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PubMed2000

Two-dimensional screening of the Wageningen chicken BAC library.

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies.

Animals↗
PubMed2004

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements.

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.

Biological Evolution↗
PubMed2003

Concomitant reiterative BAC walking and fine genetic mapping enable physical map development for the broad-spectrum late blight resistance region, RB.

The wild potato species Solanum bulbocastanum is a source of genes for potent late blight resistance. We previously mapped resistance to a single region of the S. bulbocastanum chromosome 8 and named the region RB (for "Resistance from S. Bulbocastanum"). We now report physical mapping and contig construction for the RB region via a novel reiterative method of BAC walking and concomitant fine genetic mapping. BAC walking was initiated using RFLP markers previously shown to be associated with late blight resistance. Subcontig extension was accomplished using new probes developed from BAC ends. Significantly, BAC end and partial BAC sequences were also used to develop PCR-based markers to enhance map resolution in the RB region. As they were developed from BAC clones of known position relative to RB, our PCR-based markers are known a priori to be physically closer to the resistance region. These markers allowed the efficient screening of large numbers of segregating progeny at the cotyledon stage, and permitted us to assign the resistance phenotype to a region of approximately 55 kb. Our markers also directed BAC walking efforts away from regions distantly related to RB in favor of the 55-kb region. Because the S. bulbocastanum genotype used in BAC library construction is heterozygous for RB (RB/rb), codominant PCR-based markers, originally developed for fine-scale mapping, were also used to determine homolog origins for individual BAC clones. Ultimately, BAC contigs were constructed for the RB region from both resistant (RB) and susceptible (rb) homologs.

Chromosome Walking↗
PubMed2005

Comparison of AC/O3-BAC and O3-BAC processes for removing organic pollutants in secondary effluent.

AC (activated carbon)/O3-BAC (biological activated carbon) process was employed to treat secondary effluent and compared to O3-BAC process. The effects of ozone dosages and empty bed contact time (EBCT) in BAC on dissolved organic carbon (DOC) removal were investigated. The results showed that the presence of AC improved ozone utilization and biodegradability of the effluent. DOC removal increased with ozone dosage and EBCT in BAC, however, 3 mg l(-1) ozone dosage with 15 min oxidation time and 15 min EBCT in BAC were more economical and efficient. For DOC removal, AC/O3-BAC was more efficient than O3-BAC and its synergetic effect was more than that in O3-BAC process. The biomass of the subsequent BAC unit in AC/O3-BAC process was more than that in O3-BAC process and much more than that in BAC alone. Except for organic pollutants with molecular weight (MW) >10 kDa, those of other MW range were decomposed much more by AC/O3 process than by O3 process. GC/MS analysis showed that dibutyl phthalate, bis(2-ethylhexyl) phthalate, 4-bromo-3-chloroaniline, 2-propanone-ethylhydrazone and phenol derivatives were prevalent organic compounds in the secondary effluent. Some aromatic compounds, such as 4-bromo-3-chloroaniline and 2,4-dichloro-benzenamine disappeared after AC/O3 treatment. However, some small molecules were generated, after further biological treatment by BAC, the kinds and concentration of organic compounds were greatly reduced.

Bioreactors↗
PubMed1997

Human BAC library: construction and rapid screening.

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

B-Lymphocytes↗
PubMed2004

Potential risks of providing drinking drivers with BAC information.

The objective of this paper is to discuss the benefits and risks of providing drinkers with tools that allow them to estimate their blood alcohol concentration (BAC), and to examine the field usability of one commercially available tool. Drinking and driving laws are specified in terms of the driver's BAC, and there is concern that the absence of a method for drivers to accurately estimate their BAC level limits their ability to determine whether they can drive legally. A number of devices that provide a method for the individual to estimate or measure their BAC have been developed. Although some of these devices--such as the "know your limit" (KYL) cards--have been widely distributed, their effectiveness in encouraging good driving decisions have rarely been tested. This article describes a pilot study on the field usability of the Guardian Angel (GA) personal alcohol test in a field setting. The GA test analyzes saliva samples from drinking and indicates under which BAC category they fall (.00 -.04;.04 -.08;.08+). The research examined whether drinkers could, in natural drinking environments, correctly administer and interpret the test results. The methodology involves sampling drinkers on a weekend night on and around the grounds of a large West Coast university as they traveled between off-campus parties, bars, and their dorms. They were asked to assess their own intoxication and impairment, then self-administer and interpret the Guardian Angel test. After interpreting the test, participants were asked to reassess their intoxication and impairment levels, and were given a breath BAC test using a calibrated unit. The results revealed that although the majority of drinkers' were able to administer the GA test, their interpretations did not correspond with actual BACs. The interpretations of the GA test produced false-negatives, underestimating actual BACs. Drinkers perceived themselves to be less intoxicated, on average, after interpreting the GA test results. In conclusion, this research addresses potential pros and cons of providing BAC information to drinkers. It underscores the importance of testing BAC estimation tools under field conditions and the potential risks associated with tests that do not produce accurate results.

Adult↗
PubMed1999

An Escherichia coli-Enterococcus faecalis shuttle vector as a tool for the construction of a group B Streptococcus heterologous mutant expressing the beta antigen (Bac) of the C protein complex.

Group B streptococci (GBS) represent a very important group of human pathogens. So far little is known about the mechanisms by which these bacteria can cause disease and the bacterial factors involved. One putative virulence factor is the beta antigen of the C protein complex (Bac), which can bind to the Fc region of human IgA. Its binding function might represent an important virulence mechanism. However, the genetic manipulation of this group of bacteria, necessary to prove involvement of bacterial factors in pathogenesis, is still in its infancy. We therefore tested the pAM401 vector system for its suitability in the construction of a heterologous expression mutant using the Bac protein as a model antigen. The bac gene, including its own promoter, was cloned into the Escherichia coli-Enterococcus faecalis shuttle vector pAM401 and was stably maintained extrachromosomally in the bac-deficient GBS strain 335. Expression of Bac was assessed by extracting the protein from transformed 335(pPJTU1) cells, negative controls (335 wild-type, 335(pAM401)) and other Bac-expressing GBS strains (A909, LA239). Blots of the extracted proteins probed with IgA, polyclonal sera and a monoclonal antibody raised against Bac clearly revealed expression of the 130-kDa protein in the transformed GBS 335(pPJTU1) cells. The correct processing and surface anchoring of the expressed Bac was demonstrated by binding of (125)I-labelled IgA to whole cells. Strain 335(pPJTU1) bound 12 times as much IgA compared to the parental strain LA239 and the GBS 335 negative controls, and a total of 25% compared to the high-level-expressing strain A909. Our studies show that the pAM401 shuttle vector can be used for stable heterologous expression of surface proteins in GBS. Our strategy is also of major importance for the complementation of deletion mutants in GBS and other Gram-positive human pathogens to fulfill Koch's postulates. The Bac mutant constructed in this study, 335(pPJTU1), can be used in animal models to assess the importance of Bac in GBS pathogenesis.

Antigens, Bacterial↗
PubMed2003

Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 - new vectors for in vitro and in vivo delivery.

BACKGROUND: Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells. RESULTS: We describe a series of retrofitting plasmids and a protocol for in vivo loxP/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE carries G418 resistance and oriP/EBNA-1. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice in vivo. We also show that a BAC carrying GFP, oriP and EBNA-1 can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks. CONCLUSION: The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or oriP/EBNA-1 using in vivo expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle in vivo. OriP/EBNA-1 allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks.

Animals↗
PubMed2003

Field assessment of BAC data to study late-night college drinking.

OBJECTIVE: This field study of late-night college drinking sought to (1) test the ability of the 5+/4+ measure to screen for higher levels of intoxication and (2) examine the relation between estimated and actual blood alcohol concentration (BAC). METHOD: During a 15-week spring semester, college students returning to their residence halls between 10:00 PM and 3:00 AM on Wednesday through Saturday nights were anonymously interviewed to collect BAC and self-report data (n = 1,020). RESULTS: Although 70.9% had not been drinking on Wednesday nights, a majority of the intercepted students had been drinking on the other three nights. Mean BACs on these three nights were in a moderate range (48 to 51 mg/dl), but the 5+/4+ measure classified many students as heavy episodic drinkers at relatively low BACs. For example, 66.3% of those meeting the 5+/4+ criterion for the night had BACs < 100 mg/dl. Students with BACs ranging from 70 to 90 mg/dl exhibited the greatest accuracy in estimating their BAC; those with lower BACs tended to overestimate their level of intoxication; whereas those with higher BACs tended to underestimate it. CONCLUSIONS: Field assessment of student intoxication is an important tool for examining research questions in college drinking. The 5+/4+ measure classifies many college students as heavy episodic drinkers, even though their intoxication level is below conventional thresholds used to define drunkenness. In addition, there is a discernible pattern of BAC estimation in the field that corresponds to intoxication level.

Alcohol Drinking↗
PubMed2004

Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus.

Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics. Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods. This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection. More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes. In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained. A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.

Animals↗
PubMed2001

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

Algorithms↗
PubMed2001

A bacterial artificial chromosome (BAC) library of Malus floribunda 821 and contig construction for positional cloning of the apple scab resistance gene Vf.

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5x haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.

Chromosomes, Artificial, Bacterial↗
PubMed2003

International policies on alcohol impaired driving: are legal blood alcohol concentration (BAC) limits in motorized countries compatible with the scientific evidence?

Borkenstein et al. (1974) study indicated that drivers with BACs of 0.05 to 0.09 per cent were twice as likely to crash as drivers with a zero BAC. Drivers with BACs from 0.10 to 0.14 per cent were ten times as likely to have a fatal crash in 1964. There have been numerous efforts during the history of motorized countries to control the consumption of alcohol and the problems associated with it through legislative mandate, it was not until the 1970s that acceptance of legal BAC (Blood Alcohol Concentration) limits laws became widespread. In particular, as more and more people drive automobiles, the number of traffic accidents involving drunken drivers has soared, and many of these are known to be related to the consumption of alcohol. Thus, legislators find themselves under increasing pressure to find a reasonable and fair solution to the question of alcohol impaired driving, as the scientific evidence about alcohol consumption level and psycho motor functions impairment came to clear. A landmark event in the development of policies regarding impaired driving was the establishment of the fact that consumption of alcohol does, in fact, increase the probability of traffic crashes. Legal limit laws specify a maximum permissible BAC limit for drivers. Currently, a BAC laws range from zero tolerance and 0.02 to 0.10% constitutes prima facie evidence in most countries for 'Driving under Influence of Alcohol.' This latter standard is too permissive, as driving skills deteriorate and crash involvement risk increases beginning at 0.02%. There are consequences attached to setting a BAC limit so high that a 72 kg man can drink five bottles of beer and still be under legal limit. In this sense high legal BAC limit may influence people to make bad estimates of their relative risk of injury or death while driving. Provided there is adequate political will, millions of lives could be saved in the coming years. This review is an attempt to examine in detail the available information about legal BAC limit laws, and issue of considerable interest to both policy makers and the public.

Accidents, Traffic↗
PubMed2004

Construction of BAC and BIBAC libraries and their applications for generation of SSR markers for genome analysis of chickpea, Cicer arietinum L.

Large-insert bacterial artificial chromosome (BAC) libraries, plant-transformation-competent binary BAC (BIBAC) libraries, and simple sequence repeat (SSR) markers are essential for many aspects of genomics research. We constructed a BAC library and a BIBAC library from the nuclear DNA of chickpea, Cicer arietinum L., cv. Hadas, partially digested with HindIII and BamHI, respectively. The BAC library has 14,976 clones, with an average insert size of 121 kb, and the BIBAC library consists of 23,040 clones, with an average insert size of 145 kb. The combined libraries collectively cover ca. 7.0 x genomes of chickpea. We screened the BAC library with eight synthetic SSR oligos, (GA)10, (GAA)7, (AT)10, (TAA)7, (TGA)7, (CA)10, (CAA)7, and (CCA)7. Positive BACs were selected, subcloned, and sequenced for SSR marker development. Two hundred and thirty-three new chickpea SSR markers were developed and characterized by PCR, using chickpea DNA as template. These results have demonstrated that BACs are an excellent source for SSR marker development in chickpea. We also estimated the distribution of the SSR loci in the chickpea genome. The SSR motifs (TAA)n and (GA)n were much more abundant than the others, and the distribution of the SSR loci appeared non-random. The BAC and BIBAC libraries and new SSR markers will provide valuable resources for chickpea genomics research and breeding (the libraries and their filters are available to the public at http://hbz.tamu.edu).

Base Sequence↗
PubMed2005

Construction of two BAC libraries from cucumber (Cucumis sativus L.) and identification of clones linked to yield component quantitative trait loci.

Two bacterial artificial chromosome (BAC) libraries were constructed from an inbred line derived from a cultivar of cucumber (Cucumis sativus L.). Intact nuclei were isolated and embedded in agarose plugs, and high-molecular-weight DNA was subsequently partially digested with BamHI or EcoRI. Ligation of double size-selected DNA fragments with the pECBAC1 vector yielded two libraries containing 23,040 BamHI and 18,432 EcoRI clones. The average BamHI and EcoRI insert sizes were estimated to be 107.0 kb and 100.8 kb, respectively, and BAC clones lacking inserts were 1.3% and 14.5% in the BamHI and EcoRI libraries, respectively. The two libraries together represent approximately 10.8 haploid cucumber genomes. Hybridization with a C(0)t-1 DNA probe revealed that approximately 36% of BAC clones likely carried repetitive sequence-enriched DNA. The frequencies of BAC clones that carry chloroplast or mitochondrial DNA range from 0.20% to 0.47%. Four sequence-characterized amplified region (SCAR), four simple sequence repeat, and an randomly amplified polymorphic DNA marker linked with yield component quantitative trait loci were used either as probes to hybridize high-density colony filters prepared from both libraries or as primers to screen an ordered array of pooled BAC DNA prepared from the BamHI library. Positive BAC clones were identified in predicted numbers, as screening by polymerase chain reaction amplification effectively overcame the problems associated with an overabundance of positives from hybridization with two SCAR markers. The BAC clones identified herein that are linked to the de (determinate habit) and F (gynoecy) locus will be useful for positional cloning of these economically important genes. These BAC libraries will also facilitate physical mapping of the cucumber genome and comparative genome analyses with other plant species.

Chromosomes, Artificial, Bacterial↗
PubMed2004

Phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay.

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.

Bacteria↗
PubMed2004

Coupling generation of cytomegalovirus deletion mutants and amplification of viral BAC clones.

Human cytomegalovirus (HCMV) genome manipulation has always been difficult. Recently, the introduction of full-length HCMV DNA into Escherichia coli as an artificial bacterial chromosome (BAC) clone has allowed reliable targeted mutagenesis. Here, we show the next generation of improvement in designing recombinant HCMV, which will also be applicable to other viral BAC clones. An inducible origin of replication linked with an antibiotic resistance marker was used as a cassette for targeted replacement of sequences within a HCMV BAC clone, TowneBAC. The origin of replication allowed for the induction of increased amounts BAC DNA that improved recovery, ease of use and transfections for mutant viruses. By specific deletion of UL147 and the recombinant GFP gene, we have shown that targeted deletion of a gene and selection for a recombinant genome are coupled with the ability to amplify the BAC clone DNA. These HCMV BAC clones were amplified approximately 10-fold. In the case of the removal of GFP from the clone TowneBAC shown in this study, the resulting BAC DNA preparation following amplification was used for successful primary cell transfection. Both parental and deletion BAC clone transfections gave similar levels of recombinant HCMV, and the GFP deletion virus replicated the same as the TowneBAC in a multi-step growth curve analysis.

Cells, Cultured↗
PubMed1999

High throughput direct end sequencing of BAC clones.

Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed.

Arabidopsis↗