BAC libraries and comparative genomics of aquatic chordate species

Comparative Biochemistry and Physiology, Part C 138 (2004) 233 – 244 www.elsevier.com/locate/cbpc

Review

BAC libraries and comparative genomics of aquatic chordate species

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Tsutomu Miyake*, Chris T. Amemiya Molecular Genetics Department, Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle, WA 98101, United States Received 25 March 2004; received in revised form 9 July 2004; accepted 14 July 2004

Abstract The bacterial artificial chromosome (BAC) system is useful for creating a representation of the genomes of target species. The system is advantageous in that it can accommodate exogenous inserts that are very large (N100 kilobases, kb), thereby allowing entire eukaryotic genes (including flanking regulatory regions) to be encompassed in a single clone. The interest in BACs has recently been spawned by vast improvements in high throughput genomic sequencing such that comparisons of orthologous regions from different genomes (comparative genomics) are being routinely investigated, and comprise a significant component, of all major sequencing centers. In this review, we discuss the general principles of BAC cloning, the resources that are currently available, and some of the applications of the technology. It is not intended to be an exhaustive treatise; rather our goal is to provide a primer of the BAC technology in order to make readers aware of these resources and how they may utilize them in their own research programs. D 2004 Elsevier Inc. All rights reserved. Keywords: BAC libraries; Comparative genomics; Aquatic chordate species

Contents 1. What are BAC libraries? . . . . . . . . . . . . . . 2. Who sponsors BAC library construction? . . . . . 3. What BAC libraries are currently available? . . . . 4. Utility of BAC libraries and comparative genomics 5. Summary . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. What are BAC libraries? Bacterial artificial chromosomes are based on plasmid vectors that are essentially composed of an F-factor origin of replication with a chloramphenicol resistance gene (Fig. 1) (Shizuya et al., 1992; Osoegawa et al., 1998; Amemiya et

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al., 1999). The F-factor replicon allows propagation of the bacterial plasmid as a single copy entity in Escherichia coli, thus permitting stable propagation of cloned inserts greater than 100 kilobase pairs (kb).1 The ability to accommodate such large inserts is advantageous for many applications in genome biology, including positional cloning, targeted genomic sequencing, and as vehicles for generating trans-

B

This paper is based on a presentation given at the conference: Aquatic Animal Models of Human Disease hosted by the American Type Culture Collection and the University of Miami in Manassas, Virginia, USA, September 29–October 2, 2003. * Corresponding author. Tel.: +1 206 583 6093; fax: +1 206 583 2297. E-mail address: [email protected] (T. Miyake). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.07.001

1 Note, a similar vector, P1-derived artificial chromosome (PAC) (Ioannou et al., 1994), uses a P1 bacteriophage origin of replication instead of the F-factor replicon (as used in BACs). However, for all intents and purposes, the vectors are very similar with respect to cloning of large exogenous fragments and will both be called BACs in this review.

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Fig. 1. Diagram of a typical BAC vector. This vector is a compilation of the original pBAC108L (Shizuya et al., 1992), pBACe3.6 (Frengen et al., 1999) and pCC1BACk (Epicentre Technologies). The camR gene encodes chloramphenicol antibiotic resistance. The repE, parA, parB, parC and redF genes are essential components of the single-copy F-factor replicon. In order to prepare large quantities of vector, the oriV replicon is used for multi-copy replication. Note the oriV replicon must be used in a host E. coli strain (EPI300) that allows induction of the multi-copy origin of replication via growth of the clone in arabinose-containing media.

genic animals. A flow chart for BAC cloning is given in Fig. 2. The procedure is conceptually simple although the actual generation of a library is both technically challenging and laborious. Briefly, high molecular weight genomic DNA from the target species is prepared in low melting point agarose; subsequent manipulations are done in situ in the agarose to prevent shearing of the DNA. The genomic DNA is partially digested with a restriction enzyme, extensively size-selected using a series of pulsed-field gel electrophoresis runs, and ligated to dephosphorylated BAC vector. Ligation products are electrotransformed into E. coli using a strain that has a particular cell wall defect for increasing transformation of large DNA constructs (DH10B, Invitrogen). Subsequent colonies are plated on a chloramphenicol plate and barrayedQ with a robotic picker into individual 384-well microtiter dishes, incubated overnight and ultimately stored at 80 8C in glycerol containing media. The library thus is encompassed in numerous 384-well microtiter dishes, the number of which is dependent on the genomic bcoverageQ of the library. The size range of cloned

genomic DNA in a BAC library typically varies from 100 to 200 kb and, in general, the larger the average insert size the more labor, time and expense have been afforded in generating the library. The genomic coverage of each BAC library depends on several factors, including the purpose of the library and the genome size of the target species. The most high quality BAC libraries are in the range of 10 genomic coverage with 150 kb inserts and contain fewer than 5% ghost clones.2 Insofar as the feasibility of constructing BAC libraries, most metazoan DNAs appear tractable to BAC cloning; however, certain genomes do tend to be problematic as a result of skewed base composition, high repeat frequencies, symbiont DNA contamination and excessively large genomes. The former two are due to the inability of the E. coli host system to accommodate such DNA,3 and may have a pronounced effect on both the representation of the library and whether constructing a library from that organism is even possible. The latter two biological factors will also ultimately affect the quality and representation of the library. BAC libraries containing symbiont DNA contamination will contain a lower proportion of the desired clones, a substantial problem if that library is being used for a genome sequencing project. This is less of a problem if the library is to be used only to isolate genomic regions rather than as a sequencing reagent. A large genome is a more practical issue that is difficult to deal with effectively since the number of 384-well microtiter dishes will become unmanageable for a library of usable representation. Thus certain organisms with enormous genomes (e.g., lungfishes) will never be candidates for BAC libraries. In addition to the aforementioned problems, two other issues that have been shown to influence the BAC cloning process are high endogenous nuclease activities of the tissue chosen for DNA extraction as well as presence of cloning inhibitors in the extracted DNA. Success in overcoming these problems has been achieved in some cases but not others (P. De Jong, R. Wing and C. Amemiya, unpublished). Once BAC libraries are generated, high-density colony filters (macro-arrays) are produced using a robotics workstation.4 These filters are used for hybridization screening of the libraries, and identification of positive clones is done in a very straightforward manner (Amemiya et al., 1999). Every clone in the library has a unique address, thus in one step the investigator can retrieve the clones of interest for subsequent verification and characterization. Moreover, clever bovergo-oligoQ strategies have been developed that

2 Ghost clones are, by definition, non-recombinant clones that do not have inserts. These include vector bartifactsQ from rearrangement/deletion during the BAC cloning procedure. 3 These DNAs are less clonable due to their toxicity to the E. coli host. 4 In some cases, bsmartQ DNA pools are produced for the library, allowing screening via PCR. However, this method is comparatively more tedious than hybridization-based screening and is not readily amenable to multiplexing.

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Fig. 2. Flow chart for the preparation of a large-insert BAC genomic library from vertebrates. This figure is meant to serve only as a basic overview. (1) Cells (or nuclei) are isolated, washed, and counted using a hemacytometer. (2) The cells (or nuclei) are embedded at a fixed density (roughly 20–30 Ag DNA per 80 Al plug mold) in low melting point agarose blocks. The blocks are then treated with proteolytic agents to lyse the cells (nuclei). The embedding in agarose prevents shearing of the high molecular weight (HMW) DNA during handling. (3) The blocks are extensively equilibrated with TE buffer to remove salts and other small molecules and are partially digested in situ with suitable restriction enzyme for BAC cloning. The partial digestion reactions are carried out using either a time-course experiment or methylase competition. (4) A preparative pulsed field gel is then run on the selected digests, and the region of interest is excised without staining of the DNA with ethidium bromide or exposure to UV irradiation. The region of interest is then excised in order to extract different sized DNA fractions. (5) A small piece of gel from each fraction is run on an analytical pulsed field gel to validate the size range of each fraction of DNA isolated. (6) The DNA is extracted from gel pieces via electroelution and then ligated to dephosphorylated BAC vector using conditions that foster formation of large circles. (7) Ligation products are electro-transformed into electro-competent DH10B T1 phage-resistant E. coli cells and transformants are plated on agarbased selective medium (chloramphenicol for BAC vectors) and allowed to grow. (8) Recombinant colonies are robotically picked (arrayed) into individual wells of 384-well microtiter dishes that contain media supplemented with 10% filter-sterilized glycerol, incubated at 37 8C overnight and then the plates are stored at 80 8C. Subsequent manipulations (e.g., production of filter macroarrays) utilize replicas of the library. Readers requiring more detail on the specifics of BAC library construction should consult (Amemiya et al., 1996) and (Osoegawa et al., 1999).

allow simultaneous hybridization with complex probe mixtures (multiplexing) in order to isolate large contiguous genomic regions in a single probing (Ross et al., 1999; Thomas et al., 2002; Romanov et al., 2003). With regard to time and ease of screening, BAC libraries are considerably easier to screen than lambda genomic libraries or un-arrayed cosmid libraries. In terms of clone handling, since BACs are simply large plasmid-based clones, the methods used for routine isolation and handling of plasmid DNAs (with slight modifications) can be used, albeit with lower yields than conventional high-copy plasmid clones.

from federal granting agencies (predominantly NIH and NSF). In this way, the libraries are assured to be of high quality and will be disseminated to the scientific community without sensitive intellectual property issues. Both the National Human Genome Research Institute (NHGRI) at NIH as well as the Directorate for Biological Sciences at NSF have recognized the utility of BAC resources and have been major driving forces for BAC library construction. Both have requested grant applications for BAC library construction; however, only the NHGRI program is still actively soliciting proposals for BAC libraries from a variety of organisms. Species for which NSF awarded grants can be found at: http://www.nsf.gov/bio/pubs/awards/

2. Who sponsors BAC library construction? Due to the high cost and considerable expertise required in making BAC libraries, most libraries are generated in specialty laboratories5 through contract-type mechanisms

5 It is generally not recommended that individual investigators attempt to construct BAC libraries on their own if they have not previously worked with very high molecular weight DNA and do not possess the equipment and infrastructure for carrying out this work.

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bachome.htm. These represent taxa thought to be of the highest immediate importance to a variety of biological problems and run the gamut from metazoans to plants. Although NSF no longer is specifically funding grants for generating BAC libraries, in certain programs (e.g., Tree of Life, Biological Research Collections) BAC library construction, if sufficiently justified, can be written into collaborative or investigator-initiated proposals. The NHGRI has established the NIH BAC Resource Network for constructing BAC libraries of many metazoan taxa (http://www.genome.gov/10001844). In contrast to the NSF BAC program, three specialized Centers for BAC library production were established in order to fulfill the needs of the biomedical community for BAC library construction: Benaroya Research Institute at Virginia Mason (http://www.benaroyaresearch.org, Chris Amemiya, Director), BACPAC Resources at Children’s Hospital Oakland (http://bacpac.chori.org/home.htm, Pieter de Jong, Director) and Arizona Genomics Institute at University of Arizona (http://www.genome.arizona.edu, Rod Wing, Director). The committee at the NHGRI determines which of BAC libraries from the choice of taxa were made based on bwhite paperQ submissions by the community, and assignments are then made to the respective BAC Library Production Centers. The white paper submissions are done electronically; instructions and selection criteria are specified on the NHGRI website (http://www.genome.gov/ 10001845). A BAC library so produced is of very high quality and must meet strict quality assurance standards prior to being distributed to the community. Thus, this mechanism creates a win-win situation: the NHGRI pays for a high-quality library to be generated and the proposer and community benefit greatly by gaining access to a valuable genome resource that can be utilized for addressing important biological questions. Lastly, it should be mentioned that there are a small number of biotech companies that will construct BAC libraries on a contractual basis.

3. What BAC libraries are currently available? BAC libraries have now been generated from several metazoan species, including both protostomes and deuterostomes. Table 1 lists BAC libraries from aquatic chordate species, many of these libraries being constructed in our laboratory. While it is difficult to keep an accurate accounting of all the libraries that are now in existence, BAC libraries made or being made as part of the NSF BAC program are listed at http://www.nsf.gov/bio/pubs/awards/ bachome.htm; libraries made or being made as part of the NHGRI BAC Resource Network are listed at http:// www.genome.gov/page.cfm?pageID=10001852. In addition, NHGRI provides a relatively up-to-date listing of all metazoan libraries that are available to the public at http:// www.genome.gov/11008350.

4. Utility of BAC libraries and comparative genomics BAC libraries are very useful for many applications in modern biology: isolation of intact genes or gene clusters from regions of interest (Kim et al., 2000; Chiu et al., 2002, 2004; Powers and Amemiya, 2004), whole genome physical mapping (Chen et al., 2004), elucidating gene organization (Amores et al., 1998; Strong et al., 1999), positional cloning (Brownlie et al., 1998; Donovan et al., 2000), long range DNA sequencing and anchoring (Mahairas et al., 1999; Lander et al., 2001; Ness et al., 2002; Krzywinski et al., 2004), and identification of putative cis-regulatory elements (Sumiyama et al., 2002, Sumiyama and Ruddle, 2003). In addition, because BAC clones can harbor large stretches of flanking DNA, they are exceptionally good substrates for inserting reporter genes into coding sequences for elucidating gene expression patterns in transgenic systems (Jessen et al., 1999; Sumiyama and Ruddle, 2003; Gong et al., 2003). While there are several other applications of BAC clones as described above, we will now briefly discuss what has become a major focus of the genomics community, namely to elucidate the logic of non-coding, cis-regulatory DNA (Collins et al., 2003). The recent explosion in DNA sequencing throughput is resulting in a paradigm shift in our approach to biological investigation. We no longer need to rely on bthought experimentsQ for inferring how the genome has diverged over evolutionary time and space; we are able to directly observe these changes and more clearly understand how the genome is organized and regulated, and what kinds of changes result in biological novelty. It is, after all, the genome that evolves; the phenotype must necessarily follow suit. Thus we can use btargeted comparative sequencingQ (sequencing of BACs from orthologous genomic regions between taxa) as a first approximation as to how non-coding cis-regulatory regions contribute to genome functioning (Pennacchio and Rubin, 2001, 2003; Thomas et al., 2003; Force et al., in press). Fig. 3 gives an overview of how targeted comparative sequencing is carried out; the ultimate goal is to obtain sequence information from orthologous regions of different genomes for comparison and scrutiny. We assume that embedded in the genomes of organisms are snippets of sequences (cis-regulatory elements) that, when such comparisons are made, will begin to reveal how genes and associated networks are intertwined and regulated. Fig. 4 shows an example of such long-range comparisons (graphical output of a global alignment between divergent taxa). Empirical studies still must be carried out to validate inferences made from these comparative genomic analyses as diagrammed in Fig. 3 (DiLeone et al., 2000; Mortlock et al., 2003; Juan and Ruddle, 2003). As we examine the diversity of life, it becomes evident that there are biological novelties at every major phylogenetic node, and that several of these characteristics are reminiscent of those possessed by bhigherQ vertebrates, namely humans. Table 2 lists several major chordate taxa (in

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Table 1 A list of BAC libraries generated from selected aquatic chordate speciesa Organism

Average insert size (kb)

Coverage

Site of construction and reference

Alligator mississipiensis (American alligator) Chrysemys picta (painted turtle) Silurana (Xenopus) tropicalis

Latimeria menadoensis (Indonesian coelacanth)

145 140 75 170 108 85 130 170

10 10 6.8 6.6 2 3 2 N10

Spheroides nephelus (pufferfish)

140

6–7

Takifugu rubripes (pufferfish) Chaenocephalus aceratus (Antarctic icefish) Notothenia coriiceps (Antarctic rock cod) Dissostichus mawsoni (Antarctic toothfish) Haplochromis chilotes (Lake Victoria cichlid)

80

9

130 128

7 10

150

12

65–194 190

6 – 65 10X

145 190 188

5 – 6 19.3 18.8

154 –177 115 40 (fosmid)

6.2 – 13.1 5 10

165 131 175 161

10.4 2.4 10 10.6

130 130 100 110 60 80 145 46

5–6 5 3 – 4 N20 1–2 10 13.7

80 142 130

10 17 13

VMRC, JGI (VMRC8) VMRC, JGI ISB-1 (CHORI) CHORI (CHORI-216) Research Genetics VMRC (RZPD-710) VMRC VMRC (VMRC4) (Danke et al., 2004) VMRC (RZPD-715) (Amemiya et al., 2001) HGMP-RC VMRC, in progress VMRC, in progress VMRC (VMRC13) Okada Laboratory (Watanabe et al., 2003) VMRC (VMRC14) HCGS, in progress (Katagiri et al., 2001) Volff laboratory (Froschauer et al., 2002) VMRC (RZPD-756) CHORI (CHORI-213) CHORI (CHORI-214) Reviewed in (Thorgaard et al., 2002) CHORI (CHORI-217) VMRC (RZPD-706) VMRC, in progress (sheared DNA from one animal) CHORI (CHORI-211, RZPD-736) CHORI (RZPD-728) Keygene (RZPD-735) CHORI (CHORI-212) HCGS, in progress VMRC VMRC9 VMRC VMRC VMRC CHORI, in progress VMRC (VMRC3) CHORI (CHORI-301, sheared library) CHORI, in progress VMRC (VMRC2) CHORI (CHORI-302) CUGI

Xenopus laevis Xenopus laevis-gilli

Astatotilapia burtoni (African cichlid) Metriaclima zebra (Lake Malawi zebra cichlid) Oreochromis niloticus (Nile tilapia), 4 libraries Xiphophorus maculatus (platyfish) Oryzias latipes (medaka) Gasterosteus aculatus (threespine stickleback) Salmo salar (Atlantic salmon) Oncorhynchus mykiss (rainbow trout), at least 4 libraries Oncorhynchus tshawytscha (chinook salmon) Danio rerio (zebrafish)

Ictalurus punctatus (channel catfish) Astyanax mexicanus (blind cavefish) Polypterus senegalis (bichir) Raja eglanteria (skate) Heterodontus francisci (horn shark) Petromyzon marinus (lamprey) Ciona intestinalis (tunicate) Ciona savignyi (tunicate) Oikopleura dioica (larvacean) Branchiostoma floridae (Amphioxus) Saccoglossus kowalevskii (acorn worm)

CHORI: The BACPAC Resources Center, Children’s Hospital Oakland Research Institute, Oakland, CA. www.chori.org/bacpac/home.htm. CUGI: Clemson University Genomics Institute, Clemson University, Clemson, SC. www.genome.clemson.edu. HCGS: Hubbard Center for Genomic Studies, University of New Hampshire, Durham, NH. http://hcgs.unh.edu/index.php. ISB: Institute for Systems Biology, Seattle, WA. www.systemsbiology.org. JGI: Joint Genome Institute, Walnut Creek, CA. http://www.jgi.doe.gov/programs/comparative/top_level/BAC.html. MRC HGMP-RC: UK Human Genome Mapping Project Resource Center, Cambridge, UK. www.hgmp.mrc.ac.uk. Research Genetics: Invitrogen Corporation, Carlsbad, CA.www.invitrogen.com. RZPD: Resource Center and Primary Database, Deutsches Ressourcenzentrum fur Genomforschung, Berlin, Germany. www.rzpd.de. Okada Laboratory: Department of Biological Sciences, Tokyo Institute of Technology, Tokyo, Japan. http://www.evolution.bio.titech.ac.jp/index_e.html. VMRC: Benaroya Research Institute at Virginia Mason, Seattle, WA. http://benaroyaresearch.org/bri_investigators/amemiya/default.htm. Volff Laboratory: Lehrstuhl Physiologische Chemie I, Biozentrum, University of Wurzburg, Am Hubland, Germany. http://www.biozentrum.uni-wuerzburg.de/ pc1/volff/index. a Note that this table is not an exhaustive listing of all BAC libraries and primarily lists those from the Amemiya laboratory and others that are publicly available. A compilation of all metazoan BAC libraries available to the scientific community that is updated regularly is available at http://www.genome.gov/ 11008350.

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Fig. 3. Flowchart for targeted comparative sequencing approach. In targeted comparative sequencing (Thomas et al., 2003), BAC libraries from different species are utilized in order to isolate specific regions that are orthologous to one another (1, 2). Sequences are obtained from respective BAC clones (3) and aligned and compared to the same region from other taxa (4). The results obtained in (4) allow testable inferences to be made so that functional experiments can be carried out in vivo and in vitro (5), particularly with regard to the regulation of genes and gene families. These experiments involve generation of transgenic reporter constructs and knock-in/knockout strategies for assessing functionality of genomic regions (Shashikant et al., 1998; DiLeone et al., 2000; Sumiyama et al., 2002; Mortlock et al., 2003; Juan and Ruddle, 2003).

a broad phylogenetic context) along with emergent phenotypic and/or developmental characters at each node. Can comparative genomics help to unravel evolution of morphological structures and innovations? And are we able to use comparative genomics of bancestralQ organisms in order to better understand form and function in more complex organisms such as humans? These questions lie at the heart of the newly revitalized field of evolution-developmental biology (evo-devo) and functional comparative genomics. One of the most powerful tools for evolution-developmental research is to screen and characterize gene clusters from a BAC library, including flanking regions of coding sequences that are known to contain conserved non-coding

sequences and to function as cis-regulatory elements. As shown in Fig. 3, once gene clusters are screened from a BAC library, genome-wide comparisons enable us to characterize gene clusters, to identify conserved non-coding regions and to allow functional studies of coding and noncoding sequences in animal models (Brown et al., 2002; Martinez and Amemiya, 2002; Miyake et al., 2002). There are a number of examples in which BAC libraries have been utilized to carry out a multitude of studies as described above: Hox gene and their clusters in fishes (Amores et al., 1998; Kim et al., 2000; Chiu et al., 2004; Amores et al., 2004), Dlx gene family in mammals (Miyake et al., 2002), novel immune-type receptor family in teleost fish (NITRs)

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Fig. 4. Comparative sequence analysis of an extended region in the zebrafish genome. The complete sequence of a BAC clone [BUSM 179B16 (Amemiya and Zon, 1999), 107286 bp] was obtained from GenBank (AL954668). This sequence was generated by the Sanger Centre as part of the zebrafish genome initiative. The sequence was used in a GenomeVista analysis in order to determine its syntenic relationship to other genomic sequences (Lawrence Berkeley National Laboratory; http://www-gsd.lbl.gov/vista/index.shtml), namely human, mouse and rat. The multiple VISTA plot shows a portion of the extended zebrafish region that was globally aligned with the human orthologous region on chromosome 17 (plot number 1). Two other plots are shown: (2) human vs. mouse; and (3) human vs. rat. The numbers below the plot indicate the relative position on human chromosome 17. There are two genes whose alignments are clearly shown: MYH1 and MYH2, myosin heavy chain 1 and myosin heavy chain 2, respectively. The blue boxes in each represent the exon sequences whereas the arrowheads denote transcriptional orientation. Peaks within the plots represent levels of nucleotide identity between the species being compared; blue peaks represent sequence identity within exons whereas pink peaks represent sequence identity in noncoding regions that potentially represent cisregulatory elements. As can be seen, there are considerably higher levels of noncoding sequence identity between human and mouse (2) and between human and rat (3), and that outside of the exons, no identity exists between zebrafish and human. This is not always the case and is somewhat dependent on the particular gene being examined. In some instances such as the HOX clusters, there is considerable conservation of noncoding sequences between organisms as phylogenetically disparate as sharks and tetrapods (Kim et al., 2000; Chiu et al., 2002). The functionality of the putative cis-regulatory regions must be assessed via in vitro and in vivo experiments.

(Strong et al., 1999; Yoder et al., 2001), VLR (variable lymphocyte receptor) adaptive immune system in lampreys (Pancer et al., 2004). Among the examples given above, BAC libraries have played a major role in characterizing Hox clusters from different chordate animals (Martinez and Amemiya, 2002). A discovery of seven to eight clusters of Hox genes in bony fishes has sparked intensive efforts to investigate the evolution and functional roles of genome-wide duplication of Hox genes in lower chordate species (Amores et al., 1998; Chiu et al., 2002; Force et al., in press). It has been shown that all duplicated Hox genes are not necessarily preserved—some have been lost and others been preserved as a result of the partitioning of gene subfunctions between gene duplicates (Force et al., 1999; Force et al., in press). In order to examine the genomic structure of Hox clusters in a group of phylogenetically advanced teleost fishes, the pufferfishes, characterization of BAC contigs (Amemiya et al., 2001) as well as data mining were used to reveal structural changes as a result of teleost-specific genome duplication (Amores et al., 2004). The comparative analysis of cluster organization among vertebrates revealed that a disproportionate number of Hox genes were lost in pufferfishes (and other teleosts) but that the expression patterns of

retained duplicates often exhibited notable differences between duplicates. Recently, hitherto unidentified vertebrate Hox14 genes (Hoxa14 in the Indonesian coelacanth Latimeria menadoensis and Hoxd14 in the horn shark Heterodontus francisci) were discovered as a result of sequence analysis of clusters that have been screened and characterized from BAC libraries (Danke et al., 2004; Powers and Amemiya, 2004). These genes are noteworthy as they are highly suggestive that the ancestral vertebrate HOX clusters possessed an additional bposteriorQ gene (Hox14), not the 13 paralog group genes as previously believed. The implication from all these data is that dynamic changes of Hox duplicates might have contributed to developmental diversification of morphologies in the evolution of bony fishes. Since Hox genes have been shown to regulate the axial patterning and limb development, evolutionary changes of Hox gene organization and their function will provide clues for deciphering the underlying genetic and evolutionary mechanisms for emerging phenotypes at respective evolutionary nodes (Wagner et al., 2003), i.e., the innovation of jaws in gnathostomes (jawed vertebrates) (Cohn, 2002; Takio et al., 2004) and a transition of fins to limbs in tetrapods (Clark, 2002) (Table 2).

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Table 2 Broad classification of extant chordates, with a listing of phenotypes that are emergent at each node of chordate evolution

Table 2 (continued) Taxa

Notable characteristics, including those which are germane to humans and medicine

Taxa

Notable characteristics, including those which are germane to humans and medicine

Ray-finned fishes

Hemichordates

Neural tube (Holland and Graham, 1995; Hall, 1999), notochord (Hall, 1999), pharyngeal slits. Endodermal fore- and midgut (Lehman, 1983), expression of a series of genes that indicate initial evolution of neural crest primordial cells (BMP-7, (Hammerschmidt et al., 1996; Miya et al., 1996, 2001) and development of primordial facial structures (Pax-3, Pax-7; (Wada et al., 1996), the endostyle homologous with the thyroid gland (Dehal et al., 2002), development of the heart with expression of a series of genes analogous to development of the vertebrate heart (Dehal et al., 2002). Brain developed into three or more distinct regions, metameric organization of the pharyngeal arches (Lehman, 1983), putative cranial neural crest dcellsT (Holland and Garcia-Fernandez, 1996; Wada et al., 1996; Hall, 1999), segmented axial muscles in the trunk (Lehman, 1983). Three sensory organs (a single nose, eyes without eye lens and inner ears) derived from placodes (Forey and Janvier, 1993), the earliest sign of pituitary development (Gorbman and Tamarin, 1985; Sower, 1998). Cranial skeletons derived from neural crest and mesodermal cells (Northcutt and Gans, 1983; Hall, 1998; Smith and Hall, 1990, 1993), Meckel’s cartilage in the mandibular arch (Hall, 1984, 1998), a unique set of sensory and motor cranial nerves (Forey and Janvier, 1993; Janvier, 1996), cranial muscles including extrinsic eye muscles (Miyake et al., 1992; Holland et al., 1993), at least two semicircular canals in the labyrinth, development of neuromasts, atrium and ventricle of heart, neural innervation of the heart in adults, metameric axial skeletons derived from somites (Smith and Hall, 1990, 1993), nephrons as a functional excretory organ. Development of the jaws (palatoquadrate and Meckel’s cartilage in the lower and upper jaw, respectively) (Carroll, 1988), dentine and enameloid in teeth and scales (Smith and Hall, 1990, 1993; Hall, 1998; Miyake et al., 1999), type II collagen as the major matrix protein in cartilages Miyake et al., 1999), true lymphoid organs including thymus and a tissue equivalent to bone marrow (Hansen and Zapata, 1998; Zapata and Amemiya, 2000).

Tooth bearing dermal bones on the gape of the jaws (De Beer, 1985), the palate formed by the perichondrally ossified palatoquadrate and a series of dermal bones on the oral cavity not on the gape of the upper jaw (De Beer, 1985; Schultze, 1993), enamel in scales (Polypteridae and probably Amia and gars) but lost in most of ray-fin fishes (Sire, 1989; Zylberberg et al., 1997), mineralized cranial and axial skeletons (Smith and Hall, 1990, 1993), dermal fin rays made up by lepidotrichiae, very small vertebrate genomes (Brenner et al., 1993; Baxendale et al., 1995; Elgar et al., 1996). Endoskeletal supports in pectoral fins (a transition from multiple elements to single element (dhumerusT) articulating with girdle (Cloutier and Ahlberg, 1996), enamel in teeth (Smith, 1978), lung-like structures (Helfman et al., 1997). Features adapted for life on land: limbs, cranial and axial skeletons, respiration, water balance, sensory input (Nelson, 1984; Carroll, 1988). Extraembryonic membrane: allantois, chorion and amnion (Carroll, 1988), the articular–quadrate joint, development of the tympanum (Lombard and Bolt, 1979). Feathers, true flight capability associated with morphological changes, warm-bloodedness (Carroll, 1988). Enucleated erythrocytes, a lateral temporal fenestra in the skull, fur (Carroll, 1988).

Urochordates

Cephalochordates

Hagfish

Lamprey

Chimeras, sharks and rays

Lobe-finned fishes

Amphibians

Reptiles

Birds

Mammals

In many instances, the emergent characters are shared with higher vertebrates (humans, mice).

There is no doubt that comparative genomics can unveil hidden signatures in the genome and that such signatures may serve as targets for therapy and/or drug intervention in the case of humans (Stevens et al., 2001). This realization has largely been responsible for the enthusiasm for generating BAC resources by NIH and has also resulted in a large all-encompassing initiative, ENCODE (ENCyclopedia Of DNA Elements), which seeks to delineate all the noncoding regulatory elements in the human genome (Collins et al., 2003). The initiative utilizes comparative genomics on a very large scale to initially predict those DNA elements that putatively encode functions, and to develop methods to survey whether or not the predictions are accurate. While the community has previously relied on relatively labor-intensive means to evaluate the biological activity of conserved DNA elements (i.e., transgenesis, knock-in/knockout technology), such methods are clearly insufficient for the daunting abundance of data that are being generated by these large-scale efforts. Ironically,

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although the human genome is completely sequenced, the difficult task in deciphering how the genome actually works is only in its infancy, a most exciting time indeed.

5. Summary BAC libraries are collections of large exogenous DNA inserts cloned into stable plasmid vectors and propagated in E. coli. The libraries are extremely useful for biological investigation and programs have been initiated to develop more BAC resources for the community. BACs have become exceedingly useful for the comparative genomic approach wherein orthologous regions are compared and contrasted between different taxa in order to begin to understand the logic of genome organization and regulation. This approach has garnered much interest in both the evolution and biomedical communities since it unveils a new paradigm for studying the regulation of the genome and how genomic changes in non-coding sequences may effect physiological and anatomical change.

Acknowledgements We thank the members of the Amemiya laboratory. Our laboratory is funded, in part, by National Institutes of Health (RR14085, HG02526-01), the National Science Foundation (IBN-0207870, IBN-0321461) and the United States Department of Energy (DE-FG03-01ER63273).

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