• Genomics & Informatics
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• v.4 no.2
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• pp.80-86
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• 2006

It is clear that the construction of large insert DNA libraries is important for map-based gene cloning, the assembly of physical maps, and simple screening for specific genomic sequences. The bacterial artificial chromosome (BAC) system is likely to be an important tool for map-based cloning of genes since BAC libraries can be constructed simply and analyzed more efficiently than yeast artificial chromosome (YAC) libraries. BACs have significantly expanded the size of fragments from eukaryotic genomes that can be cloned in Escherichia coli as plasmid molecules. To facilitate the isolation of molecular-biologically important genes in Ashbya gossypii, we constructed Ashbya chromosome-specific BAC libraries using pBeloBAC11 and pBACwich vectors with an average insert size of 100 kb, which is equivalent to 19.8X genomic coverage. pBACwich was developed to streamline map-based cloning by providing a tool to integrate large DNA fragments into specific sites in chromosomes. These chromosome-specific libraries have provided a useful tool for the further characterization of the Ashbya genome including positional cloning and genome sequencing.

• Journal of Microbiology and Biotechnology
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• v.32 no.7
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• pp.911-917
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• 2022

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

• Proceedings of the Zoological Society Korea Conference
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• 2000.10a
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• pp.246.2-246
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• 2000

No Abstract, See Full Text

• The Plant Pathology Journal
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• v.17 no.6
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• pp.375.1-375
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• 2001

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.256-263
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• 2006

In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a $C_{0}t-l$ DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.

• IMMUNE NETWORK
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• v.11 no.1
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• pp.1-10
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• 2011

Interleukin-7 (IL-7) is an essential cytokine for T cells. However, IL-7 is not produced by T cells themselves such that T cells are dependent on extrinsic IL-7. In fact, in the absence of IL-7, T cell development in the thymus as well as survival of naive T cells in the periphery is severely impaired. Furthermore, modulating IL-7 availability in vivo either by genetic means or other experimental approaches determines the size, composition and function of the T cell pool. Consequently, understanding IL-7 expression is critical for understanding T cell immunity. Until most recently, however, the spatiotemporal expression of in vivo IL-7 has remained obscured. Shortage of such information was partly due to scarce expression of IL-7 itself but mainly due to the lack of adequate reagents to monitor IL-7 expression in vivo. This situation dramatically changed with a recent rush of four independent studies that describe the generation and characterization of IL-7 reporter mice, all utilizing bacterial artificial chromosome transgene technology. The emerging consensus of these studies confirmed thymic stromal cells as the major producers of IL-7 but also identified IL-7 reporter activities in various peripheral tissues including skin, intestine and lymph nodes. Strikingly, developmental and environmental cues actively modulated IL-7 reporter activities in vivo suggesting that IL-7 regulation might be a new mechanism of shaping T cell development and homeostasis. Collectively, the availability of these new tools opens up new venues to assess unanswered questions in IL-7 biology in T cells and beyond.

• BMB Reports
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• v.40 no.5
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• pp.656-661
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• 2007

In this report, we describe an optimized method for generation of ephA8 BAC transgenic mice expressing the lacZ reporter gene under ephA8 regulatory sequences. First, we constructed a targeting vector that carries a 1.2 kb ephA8 DNA upstream of its first exon, a lacZ expression cassette, a kanamycin cassette, and a 0.7 kb ephA8 DNA downstream of its first exon. Second, the targeting vector was electroporated into cells containing the ephA8 BAC and pKOBEGA, in which recombinases induce a homologous recombination between the ephA8 BAC DNA and the targeting vector. Third, the FLP plasmid expressing the Flipase was electroporated into these bacteria to eliminate a kanamycin cassette from the recombinant BAC DNA. The appropriate structures of the modified ephA8 BAC DNA were confirmed by Southern analysis. Finally, BAC transgenic mouse embryos were generated by pronuclear injection of the recombinant BAC DNA. Whole mount X-gal staining revealed that the lacZ reporter expression is restricted to the anterior region of the developing midbrain in each transgenic embryo. These results indicate that the ephA8 BAC DNA contains most, if not all, regulatory sequences to direct temporal and spatial expression of the lacZ gene in vivo.

• BMB Reports
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• v.44 no.2
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• pp.113-117
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• 2011

Previous studies suggest that EphA7 plays a critical role in neural tube closure or cortical progenitor apoptosis. In this report, enhancer trap assay was used to modify various EphA7 BAC clones and screen a large genomic region spanning 570 kb downstream of the EphA7 gene. We found that the dorsal midline-specific EphA7 enhancer resides on the 457D20 EphA7 BAC clone and is localized to a 35 kb genomic region in between +345.7 kb to +379.8 kb downstream of the EphA7 transcription start site. Identification of the EphA7 BAC clone containing a long-range dorsal midline enhancer may constitute a useful tool for investigating the biological functions of EphA7 in vivo.

• Journal of Animal Science and Technology
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• v.51 no.2
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• pp.123-128
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• 2009

Recently, the $2^{nd}$ generation genome map of the Korean cattle (Hanwoo) has been constructed by comparison of the nucleotide sequence of the Korean cattle BAC clones with whole genome sequence of the bovine data-base (B_tau 2.1 build). The objective of this study was to update the $2^{nd}$ generation genome map of the Korean cattle using the similar approach. The nucleotide sequence of the Korean cattle BAC clones utilized in the construction of the $2^{nd}$ generation map was compared with the newly released bovine data-base (B_tau 3.1 build) to generate the $3^{rd}$ generation map. While, 5,105 BAC clones were localized on bovine chromosome in the $2^{nd}$ generation map, a total of 9,595 BAC clones, which spans about 37.27% of the bovine chromosome after eliminating the overlapping sequence among the clones, have been mapped on the bovine chromosome in the $3^{rd}$ generation map. Further analysis of the nucleotide sequence of the BAC clones will allow us to develop map and facilitate to pinpoint the genes that are important for the improvement of the performance in this cattle breed.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.11
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• pp.1636-1650
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• 2007

As an initial step toward a better understanding of the genome structure of Korean cattle (Hanwoo breed) and initiation of the framework for genomic research in this bovine, the bacterial artificial chromosome (BAC) end sequencing of 21,024 clones was recently completed. Among these clones, BAC End Sequences (BESs) of 20,158 clones with high quality sequences (Phred score ${\geq}20$, average BES equaled 620 bp and totaled 23,585,814 bp), after editing sequencing results by eliminating vector sequences, were used initially to compare sequence homology with the known bovine chromosomal DNA sequence by using BLASTN analysis. Blast analysis of the BESs against the NCBI Genome database for Bos taurus (Build 2.1) indicated that the BESs from 13,201 clones matched bovine contig sequences with significant blast hits (E<$e^{-40}$), including 7,075 single-end hits and 6,126 paired-end hits. Finally, a total of 5,105 clones of the Korean cattle BAC clones with paired-end hits, including 4,053 clones from the primary analysis and 1,052 clones from the secondary analysis, were mapped to the bovine chromosome with very high accuracy.