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PubMed2005

Mapping segmental and sequence variations among laboratory mice using BAC array CGH.

We used arrays of 2069 BACs (1303 nonredundant autosomal clones) to map sequence variation among Mus spretus (SPRET/Ei and SPRET/Glasgow) and Mus musculus (C3H/HeJ, BALB/cJ, 129/J, DBA/2J, NIH, FVB/N, and C57BL/6) strains. We identified 80 clones representing 74 autosomal loci of copy number variation (|log(2)ratio| >/= 0.4). These variant loci distinguish laboratory strains. By FISH mapping, we determined that 63 BACs mapped to a single site on C57BL/6J chromosomes, while 17 clones mapped to multiple chromosomes (n = 16) or multiple sites on one chromosome (n = 1). We also show that small ratio changes (Delta log(2)ratio approximately 0.1) distinguish homozygous and heterozygous regions of the genome in interspecific backcross mice, providing an efficient method for genotyping progeny of backcrosses.

Animals↗
PubMed2005

Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors.

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.

Animals↗
PubMed2005

Chromosomal mapping of 170 BAC clones in the ascidian Ciona intestinalis.

The draft genome ( approximately 160 Mb) of the urochordate ascidian Ciona intestinalis has been sequenced by the whole-genome shotgun method and should provide important insights into the origin and evolution of chordates as well as vertebrates. However, because this genomic data has not yet been mapped onto chromosomes, important biological questions including regulation of gene expression at the genome-wide level cannot yet be addressed. Here, we report the molecular cytogenetic characterization of all 14 pairs of C. intestinalis chromosomes, as well as initial large-scale mapping of genomic sequences onto chromosomes by fluorescent in situ hybridization (FISH). Two-color FISH using 170 bacterial artificial chromosome (BAC) clones and construction of joined scaffolds using paired BAC end sequences allowed for mapping of up to 65% of the deduced 117-Mb nonrepetitive sequence onto chromosomes. This map lays the foundation for future studies of the protochordate C. intestinalis genome at the chromosomal level.

Animals↗
PubMed2004

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.

Animals↗
PubMed2006

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B.

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.

Chromosomes, Artificial, Bacterial↗
PubMed2006

Characterizing the composition and evolution of homoeologous genomes in hexaploid wheat through BAC-end sequencing on chromosome 3B.

Bread wheat (Triticum aestivum) is one of the most important crops worldwide. However, because of its large, hexaploid, highly repetitive genome it is a challenge to develop efficient means for molecular analysis and genetic improvement in wheat. To better understand the composition and molecular evolution of the hexaploid wheat homoeologous genomes and to evaluate the potential of BAC-end sequences (BES) for marker development, we have followed a chromosome-specific strategy and generated 11 Mb of random BES from chromosome 3B, the largest chromosome of bread wheat. The sequence consisted of about 86% of repetitive elements, 1.2% of coding regions, and 13% remained unknown. With 1.2% of the sequence length corresponding to coding sequences, 6000 genes were estimated for chromosome 3B. New repetitive sequences were identified, including a Triticineae-specific tandem repeat (Fat) that represents 0.6% of the B-genome and has been differentially amplified in the homoeologous genomes before polyploidization. About 10% of the BES contained junctions between nested transposable elements that were used to develop chromosome-specific markers for physical and genetic mapping. Finally, sequence comparison with 2.9 Mb of random sequences from the D-genome of Aegilops tauschii suggested that the larger size of the B-genome is due to a higher content in repetitive elements. It also indicated which families of transposable elements are mostly responsible for differential expansion of the homoeologous wheat genomes during evolution. Our data demonstrate that BAC-end sequencing from flow-sorted chromosomes is a powerful tool for analysing the structure and evolution of polyploid and highly repetitive genomes.

Base Sequence↗
PubMed2006

Identification of brain neurons expressing the dopamine D4 receptor gene using BAC transgenic mice.

The dopamine D4 receptor (D4R) has received considerable interest because of its higher affinity for atypical antipsychotics, the extremely polymorphic nature of the human gene and the genetic association with attention deficit and hyperactivity disorder (ADHD). Several efforts have been undertaken to determine the D4R expression pattern in the brain using immunohistochemistry, binding autoradiography and in situ hybridization, but the overall published results present large discrepancies. Here, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the mouse dopamine D4 receptor gene (Drd4). Immunohistochemical analysis performed in brain sections of Drd4-EGFP transgenic mice using an anti-EGFP polyclonal antibody showed that transgenic expression was predominant in deep layer neurons of the prefrontal cortex, particularly in the orbital, prelimbic, cingulate and rostral agranular portions. In addition, discrete groups of Drd4-EGFP labelled neurons were observed in the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. EGFP was not detected in the striatum, hippocampus or midbrain as described using other techniques. Given the fine specificity of EGFP expression in BAC transgenic mice and the high sensitivity of the EGFP antibody used in this study, our results indicate that Drd4 expression in the adult mouse brain is limited to a more restricted number of areas than previously reported. Its leading expression in the prefrontal cortex supports the importance of the D4R in complex behaviours depending on cortical dopamine (DA) transmission and its possible role in the etiopathophysiology of ADHD.

Animals↗
PubMed1998

Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene.

The shaker-2 mouse mutation, the homolog of human DFNB3, causes deafness and circling behavior. A bacterial artificial chromosome (BAC) transgene from the shaker-2 critical region corrected the vestibular defects, deafness, and inner ear morphology of shaker-2 mice. An unconventional myosin gene, Myo15, was discovered by DNA sequencing of this BAC. Shaker-2 mice were found to have an amino acid substitution at a highly conserved position within the motor domain of this myosin. Auditory hair cells of shaker-2 mice have very short stereocilia and a long actin-containing protrusion extending from their basal end. This histopathology suggests that Myo15 is necessary for actin organization in the hair cells of the cochlea.

Amino Acid Sequence↗
PubMed2000

A BAC-based physical map of the major autosomes of Drosophila melanogaster.

We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing.

Animals↗
PubMed2004

Comparative analysis of a Brassica BAC clone containing several major aliphatic glucosinolate genes with its corresponding Arabidopsis sequence.

We compared the sequence of a 101-kb-long bacterial artificial chromosome (BAC) clone (B21H13) from Brassica oleracea with its homologous region in Arabidopsis thaliana. This clone contains a gene family involved in the synthesis of aliphatic glucosinolates. The A. thaliana homologs for this gene family are located on chromosome IV and correspond to three 2-oxoglutarate-dependent dioxygenase (AOP) genes. We found that B21H13 harbors 23 genes, whereas the equivalent region in Arabidopsis contains 37 genes. All 23 common genes have the same order and orientation in both Brassica and Arabidopsis. The 16 missing genes in the broccoli BAC clone were arranged in two major blocks of 5 and 7 contiguous genes, two singletons, and a twosome. The 118 exons comprising these 23 genes have high conservation between the two species. The arrangement of the AOP gene family in A. thaliana is as follows: AOP3 (GS-OHP) - AOP2 (GS-ALK) - pseudogene - AOP1. In contrast, in B. oleracea (broccoli and collard), two of the genes are duplicated and the third, AOP3, is missing. The remaining genes are arranged as follows: Bo-AOP2.1 (BoGSL-ALKa) - pseudogene - AOP2.2 (BoGSL-ALKb) - AOP1.1 - AOP1.2. When the survey was expanded to other Brassica accessions, we found variation in copy number and sequence for the Brassica AOP2 homologs. This study confirms that extensive rearrangements have taken place during the evolution of the Brassicacea at both gene and chromosomal levels.

Arabidopsis↗
PubMed2001

Isolation, characterization and FISH assignments of horse BAC clones containing type I and II markers.

In order to increase the number of markers on the horse cytogenetic map and expand the integration with the linkage map, an equine BAC library was screened for genes and for microsatellites. Eighty-nine intra-exon primers were designed from consensus gene sequences in documented species. After PCR screening, 38 clones containing identified genes were isolated and FISH mapped. These data allowed us to refine the available Zoo-FISH results, to define ten new conserved cytogenetic segments and expand two others, thus leading to the identification of a total of 26 conserved segments between horse and human. Interestingly, a new homeology segment was detected between ECA6p and HSA2q. Screening BAC clones for dinucleotide repeats led to the isolation of 33 microsatellites. Ten of the clones each contained at least a polymorphic microsatellite and one specific gene. The success of the approach in the production of integrative anchor loci and their potential use in localization and analysis of traits of interest by the candidate gene and positional cloning approach, are discussed.

Alleles↗
PubMedDate not supplied

Cytogenetical anchoring of sheep linkage map and syntenic groups using a sheep BAC library.

In order to simultaneously integrate linkage and syntenic groups to the ovine chromosomal map, a sheep bacterial artificial chromosome (BAC) library was screened with previously assigned microsatellites using a sheep-hamster hybrid panel and genetic linkage. Thirty-three BACs were obtained, fluorescently labelled and hybridised on sheep-goat hybrid metaphases (2n = 57). This study allowed us, (i), to anchor all linkage groups on sheep chromosomes, (ii), to give information on the probable position of the centromere on the linkage map for the centromeric chromosomes, (iii), to contradict the previous orientation of the ovine X linkage group by the mapping of BMS1008 on OARXq38. Concerning our somatic cell hybrid panel, this study resulted in the assignment of all the previously unassigned groups to ovine chromosomes and a complete characterisation of the hybrid panel. In addition, since hybridisations were performed on a sheep-goat hybrid, new marker/anchoring points were added to the caprine cytogenetic map.

Journal Article↗
PubMedDate not supplied

Substitution of the alpha-lactalbumin transcription unit by a CAT cDNA within a BAC clone silenced the locus in transgenic mice without affecting the physically linked Cyclin T1 gene.

We recently reported that a goat bacterial artificial chromosome (BAC) clone conferred site-independent expression in transgenic mice of the two loci present within its insert, the ubiquitously expressed Cyclin T1 and the mammary specific alpha-lactalbumin (alphalac) genes. To assess if this vector could target mammary-restricted expression of cDNA, the CAT ORF was introduced by homologous recombination in Escherichia coli in place of the alphalac transcription unit. The insert of this modified BAC was injected into mice and three transgenic lines were derived. None of these lines expressed the CAT gene suggesting that the use of long genomic inserts is not sufficient to support the expression of intron-less transgenes. The physically linked goat Cyclin T1 locus was found to be active in all three lines. This observation reinforced the hypothesis that the two loci are localised in two separate chromatin domains.

Animals↗
PubMed2005

Comparative analysis of a BAC contig of porcine chromosome 13q31-q32 and human chromosome 3q21-q22.

BACKGROUND: The gene(s) encoding the ETEC F4ab/ac receptors, involved in neonatal diarrhoea in pigs (a disease not yet described in humans), is located close to the TF locus on Sscr13. In order to reveal and characterize possible candidate genes encoding these receptors, a porcine physical map of the TF region is indispensable. RESULTS: A contig of 33 BAC clones, covering approximately 1.35 Mb surrounding the TF locus on Sscr13q31-q32, was built by chromosome walking. A total of 22,552 bp from the BAC contig were sequenced and compared with database sequences to identify genes, ESTs and repeat sequences, and to anchor the contig to the syntenic region of the human genome sequence (Hsap3q21-q22). The contig was further annotated based on this human/porcine comparative map, and was also anchored to the Sanger porcine framework map and the integrated map of Sscr13 by RH mapping. CONCLUSION: The annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage (gene order and orientation) with the human genome sequence, based on 46 anchor points. This underlines the importance of the human/porcine comparative map for the identification of porcine genes associated with genetic defects and economically important traits, and for assembly of the porcine genome sequence.

Animals↗
PubMed2006

BAC-FISH refutes report of an 8p22-8p23.1 inversion or duplication in 8 patients with Kabuki syndrome.

BACKGROUND: Kabuki syndrome is a multiple congenital anomaly/mental retardation syndrome. The syndrome is characterized by varying degrees of mental retardation, postnatal growth retardation, distinct facial characteristics resembling the Kabuki actor's make-up, cleft or high-arched palate, brachydactyly, scoliosis, and persistence of finger pads. The multiple organ involvement suggests that this is a contiguous gene syndrome but no chromosomal anomalies have been isolated as an etiology. Recent studies have focused on possible duplications in the 8p22-8p23.1 region but no consensus has been reached. METHODS: We used bacterial artificial chromosome-fluorescent in-situ hybridization (BAC-FISH) and G-band analysis to study eight patients with Kabuki syndrome. RESULTS: Metaphase analysis revealed no deletions or duplications with any of the BAC probes. Interphase studies of the Kabuki patients yielded no evidence of inversions when using three-color FISH across the region. These results agree with other research groups' findings but disagree with the findings of Milunsky and Huang. CONCLUSION: It seems likely that Kabuki syndrome is not a contiguous gene syndrome of the 8p region studied.

Abnormalities, Multiple↗
PubMed2005

Frequency of prenatal care visits by ethnic minority mothers and association with infant birthweight in Bac Kan Province, Vietnam.

The objective of this cross-sectional study was to evaluate the association between prenatal care visits and infant birthweight among ethnic minority mothers in the mountainous Bac Kan province. This was done by comparing the frequency and timing of first prenatal care visit of 32 mothers with low birthweight (LBW) infants and 32 mothers with normal birthweight (NBW) infants. During pregnancy, mothers of NBW infants underwent 3.4+/-1 (mean) prenatal care visits and mothers of LBW infants 2.8+/-0.9 (P = 0.02). Mothers of NBW infants underwent their first prenatal care visit at 13.1+/-5.7 weeks of gestation, one week earlier than mothers of LBW infants. The frequency of prenatal care visit are probably associated with a decreased risk of LBW among ethnic minority mothers in Bac Kan province.

Adult↗
PubMed2003

Construction of a BAC library for Haplochromis chilotes, a cichlid fish from Lake Victoria.

Cichlid fishes in Lake Victoria are model organisms for studying rapid radiation and speciation. On the way to examine the molecular basis of how these cichlid fishes achieved such a remarkable morphological diversification, we constructed a bacterial artificial chromosome (BAC) library derived from a cichlid species, Haplochromis chilotes, from Lake Victoria. The library includes 157,056 clones with the average insert size of 128 kb, corresponding to a 10-fold coverage of the H. chilotes genome. Given that the cichlid fishes endemic to Lake Victoria are closely related to one another phylogenetically and their genomes are nearly identical, this BAC library can be utilized to isolate genes from the more than 200 Haplochromine cichlid species in Lake Victoria.

Animals↗
PubMed1998

Construction of a BAC library of the rice blast fungus Magnaporthe grisea and finding specific genome regions in which its transposons tend to cluster.

We have constructed a BAC library of the rice blast fungus Magnaporthe grisea consisting of 5760 clones. The insert size ranged from 35 to 175 kbp, with an average of 120 kbp. The library is about 18 genomes equivalent, therefore covering more than 99.999% of the genome. This library is the first to be constructed using a rice pathogenic wild type isolate. Improved high molecular weight DNA size fractionating helped to construct the library with high efficiency. Total library clones were arranged onto two nylon membranes for efficient screening. Test hybridization with a single-copy RFLP marker showed ten positive clones, of which restriction patterns indicated no chimerality or deletions. As a model case of application of this library, the distribution of the well-studied fungal retrotransposons MGSR1, MGR583, and MAGGY and DNA transposons MGR586 and Pot2 was analyzed. Of all the BAC clones, 10%, 13%, 18%, 12%, and 23% contained MGSR1, MGR583, MAGGY, MGR586 and Pot2, respectively. The percentage of clones possessing more than five kinds of transposons was 1.4%, 215 times greater than the expected number. The results show that these transposons were distributed in clusters in the M. grisea genome.

Ascomycota↗