scholarly journals Characterization of microsatellites revealed by genomic sequencing of Populus trichocarpa

2004 ◽  
Vol 34 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Gerald A Tuskan ◽  
Lee E Gunter ◽  
Zamin K Yang ◽  
TongMing Yin ◽  
Mitchell M Sewell ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Populus Trichocarpa ◽  
Repeat Unit ◽  
Artificial Chromosome ◽  
Genetic Maps ◽  
Bacterial Artificial Chromosomes ◽  
Tetranucleotide Repeat ◽  
Artificial Chromosomes ◽  
High Utility ◽  
Populus Species

Microsatellites or simple sequence repeats (SSRs) are highly polymorphic, codominant markers that have great value for the construction of genetic maps, comparative mapping, population genetic surveys, and paternity analyses. Here, we report the development and testing of a set of SSR markers derived from shotgun sequencing from Populus trichocarpa Torr. & A. Gray, a nonenriched genomic DNA library, and bacterial artificial chromosomes. Approximately 23% of the 1536 genomic clones and 48% of the 768 bacterial artificial chromosome subclones contained an SSR. Of the sequences containing an SSR, 72.4% contained a dinucleotide, 19.5% a trinucleotide, and 8.1% a tetranucleotide repeat unit; 26.6% of the sequences contained multiple SSR motifs in a complex or compound repeat structures. A survey of the genome sequence database revealed very similar proportional distribution, indicating that our limited rapid, shallow sequencing effort is representative of genome-wide patterns. In total, 492 primer pairs were designed and these yielded 77 markers that were mapped in an F2 pedigree, including 26 that were sufficiently informative to be included in a Populus framework map. SSRs with GC-rich motifs mapped at a significantly higher frequency than expected, although AT-rich SSRs accounted for the majority of mapped markers due to their higher representation in the genome. SSR markers developed from P. trichocarpa showed high utility throughout the genus, with amplification rates in excess of 70% for all Populus species tested. Finally, at least 30% of the markers amplified in several willow species, suggesting that some of these SSRs will be transferable across genera.

scholarly journals Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8320-8329 ◽  
Author(s):  
Eva-Maria Borst ◽  
Gabriele Hahn ◽  
Ulrich H. Koszinowski ◽  
Martin Messerle
Keyword(s):  
Escherichia Coli ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Reading Frame ◽  
E Coli ◽  
Artificial Chromosomes ◽  
Internal Repeat ◽  
The Stability

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

scholarly journals Chimeras Link to Tandem Repeats and Transposable Elements in Tetraploid Hybrid Fish

2016 ◽  
Author(s):  
Lihai Ye ◽  
Xiaojun Tang ◽  
Yiyi Chen ◽  
Li Ren ◽  
Fangzhou Hu ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Tandem Repeats ◽  
Artificial Chromosome ◽  
Strand Break ◽  
Bacterial Artificial Chromosomes ◽  
Distant Hybridization ◽  
Artificial Chromosomes ◽  
Hybrid Fish ◽  
The Relationship ◽  
Allotetraploid Hybrid

AbstractThe formation of the allotetraploid hybrid lineage (4nAT) encompasses both distant hybridization and polyploidization processes. The allotetraploid offspring have two sets of sub-genomes inherited from both parental species and therefore it is important to explore its genetic structure. Herein, we construct a bacterial artificial chromosome library of allotetraploids, and then sequence and analyze the full-length sequences of 19 bacterial artificial chromosomes. Sixty-eight DNA chimeras are identified, which are divided into four models according to the distribution of the genomic DNA derived from the parents. Among the 68 genetic chimeras, 44 (64.71%) are linked to tandem repeats (TRs) and 23 (33.82%) are linked to transposable elements (TEs). The chimeras linked to TRs are related to slipped-strand mispairing and double-strand break repair while the chimeras linked to TEs are benefit from the intervention of recombinases. In addition, TRs and TEs are linked not only with the recombinations, but also with the insertions/deletions of DNA segments. We conclude that DNA chimeras accompanied by TRs and TEs coordinate a balance between the sub-genomes derived from the parents which reduces the genomic shock effects and favors the evolutionary and adaptive capacity of the allotetraploidization. It is the first report on the relationship between formation of the DNA chimeras and TRs and TEs in the polyploid animals.

FISH of a maize sh2-selected sorghum BAC to chromosomes of Sorghum bicolor

Genome ◽  
1997 ◽  
Vol 40 (4) ◽  
pp. 475-478 ◽  
Author(s):  
Martha I. Gómez ◽  
M. Nurul Islam-Faridi ◽  
Sung-Sick Woo ◽  
Don Czeschin Jr. ◽  
Michael S. Zwick ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Sorghum Bicolor ◽  
Fluorescence In Situ Hybridization ◽  
Artificial Chromosome ◽  
Single Copy ◽  
Bacterial Artificial Chromosomes ◽  
Extra Copy ◽  
Artificial Chromosomes ◽  
Sorghum Plant

Fluorescence in situ hybridization (FISH) of a 205 kb Sorghum bicolor bacterial artificial chromosome (BAC) containing a sequence complementary to maize sh2 cDNA produced a large pair of FISH signals at one end of a midsize metacentric chromosome of S. bicolor. Three pairs of signals were observed in metaphase spreads of chromosomes of a sorghum plant containing an extra copy of one arm of the sorghum chromosome arbitrarily designated with the letter D. Therefore, the sequence cloned in this BAC must reside in the arm of chromosome D represented by this monotelosome. This demonstrates a novel procedure for physically mapping cloned genes or other single-copy sequences by FISH, sh2 in this case, by using BACs containing their complementary sequences. The results reported herein suggest homology, at least in part, between one arm of chromosome D in sorghum and the long arm of chromosome 3 in maize.Key words: sorghum, maize, shrunken locus, physical mapping, fluorescence in situ hybridization, bacterial artificial chromosomes.

scholarly journals Using Bacterial Artificial Chromosomes in Leukemia Research: The Experience at the University Cytogenetics Laboratory in Brest, France

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Etienne De Braekeleer ◽  
Nathalie Douet-Guilbert ◽  
Audrey Basinko ◽  
Frédéric Morel ◽  
Marie-Josée Le Bris ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Artificial Chromosome ◽  
Genome Project ◽  
Comparative Genomic ◽  
Cancer Genes ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Dna Libraries ◽  
Conventional Cytogenetics

The development of the bacterial artificial chromosome (BAC) system was driven in part by the human genome project in order to construct genomic DNA libraries and physical maps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300 kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions.

scholarly journals An Overlapping Bacterial Artificial Chromosome System That Generates Vectorless Progeny for Channel Catfish Herpesvirus

2008 ◽  
Vol 82 (8) ◽  
pp. 3872-3881 ◽  
Author(s):  
Dusan Kunec ◽  
Larry A. Hanson ◽  
Sandra van Haren ◽  
I. F. Nieuwenhuizen ◽  
Shane C. Burgess
Keyword(s):  
Channel Catfish ◽  
Infectious Clone ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Targeted Mutation ◽  
Infectious Clones ◽  
Viral Progeny ◽  
Herpesvirus 1

ABSTRACT Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild-type (wt) virus are extremely valuable tools for elucidating the roles of specific genes in virus pathophysiology as well as for making vaccines. Ictalurid herpesvirus 1 (channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here, we describe the cloning of the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BACs). These clones allowed us to regenerate vectorless wt CCVs with a phenotype that is indistinguishable from that of the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full-length CCV BAC by replacing the CCV ORF5 with the BAC cassette and cotransfecting CCO cells. The viral progeny that we used to transform Escherichia coli and the resulting BAC had only one of the 18-kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of the generation of an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants.

scholarly journals Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during CultureIn Vitro

2016 ◽  
Vol 90 (8) ◽  
pp. 3929-3943 ◽  
Author(s):  
Isa Murrell ◽  
Gavin S. Wilkie ◽  
Andrew J. Davison ◽  
Evelina Statkute ◽  
Ceri A. Fielding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Clinical Strain ◽  
Minimal Risk ◽  
Bacterial Artificial Chromosomes ◽  
Wild Type ◽  
Artificial Chromosomes

ABSTRACTClinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb′ (UL/b′) region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L.IMPORTANCEResearchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagatedin vitrowith minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique

Genome ◽  
2006 ◽  
Vol 49 (5) ◽  
pp. 432-444 ◽  
Author(s):  
Michiya Koike ◽  
Kanako Kawaura ◽  
Yasunari Ogihara ◽  
Atsushi Torada
Keyword(s):  
Ssr Markers ◽  
Common Wheat ◽  
Genomic Dna ◽  
Genomic Library ◽  
Artificial Chromosome ◽  
Triticum Aestivum L ◽  
Pcr Screening ◽  
Artificial Chromosomes ◽  
Isolation And Characterization ◽  
Simple Sequence

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.Key words: common wheat, simple sequence repeat (SSR), PCR screening, LTR-retrotransposon, TAC clone.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

Homologous Recombination Using Bacterial Artificial Chromosomes

2015 ◽  
Vol 2015 (2) ◽  
pp. pdb.prot072397
Author(s):  
Cary Lai ◽  
Tobias Fischer ◽  
Elizabeth Munroe
Keyword(s):  
Homologous Recombination ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes
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