Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey
© Luo et al; licensee BioMed Central Ltd. 2006
Received: 24 December 2005
Accepted: 03 May 2006
Published: 03 May 2006
Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates.
In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum.
The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible.
We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.
Bacterial artificial chromosome (BAC) libraries are indispensable for many applications in genomic studies [1–3]. BAC-end sequences have been used to develop sequence-tagged connector (STC) frameworks [4, 5], to survey genome structures , and for comparative analysis of gene structure and synteny. Although the whole-genome shotgun method has been used to produce genome draft sequences , mapped BACs usually have been required to provide a framework for sequence assembly and templates to complete the sequences of complex genomes [3, 5, 8]. BACs and BAC-based maps have also been used in sequencing of targeted genome regions , chromosomal landing or positional cloning , genome function investigation , and evolutionary and comparative studies . Through BAC sequencing, a full-length avian androgen receptor gene was identified, which had not been detected with conventional methods .
Sharks belong to the phylogenetic taxon comprising the oldest jawed vertebrates, the cartilaginous fish, which diverged from the common ancestor of all other jawed vertebrates 460–520 million years ago . Genomic and genetic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. This ancient taxon is of particular interest to us since it is the oldest group of living animals having an adaptive immune system with underlying molecules and mechanisms similar to those of mammals . While most sharks and other cartilaginous fish have large genome sizes (~80% of species studied have larger genome sizes than human, with some up to 5 times as large), the nurse shark genome size is relatively small at 4 × 109 bp/haploid genome, only slightly larger than that of humans (3.4 × 109). Thus, the nurse shark is a candidate to become a model species in the biomedical and genomics fields. However, most of the shark genes and intergenic regions are much larger than those of mammals, and thus large-insert genomic libraries are essential to obtain sufficient genomic information. Previously, BAC libraries of other cartilaginous fish (clearnose skate and horn shark)were described by Miyake and Amemiya . In this paper we report the construction and characterization of a publicly-available nurse shark BAC library and carry out a preliminary genome survey.
Results and discussion
BAC library construction
We constructed a nurse shark BAC library, consisting of a total of 313,344 clones that were deposited in 816 barcode-ordered 384-well microtiter plates. To complete this large library, approximately 20 ligations were performed. Since colonies with a diameter of <1.5 mm did not grow well in the freezing media, only those colonies with a diameter of >1.5 mm on selection-agar plates were picked.
Insert size distribution
BAC end sequence analysis
We sequenced the first 96 of the above 408 sampling clones at both ends. One hundred seventy-seven BAC end sequences (BES) were obtained with an average high-quality base-pair (bp) number of 548 bp ([GenBank:CZ549372~CZ579549], excluding [GenBank:CZ549507] which is the vector sequence), covering a total of ~100,000 bp. The average GC content of the BES is 41%, ranging from 26–66%. Sequences from other parts of the nurse shark genome (~300 kb) have an average of 42–45% GC content (YO, MFF; personal observation), which is consistent with this finding. It is worth noting that the GC content was calculated in windows of the BES lengths, and some smaller regions may contain higher GC content. Indeed, we previously detected simple repeats with a high GC content (70–80%) in a region of the Major Histocompatibility Complex (MHC) class I gene . Analysis of the BES also revealed 4 sequences containing significantly long microsatellite repeats (simple sequence repeats, SSRs): [GenBank:CZ549546] (AG)9, [GenBank:CZ549467] (AG)19, [GenBank:CZ549532] (AT)9 and [GenBank:CZ549547] (AC)14. However, no significant tri-nucleotide or longer motifs of SSRs were found in our BES. The CpG dinucleotide frequency was under-represented (the observed value is only 1/4 of the expected one) while the AA and TT dinucleotide frequencies were over-represented in this set of BES.
The classification of BES.
Number of BES
NSRE1 and NSRE2 analysis
BAC library screening with gene-specific probes
Number of gene-specific clones in the nurse shark BAC library Single copy genes.
Single copy genes
Number of positive clones
Colony hyb./Library scr.
MHC class I
Possibly two genes in tandem or close vicinity
Colony hyb./Library scr.
MHC class IIα
MHC class IIβ
Colony hyb./Library scr.
Unlike most other species, the nurse shark MHC genes are much (3–5 times) larger than those of mammals and have even larger distances in intergenic regions . Previously, we constructed a genomic cosmid library with an average insert size of ~40 kb. However, most cosmid clones contain at most a single gene (data not shown). In this study, we used several linked genes (TAP1, MHC class I, class II, Factor B, LMP7, LMP2 [17, 24], and Ring3 (unpublished data)) to quickly glean the number of genes in a single BAC clone; we found up to four genes in a single BAC clone, making physical mapping possible. Thus far, our analysis has convincingly shown that the BAC library is a useful tool (and perhaps the only way) to obtain genetic information for this species.
We report in this paper a large insert, deep-coverage and high-quality BAC library for a cartilaginous fish that will be very useful to the scientific community for gene isolation, genetic analysis, and comparative genomics. We found two new groups of repetitive elements, designated as NSRE1 and NSRE2, which are specific to the nurse shark genome. These repetitive elements may contribute to the architecture and evolution of the nurse shark genome. The BAC library, HDR filters and individual clones are available to the public from the Arizona Genomics Institute's BAC/EST Resource Center .
Isolation of high molecular weight DNA
Blood was obtained from the nurse shark individual "Yellow" using a Heparinized 18G 1 1/2" needle from the caudal vein. To obtain ~30 micrograms of DNA per 80-μl agarose plug, approximately 4 × 106 erythrocytes were embedded in 1% InCert agarose (FMC, Rockland, ME) prepared in 1/2x PBS and molded in ~200 Plug Molds (BIO-RAD, Hercules, CA). Twenty plugs were then submerged in 50 ml of cell lysis solution (1% lithium dodecyl sulfate, 10 mM Tris, pH8.0, 100 mM EDTA, pH8.0) and incubated overnight at 37°C with occasional swirling. The cell lysis solution was replaced with 50 ml 20% NDS (0.2% N-lauroylsarcosine, 2 mM Tris, pH9.0, 0.14M EDTA, pH9.0), and DNA plugs were shaken gently at room temperature for two hours, and then kept at 4°C .
BAC vector preparation
We used a modified version of the BAC vector pBeloBAC11, pIndigoBAC536Swa. A first modified version of pBeloBAC11 [GenBank:U51113], pIndigoBAC536, was a gift from Dr. H. Shizuya of Caltech. pIndigoBAC536 has the internal Eco R1 site of pBeloBAC11 destroyed so that the unique Eco R1 site in the multiple cloning sites can be used for cloning, and also contains a random point mutation in the lac Z gene that provides colonies with a darker blue-color on X-gal/IPTG selection. We further inserted two Swa I sites (ATTTAAAT) near and internal to the two Not I sites of pIndigoBAC536 (this new version is named pIndigoBAC536Swa) to facilitate insert-size estimation of clones from GC-rich organisms (Luo et al, unpublished data). We then cloned this single-copy BAC vector pIndigoBAC536Swa into the high-copy vector pGEM-4Z (this composed high-copy plasmid is named pAGIBAC1) to facilitate the preparation of the single-copy BAC vector as we did for pIndigoBAC536 (the composed high-copy pIndigoBAC536-pGEM-4Z plasmid is named pCUGIBAC1) . pCUGIBAC1 is available through Clemson University Genomics Institute  and pAGIBAC1 is available through Arizona Genomics Institute . The linearized and dephosphorylated single-copy BAC vector pIndigoBAC536Swa can be prepared from the high-copy pAGIBAC1 according to our previously published method .
Generation and size selection of large DNA fragments for BAC cloning
Large genomic DNA fragments for BAC cloning were prepared according to our previously published method . The DNA-agarose plugs were washed thoroughly with TE buffer (10 mM Tris/1 mM EDTA, pH8.0) and stored in 70% ethanol at -20°C. A desired number of DNA plugs were transferred to TE buffer the day before use and kept at 4°C overnight. The DNA plugs were test-digested with various amount of Hin dIII (1–50U) for 20 minutes at 37°C to optimize partial digestion conditions and the fragmented DNAs were separated on 1% agarose gels by Pulsed Field Gel Electrophoresis (PFGE) (CHEF Mapper, BIO-RAD) at 1–50 sec linear ramp, 6 volts/cm, 14°C in 0.5X TBE buffer for 18–20 hours. Bulk digestions were then carried out using the conditions that produced the most DNA fragments in the range of 100–400 kb. Fragmented DNAs were separated on a 1% CHEF gel in the same conditions described above. DNA fractions ranging from 150–250 kb and 250–350 kb were excised from the gel and subjected to a second size selection on a 1% CHEF gel at 4 sec constant time, 6 volts/cm, 14°C in 0.5X TBE buffer for 18–20 hours. DNA fragments were electroeluted with dialysis tubing as described by Strong et al  or with an Electro-eluter Model 422 (BIO-RAD) following the manufacture's instructions. DNA concentrations were determined on subsequent agarose gels.
Ligation and transformation
One hundred to two hundred nanograms of size-selected DNA fragments were ligated with 20 nanograms of dephosphorylated BAC vector in a 100 μl of volume at 16°C overnight. The ligation reactions were terminated at 65°C for 15 min, and the ligation products were desalted in 0.1 M glucose : 1% agarose cones for 1.5 hours on ice as described by Atrazhev and Elliott  and electroporated into the E. coli strain DH10B T1 phage resistant (F-mcr A Δ(mrr-hsd RMS-mcr BC) φ80dlac Z ΔM15 Δlac X74 deo R rec A1 end A1 ara D139 Δ(ara, leu) 7697 gal U gal K λ-rps L nup G) electrocompetent cells (Invitrogen, Carlsbad, CA). Transformants were grown on LB plates supplemented with 12.5 mg/L chloramphenicol, 80 mg/L X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside or 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and 100 mg/L IPTG (Isopropyl-beta-D-thiogalactoside or Isopropyl-beta-D-thiogalactopyranoside) at 37°C overnight.
Library arraying and high density replica (HDR) filters
A total of 313,344 individual recombinant clones (white color on X-gal plates) were picked robotically (Genetix, New Milton, UK) and arrayed into 816 barcode-ordered 384-well microtiter plates containing freezing media (10 g/L Bacto tryptone, 5 g/L Bacto yeast extract, 10 g/L NaCl, 36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM Na-citrate, 6.8 mM (NH4)2SO4, 4.4% glycerol, autoclaved and added filter-sterilized MgSO4 solution to the final concentration of 0.4 mM) supplemented with 12.5 mg/L of chloramphenicol. After an overnight incubation at 37°C, empty wells were back-filled manually and duplicate copies were replicated. The master library and the two copies were then stored in -80°C freezers at different locations. The whole BAC library was gridded onto 17 22.5 cm × 22.5 cm Hybond N+ membrane filters (Amersham, Piscataway, NJ) in high density, double spots, and 4 × 4 patterns with Genetix Q-bots (Genetix). Each 22.5 cm × 22.5 cm filter supports 18,432 clones in duplicate in 6 fields. The filters were placed on LB media supplemented with 12.5 mg/L of chloramphenicol and incubated overnight at 37°C. The filters were then soaked in 0.5N NaOH/1.5M NaCl for 7 min, in 1.5M NaCl/0.5M Tris-HCl (pH8) for 7 min, air dried for 1–2 hours, soaked in 0.4N NaOH for 20 min, in 20x SSPE for 7 min, and air-dried overnight.
DNA analysis of BAC clones
BAC DNAs were extracted with Tomtec Quadra 96 model 320 (Tomtec, Hamden, CT) in a 96-well format at AGI. The 408 sampling BAC clones were selected by picking one clone from A01 position of every other 384-well plate of the library and arranged in the same order of the library plates. Inserts were liberated by digesting with Not I or Swa I and their sizes were determined on CHEF gels.
BAC end sequencing
BAC DNAs were sequenced at both ends using BigDye Terminator v.3 (Applied Biosystems, ABI, Foster City, CA) according to manufacturer's instruction. The T7 primer (5' TAA TAC GAC TCA CTA TAG GG 3') was used as the "forward" primer and the BES_HR primer (5' CAC TCA TTA GGC ACC CCA 3') was used as the "reverse" primer. Cycle sequencing was performed using PTC-200 thermal cyclers (MJ Research, Waltham, MA) in a 384-well format with the following regime: 150 cycles of 10 sec at 95°C, 5 sec at 55°C, and 2.5 min at 60°C. After the cycle-sequencing step, the DNA was purified by magnetic beads, CleanSeq (Agencourt, Beverly, MA) according to manufacturer's instruction. Samples were eluted into 20 μl of water and separated on ABI 3730xl DNA capillary sequencers with default conditions. Sequence data was collected by data collection software (Applied Biosystems), extracted using sequence analysis software (Applied Biosystems) and transferred to a UNIX workstation. Sequences were base-called using the program Phred [31, 32]; vector and low-quality (Phred value <16) sequences were removed by CROSS_MATCH [31, 32].
Bioinformatics analyses of sequences
Similarity searches against public GenBank and in-house database were carried out using the Blast algorithm. Composition analyses as well as searches for inverted repeats were done using the programs "composition" and "palindrome" respectively, both of which are included in the package EMBOSS . SSR were searched using the software "Sputnik" .
The nucleotide sequences of both NSRE1 and NSRE2 were aligned using ClustalW. NSRE1 motifs were extracted from BES and full-length BES sequences were aligned for NSRE2.
Southern blotting for NSREs
Five μg genomic DNAs were digested with 80 units of restriction enzyme Hin dIII for 6 hours at 37°C. DNA fragments were separated in a 0.8% agarose gel by electrophoresis and blotted onto a nylon membrane. Overlapping oligonucleotide (overgo) hybridization was performed according to Ross et al  with modifications. The NSRE1 and NSRE2 overgo probes were designed from the sequence [GenBank:AF357922] nt positions 2–103 region and sequence [GenBank:AF357928] nt position 4406–4529 region respectively. Primers used for NSRE1 are: 5' TCT CGG CCC GAA ACG TCA GCT TTC 3' and 5' AGC ATC AGA GGA GCA CGA AAG CTG 3'. Primers used for NSRE2 are: 5' TGC TGT TCC TGC AAC CTT CGG GTA 3' and 5' AAT GCC ACA ACG ACG CTA CCC GAA 3'. Each set of primers overlaps by 8 base pairs. Probes were labeled with both 32P-dCTP and 32P-dATP using Klenow enzyme. Hybridization was carried out overnight in a solution containing 1% Bovine Serum Albumin (BSA), 1 mM EDTA pH8.0, 7% SDS, 0.5M sodium phosphate at 60°C. Membranes were washed in 4x SSC, 0.1% SDS at room temperature, followed by 1.5x SSC, 0.1% SDS at 60°C. Membranes were exposed to screens and scanned using the phosphor imager.
BAC sequencing and assembly
The BAC clone GC_Ba0754I06 that covered 170 kb was bidirectionally shotgun sequenced with an average redundancy of about 6, which was sufficient for assembly and analysis of the entire sequence using previously established procedures . The draft sequence was searched using Blast2 for NSRE1 and NSRE2, BlastN and BlastX against the 'nr' database in GenBank for SINEs/LINEs and CR1-like SINEs/LINEs, respectively. During the BlastX search, at least 27 exons were identified with significant similarity to other species' fatty acid synthase (FASN) exons (E-values of 3e-56 and 3e-46 to chicken [GenBank:AAB46389] and rat [GenBank:AAA41145], respectively).
BAC library screening
The seventeen HDR filters of the BAC library were pre-hybridized in high-stringency hybridization solution (50% Formamide, 6x SSC, 0.5% SDS, 5x Denhardt's solution)  supplemented with 100μg/ml of denatured salmon sperm DNA for ~4 hours at 42°C. Probes (50 ng) were radiolabeled using the random-priming method (Roche, Indianapolis, IN) or by incorporating α32P-dCTP in the polymerase chain reaction (PCR) . Hybridization was performed overnight at 42°C and membranes were washed at room temperature in pre-warmed (42°C) 2x SSC/1% SDS for 20 minutes, followed by washing in 0.2x SSC/0.1% SDS for 20 minutes at 65°C. Membranes were exposed to X-ray film for various lengths of time to obtain positive signals and the desired background. General protocols for high-density BAC library filter screening and address determination of positive signals are publicly available from our website . Putative positive clones were re-spotted on nylon membranes for colony hybridization to confirm true positives. For hybridization with NSRE overgo probes, we performed in the same hybridization solution as described above for Southern Blotting. Filters were washed in 4x SSC, 0.1% SDS at room temperature, followed by 0.75x SSC, 0.1% SDS at 60°C, exposed to screens, and scanned using the phosphor imager.
We thank Samina Makda, Miriam Eaton, Olin Feuerbacher, Marina Wissotski, Michaela Byrne, Daniel Smart, Diana Stum-Partney and Angelina Angelova for technical assistance and Kiran Rao for data base management. This work was supported by grants from NIH (grants U1HG02525A and AI27877).
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