Isolation and chromosomal assignment of canine genomic BAC clones representing 25 cancer-related genes

An extensive number of genes have been implicated in the initiation and progression of human cancers, aiding our understanding of the genetic aetiology of this highly heterogeneous disease. In order to facilitate extrapolation of such information between species, we have isolated and physically mapped the canine orthologues of 25 well-characterised human cancer-related genes. The identity of PCR products representing each canine gene marker was first confirmed by DNA sequencing analysis. Each product was then radiolabelled and used to screen a genomic BAC library for the domestic dog. The chromosomal location of each positive clone in the canine karyotype was determined by fluorescence in situ hybridisation (FISH) onto canine metaphase preparations. Of the 25 genes, the FISH localisation of 21 correlated fully with that expected on the basis of known regions of conserved synteny between the human and canine genomes. Three correlated less closely, and the chromosomal location of the remaining marker showed no apparent correlation with current comparative mapping data. In addition to generating useful comparative mapping information, this panel of markers will act as a valuable resource for detailed study of candidate genes likely to be involved in tumourigenesis, and also forms the basis of a canine cancer-gene genomic microarray currently being developed for the study of unbalanced genomic aberrations in canine tumours.     

Rapid chromosomal assignment of multiple genomic clones in tomato using primary trisomics.

In order to rapidly assign relatively large numbers of tomato genomic clones to specific chromosomes, we have developed the following approach: groups of five to eight clones from a single copy tomato library are pooled, nick-translated, and utilized as probes against Southern blots consisting of a panel of trisomic DNAs. Since the trisomic DNAs are digested with the same enzyme used to produce the genomic library (PstI), each hybridizing band can be related to a specific genomic clone and the relative intensities of the bands can be used to assign each to a specific chromosome. With this technique, we have assigned 52 clones to specific chromosomes and verified the assignment of 21 out of 23 by genetic mapping in a segregating F2 population. In addition to selecting clones according to chromosome, the idea of using multiple clones or "molecular darts" may have broader applications, such as screening for differences between the genomes of nearly isogenic lines.     

FISH mapping and sequence analysis of 87 porcine BAC clones

Ninety-one bacterial artificial chromosomes (BAC) clones, selected effectively at random from our library, were used as probes for fluorescence in situ hybridization. Of these, 87 clones gave a specific signal in one, two or three different pair(s) of swine metaphase chromosomes. The ends of 35 BAC clones were sequenced in order to obtain information for comparative mapping. Fifteen of them gave useful comparative mapping information.     

Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers

A horse BAC library was constructed with about 40, 000 clones and mean insert size of 110 kb representing a 1.5 genome equivalent coverage and a probability of finding a single sequence of 0.75. It was characterized by PCR screening of about 130 sequences of horse microsatellites and exonic gene sequences retrieved from databases. BACs containing 8 microsatellites and 12 genes were subsequently localized by fluorescent in situ hybridization (FISH) on chromosomes. Two linkage groups were newly assigned to chromosomes: LG2 to ECA3 and LG5 to ECA24, and five linkage groups were also oriented—LG3, LG4, LG5, LG8, and LG12—leaving only three groups unassigned. This work showed how this library makes an integrated map a realistic objective for the near future and how it can make comparative mapping more efficient in a search for candidate genes of interest. Received: 9 January 1998 / Accepted: 13 April 1998     

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-Pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.     

Construction and characterization of a peanut HindIII BAC library

Bacterial artificial chromosome (BAC) libraries have been an essential tool for physical analyses of genomes of many crops. We constructed and characterized the first large-insert DNA library for Arachis hypogaea L. The HindIII BAC library contains 182,784 clones; only 5,484 (3%) had no inserts; and the average insert size is 104.05 kb. Chloroplast DNA contamination was very low, only nine clones, and r-DNA content was 1,208, 0.66% of clones. The depth of coverage is estimated to be 6.5 genome-equivalents, allowing the isolation of virtually any single-copy locus. This rate of coverage was confirmed with the application of 20 overgos, which identified 305 positive clones from the library. The identification of multiple loci by most probes in polyploids complicates anchoring of physical and genetic maps. We explored the practicality of a hybridization-based approach for determination of map locations of BAC clones in peanut by analyzing 94 clones detected by seven different overgos. The banding patterns on Southern blots were good predictors of contig composition; that is, the clones that shared the same size bands and ascribed to the same overgos usually also located in the same contigs. This BAC library has great potential to advance future research about the peanut genome.Requests for the BAC library (or subsets) should be directed to Dr. A. Paterson (paterson@uga.edu).     

Construction and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).     

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.     

Construction of an arabidopsis BAC library and isolation of clones hybridizing with disease-resistance,gene-like sequences

A bacterial artificial chromosome (BAC) library consisting of 9,000 clones (representing a 4.5 genome equivalents) with an average DNA insert size of 60 kb was constructed from arabidopsis nuclear DNA. We have demonstrated the usefulness of this library by identifying one BAC clone that hybridizes with the arabidopsisPHYB gene and seven clones, representing four distinct classes, that hybridize to a putative disease-resistance gene. This library represents an additional resource for map-based cloning and physical mapping in arabidopsis.     

Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

 To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18 432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364 kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby. Received: 15 November 1996 / Accepted: 24 January 1997     

Construction and characterization of the IGF Arabidopsis BAC library

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10?752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100?kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180?bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80?kb?<85% <120?kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.     

Analysis of zebrafish Mhc using BAC clones

 Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes. Received: 14 August 1997 / Revised: 16 September 1997     

Construction and characterization of a Lipotes vexillifer genomic DNA BAC library

We constructed a genomic DNA library for Lipotes vexillifer (L. vexillifer), the Baiji or Yangtze River dolphin, one of the most endangered mammals in the world. The library consists of 149,000 BAC clones, with an average insert size of 83 kb, representing approximately 3.4 haploid genome equivalents. PCR amplification of four known L. vexillifer genes yielded two to four positive clones each. To demonstrate the utility of this library, we isolated and sequenced the L. vexillifer alpha lactalbumin gene, which is a gene specific to mammals and one which has been widely used as molecular tool in phylogenetic analysis. We also end-sequenced 20 randomly selected clones, resulting in the identification of at least five new L. vexillifer genes, five SSR loci, and one SINE locus. These results suggest that this library is a valuable resource for candidate gene cloning, physical mapping, and genome sequencing of this important and threatened species.     

Construction of BAC library for the amphibian Xenopus tropicalis

Xenopus tropicalis has become an alternative model to the amphibian Xenopus laevis because it is better suited for genetic and genomic studies. We have constructed a genomic BAC library consisting of over 100,000 clones from sperm of Xenopus tropicalis. Analysis by pulsed field gel electrophoresis of representative BAC clones indicated the average size of insert DNA to be 100 kb, and we estimated the library covers 6 times the Xenopus tropicalis genome of 1.7 x 10(9) base pairs. To evaluate the BAC library, we attempted to isolate BAC clones which contain a protocadherin gamma (Pcdh gamma) gene and found that the isolated BAC clones are assembled as two separate contigs. This result suggests the presence of at least two clusters for the Pcdh gamma gene in the genome of X. tropicalis.     

Construction and characterization of a sheep BAC library of three genome equivalents

A sheep BAC library of over three genome equivalents was constructed and arrayed in superpools and row, column, and plate pools. The library contains 90,000 clones distributed in 39 superpools. The average insert size was estimated at 123 kb. The library was screened by PCR with 77 primer pairs corresponding to ovine microsatellites distributed throughout the genome. The probability of finding a random sequence in the library could be estimated at 0.96. Received: 2 November 1998 / Accepted: 29 January 1999     

Construction of a BAC library of pearl millet, Pennisetum glaucum

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe. Received: 30 January 2000 / Accepted: 16 October 2000     

FISH mapping of 57 BAC clones reveals strong conservation of synteny between Galliformes and Anseriformes

Karyotypes of chicken (Gallus gallus domesticus; 2n = 78) and mallard duck (Anas platyrhynchos; 2n = 80) share the typical organization of avian karyotypes including a few macrochromosome pairs, numerous indistinguishable microchromosomes, and Z and W sex chromosomes. Previous banding studies revealed great similarities between chickens and ducks, but it was not possible to use comparative banding for the microchromosomes. In order to establish precise chromosome correspondences between these two species, particularly for microchromosomes, we hybridized 57 BAC clones previously assigned to the chicken genome to duck metaphase spreads. Although most of the clones showed similar localizations, we found a few intrachromosomal rearrangements of the macrochromosomes and an additional microchromosome pair in ducks. BAC clones specific for chicken microchromosomes were localized to separate duck microchromosomes and clones mapping to the same chicken microchromosome hybridized to the same duck microchromosome, demonstrating a high conservation of synteny. These results demonstrate that the evolution of karyotypes in avian species is the result of fusion and/or fission processes and not translocations.     

Construction and characterization of a bovine BAC library with four genome-equivalent coverage

A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents.