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At least 235 records · Page 13Linked to original sources
PubMed1998

Construction of a bacterial artificial chromosome (BAC) contig across the minimally deleted region in 13q14.3 in B-cell chronic lymphocytic leukemia.

Loss of heterozygosity (LOH) analysis in B-cell chronic lymphocytic leukemia (BCLL) has indicated that a frequent genetic event is loss of alleles from an approximately 500 kb region in 13q14.3, distal to the retinoblastoma gene. We have used DNA markers from this region to isolate and characterize a series of bacterial artificial chromosomes (BACs) which span the region between markers D13S319 and D13S25, which represents the common region of LOH. This entire region appears to be contained within only two minimally overlapping BACs, representing a maximum distance of approximately 350 kb. This BAC contig has been used to position known STS, EST and gene markers within the region. We have also used a modified differential display/RNA fingerprinting procedure designed to isolated transcribed sequences from YACs to isolate two transcribed units from the region which have also been positioned within the contig. The construction of a BAC contig with minimal redundancy provides the ideal resources from which to begin to identify candidate genes related to BCLL.

Blotting, Southern↗
PubMed2000

Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus.

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms.

Animals↗
PubMed2006

Digital Screening for Blood Alcohol Concentration (BAC) in a Southern Nigeria City.

OBJECTIVE: Blood alcohol concentration (BAC) was digitally determined for transport operators, passengers, and pedestrians in a southern Nigeria city. METHODS: The subjects were screened with a digital breathalyzer, Alco Scan CA 2000, for BAC along major accident-prone highways, resting spots, and hospitals. The mouthpieces were complemented with disposable straws for rapid and hygienic screening of the subjects. RESULTS: Based on convenience sampling, a significant proportion of Nigerians has a BAC level above 0.06 on accident-prone highways (44.60%), at resting spots (54.50%), and in hospitals (47.80%). CONCLUSION: This research confirms that digital breathalyzer BAC levels are reliable and empirical means for determining incidents of alcohol-impaired driving.

Accidents, Traffic↗
PubMed1997

Bacterial artificial chromosome (BAC) library as a tool for physical mapping of the archaeon Methanosarcina thermophila TM-1.

We have used a variety of methods to characterize the genome of the archaeon Methanosarcina thermophila TM-1. Pulsed-field gel analysis indicates a genome size of 2.8 Mb. We have constructed a bacterial artificial chromosome (BAC) library of M. thermophila and have used it to generate physical maps for this organism. The library is made up of 384 clones with an average insert size of 58 kb representing 8.0 genome equivalents. The utility of the library for low-resolution physical mapping was shown by identifying NotI linking clones and using these to order the NotI macrorestriction fragments of M. thermophila into a 2.8 Mb map. Hybridization of nine single copy genes and a 16S rRNA sequence to these macrorestriction fragments forms the basis for the first genetic map in this organism. High-resolution physical maps, consisting of overlapping clones, have been created using HindIII fingerprints of BAC clones. In this way, we identified a minimal path of five clones that span a 270 kb NotI fragment. The ease of manipulating BAC clones makes the BAC system an excellent choice for the construction of low-resolution and high-resolution physical and genetic maps of archaeal genomes. It also provides a substrate for future genome-sequencing efforts.

Chromosome Mapping↗
PubMed2000

Human BAC ends.

The Human BAC Ends database includes all non-redundant human BAC end sequences (BESs) generated by The Institute for Genomic Research (TIGR), the University of Washington (UW) and California Institute of Technology (CalTech). It incorporates the available BAC mapping data from different genome centers and the annotation results of each end sequence for the contents of repeats, ESTs and STS markers. For each BAC end the database integrates the sequence, the phred quality scores, the map and the annotation, and provides links to sites of the library information, the reports of GenBank, dbGSS and GDB, and other relevant data. The database is freely accessible via the web and supports sequence or clone searches and anonymous FTP. The relevant sites and resources are described at http://www.tigr.org/ tdb/humgen/bac_end_search/bac_end_intro.html

Chromosome Mapping↗
PubMed2000

Anchoring of rice BAC clones to the rice genetic map in silico.

A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases. Integration of genetic and physical maps involves labor-intensive empirical experiments. To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases. We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.

Chromosome Mapping↗
PubMed2001

GenMapDB: a database of mapped human BAC clones.

GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9-22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequences of the clones. GenMapDB is freely available to the public. The main purpose of GenMapDB is to organize the mapping data and to allow the research community to search for mapped BAC clones that can be used in gene mapping studies and chromosomal mutation analysis projects.

Chromosome Mapping↗
PubMed2005

Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays.

Efficient construction of BAC-based human artificial chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.

Autoantigens↗
PubMed2003

A simple two-step, 'hit and fix' method to generate subtle mutations in BACs using short denatured PCR fragments.

The bacteriophage lambda recombination system has proven to be a valuable tool for engineering bacterial artificial chromosomes (BAC). Due to its high efficiency, subtle alterations in the BACs can be generated using oligonucleotides as targeting vectors. Since no selection marker is used, recombinant clones are identified utilizing a selective PCR screening method. However, occasionally the selective PCR screening is not feasible. We describe here a two-step 'hit and fix' method that can be reliably used for generating any subtle alteration in BACs using short denatured PCR fragments as targeting vectors. In the first step of this method, 6-20 nucleotides are changed around the base where the mutation has to be generated. In the second step, these altered nucleotides are reverted to the original sequence and simultaneously a subtle alteration is introduced. Since in each step several nucleotides are changed, PCR primers specific for such alterations can be designed. This two-step method provides a simple and efficient tool for generating subtle alterations in BACs that can be very valuable for functional analysis of genes.

Chromosomes, Artificial, Bacterial↗
PubMed2005

Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms.

A strategy is presented to select, pool and spot human BAC clones on an array in such a way that each spot contains five well performing BAC clones, covering one chromosome arm. A mini-array of 240 spots was prepared representing all human chromosome arms in a 5-fold as well as some controls, and used for comparative genomic hybridization (CGH) of 10 cell lines with aneusomies frequently found in clinical cytogenetics and oncology. Spot-to-spot variation within five replicates was below 6% and all expected abnormalities were detected 100% correctly. Sensitivity was such that replacing one BAC clone in a given spot of five by a BAC clone from another chromosome, thus resulting in a change in ratio of 20%, was reproducibly detected. Incubation time of the mini-array was varied and the fluorescently labelled target DNA was diluted. Typically, aneusomies could be detected using 30 ng of non-amplified random primed labelled DNA amounts in a 4 h hybridization reaction. Potential application of these mini-arrays for genomic profiling of disseminated tumour cells or of blastomeres for preimplantation genetic diagnosis, using specially designed DNA amplification methods, are discussed.

Aneuploidy↗
PubMed2002

Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome

Arabinose↗
PubMed2003

A cattle-human comparative map built with cattle BAC-ends and human genome sequence.

As a step toward the goal of adding the cattle genome to those available for multispecies comparative genome analysis, 40,224 cattle BAC clones were end-sequenced, yielding 60,547 sequences (BAC end sequences, BESs) after trimming with an average read length of 515 bp. Cattle BACs were anchored to the human and mouse genome sequences by BLASTN search, revealing 29.4% and 10.1% significant hits (E < e-5), respectively. More than 60% of all cattle BES hits in both the human and mouse genomes are located within known genes. In order to confirm in silico predictions of orthologyand their relative position on cattle chromosomes, 84 cattle BESs with similarity to sequences on HSA11 were mapped using a cattle-hamster radiation hybrid (RH) panel. Resulting RH maps of BTA15 and BTA29 cover approximately 85% of HSA11 sequence, revealing a complex patchwork shuffling of segments not explained by a simple translocation followed by internal rearrangements. Overlay of the mouse conserved syntenies onto HSA11 revealed that segmental boundaries appear to be conserved in all three species. The BAC clone-based comparative map provides a foundation for the evolutionary analysis of mammalian karyotypes and for sequencing of the cattle genome.

Animals↗
PubMed2001

Mouse BAC ends quality assessment and sequence analyses.

A large-scale BAC end-sequencing project at The Institute for Genomic Research (TIGR) has generated one of the most extensive sets of sequence markers for the mouse genome to date. With a sequencing success rate of >80%, an average read length of 485 bp, and ABI3700 capillary sequencers, we have generated 449,234 nonredundant mouse BAC end sequences (mBESs) with 218 Mb total from 257,318 clones from libraries RPCI-23 and RPCI-24, representing 15x clone coverage, 7% sequence coverage, and a marker every 7 kb across the genome. A total of 191,916 BACs have sequences from both ends providing 12x genome coverage. The average Q20 length is 406 bp and 84% of the bases have phred quality scores > or = 20. RPCI-24 mBESs have more Q20 bases and longer reads on average than RPCI-23 sequences. ABI3700 sequencers and the sample tracking system ensure that > 95% of mBESs are associated with the right clone identifiers. We have found that a significant fraction of mBESs contains L1 repeats and approximately 48% of the clones have both ends with > or = 100 bp contiguous unique Q20 bases. About 3% mBESs match ESTs and > 70% of matches were conserved between the mouse and the human or the rat. Approximately 0.1% mBESs contain STSs. About 0.2% mBESs match human finished sequences and > 70% of these sequences have EST hits. The analyses indicate that our high-quality mouse BAC end sequences will be a valuable resource to the community.

Animals↗
PubMed2002

BAC library of T. pallidum DNA in E. coli.

Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidum chromosome was missing in the BAC library, between bp 248727 and 264323. In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene. Altogether, 13 predicted T. pallidum ORFs (1.25% of the total) were incomplete or missing in the library. Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert. Of mapped clones, 19 were selected to represent the minimal set of E. coli BAC clones covering 1026 of the total 1040 (98.7%) predicted T. pallidum ORFs. Using this minimal set of clones, at least 12 T. pallidum proteins were shown to react with pooled sera from rabbits immunized with T. pallidum, indicating that at least some T. pallidum genes are transcribed and expressed in E. coli.

Animals↗
PubMed2005

The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza.

Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.

Base Sequence↗
PubMed1997

Retrofitting vectors for Escherichia coli-based artificial chromosomes (PACs and BACs) with markers for transfection studies.

P1-based artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have significantly expanded the size of fragments from eukaryotic genomes that can be stably cloned in Escherichia coli as plasmid molecules. Functional characterization of a gene within a given PAC or BAC clone often requires transferring the DNA into eukaryotic cells for transient or long-term expression. To facilitate transfection studies, we have developed protocols using the Notl restriction sites of any PAC or BAC clone to introduce a transfection reporter gene, lacZ, together with a selectable marker, neo. This enables transfected cells to be detected by X-Gal staining to verify DNA uptake, and clones of stably transformed cells may be selected for in the presence of the antibiotic G418. The same retrofitting protocols may be applied with other markers of interest to extend the functionality of PAC and BAC libraries, and specialized aspects of such manipulation of E. coli-based artificial chromosomes are outlined.

Bacteriophage P1↗
PubMed1999

Human genome anatomy: BACs integrating the genetic and cytogenetic maps for bridging genome and biomedicine.

Human genome sequencing is accelerating rapidly. Multiple genome maps link this sequence to problems in biology and clinical medicine. Because each map represents a different aspect of the structure, content, and behavior of human chromosomes, these fundamental properties must be integrated with the genome to understand disease genes, cancer instability, and human evolution. Cytogenetic maps use 400-850 visible band landmarks and are the primary means for defining prenatal defects and novel cancer breakpoints, thereby providing simultaneous examination of the entire genome. Recent genetic, physical, and transcript maps use PCR-based landmarks called sequence-tagged sites (STSs). We have integrated these genome maps by anchoring the human cytogenetic to the STS-based genetic and physical maps with 1021 STS-BAC pairs at an average spacing of approximately 1 per 3 Mb. These integration points are represented by 872 unique STSs, including 642 polymorphic markers and 957 bacterial artificial chromosomes (BACs), each of which was localized on high resolution fluorescent banded chromosomes. These BACs constitute a resource that bridges map levels and provides the tools to seamlessly translate questions raised by genomic change seen at the chromosomal level into answers based at the molecular level. We show how the BACs provide molecular links for understanding human genomic duplications, meiosis, and evolution, as well as reagents for conducting genome-wide prenatal diagnosis at the molecular level and for detecting gene candidates associated with novel cancer breakpoints.

Chromosome Mapping↗
PubMed2004

Characterization and mapping of canine microsatellites isolated from BAC clones harbouring DNA sequences homologous to seven human genes.

Human primers specific for the genes LEP, HBB, PAX3, ESR2, TPH1, ABCA4 and ATP2A2 were used to identify clones in a canine BAC library. Subcloning of the positive BACs in plasmids, screening with microsatellite motifs and subsequent sequencing allowed for the identification of eight novel microsatellites. The presence of the gene of interest was confirmed by sequencing the polymerase chain reaction (PCR) products amplified in the positive BACs. Fluorescent in situ hybridization (FISH) using the positive BACs as probes allowed for the chromosomal localization of the insert DNAs in two canid species, dog (Canis familiaris) and red fox (Vulpes vulpes). The use of gene-associated microsatellites may accelerate the identification of candidate genes for phenotypic traits in linkage studies.

Animals↗