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At least 379 records · Page 21Linked to original sources
PubMed2003

Unmasking Kabuki syndrome: chromosome 8p22-8p23.1 duplication revealed by comparative genomic hybridization and BAC-FISH.

Kabuki syndrome (KS) is a multiple congenital anomalies/mental retardation syndrome that heretofore has had an unknown etiology. Although several cases with KS features have been reported with different chromosome anomalies, none have had an autosomal cytogenetic aberration in common. We found an 8p22-8p23.1 duplication, using comparative genomic hybridization (CGH) in six unrelated patients diagnosed with KS. This observation was confirmed using BAC-FISH in all cases that delimited the duplicated region to approximately 3.5 Mb. No duplication of this region was found in two parents or 20 controls by either CGH or BAC-FISH. Two out of two mothers of KS patients and one out of 20 controls were found to have a heterozygous submicroscopic inversion at 8p23.1. As the six patients with KS represent different races, this duplication may represent a common etiologic basis for this disorder.

Abnormalities, Multiple↗
PubMed2001

Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

ATP Binding Cassette Transporter 1↗
PubMed1986

Comparison of visual and photometric Bac-T-Screen results.

The Bac-T-Screen (Marion Laboratories, Kansas City, MO) was used to screen 826 urine specimens. Of these, 85 either pigmented or clogged the Bac-T-Screen filter and could not be evaluated. Results for the remaining 741 specimens were examined both visually and photometrically by a newly developed photometric card reader. The results were then compared. Screening results for all urines containing greater than or equal to 10(5) pathogens/mL were equivalent for both methods, with sensitivity and predictive negative values of greater than 98% and greater than 99%, respectively. The predictive values of positive tests were also equivalent at 57.5% for visual and 59.6% by photometry. The overall agreement varied with the card reader value used because the photometric card-reader procedure allows the user to select desired sensitivity and specificity levels.

Bacteriological Techniques↗
PubMed1998

A physical map of Arabidopsis thaliana chromosome 3 represented by two contigs of CIC YAC, P1, TAC and BAC clones.

We have constructed a physical map of Arabidopsis thaliana chromosome 3 by ordering the clones from CIC YAC, P1, TAC and BAC libraries using the sequences of a variety of genetic and EST markers and terminal sequences of clones. The markers used were 112 DNA markers, 145 YAC end sequences, and 156 end sequences of P1, TAC and BAC clones. The entire genome of chromosome 3, except for the centromeric and telomeric regions, was covered by two large contigs, 13.6 Mb and 9.2 Mb long. This physical map will facilitate map-based cloning experiments as well as genome sequencing of chromosome 3. The map and end sequence information are available on the KAOS (Kazusa Arabidopsis data Opening Site) web site at http://www.kazusa.or.jp/arabi/.

Arabidopsis↗
PubMed2006

Tuatara (Sphenodon) genomics: BAC library construction, sequence survey, and application to the DMRT gene family.

The tuatara (Sphenodon punctatus) is of "extraordinary biological interest" as the most distinctive surviving reptilian lineage (Rhyncocephalia) in the world. To provide a genomic resource for an understanding of genome evolution in reptiles, and as part of a larger project to produce genomic resources for various reptiles (evogen.jgi.doe.gov/second_levels/BACs/our_libraries.html), a large-insert bacterial artificial chromosome (BAC) library from a male tuatara was constructed. The library consists of 215 424 individual clones whose average insert size was empirically determined to be 145 kb, yielding a genomic coverage of approximately 6.3x. A BAC-end sequencing analysis of 121 420 bp of sequence revealed a genomic GC content of 46.8%, among the highest observed thus far for vertebrates, and identified several short interspersed repetitive elements (mammalian interspersed repeat-type repeats) and long interspersed repetitive elements, including chicken repeat 1 element. Finally, as a quality control measure the arrayed library was screened with probes corresponding to 2 conserved noncoding regions of the candidate sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig spanning nearly 300 kb was generated, supporting the deep coverage and utility of the library for exploring tuatara genomics.

Animals↗
PubMed1997

Efficient Cre-lox linearisation of BACs: applications to physical mapping and generation of transgenic animals.

Due to the size of BAC, PAC and P1 clones, it is often difficult to construct detailed restriction maps, with large number of restriction fragments leading to ambiguity of mapping data. We report the use of Cre recombinase to linearise and asymmetrically introduce label at the unique loxP site of large loxP-containing clones. Subsequent partial digestion allows the direct ordering of restriction fragments. Additionally, BAC DNA linearised using the Cre-lox system has been used successfully to generate transgenic animals.

Animals↗
PubMed1997

Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.

We describe a simple electroelution method for purifying large, gel-fractionated DNA molecules that alleviates the need for melting of the agarose and subsequent enzymatic agarose digestion. The method yields DNA that is visibly more intact than that purified from a standard agarose-digestion protocol and is more amenable to large-fragment cloning with PAC and BAC vectors. These findings are notable in that PAC and BAC library construction is a very labor-intensive and costly procedure, such that any net improvement in cloning efficiency is highly advantageous. This method also should prove useful towards other applications which require purification of very large DNA molecules, such as YAC cloning.

Animals↗
PubMed2000

Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli.

We have used GET Recombination, an inducible homologous recombination system for Escherichia coli, to insert one of the most common thalassaemia mutations into the intact beta-globin locus in a second generation BAC vector. We first inserted a PCR fragment carrying the tetracycline resistance gene (TetR) into the beta-globin gene. All recombinant clones examined contained the TetR gene at the correct target site. Next, a PCR fragment with the IVS I-110 G-->A splicing mutation but no selectable marker was used to replace the TetR gene in a second round of GET Recombination. Recombinant clones were selected by plating on medium containing chlorotetracycline and fusaric acid. Although counterselection for the TetR gene is not very efficient, four recombinant colonies with the IVS I-110 mutation were identified among 480 clones screened. Analysis of the recombinant clones did not show any other modifications or rearrangements. Thus the TetR gene can be used in combination with GET Recombination to introduce point mutations and other modifications in BACs without leaving behind any operational sequences, in order to generate accurate cell and transgenic mouse models for various diseases.

Chromosomes, Bacterial↗
PubMed2000

Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination.

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.

Cloning, Molecular↗
PubMed2002

A reliable and efficient method for deleting operational sequences in PACs and BACs.

P1-derived artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have become very useful as tools to study gene expression and regulation in cells and in transgenic mice. They carry large fragments of genomic DNA (> or =100 kb) and therefore may contain all of the cis-regulatory elements required for expression of a gene. Because of this, even when inserted randomly in the genome, they can emulate the native environment of a gene resulting in a tightly regulated pattern of expression. Because these large genomic clones often contain DNA sequences which can manipulate chromatin at the local level, they become immune to position effects which affect expression of smaller transgenes, and thus their expression is proportional to copy number. Transgenic mice containing large BACs and PACs have become excellent models to examine the regulation of gene expression. Their usefulness would certainly be increased if easy and efficient methods are developed to manipulate them. We describe herein a method to make deletion mutations reliably and efficiently using a novel modification of the Chi-stimulated homologous recombination method. Specifically, we generated and employed a Lox511 'floxed' CAM resistance marker that first affords selection for homologous recombination in Escherichia coli, and then can be easily deleted leaving only a single Lox511 site as the footprint.

Bacteriophage P1↗
PubMed2006

BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH).

The array CGH technique (Array Comparative Genome Hybridization) has been developed to detect chromosomal copy number changes on a genome-wide and/or high-resolution scale. It is used in human genetics and oncology, with great promise for clinical application. Until recently primarily PCR amplified bacterial artificial chromosomes (BACs) or cDNAs have been spotted as elements on the array. The large-scale DNA isolations or PCR amplifications of the large-insert clones necessary for manufacturing the arrays are elaborate and time-consuming. Lack of a high-resolution highly sensitive (commercial) alternative has undoubtedly hindered the implementation of array CGH in research and diagnostics. Recently, synthetic oligonucleotides as arrayed elements have been introduced as an alternative substrate for array CGH, both by academic institutions as well as by commercial providers. Oligonucleotide libraries or ready-made arrays can be bought off-the-shelf saving considerable time and efforts. For RNA expression profiling, we have seen a gradual transition from in-house printed cDNA-based expression arrays to oligonucleotide arrays and we expect a similar transition for array CGH. This review compares the different platforms and will attempt to shine a light on the 'BAC to the future' of the array CGH technique.

Chromosome Aberrations↗
PubMed2006

An improved method to identify BAC clones using pooled overgos.

Hybridization using overgo probes is an established approach for screening arrayed bacterial artificial chromosome (BAC) libraries. We have improved the use of overgos by increasing the yield of positive clones using reduced levels of radioisotopes and enzyme. The strategy involves labeling with all four radiolabeled nucleotides in a hot pulse followed by a cold nucleotide chase and then extending the exposure time to compensate for reduced specific activity of the probes. The resulting cost savings and reduced human exposure to radiation make the use of highly pooled overgo probes a more attractive approach for screening of BAC libraries from organisms with large genomes.

Chromosomes, Artificial, Bacterial↗
PubMed2004

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

Cell Line, Tumor↗
PubMed2005

Simple and highly efficient BAC recombineering using galK selection.

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (lambda) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive lambda prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.

Base Sequence↗
PubMed2003

A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

Animals↗
PubMed2004

A BAC- and BIBAC-based physical map of the soybean genome.

Genome-wide physical maps are crucial to many aspects of advanced genome research. We report a genome-wide, bacterial artificial chromosome (BAC) and plant-transformation-competent binary large-insert plasmid clone (hereafter BIBAC)-based physical map of the soybean genome. The map was constructed from 78001 clones from five soybean BAC and BIBAC libraries representing 9.6 haploid genomes and three cultivars, and consisted of 2905 BAC/BIBAC contigs, estimated to span 1408 Mb in physical length. We evaluated the reliability of the map contigs using different contig assembly strategies, independent contig building methods, DNA marker hybridization, and different fingerprinting methods, and the results showed that the contigs were assembled properly. Furthermore, we tested the feasibility of integrating the physical map with the existing soybean composite genetic map using 388 DNA markers. The results further confirmed the nature of the ancient tetraploid origin of soybean and indicated that it is feasible to integrate the physical map with the linkage map even though greater efforts are needed. This map represents the first genome-wide, BAC/BIBAC-based physical map of the soybean genome and would provide a platform for advanced genome research of soybean and other legume species. The inclusion of BIBACs in the map would streamline the utility of the map for positional cloning of genes and QTLs, and functional analysis of soybean genomic sequences.

Chromosomes, Artificial, Bacterial↗
PubMed2001

Advanced integrated mouse YAC map including BAC framework.

Functional characterization of the mouse genome requires the availability of a comprehensive physical map to obtain molecular access to chromosomal regions of interest. Positional cloning remains a crucial way of linking phenotype with particular genes. A key step and frequent stumbling block in positional cloning is making a contig of a genetically defined candidate region. The most efficient first step is isolating YAC (Yeast Artificial Chromosome) clones. A robust, detailed YAC contig map is thus an important tool. Employing Interspersed Repetitive Sequence (IRS)-PCR genomics, we have generated an advanced second-generation YAC contig map of the mouse genome that doubles both the depth of clones and the density of markers available. In addition to the primarily YAC-based map, we located 1942 BAC (Bacterial Artificial Chromosome) clones. This allows us to present for the first time a dense framework of BACs spanning the genome of the mouse, which, for instance, can serve as a nucleus for genomic sequencing. Four large-insert mouse YAC libraries from three different strains are included in our data, and our analysis incorporates the data of Hunter et al. and Nusbaum et al. There is a total of 20,205 markers on the final map, 12,033 from our own data, and a total of 56,093 YACs, of which 44,401 are positive for more than one marker.

Algorithms↗