CHAPTER 1

With the creation of yeast artificial chromosomes (YACs) in the late 1980s (Burke et al. 1987), cloning of megabase-sized DNA fragments became possible, and library-based exploration of even the largest genomes appeared practicable. However, YACs have some serious drawbacks as cloning vectors (Anderson 1993). For example, roughly 50% of YAC clones are chimeric or possess insert rearrangements (Burke 1990; Neil et al. 1990; Green et al. 1991; Anderson 1993; Venter et al. 1996; Cai et al. 1998). Such clones are unsuitable for sequencing and mapping research, and a great deal of time is devoted to "weeding out" chimeras and clones with rearranged inserts (Green et al. 1991; Anderson 1993; Venter et al. 1996). Additionally, manipulation and isolation of YAC inserts is difficult and time consuming (O'Conner et al. 1989; Woo et al. 1994).

In the early 1990s, "bacterial artificial chromosomes" (BACs) emerged as an alternative to YACs (Shizuya et al. 1992). Contrary to their name, BACs are not really artificial chromosomes per se, but rather are modified bacterial F factors. Though they can carry inserts approaching 500 kb in length, insert sizes between 80 and 200 kb are more typical (e.g., Shizuya et al. 1992; Woo et al. 1994; Cai et al. 1995; Choi et al. 1995; Kim et al. 1996; Zhang et al. 1996; Yang et al. 1997; Tomkins et al. 1999a, Tomkins et al. 1999b). Most BAC vectors possess traditional plasmid selection features such as an antibiotic resistance gene and a polycloning site within a reporter gene (allowing insertional inactivation) (see Choi and Wing 1999 for a review of BAC vectors and FIGURE 1.1 for a diagram of the most common BAC vector, pBeloBAC11). BAC clones have several notable advantages over YACs. In particular, BACs are relatively immune to chimerism and insert rearrangements (Woo et al. 1994; Cai et al. 1995; Kim et al. 1996; Boysen et al. 1997; Venter et al. 1996; Venter et al. 1998). The stability of BAC inserts appears to be due, in part, to F factor genes (parA and parB) that prevent more than one BAC from simultaneously inhabiting a bacterium (Willetts and Skurray 1987; Shizuya et al. 1992; Cai et al. 1998). An additional advantage of BAC clones is that they are relatively easy to manipulate and propagate compared to viral- or yeast-based clones (O'Conner et al. 1989; Burke and Olsen 1991; Paterson 1996; Marra et al. 1997). Consequently, BACs have supplanted YACs as the dominant vector used in large-scale physical mapping and sequencing (Cai et al. 1998; Kelley et al. 1999)

BAC libraries in which each clone is stored and archived individually (i.e., ordered libraries) are rapidly becoming a central tool in modern genetics research. Such libraries have been made for a host of taxa (e.g., TABLE 1.1; Cai et al. 1995; Choi et al. 1995; Kim et al. 1996; Wang et al. 1996; Frijters et al. 1997; Marec and Shoemaker 1997; Nakamura et al. 1997; Yang et al. 1997; Danesh et al. 1998; Vinatzer et al. 1998; Moullet et al. 1999; Nam et al. 1999; Salimath and Bhattacharyya 1999), and employed in a variety of applications. For example:

1. The suitability of BACs as DNA sequencing/PCR templates has led to the development of BAC-end sequencing (Venter et al. 1996; Boysen et al. 1997; Rosenblum et al. 1997), fostered advances in STS-based mapping (Venter et al. 1996, Venter et al. 1998), and provided a means to quickly search well-defined genomic regions for phenotypically-significant genes (Bouck et al. 1998).
2. The facility of BACs as a large DNA cloning vector (Shizuya et al. 1992) combined with the development of methods for high-throughput DNA fingerprinting (Marra et al. 1997), contig assembly (Gillett et al. 1996; Soderlund et al. 1997; Ding et al. 1999), BAC-end sequencing, and STS-based mapping have helped investigators bridge gaps between DNA markers in physically-large genomes (i.e., physical mapping). Consequently, many interesting and important genes have been isolated (Wang et al. 1996; Nakamura et al. 1997; Yang et al. 1997; Cai et al. 1998; Danesh et al. 1998; Yang et al. 1998; Folkertsma et al. 1999; Moullet et al. 1999; Nam et al. 1999; Patocchi et al. 1999; Salimath and Bhattacharyya 1999; Sanchez et al. 1999). High-throughput physical mapping already has resulted in the construction of BAC contigs encompassing entire chromosomes and/or complete chromosome sets (Mozo et al. 1999).
3. Many of the DNA probes used to make genetic maps can be localized to specific BACs, providing a means of superimposing genetic maps directly onto BAC-based physical maps (e.g., Yang et al. 1997; Mozo et al. 1999; Draye et al., submitted). This feature also facilitates map-based cloning of genes responsible for specific phenotypes (Danesh et al. 1998; Nam et al. 1999; Patocchi et al. 1999; Sanchez et al. 1999).
4. BAC-based physical mapping enjoys the fundamental advantage of somatic cell genetics in that it does not require DNA polymorphism (Lin et al. 2000). Therefore it provides an alternative to radiation hybrid mapping in which chromosomes are broken by radiation and propagated in cell cultures (see Goss and Harris 1975; Deloukas et al. 1998). Of particular interest to botanists, this feature has also spawned efficient methods to determine the locus-specificity of individual BACs that correspond to multi-locus DNA probes in a manner that can efficiently be applied on a large scale (Lin et al. 2000).
5. BAC-based mapping in conjunction with efficient multiplex screening methods (Cai et al. 1998) may open the door to the development of comprehensive "gene maps" (Hudson et al. 1995) for numerous genomes, conferring many of the advantages of complete genome sequencing decades before complete sequences are likely to be available.
6. BACs have successfully been employed as probes in fluorescence in situ hybridization (FISH) (Cai et al. 1995; Hanson et al. 1995; Jiang et al. 1995; Lapitan et al. 1997; Gómez et al. 1997; Morisson et al. 1998; Godard et al. 1999). FISH-based localization of cloned DNA sequences on chromosomes allows molecular and physical maps to be directly superimposed onto the framework of chromosomes, and subsequently provides useful information on the relationship between chromosome structure, DNA sequence, and recombination (Peterson et al. 1999).
7. Full-scale BAC-based genome sequencing efforts are underway (Venter et al. 1998).

Current published protocols for constructing BAC libraries are not particularly detailed, making it difficult for investigators without previous experience in BAC library construction to create BAC libraries de novo. Additionally, creation of plant BAC libraries has been limited because plant cells possess certain natural features that make isolation of "clean", high molecular weight DNA difficult (e.g., cell walls, stored carbohydrates, and volatile secondary compounds). Collectively, we (the authors of this guide) have been involved in the construction of > 20 plant BAC libraries including libraries for species in which secondary compounds, carbohydrates, and/or endogenous nucleases are known to be a problem (TABLE 1.1). Through this document we seek to introduce the new practitioner to efficient BAC cloning of plant DNA, and also to help the experienced investigator streamline the cloning process. We hope that by reducing the obstacles to BAC cloning in plants, we will foster new and accelerated progress in plant genomics, and contribute to the rapid growth in the plant genomic infrastructure that is opening the door to a new era of botanical discovery.

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