Bacterial Artificial Chromosome Sequences Research Articles

Rapid generation of markerless recombinant MVA vaccines by en passant recombineering of a self-excising bacterial artificial chromosome

The non-replicating poxviral vector modified vaccinia virus Ankara (MVA) is currently a leading candidate for development of novel recombinant vaccines against globally important diseases. The 1980s technology for making recombinant MVA (and other poxviruses) is powerful and robust, but relies on rare recombination events in poxviral-infected cells. In the 21st century, it has become possible to apply bacterial artificial chromosome (BAC) technology to poxviruses, as first demonstrated by B. Moss’ lab in 2002 for vaccinia virus. A similar BAC clone of MVA was subsequently derived, but while recombination-mediated genetic engineering for rapid production was used of deletion mutants, an alternative method was required for efficient insertion of transgenes. Furthermore “markerless” viruses, which carry no trace of the selectable marker used for their isolation, are increasingly required for clinical trials, and the viruses derived via the new method contained the BAC sequence in their genomic DNA. Two methods are adapted to MVA–BAC to provide more rapid generation of markerless recombinants in weeks rather than months. “ En passant” recombineering is applied to the insertion of a transgene expression cassette and the removal of the selectable marker in bacteria; and a self-excising variant of MVA–BAC is constructed, in which the BAC cassette region is rapidly and efficiently lost from the viral genome following rescue of the BAC into infectious virus. These methods greatly facilitate and accelerate production of recombinant MVA, including markerless constructs.

Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes

BackgroundSugarcane (Saccharum spp.) has become an increasingly important crop for its leading role in biofuel production. The high sugar content species S. officinarum is an octoploid without known diploid or tetraploid progenitors. Commercial sugarcane cultivars are hybrids between S. officinarum and wild species S. spontaneum with ploidy at ~12×. The complex autopolyploid sugarcane genome has not been characterized at the DNA sequence level.ResultsThe microsynteny between sugarcane and sorghum was assessed by comparing 454 pyrosequences of 20 sugarcane bacterial artificial chromosomes (BACs) with sorghum sequences. These 20 BACs were selected by hybridization of 1961 single copy sorghum overgo probes to the sugarcane BAC library with one sugarcane BAC corresponding to each of the 20 sorghum chromosome arms. The genic regions of the sugarcane BACs shared an average of 95.2% sequence identity with sorghum, and the sorghum genome was used as a template to order sequence contigs covering 78.2% of the 20 BAC sequences. About 53.1% of the sugarcane BAC sequences are aligned with sorghum sequence. The unaligned regions contain non-coding and repetitive sequences. Within the aligned sequences, 209 genes were annotated in sugarcane and 202 in sorghum. Seventeen genes appeared to be sugarcane-specific and all validated by sugarcane ESTs, while 12 appeared sorghum-specific but only one validated by sorghum ESTs. Twelve of the 17 sugarcane-specific genes have no match in the non-redundant protein database in GenBank, perhaps encoding proteins for sugarcane-specific processes. The sorghum orthologous regions appeared to have expanded relative to sugarcane, mostly by the increase of retrotransposons.ConclusionsThe sugarcane and sorghum genomes are mostly collinear in the genic regions, and the sorghum genome can be used as a template for assembling much of the genic DNA of the autopolyploid sugarcane genome. The comparable gene density between sugarcane BACs and corresponding sorghum sequences defied the notion that polyploidy species might have faster pace of gene loss due to the redundancy of multiple alleles at each locus.

A major QTL for resistance to Gibberella stalk rot in maize

Fusarium graminearum Schwabe, the conidial form of Gibberella zeae, is the causal fungal pathogen responsible for Gibberella stalk rot of maize. Using a BC(1)F(1) backcross mapping population derived from a cross between '1145' (donor parent, completely resistant) and 'Y331' (recurrent parent, highly susceptible), two quantitative trait loci (QTLs), qRfg1 and qRfg2, conferring resistance to Gibberella stalk rot have been detected. The major QTL qRfg1 was further confirmed in the double haploid, F(2), BC(2)F(1), and BC(3)F(1) populations. Within a qRfg1 confidence interval, single/low-copy bacterial artificial chromosome sequences, anchored expressed sequence tags, and insertion/deletion polymorphisms, were exploited to develop 59 markers to saturate the qRfg1 region. A step by step narrowing-down strategy was adopted to pursue fine mapping of the qRfg1 locus. Recombinants within the qRfg1 region, screened from each backcross generation, were backcrossed to 'Y331' to produce the next backcross progenies. These progenies were individually genotyped and evaluated for resistance to Gibberella stalk rot. Significant (or no significant) difference in resistance reactions between homozygous and heterozygous genotypes in backcross progeny suggested presence (or absence) of qRfg1 in '1145' donor fragments. The phenotypes were compared to sizes of donor fragments among recombinants to delimit the qRfg1 region. Sequential fine mapping of BC(4)F(1) to BC(6)F(1) generations enabled us to progressively refine the qRfg1 locus to a ~500-kb interval flanked by the markers SSR334 and SSR58. Meanwhile, resistance of qRfg1 to Gibberella stalk rot was also investigated in BC(3)F(1) to BC(6)F(1) generations. Once introgressed into the 'Y331' genome, the qRfg1 locus could steadily enhance the frequency of resistant plants by 32-43%. Hence, the qRfg1 locus was capable of improving maize resistance to Gibberella stalk rot.

The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination

During immunoglobulin heavy chain (Igh) V(D)J recombination, D to J precedes V to DJ recombination in an ordered manner, controlled by differential chromatin accessibility of the V and DJ regions and essential for correct antibody assembly. However, with the exception of the intronic enhancer Emu, which regulates D to J recombination, cis-acting regulatory elements have not been identified. We have assembled the sequence of a strategically located 96-kb V-D intergenic region in the mouse Igh and analyzed its activity during lymphocyte development. We show that Emu-dependent D antisense transcription, proposed to open chromatin before D to J recombination, extends into the V-D region for more than 30 kb in B cells before, during, and after V(D)J recombination and in T cells but terminates 40 kb from the first V gene. Thus, subsequent V antisense transcription before V to DJ recombination is actively prevented and must be independently activated. To find cis-acting elements that regulate this differential chromatin opening, we identified six DNase I-hypersensitive sites (HSs) in the V-D region. One conserved HS upstream of the first D gene locally regulates D genes. Two further conserved HSs near the D region mark a sharp decrease in antisense transcription, and both HSs bind CTCF in vivo. Further, they both possess enhancer-blocking activity in vivo. Thus, we propose that they are enhancer-blocking insulators preventing Emu-dependent chromatin opening extending into the V region. Thus, they are the first elements identified that may control ordered V(D)J recombination and correct assembly of antibody genes.

Dissecting the telomere region of barley chromosome 5HL using rice genomic sequences as references: new markers for tracking a complex region in breeding

The terminal region of barley chromosome 5HL controls malt extract, diastatic power, free amino acid nitrogen, alpha-amylase activity, seed dormancy and pre-harvest sprouting. Comparative analysis of the barley and rice maps has established that the terminal region of barley chromosome 5HL is syntenic to rice chromosome 3L near the telomere end. The rice BAC (Bacterial Artificial Chromosome) sequences covering the region of chromosome 3L were used to search barley expressed sequenced tags database. Thirty-three genes were amplified by PCR (polymerase chain reaction) with the primers designed from barley ESTs (expressed sequence tag). Comparison of the sequences of the PCR generated DNA fragments revealed polymorphisms including single nucleotide polymorphism (SNP), insertions or deletions between the barley varieties. Seven new PCR based molecular markers were developed and mapped within 10 cM in three doubled haploid barley populations (Stirling × Harrington, Baudin × AC Metcalfe and Chebec × Harrington). The mapped genes maintain the micro-syntenic relationship between barley and rice. These gene specific markers provide simple and efficient tools for germplasm characterization and marker-assisted selection for barley malting quality, and ultimately lead to isolation and identification of the major gene(s) controlling multiple quality traits on barley chromosome 5HL.

Identification and Functional Characterization of Monofunctionalent-Copalyl Diphosphate andent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

Fine mapping of pepper trichome locus 1 controlling trichome formation in Capsicum annuum L. CM334

Trichomes are present on nearly all land plants and protect plants against insect herbivores, drought and UV radiation. The trichome-bearing phenotype is conferred by the dominant allele of the pepper trichome locus 1 (Ptl1) in Capsicum annuum, Mexican 'Criollo de Morelos-334' (CM334). A genetic analysis using simple sequence repeats from pepper cDNA identified the HpmsE031 marker as tightly linked to Ptl1 in 653 individuals of an F(2) population derived from a cross between CM334 and Chilsungcho varieties. A bacterial artificial chromosome (BAC) library from CM334 covering 12x of the genome was screened using the HpmsE031 SSR marker as a probe and three BAC clones were identified. The Ptl1 region was covered by one 80 kb BAC clone, TT1B7. Fluorescence in situ hybridization (FISH) confirmed that TT1B7 localized to pepper chromosome 10. One co-dominant marker, Tco, and one dominant marker, Tsca, were successfully developed from the TT1B7 BAC sequence. Tco mapped 0.33 cM up from Ptl1 and Tsca mapped 0.75 cM down from Ptl1. Analysis of the BAC sequence predicts the presence of 14 open reading frames including 60S ribosomal protein L21-like protein (Solanum demissum), protein kinase 2 (Nicotiana tabacum), hypothetical proteins, and unnamed protein products. These results will provide not only useful information for map-based cloning of Ptl1 in Capsicum but also the starting points for analysis of R-gene cluster inked with Ptl1.

Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constraints. We introduced the 160-kb human herpes virus 6A (HHV-6A) genome into BACs by digesting the viral DNA replicative intermediates with the Sfil enzyme that cleaves the viral genome in a single site. The recombinant BACs contained also the puromycin selection gene, GFP, and LoxP sites flanking the BAC sequences. The HHV-6A-BAC vectors were retained stably in puromycin selected 293T cells. In the presence of irradiated helper virus, supplying most likely proteins enhancing gene expression they expressed early and late genes in SupT1 T cells. The method is especially attractive for viruses that replicate inefficiently and for viruses propagated in suspension cells. We have used the fact that the BAC cloning "freezes" the viral DNA replication intermediates to analyze their structure. The results revealed that HHV-6A-BACs contained a single direct repeat (DR) rather than a DR-DR sequence, predicted to arise by circularization of parental genomes with a DR at each terminus. HHV-6A DNA molecules prepared from the infected cells also contained DNA molecules with a single DR. Such forms were not previously described for HHV-6 DNA.

Development of SNP markers linked to the L locus in Capsicum spp. by a comparative genetic analysis

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L 4 gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L 4 - and L 3 -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L 3 , was located on the opposite side of 087H3T7, about 0.7 cM away from L 4 . This supports the idea that L 3 and L 4 may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L 4 to elite germplasm against most aggressive tobamoviruses pathotype P1,2,3 is discussed.

A highly annotated whole-genome sequence of a Korean individual

Recent advances in sequencing technologies have initiated an era of personal genome sequences. To date, human genome sequences have been reported for individuals with ancestry in three distinct geographical regions: a Yoruba African, two individuals of northwest European origin, and a person from China. Here we provide a highly annotated, whole-genome sequence for a Korean individual, known as AK1. The genome of AK1 was determined by an exacting, combined approach that included whole-genome shotgun sequencing (27.8x coverage), targeted bacterial artificial chromosome sequencing, and high-resolution comparative genomic hybridization using custom microarrays featuring more than 24 million probes. Alignment to the NCBI reference, a composite of several ethnic clades, disclosed nearly 3.45 million single nucleotide polymorphisms (SNPs), including 10,162 non-synonymous SNPs, and 170,202 deletion or insertion polymorphisms (indels). SNP and indel densities were strongly correlated genome-wide. Applying very conservative criteria yielded highly reliable copy number variants for clinical considerations. Potential medical phenotypes were annotated for non-synonymous SNPs, coding domain indels, and structural variants. The integration of several human whole-genome sequences derived from several ethnic groups will assist in understanding genetic ancestry, migration patterns and population bottlenecks.

A BAC-based physical map of the Hessian fly genome anchored to polytene chromosomes

BackgroundThe Hessian fly (Mayetiola destructor) is an important insect pest of wheat. It has tractable genetics, polytene chromosomes, and a small genome (158 Mb). Investigation of the Hessian fly presents excellent opportunities to study plant-insect interactions and the molecular mechanisms underlying genome imprinting and chromosome elimination. A physical map is needed to improve the ability to perform both positional cloning and comparative genomic analyses with the fully sequenced genomes of other dipteran species.ResultsAn FPC-based genome wide physical map of the Hessian fly was constructed and anchored to the insect's polytene chromosomes. Bacterial artificial chromosome (BAC) clones corresponding to 12-fold coverage of the Hessian fly genome were fingerprinted, using high information content fingerprinting (HIFC) methodology, and end-sequenced. Fluorescence in situ hybridization (FISH) co-localized two BAC clones from each of the 196 longest contigs on the polytene chromosomes. An additional 70 contigs were positioned using a single FISH probe. The 266 FISH mapped contigs were evenly distributed and covered 60% of the genome (95,668 kb). The ends of the fingerprinted BACs were then sequenced to develop the capacity to create sequenced tagged site (STS) markers on the BACs in the map. Only 3.64% of the BAC-end sequence was composed of transposable elements, helicases, ribosomal repeats, simple sequence repeats, and sequences of low complexity. A relatively large fraction (14.27%) of the BES was comprised of multi-copy gene sequences. Nearly 1% of the end sequence was composed of simple sequence repeats (SSRs).ConclusionThis physical map provides the foundation for high-resolution genetic mapping, map-based cloning, and assembly of complete genome sequencing data. The results indicate that restriction fragment length heterogeneity in BAC libraries used to construct physical maps lower the length and the depth of the contigs, but is not an absolute barrier to the successful application of the technology. This map will serve as a genomic resource for accelerating gene discovery, genome sequencing, and the assembly of BAC sequences. The Hessian fly BAC-clone assembly, and the names and positions of the BAC clones used in the FISH experiments are publically available at .

Efficient frequency-based de novo short-read clustering for error trimming in next-generation sequencing

Novel massively parallel sequencing technologies provide highly detailed structures of transcriptomes and genomes by yielding deep coverage of short reads, but their utility is limited by inadequate sequencing quality and short-read lengths. Sequencing-error trimming in short reads is therefore a vital process that could improve the rate of successful reference mapping and polymorphism detection. Toward this aim, we herein report a frequency-based, de novo short-read clustering method that organizes erroneous short sequences originating in a single abundant sequence into a tree structure; in this structure, each "child" sequence is considered to be stochastically derived from its more abundant "parent" sequence with one mutation through sequencing errors. The root node is the most frequently observed sequence that represents all erroneous reads in the entire tree, allowing the alignment of the reliable representative read to the genome without the risk of mapping erroneous reads to false-positive positions. This method complements base calling and the error correction of making direct alignments with the reference genome, and is able to improve the overall accuracy of short-read alignment by consulting the inherent relationships among the entire set of reads. The algorithm runs efficiently with a linear time complexity. In addition, an error rate evaluation model can be derived from bacterial artificial chromosome sequencing data obtained in the same run as a control. In two clustering experiments using small RNA and 5'-end mRNA reads data sets, we confirmed a remarkable increase ( approximately 5%) in the percentage of short reads aligned to the reference sequence.

Genome‐wide genetic diversity of ‘Nici’, the DNA source for the CHORI‐260 turkey BAC library and candidate for whole genome sequencing

Vertebrate whole genome sequence assembly can benefit from a priori knowledge of variability in the target genome, with researchers often selecting highly inbred individuals for sequencing. However, for most species highly inbred research lines are lacking, requiring the use of an outbred individual(s). Here we examined the source DNA [Nicholas inbred (Nici)] of the CHORI-260 turkey bacterial artificial chromosome (BAC) library through analysis of microsatellites and BAC sequences. Heterozygosity of Nici was compared with that of individuals from several breeder lines. Seventy-eight microsatellites were screened for polymorphism in a total of 43 birds, identifying an average individual heterozygosity of 0.39, with Nici at 0.35. Additional loci (total of 147) were examined on a subset of individuals to obtain better genome coverage. The mean heterozygosity for this subset was 0.33 with Nici at 0.31. Examination of approximately 200 kb of genome sequence identified SNPs in the order of one per 200 bp in Nici. These data suggest that the heterozygosity of Nici is comparable to other birds of selected breeder lines and that whole genome sequencing would result in an abundant resource of genome-wide polymorphisms.

Haplotype analyses in wheat for complex traits: tracking the chromosome 3B and 7B regions associated with late maturity alpha amylase (LMA) in breeding programs

The quantitative trait loci (QTLs) on chromosomes 7BL and 3BS from Halberd have been used as a major source of tolerance to late maturity α amylase (LMA) within Australian wheat breeding programs. New simple sequence repeat (SSR) markers identified from the sequencing of Bacterial Artificial Chromosome (BAC) clones from the wheat cv. Renan library, and known SSRs, were used to characterise these major QTLs. The reduction or elimination of the LMA defect in wheat cultivars is a major goal for wheat breeding programs and is confounded by the complexity in measuring the trait unambiguously. In this haplotyping study focussing on two significant chromosomal regions, markers and combinations of markers were investigated for their ability to discriminate between 39 Australian and Mexican wheat lines differing in levels of LMA. Genetic relationships among these wheat lines estimated by cluster analysis of molecular marker data were combined with phenotypic information in order to calibrate the genotypes of the wheat lines against their LMA phenotype. It was evident that some SSRs from the respective QTLs had greater discriminating power than others to identify LMA phenotypes. Discrimination was not, however, absolute and a statistical analysis of the data defined a risk factor associated with particular combinations of alleles, for use in early selection or backcrossing.

A Bioinformatics Resource for Crop Functional Genomics: GFSelector Module in Automated Annotation System, RiceGAAS

GFSelector (Gene Function Selector, http://alnilam.mi.mss.co.jp/rgadb/) has been developed to perform computational classification of gene models and assignment of unique biological function. It has been incorporated in RiceGAAS (http://ricegaas.dna.affrc.go.jp/usr/) which was designed to provide an analysis pipeline for user submitted genome sequences and comprehensive database for all rice gene models. The combined system facilitates accurate modelling of predicted rice genes, classification of gene structure, and assigning of function and GO (gene ontology) terms to the gene models. The reliability and accuracy are enhanced by integrating several reference databases into the system and generating multiple candidates for determining the function of the gene models. The pipeline is also fully automated thereby facilitating regularly updates of the rice gene models using the latest reference databases. Annotation of soybean, wheat and banana BAC (bacterial artificial chromosome) sequences was performed to test the applicability of the pipeline to other crops. As compared with the GenBank CDS (coding sequence) features, more than 83% of nucleotide-level sensitivity was obtained for the gene modelling by the pipeline. It was also confirmed that 95% of functional annotation by the pipeline was nearly equal or better than the corresponding GenBank CDS feature.

Haplotype divergence in Beta vulgaris and microsynteny with sequenced plant genomes

We characterized two overlapping sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) clones representing different haplotypes. A total of 254 kbp of the genomic sequence was determined, of which the two BACs share 92 kbp. Eleven of 15 genes discovered in the sequenced interval locate to the overlap region. The haplotypes differ in exons by 1% (nucleotide level) and in non-coding regions by 9% (6% mismatches, 3% gaps; alignable regions only). Large indels or high sequence divergence comprised 11% of either sequence. Of such indels, 68 and 45%, respectively, could be attributed to haplotype-specific integration of transposable elements. We identified novel repeat candidates by comparing the two BAC sequences to a set of genomic sugar beet sequences. Synteny was found with Arabidopsis chromosome 1 (At1), At2 and At4, Medicago chromosome 7, Vitis chromosome 15 and paralogous regions on poplar chromosomes II and XIV.

The Pig Genome Database (PiGenome): an integrated database for pig genome research

We established the Pig Genome Database (PiGenome) for pig genome research. The PiGenome integrates and analyzes all publicly available genome-wide data on pigs, including UniGenes, sequence tagged sites (STS) markers, quantitative trait loci (QTLs) data, and bacterial artificial chromosome (BAC) contigs. In addition, we produced 69,545 expressed sequence tags (ESTs) from the full-length enriched cDNA libraries of six tissues and 182 BAC contig sequences, which are also included in the database. QTLs, genetic markers, and BAC end-sequencing information were collected from public databases. The full-length enriched EST data were clustered and assembled into unique sequences, contigs, and singletons. The PiGenome provides functional annotation, identification of transcripts, mapping of coding sequences, and SNP information. It also provides an advanced search interface, a disease browser, alternative-splicing events, and a comparative gene map of the pig. A graphical map view and genome browser can map ESTs, contigs, BAC contigs (from the National Institute of Animal Science), Sino-Danish Pig Genome Project transcripts, and UniGene onto pig genome sequences which include our 182 BAC contigs and publically available BAC sequences of the Wellcome Trust Sanger Institute. The PiGenome is accessible at http://pigenome.nabc.go.kr/ .

Lineage-specific diversification of killer cell Ig-like receptors in the owl monkey, a New World primate

Killer cell Ig-like receptors (KIRs) modulate the cytotoxic effects of natural killer cells. In primates, the KIRs are highly diverse as a consequence of variation in gene content, alternative domain composition, and loci polymorphism. We analyzed a bacterial artificial chromosome (BAC) clone draft sequence spanning the owl monkey KIR cluster. The draft sequence had seven ordered yet unconnected contigs containing six full-length and two partial gene models, flanked by the LILRB and FcAR framework genes. Gene models were predicted to encode KIRs with inhibitory, activating, or dual functionality. Four gene models encoded three Ig domain receptors, while three others encoded molecules with four Ig domains. The additional domain resulted from an insertion in tandem of a 2,101 bp fragment containing the last 289 bp of intron 2, exon 3, and intron 3, resulting in molecules with two D0 domains. Re-screening of the owl monkey BAC library and sequencing of partial cDNAs from an owl monkey yielded five additional KIRs, four of which encoded receptors with short cytoplasmic domains with premature stop codons due to either a single nucleotide substitution or deletion or the absence of exon 8. Phylogenetic analysis by domains showed that owl monkey KIRs were monophyletic, clustering independently from other primate KIR lineages. Retroelements found in introns, however, were shared by KIRs from different primate lineages. This suggests that the owl monkey inherited a KIR cluster with a rich history of exon shuffling upon which positive selection for ligand binding operated to diversify the receptors in a lineage-specific fashion.

A Gateway® recombination herpesvirus cloning system with negative selection that produces vectorless progeny

Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines.

Optimal pooling for genome re-sequencing with ultra-high-throughput short-read technologies

New generation sequencing technologies offer unique opportunities and challenges for re-sequencing studies. In this article, we focus on re-sequencing experiments using the Solexa technology, based on bacterial artificial chromosome (BAC) clones, and address an experimental design problem. In these specific experiments, approximate coordinates of the BACs on a reference genome are known, and fine-scale differences between the BAC sequences and the reference are of interest. The high-throughput characteristics of the sequencing technology makes it possible to multiplex BAC sequencing experiments by pooling BACs for a cost-effective operation. However, the way BACs are pooled in such re-sequencing experiments has an effect on the downstream analysis of the generated data, mostly due to subsequences common to multiple BACs. The experimental design strategy we develop in this article offers combinatorial solutions based on approximation algorithms for the well-known max n-cut problem and the related max n-section problem on hypergraphs. Our algorithms, when applied to a number of sample cases give more than a 2-fold performance improvement over random partitioning.Contact:cenk@cs.sfu.ca