Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2)

Genome ◽  
2001 ◽  
Vol 44 (2) ◽  
pp. 154-162 ◽  
Author(s):  
Meizhong Luo ◽  
Yi-Hong Wang ◽  
David Frisch ◽  
Tarek Joobeur ◽  
Rod A Wing ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Fusarium Wilt ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Microtiter Plates ◽  
Bac Libraries ◽  
Average Size ◽  
Improved Methods

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.Key words: bacterial artificial chromosome (BAC) library, Fusarium wilt, melon, pCUGIBAC1, resistant gene.

Transgenic modification of cows milk for value-added processing

1998 ◽  
Vol 10 (8) ◽  
pp. 671 ◽  
Author(s):  
Kurt A. Zuelke
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Dairy Cattle ◽  
Dna Sequences ◽  
Gene Locus ◽  
Bac Library ◽  
Value Added ◽  
Artificial Chromosome ◽  
Coordinated Expression ◽  
Casein Genes ◽  
Transgenic Technologies

The application of transgenic technologies in dairy cattle has been restricted largely to producing potential pharmaceutical or nutriceutical products in the mammary gland. Broader application of transgenesis in dairy cattle production will require identifying target traits that are both amenable to transgenic modification and economically important to the dairy industry. The casein proteins are the most valuable component of cows milk destined for value-added processing. The four bovine casein genes lie within a single, multi-gene locus of approximately 200 kb in length. The working hypothesis is that this multi-gene locus contains all of the DNA sequences required to regulate the coordinated expression of all four individual casein genes (i.e. a locus control region or LCR). The initial research aim is to clone the entire casein locus into a bacterial artificial chromosome (BAC) vector, thus preserving the extended 5′and 3′ regions that flank the locus, as well as maintaining the spatial integrity of the four individual casein genes that comprise the locus. The author's laboratory has prepared a bacterial artificial chromosome (BAC) library of genomic DNA from elite dairy cattle. Partial, non-elite BAC clones of the casein gene locus are being tested in transgenic mice to establish proof of concept. Advances in nuclear transfer of transfected somatic cells should improve the efficiency of producing transgenic calves that possess a BAC casein construct introduced into an elite genetic background.

Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi)

Genome ◽  
2004 ◽  
Vol 47 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Yaping Qian ◽  
Li Jin ◽  
Bing Su
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Large Scale ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Spider Monkey ◽  
Ateles Geoffroyi ◽  
Comparative Genomic ◽  
Average Insert Size ◽  
Bac Clones ◽  
Genomic Study

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey (Ateles geoffroyi). A total of 193 152 BAC clones were generated in this library. The average insert size of the BAC clones was estimated to be 184.6 kb with the small inserts (50-100 kb) accounting for less than 3% and the non-recombinant clones only 1.2%. Assuming a similar genome size with humans, the spider monkey BAC library has about 11× genome coverage. In addition, by end sequencing of randomly selected BAC clones, we generated 367 sequence tags for the library. When blasted against human genome, they showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the library. This black-handed spider monkey BAC library would serve as a valuable resource in comparative genomic study and large-scale genome sequencing of nonhuman primates.Key words: black-handed spider monkeys, Ateles geoffroyi, BAC library.

scholarly journals Use of a Mycobacterium tuberculosisH37Rv Bacterial Artificial Chromosome Library for Genome Mapping, Sequencing, and Comparative Genomics

1998 ◽  
Vol 66 (5) ◽  
pp. 2221-2229 ◽  
Author(s):  
Roland Brosch ◽  
Stephen V. Gordon ◽  
Alain Billault ◽  
Thierry Garnier ◽  
Karin Eiglmeier ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Bacterial Artificial Chromosome ◽  
Genome Mapping ◽  
Sequence Data ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Excellent Correlation ◽  
Sequencing Project ◽  
Polysaccharide Biosynthesis ◽  
Bac Clones

ABSTRACT The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosisgenome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of ∼150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

scholarly journals A new targeted capture method using bacterial artificial chromosome (BAC) libraries as baits for sequencing relatively large genes

2018 ◽  
Author(s):  
Kae Koganebuchi ◽  
Takashi Gakuhari ◽  
Hirohiko Takeshima ◽  
Kimitoshi Sato ◽  
Kiyotaka Fujii ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Data Quality ◽  
Susceptibility Gene ◽  
Artificial Chromosome ◽  
Individual Genome ◽  
Genome Region ◽  
Capture Method ◽  
Bac Libraries ◽  
Targeted Capture ◽  
Commercial Kit

AbstractTo analyze a specific genome region using next-generation sequencing technologies, the enrichment of DNA libraries with targeted capture methods has been standardized. For enrichment of mitochondrial genome, a previous study developed an original targeted capture method that use baits constructed from long-range polymerase chain reaction (PCR) amplicons, common laboratory reagents, and equipment. In this study, a new targeted capture method is presented, that of bacterial artificial chromosome (BAC) double capture (BDC), modifying the previous method, but using BAC libraries as baits for sequencing a relatively large gene. We applied the BDC approach for the 214 kb autosomal region, ring finger protein 213, which is the susceptibility gene of moyamoya disease (MMD). To evaluate the reliability of BDC, cost and data quality were compared with those of a commercial kit. While the ratio of duplicate reads was higher, the cost was less than that of the commercial kit. The data quality was sufficiently the same as that of the kit. Thus, BDC can be an easy, low-cost, and useful method for analyzing individual genome region with substantial length.

scholarly journals Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Mikhail Nefedov ◽  
Lucia Carbone ◽  
Matthew Field ◽  
Jacquie Schein ◽  
Pieter J. de Jong
Keyword(s):  
Homologous Recombination ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Pcr Assay ◽  
New Approach ◽  
E Coli ◽  
Functional Studies ◽  
Kanamycin Cassette ◽  
Orangutan Genome ◽  
Bac Libraries

We have developed a new approach to screen bacterial artificial chromosome (BAC) libraries by recombination selection. To test this method, we constructed an orangutan BAC library using anE. colistrain (DY380) with temperature inducible homologous recombination (HR) capability. We amplified one library segment, induced HR at42∘C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.

Construction, characterization, and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.)

Genome ◽  
2007 ◽  
Vol 50 (4) ◽  
pp. 412-421 ◽  
Author(s):  
Melanie Febrer ◽  
Foo Cheung ◽  
Christopher D. Town ◽  
Steven B. Cannon ◽  
Nevin D. Young ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
White Clover ◽  
Trifolium Repens ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Sequencing Analysis ◽  
Average Insert Size ◽  
Model Legume ◽  
Bac End Sequencing ◽  
Trifolium Repens L

White clover ( Trifolium repens L.) is a forage legume widely used in combination with grass in pastures because of its ability to fix nitrogen. We have constructed a bacterial artificial chromosome (BAC) library of an advanced breeding line of white clover. The library contains 37 248 clones with an average insert size of approximately 85 kb, representing an approximate 3-fold coverage of the white clover genome based on an estimated genome size of 960 Mb. The BAC library was pooled and screened by polymerase chain reaction (PCR) amplification using both white clover microsatellites and PCR-based markers derived from Medicago truncatula , resulting in an average of 6 hits per marker; this supports the estimated 3-fold genome coverage in this allotetraploid species. PCR-based screening of 766 clones with a multiplex set of chloroplast primers showed that only 0.5% of BAC clones contained chloroplast-derived inserts. The library was further evaluated by sequencing both ends of 724 of the clover BACs. These were analysed with respect to their sequence content and their homology to the contents of a range of plant gene, expressed sequence tag, and repeat element databases. Forty-three microsatellites were discovered in the BAC-end sequences (BESs) and investigated as potential genetic markers in white clover. The BESs were also compared with the partially sequenced genome of the model legume M. truncatula with the specific intention of identifying putative comparative-tile BACs, which represent potential regions of microsynteny between the 2 species; 14 such BACs were discovered. The results suggest that a large-scale BAC-end sequencing strategy has the potential to anchor a significant proportion of the genome of white clover onto the gene-space sequence of M. truncatula.

scholarly journals Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp.

2008 ◽  
Vol 74 (6) ◽  
pp. 1892-1901 ◽  
Author(s):  
Torsten Hain ◽  
Sonja Otten ◽  
Ulrich von Both ◽  
Som S. Chatterjee ◽  
Ulrike Technow ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Functional Genomics ◽  
Bac Library ◽  
Virulence Gene ◽  
Artificial Chromosome ◽  
Broad Host Range ◽  
Actin Assembly ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Bacterial Artificial Chromosome Vector

ABSTRACT Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.

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