Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.
- MeSH
- DNA fungální genetika MeSH
- genom fungální * MeSH
- genomová knihovna * MeSH
- místa se sekvenční adresou MeSH
- otevřené čtecí rámce MeSH
- proteom genetika MeSH
- proteomika MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvenční analýza DNA MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
- Názvy látek
- DNA fungální MeSH
- proteom MeSH
- Saccharomyces cerevisiae - proteiny MeSH
Invasive meningococcal disease surveillance in Europe combines isolate characterisation and epidemiological data to support public health intervention. A representative European Meningococcal Strain Collection (EMSC) of IMD isolates was obtained, and whole genome sequenced to characterise 799 EMSC isolates from the epidemiological year July 2011-June 2012. To establish a genome library (GL), the isolate information was deposited in the pubMLST.org/neisseria database. Genomes were curated and annotated at 2,429 meningococcal loci, including those defining clonal complex, capsule, antigens, and antimicrobial resistance. Most genomes contained genes encoding B (n = 525; 65.7%) or C (n = 163; 20.4%) capsules; isolates were genetically highly diverse, with >20 genomic lineages, five of which comprising 60.7% (n = 485) of isolates. There were >350 antigenic fine-types: 307 were present once, the most frequent (P1.7-2,4:F5-1) comprised 8% (n = 64) of isolates. Each genome was characterised for Bexsero Antigen Sequence Typing (BAST): 25.5% (n = 204) of isolates contained alleles encoding the fHbp and/or the PorA VR1 vaccine component, but most genomes (n = 513; 64.2%) did not contain the NadA component. EMSC-GL will support an integrated surveillance of disease-associated genotypes in Europe, enabling the monitoring of hyperinvasive lineages, outbreak identification, and supporting vaccine programme implementation.
- Klíčová slova
- Neisseria meningitidis, genome library, monitor vaccine coverage, surveillance, track antimicrobial susceptibility,
- MeSH
- bakteriální geny genetika MeSH
- genetická variace MeSH
- genetické lokusy MeSH
- genom bakteriální MeSH
- genomika MeSH
- genomová knihovna * MeSH
- genotyp MeSH
- lidé MeSH
- meningokoková meningitida genetika mikrobiologie MeSH
- meningokokové infekce genetika mikrobiologie MeSH
- molekulární epidemiologie MeSH
- Neisseria meningitidis séroskupiny B genetika MeSH
- Neisseria meningitidis klasifikace genetika izolace a purifikace MeSH
- sekvenování celého genomu * MeSH
- séroskupina MeSH
- surveillance populace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.
- Klíčová slova
- BAC library, BAC vector, Chromosomes, Clone, DNA cloning, High molecular weight (HMW) DNA, Pulse-field gel electrophoresis (PFGE),
- MeSH
- chromozomy rostlin * MeSH
- genom rostlinný MeSH
- genomová knihovna * MeSH
- průtoková cytometrie metody MeSH
- rostliny genetika MeSH
- umělé bakteriální chromozomy * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phylogenetic analysis was conducted to examine ruminal bacteria in two ruminal fractions (adherent fraction vs. liquid fraction) collected from cattle fed with two different diets: forage alone vs. forage plus concentrate. One hundred forty-four 16S rRNA gene (rrs) sequences were obtained from clone libraries constructed from the four samples. These rrs sequences were assigned to 116 different operational taxonomic units (OTUs) defined at 0.03 phylogenetic distance. Most of these OTUs could not be assigned to any known genus. The phylum Firmicutes was represented by approximately 70% of all the sequences. By comparing to the OTUs already documented in the rumen, 52 new OTUs were identified. UniFrac, SONS, and denaturing gradient gel electrophoresis analyses revealed difference in diversity between the two fractions and between the two diets. This study showed that rrs sequences recovered from small clone libraries can still help identify novel species-level OTUs.
- MeSH
- bachor mikrobiologie MeSH
- Bacteria klasifikace genetika izolace a purifikace MeSH
- biodiverzita MeSH
- fylogeneze MeSH
- genomová knihovna MeSH
- krmivo pro zvířata mikrobiologie MeSH
- mikrobiální společenstva genetika MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- ribotypizace MeSH
- RNA ribozomální 16S analýza MeSH
- sekvenční analýza DNA MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- RNA ribozomální 16S MeSH
BACKGROUND: Genomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C approximately 7,900 Mbp) with prevalence of DNA repeats (> 90%). An attractive possibility is to dissect the genome to small parts after flow sorting particular chromosomes and chromosome arms. To test this approach, we have chosen 1RS chromosome arm, which represents only 5.6% of the total rye genome. The 1RS arm is an attractive target as it carries many important genes and because it became part of the wheat gene pool as the 1BL.1RS translocation. RESULTS: We demonstrate that it is possible to sort 1RS arm from wheat-rye ditelosomic addition line. Using this approach, we isolated over 10 million of 1RS arms using flow sorting and used their DNA to construct a 1RS-specific BAC library, which comprises 103,680 clones with average insert size of 73 kb. The library comprises two sublibraries constructed using HindIII and EcoRI and provides a deep coverage of about 14-fold of the 1RS arm (442 Mbp). We present preliminary results obtained during positional cloning of the stem rust resistance gene SrR, which confirm a potential of the library to speed up isolation of agronomically important genes by map-based cloning. CONCLUSION: We present a strategy that enables sorting short arms of several chromosomes of rye. Using flow-sorted chromosomes, we have constructed a deep coverage BAC library specific for the short arm of chromosome 1R (1RS). This is the first subgenomic BAC library available for rye and we demonstrate its potential for positional gene cloning. We expect that the library will facilitate development of a physical contig map of 1RS and comparative genomics of the homoeologous chromosome group 1 of wheat, barley and rye.
- MeSH
- chromozomy rostlin genetika MeSH
- DNA rostlinná genetika izolace a purifikace MeSH
- genom rostlinný MeSH
- genomová knihovna MeSH
- hybridizace in situ fluorescenční MeSH
- karyotypizace MeSH
- nemoci rostlin genetika MeSH
- průtoková cytometrie MeSH
- pšenice genetika MeSH
- translokace genetická MeSH
- umělé bakteriální chromozomy genetika MeSH
- žito genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA rostlinná MeSH
BACKGROUND: The genome of classical laboratory strains of mice is an artificial mosaic of genomes originated from several mouse subspecies with predominant representation (>90%) of the Mus m. domesticus component. Mice of another subspecies, East European/Asian Mus m. musculus, can interbreed with the classical laboratory strains to generate hybrids with unprecedented phenotypic and genotypic variations. To study these variations in depth we prepared the first genomic large insert BAC library from an inbred strain derived purely from the Mus m. musculus-subspecies. The library will be used to seek and characterize genomic sequences controlling specific monogenic and polygenic complex traits, including modifiers of dominant and recessive mutations. RESULTS: A representative mouse genomic BAC library was derived from a female mouse of the PWD/Ph inbred strain of Mus m. musculus subspecies. The library consists of 144,768 primary clones from which 97% contain an insert of 120 kb average size. The library represents an equivalent of 6.7 x mouse haploid genome, as estimated from the total number of clones carrying genomic DNA inserts and from the average insert size. The clones were arrayed in duplicates onto eight high-density membranes that were screened with seven single-copy gene probes. The individual probes identified four to eleven positive clones, corresponding to 6.9-fold coverage of the mouse genome. Eighty-seven BAC-ends of PWD/Ph clones were sequenced, edited, and aligned with mouse C57BL/6J (B6) genome. Seventy-three BAC-ends displayed unique hits on B6 genome and their alignment revealed 0.92 single nucleotide polymorphisms (SNPs) per 100 bp. Insertions and deletions represented 0.3% of the BAC end sequences. CONCLUSION: Analysis of the novel genomic library for the PWD/Ph inbred strain demonstrated coverage of almost seven mouse genome equivalents and a capability to recover clones for specific regions of PWD/Ph genome. The single nucleotide polymorphism between the strains PWD/Ph and C57BL/6J was 0.92/100 bp, a value significantly higher than between classical laboratory strains. The library will serve as a resource for dissecting the phenotypic and genotypic variations between mice of the Mus m. musculus subspecies and classical laboratory mouse strains.
- MeSH
- delece genu MeSH
- DNA metabolismus MeSH
- druhová specificita MeSH
- fenotyp MeSH
- genom MeSH
- genomová knihovna * MeSH
- genotyp MeSH
- genová knihovna MeSH
- hybridizace nukleových kyselin MeSH
- jednonukleotidový polymorfismus MeSH
- komplementární DNA metabolismus MeSH
- mapování chromozomů MeSH
- modely genetické MeSH
- mutace MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- sekvenční analýza DNA MeSH
- umělé bakteriální chromozomy * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- DNA MeSH
- komplementární DNA MeSH
The analysis of the hexaploid wheat genome (Triticum aestivum L., 2 n=6 x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 10(7) of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.
We cloned and sequenced the Vicia sativa 25S-18S rDNA intergenic spacer (IGS) and the satellite repeat S12, thought to be related to the spacer sequence. The spacer was shown to contain three types of subrepeats (A, B, and C) with monomers of 173 bp (A), 10 bp (B), and 66 bp (C), separated by unique or partially duplicated sequences. Two spacer variants were detected in V. sativa that differed in length (2990 and 3168 bp) owing to an extra copy of the subrepeat A. The A subrepeats were also shown to be highly homologous to the satellite repeat S12, which is located in large clusters on chromosomes 4, 5, and 6, and is not associated with the rDNA loci. Sequencing of additional S12 clones retrieved from a shotgun genomic library allowed definition of three subfamilies of this repeat based on minor differences in their nucleotide sequences. Two of these subfamilies could be discriminated from the rest of the S12 sequences as well as from the IGS A subrepeats using specific oligonucleotide primers that labeled only a subset of the S12 loci when used in the primed in situ DNA labeling (PRINS) reaction on mitotic chromosomes. These experiments showed that, in spite of the high overall similarity of the IGS A subrepeats and the S12 satellite repeats, there are S12 subfamilies that are divergent from the common consensus and are present at only some of the chromosomes containing the S12 loci. Thus, the subfamilies may have evolved at these loci following the spreading of the A subrepeats from the IGS to genomic regions outside the rDNA clusters.
- MeSH
- chromozomy rostlin genetika MeSH
- DNA primery MeSH
- fluorescenční mikroskopie MeSH
- genetické komponenty MeSH
- genomová knihovna MeSH
- in situ značení DNA s primerem MeSH
- mezerníky ribozomální DNA genetika MeSH
- molekulární sekvence - údaje MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční seřazení MeSH
- tandemové repetitivní sekvence genetika MeSH
- Vicia sativa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA primery MeSH
- mezerníky ribozomální DNA MeSH
Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.
- MeSH
- bakteriální chromozomy genetika MeSH
- Escherichia genetika metabolismus patogenita MeSH
- genomová knihovna MeSH
- inzerční mutageneze MeSH
- kosmidy MeSH
- kyseliny hydroxamové metabolismus MeSH
- mapování chromozomů * MeSH
- molekulární sekvence - údaje MeSH
- proteiny vnější bakteriální membrány genetika metabolismus MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- Southernův blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
- Názvy látek
- aerobactin receptor MeSH Prohlížeč
- aerobactin MeSH Prohlížeč
- kyseliny hydroxamové MeSH
- proteiny vnější bakteriální membrány MeSH
- proteiny z Escherichia coli MeSH
Yeast cells growing on solid media organize themselves into multicellular structures, colonies, exhibiting patterns specific for particular yeast strains. With the aim of identifying genes involved in regulations of the colony formation, we applied a new approach enabling the extensive screening of Saccharomyces cerevisiae genes, the expression of which is changed during colony development. We used the library of S. cerevisiae DNA fragments inserted in front of the lacZ gene lacking its own promoter. Colonies of transformants with a blue/white patterned morphotype, implying that the expression of the lacZ gene from the inserted yeast promoter is switched on and off during the colony formation, were isolated. We identified several genes with variable expression during colony morphogenesis, including CCR4, PAM1, MEP3, ADE5,7 and CAT2. S. cerevisiae strain deleted in the CCR4 gene forms colonies with less organized morphology when compared with the isogenic parental strain. The synchronization of the expression patterns of some of the isolated genes in neighboring colonies was observed.
- MeSH
- beta-galaktosidasa genetika MeSH
- buněčná diferenciace genetika MeSH
- buněčné dělení genetika MeSH
- delece genu MeSH
- fungální proteiny genetika MeSH
- genetická transkripce genetika MeSH
- genomová knihovna MeSH
- karboxylyasy genetika MeSH
- katalasa genetika MeSH
- kultivační média farmakologie MeSH
- mapování chromozomů metody MeSH
- multienzymové komplexy genetika MeSH
- mutace genetika MeSH
- oxygenasy se smíšenou funkcí genetika MeSH
- peptidsynthasy genetika MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) genetika MeSH
- proteiny přenášející kationty * MeSH
- regulace genové exprese genetika MeSH
- reportérové geny genetika MeSH
- ribonukleasy * MeSH
- Saccharomyces cerevisiae - proteiny * MeSH
- Saccharomyces cerevisiae cytologie genetika metabolismus MeSH
- transformace genetická genetika MeSH
- transkripční faktory genetika MeSH
- transportní proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- beta-galaktosidasa MeSH
- CCR4 protein, S cerevisiae MeSH Prohlížeč
- fungální proteiny MeSH
- karboxylyasy MeSH
- katalasa MeSH
- kultivační média MeSH
- MEP3 protein, S cerevisiae MeSH Prohlížeč
- multienzymové komplexy MeSH
- oxygenasy se smíšenou funkcí MeSH
- peptidsynthasy MeSH
- peptidylglycine monooxygenase MeSH Prohlížeč
- proteiny přenášející kationty * MeSH
- ribonukleasy * MeSH
- Saccharomyces cerevisiae - proteiny * MeSH
- transkripční faktory MeSH
- transportní proteiny MeSH
