists, of 4,416 clones with a combined insert size average of 390 kb (ranging from 25 to 725 kb). At least 9596 of the BAC clones contain inserts. We estimate this library to represent a 0.8-fold coverage of the chicken genome. Large chicken BAC inserts were stably propagated for at least 120 cell generations, giving BACs the advantages of yeast artificial chromosomes (YACs) as cloning vehicles, with the practical advantages of bacterial plasmids.;For screening the large-clone male chicken BAC library, whole Z-chromosome painting probes (WCPs)) were devised using chromosome microdissection. A simple method was used to adapt a standard light microscope for the collection of chicken Z chromosomes from mitotic metaphase spreads. The DNA was amplified using a partially degenerate primer in a degenerate-oligonucleotide-shuttle PCR strategy. The resulting sequences, within a size range of approximately 200-800 bp, were used directly as WCPs. Collectively the WCPs provide uniform hybridization signals along the entire length of the chicken Z chromosome in FISH reactions. The WCPs were used as probes in a preliminary screening of colony blots of the BAC library: one clone (C3) carrying a 250 kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Alternatively, the PCR-amplified Z-chromosome sequences were cloned to give a microcloning library containing approximately 1,250 clones. The size range of the cloned inserts was 250-800 bp, with an average of 480 bp (176 clones examined). The Z-chromosome origin of a selected microclone was verified in a semi-quantitative Southern blot hybridization that showed positive signals with intensities approximately twice as strong for male (ZZ) as for female (ZW) chicken genomic DNA when the clone was used as a probe. In the future, microclones containing polymorphic microsatellites can be selected and sequenced for subsequent primer design and generation of Z-specific sequence-tagged-site markers (STSs). Preliminary analysis of several microsatellite-containing Z microclones indicated the feasibility of this approach. (Abstract shortened by UMI.).