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Sex Chromosome Evolution in Amniotes: Applications for Bacterial Artificial Chromosome Libraries

2011-01-01 14:48:42

Journal Of Biomedicine & Biotechnology; 2011; 2011:pp.132975


Daniel E. Janes, Nicole Valenzuela, Tariq Ezaz, Chris Amemiya, and Scott V. Edwards



Introduction



Studies of the evolutionary history and functional dynamics of amniote sex chromosomes have been enabled by bacterial artificial chromosome (BAC) libraries. Here, we review several aspects of amniote sex chromosome evolution as characterized by experimentation before and after the construction of amniote BAC libraries and we suggest that the rate of discovery and reach of comparisons have been increased by BAC resources, especially for birds and reptiles in which variability of sex chromosome organization offers great opportunities for evolutionary research. In addition to describing BAC-enabled research published within the last decade, we also describe experiments that will allow researchers to use BAC libraries to describe rates of evolution, dynamics of intrachromosomal variations, and frequency of independent origins of sex chromosomes.



A genomic BAC library consists of many DNA fragments representing the whole genome of an individual. Each BAC clone contains 100-200kb of contiguous genomic sequences, providing probes for in situ hybridizations on chromosomes and for determining long-range organization of genes. The number of BAC clones in a library varies depending on the genome size of the species as well as the depth of the library representing the whole genome. For the construction of a BAC library, DNA is isolated and fragmented by hydroshearing, nebulization, or partial restriction digestion followed by preparative pulsed field gel fractionation. Fragments are size selected, ligated into a specialized vector, and maintained in Escherichia coli that are archived in 384-well plates at -80°C. Colonies representing the library are gridded onto high-density nylon filters, and respective clone DNAs are anchored to filters by cross-linking. High-density nylon filters serve as maps to BAC libraries. Radio-labeled probes can be hybridized to filters to target clones bearing sequences of interest (Figure 1). Alternatively, “smart” pooling strategies can be employed to screen BAC libraries via polymerase chain reaction.



Several BAC libraries and associated resources are available through the Children’s Hospital Oakland Research Institute (CHORI), the Arizona Genomics Institute, the Clemson University Genomics Institute, Amplicon Express, Empire Genomics, and other vendors. Typically, whole plates, individual clones, or high-density nylon filters are available for purchase. The past decade has been a period of great discovery regarding amniote sex chromosomes, and the next ten years should be even more productive as more laboratories incorporate BAC technology.



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