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PubMed1998

BAC representation of two low-copy regions of the genome of Arabidopsis thaliana.

Two regions of Arabidopsis chromosome 4, totalling 4.7 Mb, were assayed for representation in the TAMU and IGF BAC libraries. A directed approach to BAC identification was developed. Gel-purified DNA samples of YACs selected from the YAC-based physical map of chromosome 4 were used to probe high-density colony arrays of the BAC libraries. Strategies were developed that allowed the efficient construction of restriction maps and BAC contigs. Four hundred and sixty-four BACs were mapped, assembled into two complete contigs and used to analyse genomic representation. These BACs provided a mean of 9.4-fold redundant coverage, with a range of 2- to 22-fold. The representation provided by the two libraries showed almost coincident peaks and troughs, with a periodicity of approximately 200 kb. These results demonstrate that, provided both TAMU and IGF libraries are used in their entirety, BACs should provide an excellent resource for both physical mapping and sequencing of the Arabidopsis genome.

Arabidopsis↗
PubMed2004

Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Animals↗
PubMed2004

Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert size.

The construction of bacterial artificial chromosome (BAC) libraries remains relatively complex and laborious, such that any technological improvement is considered to be highly advantageous. In this study, we addressed several aspects that improved the quality and efficiency of cloning of plant genomes into BACs. We set the 'single tube vector' preparation method with no precipitation or gel electrophoresis steps, which resulted in less vector DNA damage and a remarkable two- to threefold higher transformation efficiency compared with other known vector preparation methods. We used a reduced amount of DNA for partial digestion (up to 5 microg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 x TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 x TBE, which resulted in larger and more uniformly sized BAC inserts and, surprisingly, a two- to threefold higher transformation efficiency, probably due to less contamination with borate ions. We adopted a triple size selection that resulted in an increased mean insert size of up to 70 kb and a transformation efficiency comparable with that of double size selection. Overall, the improved protocol presented in this study resulted in a five- to sixfold higher cloning efficiency and larger and more uniformly sized BAC inserts. BAC libraries with the desired mean insert size (up to 200 kb) were constructed from several plant species, including hexaploid wheat. The improved protocol will render the construction of BAC libraries more available in plants and will greatly enhance genome analysis, gene mapping and cloning.

Journal Article↗
PubMed1988

Evaluation of the modified Bac-T-Screen and FiltraCheck-UTI urine screening systems for detection of clinically significant bacteriuria.

Previous evaluations of the Bac-T-Screen system (Vitek Systems, Inc., Hazelwood, Mo.) demonstrated excellent sensitivity with specimens from patients with clinically significant bacteriuria (including infections with small numbers of uropathogens) but poor specificity with specimens from noninfected patients. In the study reported here, the sensitivity and specificity of the Bac-T-Screen system with a modified decolorizing reagent were evaluated. A manual filtration system, FiltraCheck-UTI (Vitek Systems), for screening urine specimens, Gram stains of mixed urine specimens, and quantitative cultures were also evaluated. The test sensitivity for clinically significant bacteriuria was greater than 96% with the original Bac-T-Screen system as well as the modified system and the manual system. In comparison, the sensitivities of the Gram stains and quantitative cultures (greater than or equal to 10(5) CFU/ml) were 82 and 77%, respectively. Of the 375 patients classified as noninfected by clinical parameters, 34% had positive screening tests with the original Bac-T-Screen system, as compared with 13 and 11% with the modified Bac-T-Screen and FiltraCheck-UTI systems, respectively. Thus, the modified Bac-T-Screen system and the manual FiltraCheck-UTI system have sensitives comparable to that of the original Bac-T-Screen system and markedly improved test specificities.

Bacteria↗
PubMed1988

Visual and clinical analysis of Bac-T-Screen urine screen results.

Of 337 urine specimens evaluated, 75 of the 113 that showed positive readings on the Bac-T-Screen urine-screening instrument were found by subsequent semiquantitative culture to yield either less than 10,000 CFU of mixed bacteria per ml or no growth (less than 100 CFU/ml by our criteria). We tried to determine what factors contributed to the positive Bac-T-Screen results by examining the 75 Bac-T-Screen-positive urine specimens with three visual methods: Gram staining, hemacytometer chamber counting, and filtering through a 5.0-microns-pore-size nitrocellulose filter with subsequent microscopic examination of the stained filter. Somatic cells and other particles present in those urine specimens that yielded positive readings by the Bac-T-Screen included epithelial cells in 43%, crystals and amorphous material in 33%, and leukocytes in 17% of the specimens. There was no relationship between the numbers of particles seen in urine and the magnitude of the relative absorbance reading obtained with the Bac-T-Screen. A retrospective chart review was conducted for patients with positive Bac-T-Screen results and negative cultures. Of the 75 patients, 6 were thought to have urinary tract infections on the basis of clinical criteria; the majority of the remaining 69 patients had clinical histories revealing systemic or urogenital conditions consistent with shedding of particles in the urine. A positive reading by the Bac-T-Screen system seemed to be related to the presence of somatic cells and other particles in urine; bacteriuria was not always detectable in these cases.

Bacteriuria↗
PubMed2003

Integration of animal linkage and BAC contig maps using overgo hybridization.

The alignment of genome linkage maps, defined primarily by segregation of sequence-tagged site (STS) markers, with BAC contig physical maps and full genome sequences requires high throughput mechanisms to identify BAC clones that contain specific STS. A powerful technique for this purpose is multi-dimensional hybridization of "overgo" probes. The probes are chosen from available STS sequence data by selecting unique probe sequences that have a common melting temperature. We have hybridized sets of 216 overgo probes in subset pools of 36 overgos at a time to filter-spotted chicken BAC clone arrays. A four-dimensional pooling strategy, including one degree of redundancy, has been employed. This requires 24 hybridizations to completely assign BACs for all 216 probes. Results to date are consistent with about a 10% failure rate in overgo probe design and a 15-20% false negative detection rate within a group of 216 markers. Three complete rounds of overgo hybridization, each to sets of about 39,000 BACs (either BAMHI or ECORI partial digest inserts) generated a total of 1853 BAC alignments for 517 mapped chicken genome STS markers. These data are publicly available, and they have been used in the assembly of a first generation BAC contig map of the chicken genome.

Animals↗
PubMed1992

The limited utility of BAC for identifying alcohol-related problems among DWI offenders.

To see if blood alcohol concentration (BAC) is a significant indicator of problem drinking or an alcohol-related diagnosis, the relationships between BAC at arrest for drinking and driving, typical drinking, alcohol-related problems, problem drinking and an alcohol abuse or dependence diagnosis were examined for 235 drinking and driving offenders referred for alcoholism evaluation. BAC and typical drinking correlated weakly and no significant relationship between alcohol-related problems and BAC was found. Further, no significant relationship was found between BAC and alcohol abuse or dependence diagnoses, or between problem drinking and BAC. These findings cast doubt on the usefulness of a single report of BAC for diagnostic and screening purposes.

Accidents, Traffic↗
PubMed2000

A deep-coverage tomato BAC library and prospects toward development of an STC framework for genome sequencing.

Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs) was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of <10(-6), and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed.

Chromosomes, Bacterial↗
PubMed2001

Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences.

The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed.

Chromosomes, Artificial, Bacterial↗
PubMed2003

[Establishment of BAC mediated transgenic mice containing 97 kb beta-globin gene cluster].

OBJECTIVE: To delete IL-11 receptor alpha chain gene from the Bacterial Artificial Chromosome (BAC) chimeric DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole beta-globin gene cluster. METHODS: Two 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor alpha chain gene respectively were cloned into the Hind III and Xba I sites of pBV vector, then the 1 kb A + B fragment was recovered from the building vector and inserted into the Sal I site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor alpha chain gene was finally deleted from the BAC DNA through chloramphenicol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice. RESULTS: By RecA protein mediated homologous recombination method, we deleted the IL-11 receptor alpha chain gene from the BAC DNA containing the complete beta-globin Gene Cluster and established 3 respective transgenic mice lines. CONCLUSION: Human beta-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.

Animals↗
PubMed1997

BAC and PAC contigs covering 3.5 Mb of the Down syndrome congenital heart disease region between D21S55 and MX1 on chromosome 21.

Chromosome 21 is a model for the study of human chromosomal aneuploidy, and the construction of its physical and transcriptional maps is a necessary step in understanding the molecular basis of aneuploidy-dependent phenotypes. To identify the gene(s) responsible for Down syndrome congenital heart disease (DS-CHD), we constructed a physical map of the D21S55 to MX1 region. A bacterial artificial chromosome (BAC) library was screened using several YACs spanning the interval, and a P1-derived artificial chromosome (PAC) library was screened using radiolabeled STS PCR products and whole BACs in gap-filling initiatives. FISH confirmed the location of all BAC and PAC clones to 21q22.2-q22.3. Overlaps were established using clone-to-clone Southerns and 24 new STSs, generated from the direct sequencing of BAC and PAC ends, along with 35 preexisting STSs. Approximately 3.5 Mb of the 4- to 5-Mb D21S55 to MX1 interval is covered in 85 BACs and 24 PACs, representing fourfold coverage within the contigs. These BAC and PAC contigs are valuable reagents for isolating the genes for DS-CHD.

Base Sequence↗
PubMed2000

BAC trimming: minimizing clone overlaps.

Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA(-) BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases.

Arabidopsis↗
PubMed2001

Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli.

Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCMV) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette introduced into the HindIII "N" region of the viral genome. This virus had replication kinetics identical to wild-type virus. We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII "N" to generate another GPCMV recombinant, vAMBGPCMV. Circular viral DNA isolated from vAMBGPCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies.

Animals↗
PubMed2004

High-resolution mapping and chromosome landing at the root-know nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library.

The Ma gene for root-knot nematode (RKN)resistance from Myrobalan plum (Prunus cerasifera L.)confers a complete-spectrum and a heat-stable resistance to Meloidogvne spp., conversely to Mi-I from tomato,which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designatedSCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175,Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175,totaling 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing. In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species.

Animals↗
PubMed2005

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines.

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

Blotting, Southern↗
PubMed2006

Construction of a watermelon BAC library and identification of SSRs anchored to melon or Arabidopsis genomes.

A bacterial artificial chromosome (BAC) library was constructed for watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly distributed on the genomes of melon or Arabidopsis. Twenty eight probes (representing 66% of the tested probes) from melon and 30 probes (65%) from Arabidopsis identified positive BAC clones. Two methods were implemented to identify SSRs from the positively hybridizing BAC clones. First, analysis of BAC end sequences revealed 37 SSRs. For the second method, pooled DNA of BACs identified by the melon probes was used to develop a shotgun library. The library was then screened with synthetic SSR oligonucleotides by hybridization. Sequence analysis of positively hybridizing shotgun clones revealed 142 different SSRs. Thirty eight SSRs were characterized using three watermelon cultivars, five plant introduction (PI) accessions of C. lanatus var lanatus and four PIs of C. lanatus var citroides. Of these, 36 (95%) were found to be polymorphic with up to six alleles per marker. Polymorphism information content values for polymorphic markers varied between 0.22 and 0.79 with an average of 0.53. The methods described herein will be valuable for the construction of a watermelon linkage map with SSRs evenly distributed on its genome that is anchored to the genomes of melon and Arabidopsis.

Alleles↗
PubMed2006

Comparative sequence and genetic analyses of asparagus BACs reveal no microsynteny with onion or rice.

The Poales (includes the grasses) and Asparagales [includes onion (Allium cepa L.) and asparagus (Asparagus officinalis L.)] are the two most economically important monocot orders. The Poales are a member of the commelinoid monocots, a group of orders sister to the Asparagales. Comparative genomic analyses have revealed a high degree of synteny among the grasses; however, it is not known if this synteny extends to other major monocot groups such as the Asparagales. Although we previously reported no evidence for synteny at the recombinational level between onion and rice, microsynteny may exist across shorter genomic regions in the grasses and Asparagales. We sequenced nine asparagus BACs to reveal physically linked genic-like sequences and determined their most similar positions in the onion and rice genomes. Four of the asparagus BACs were selected using molecular markers tightly linked to the sex-determining M locus on chromosome 5 of asparagus. These BACs possessed only two putative coding regions and had long tracts of degenerated retroviral elements and transposons. Five asparagus BACs were selected after hybridization of three onion cDNAs that mapped to three different onion chromosomes. Genic-like sequences that were physically linked on the cDNA-selected BACs or genetically linked on the M-linked BACs showed significant similarities (e < -20) to expressed sequences on different rice chromosomes, revealing no evidence for microsynteny between asparagus and rice across these regions. Genic-like sequences that were linked in asparagus were used to identify highly similar (e < -20) expressed sequence tags (ESTs) of onion. These onion ESTs mapped to different onion chromosomes and no relationship was observed between physical or genetic linkages in asparagus and genetic linkages in onion. These results further indicate that synteny among grass genomes does not extend to a sister order in the monocots and that asparagus may not be an appropriate smaller genome model for plants in the Asparagales with enormous nuclear genomes.

Asparagus Plant↗
PubMed2001

Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.).

A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.

Blotting, Southern↗