• Genomics & Informatics
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• 제15권4호
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• pp.128-135
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• 2017

As next-generation sequencing technologies have advanced, enormous amounts of whole-genome sequence information in various species have been released. However, it is still difficult to assemble the whole genome precisely, due to inherent limitations of short-read sequencing technologies. In particular, the complexities of plants are incomparable to those of microorganisms or animals because of whole-genome duplications, repeat insertions, and Numt insertions, etc. In this study, we describe a new method for detecting misassembly sequence regions of Brassica rapa with genotyping-by-sequencing, followed by MadMapper clustering. The misassembly candidate regions were cross-checked with BAC clone paired-ends library sequences that have been mapped to the reference genome. The results were further verified with gene synteny relations between Brassica rapa and Arabidopsis thaliana. We conclude that this method will help detect misassembly regions and be applicable to incompletely assembled reference genomes from a variety of species.

• Asian-Australasian Journal of Animal Sciences
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• 제20권11호
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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.

• Journal of Plant Biotechnology
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• 제33권3호
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• pp.153-160
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• 2006

유전체 연구란 목적하는 유전체의 구조를 밝히고 가지고 있는 모든 유전자의 기능 및 진화과정을 망라하여 이해하고자 하는 것이다. 계통발생학상 애기장대와 연관되어 있는 Brassica rapa는 채소, 유지 및 사료로 이용되는 중요한 작물의 하나이다. Brassica rapa의 유전체 연구를 착수하는 데는 적합한 유전학적 재료 및 유전체 재료가 있어야 한다. 우리는 배추 (Brassica rapa spp. pekinensis)를 재료로 하여 표준 mapping 집단을 개발하여, 78계통으로 구성된 DH집단과 약 250 계통으로 구성된 RI집단을 개발하였다. 2가지 제한효소 (HintIII, BamHI)를 이용해 세균인공염색체 (BAC) library (KBrH, KBrB)를 만들었고, 이들은 각각 56,592개와 50,688개의 클론으로 구성되어 있다. 또한 배추의 각기 다른 부위를 이용하여 만든 22가지의 cDNA library를 이용하여 평균 575bp의 길이를 가지는 104,914개의 EST 분석을 실시 하였다. 세계적으로 'Multinational Brassica Genome Project (MBGP)' 조직이 구성되었고 배추의 전 염기서열 분석을 하기로 2003년 결정되었다. 그 첫 단계로 104,914개의 BAC 클론의 BAC-end 염기서열분석이 제안되어 2006년 9월 5개국 공동 프로젝트로 추진하여 완성하게 되었다. 이러한 BAC-end 염기서열분석의 결과는 유전자의 염기서열 해석, 및 풍부하게 존재하는 반복염기서열 DNA를 분석함으로써 배추의 유전체 구조를 이해할 수 있는 실마리를 주었다. BAC 클론의 전체 염기서열분석은, 비록 단편 내에 유전자의 결실이 변화무쌍하게 일어나지만 배추 DNA 단편이 유전체에서 광범위하게 삼중복으로 존재함을 나타냈다. 이러한 BAC-end 염기서열을 아기장대 염기서열에 비교하여 629개의 종자 BAC을 선정하게 되었고, 이들의 염기서열 분석을 완성하였다. MBGP에서는2단계로서 배추의 전 유전체 염기서열 분석을 추진하게 되었고, 유전자지도에 위치한 종자 BAC을 이용하여 인접한 BAC 클론을 찾아 염기서열 분석하는 BAC-to-BAC 방법을 추진하고 있으며 8개국에서 참여하여 현재 염기서열 분석을 추진 중 이다. 최근에 각 국에서는 생물정보학기법을 활용한 염기서열 분석 기반에 대하여 많은 토론이 진행되고 있다. 앞으로 다양한 유전체 정보가 축적됨에 따라 배추의 유전체 구조를 이해하고 농업적으로 적용하고자 하는데 기여를 할 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권3호
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• pp.316-327
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• 2007

In this study, a Korean native cattle strain (Hanwoo) evidencing high performance in terms of both meat quality and quantity was employed in the generation of 150,000 BAC clones with an average insert size of 140 kb, and corresponding to about a 6X coverage of bovine chromosomal DNA. The BAC clones were pooled in a mini-scale via three rounds of a pooling protocol, and the efficiency of this pooling protocol was evaluated by testing the accuracy of accessibility to the positive clones, via a PCR-based screening method. Two sets of primers designed from each of two known genes were tested, and each yielded 2 or 3 positive clones for each gene, thereby indicating that the BAC library pooling system was appropriate with regard to the accession of the target BAC clones. Analyses of $3.3{\times}10^6$ base pairs obtained from the 7,090 BAC end sequence (BES) showed that 34.88% of the DNA sequence harbored the repetition sequence. Analysis of the 7,090 BES to the $1^{st}$ and $2^{nd}$ generation radiation hybrid map of the cattle genome, using the COMPASS program designed for the construction of a cattle-human comparative mapping, resulted in the localization of a total of 1,374 clones proximal to 339 $1^{st}$ generation markers, and 1,721 clones proximal to 664 $2^{nd}$ generation markers. Collectively, the BAC library and pooling system of the BAC clones from the Korean cattle, coupled with the chromosome-localized BAC clones, will provide us with novel tools for the excavation of desired clones for genome mapping and sequencing, and will also furnish us with additional information regarding breed differences in cattle.

• BMB Reports
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• 제39권4호
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• pp.464-467
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• 2006

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.

• Ocean and Polar Research
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• 제27권2호
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• pp.205-214
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• 2005

From ancient Antarctic glacier ice, we extracted total genomic DNA that was suitable for prokaryotic 16S rDNA gene cloning and sequencing, and bacterial artificial chromosome (BAC) library and end-sequencing. The ice samples were from the Dry Valley region. Age dating by $^{40}Ar/^{39}Ar$ analysis on the volcanic ashes deposited in situ indicated the ice samples are minimum 100,000-300,000 yr (sample DLE) and 8 million years (sample EME) old. Further assay proved the ice survived freeze-thaw cycles or other re-working processes. EME, which was from a small lobe of the basal Taylor glacier, is the oldest known ice on Earth. Microorganisms, preserved frozen in glacier ice and isolated from the rest of the world over a geological time scale, can provide valuable data or insight for the diversity, distribution, survival strategy, and evolutionary relationships to the extant relatives. From the 16S gene cloning study, we detected no PCR amplicons with Archaea-specific primers, however we found many phylotypes belonging to Bacteria divisions, such as Actinobacteria, Acidobacteria, Proteobacteria $({\alpha},\;{\beta},\;and\;{\gamma})$, Firmicutes, and Cytophaga-Flavobacterium-Bacteroid$. BAC cloning and sequencing revealed protein codings highly identical to phenylacetic acid degradation protein paaA, chromosome segregation ATPases, or cold shock protein B of present day bacteria. Throughput sequencing of the BAC clones is underway. Viable and culturable cells were recovered from the DLE sample, and characterized by their 16S rDNA sequences. Further investigation on the survivorship and functional genes from the past should help unveil the evolution of life on Earth, or elsewhere, if any.

• Molecules and Cells
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• 제22권3호
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• pp.300-307
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• 2006

Brassica rapa ssp. pekinensis (Chinese cabbage) is an economically important crop and a model plant for studies on polyploidization and phenotypic evolution. To gain an insight into the structure of the B. rapa genome we analyzed 12,017 BAC-end sequences for the presence of transposable elements (TEs), SSRs, centromeric satellite repeats and genes, and similarity to the closely related genome of Arabidopsis thaliana. TEs were estimated to occupy 14% of the genome, with 12.3% of the genome represented by retrotransposons. It was estimated that the B. rapa genome contains 43,000 genes, 1.6 times greater than the genome of A. thaliana. A number of centromeric satellite sequences, representing variations of a 176-bp consensus sequence, were identified. This sequence has undergone rapid evolution within the B. rapa genome and has diverged among the related species of Brassicaceae. A study of SSRs demonstrated a non-random distribution with a greater abundance within predicted intergenic regions. Our results provide an initial characterization of the genome of B. rapa and provide the basis for detailed analysis through whole-genome sequencing.

• Molecules and Cells
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• 제23권2호
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• pp.145-153
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• 2007

Elucidation of the roles of circadian associated factors requires a better understanding of the molecular mechanisms of circadian rhythms, control of flowering time through photoperiodic pathways, and photosensory signal transduction. In Arabidopsis, the APRR1 quintet, APRRs 1, 3, 5, 7, and 9, are known as central oscillator genes. Other plants may share the molecular mechanism underlying the circadian rhythm. To identify and characterize these circadian response genes in Brassica crops whose genome was triplicated after divergence from Arabidopsis, we identified B. rapa BAC clones containing these genes by BLAST analysis of B. rapa BAC end sequences against the five corresponding Arabidopsis regions. Subsequent fingerprinting, Southern hybridization, and PCR allowed identification of five BAC clones, one for each of the five circadian-related genes. By draft shotgun sequencing of the BAC clones, we identified the complete gene sequences and cloned the five expressed B. rapa circadian-associated gene members, BrPRRs 1, 3, 5, 7, and 9. Phylogenetic analysis revealed that each BrPRR was orthologous to the corresponding APRR at the sequence level. Northern hybridization revealed that the five genes were transcribed at distinct points in the 24 hour period, and Southern hybridization revealed that they are present in 2, 1, 2, 2, and 1 copies, respectively in the B. rapa genome, which was triplicated and then diploidized during the last 15 million years.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2001년도 제2회 생물정보 워크샵 (DNA Chip Bioinformatics)
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• pp.61-86
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• 2001

All cancers are caused by abnormalities in DNA sequence. Throughout life, the DNA in human cells is exposed to mutagens and suffers mistakes in replication, resulting in progressive, subtle changes in the DNA sequence in each cell. Since the development of conventional and molecular cytogenetic methods to the analysis of chromosomal aberrations in cancers, more than 1,800 recurring chromosomal breakpoints have been identified. These breakpoints and regions of nonrandom copy number changes typically point to the location of genes involved in cancer initiation and progression. With the introduction of molecular cytogenetic methodologies based on fluorescence in situ hybridization (FISH), namely, comparative genomic hybridization (CGH) and multicolor FISH (m-FISH) in carcinomas become susceptible to analysis. Conventional CGH has been widely applied for the detection of genomic imbalances in tumor cells, and used normal metaphase chromosomes as targets for the mapping of copy number changes. However, this limits the mapping of such imbalances to the resolution limit of metaphase chromosomes (usually 10 to 20 Mb). Efforts to increase this resolution have led to the "new"concept of genomic DNA chip (1 to 2 Mb), whereby the chromosomal target is replaced with cloned DNA immobilized on such as glass slides. The resulting resolution then depends on the size of the immobilized DNA fragments. We have completed the first draft of its Korean Genome Project. The project proceeded by end sequencing inserts from a library of 96,768 bacterial artificial chromosomes (BACs) containing genomic DNA fragments from Korean ethnicity. The sequenced BAC ends were then compared to the Human Genome Project′s publicly available sequence database and aligned according to known cancer gene sequences. These BAC clones were biotinylated by nick translation, hybridized to cytogenetic preparations of metaphase cells, and detected with fluorescein-conjugated avidin. Only locations of unique or low-copy Portions of the clone are identified, because high-copy interspersed repetitive sequences in the probe were suppressed by the addition of unlabelled Cotl DNA. Banding patterns were produced using DAPI. By this means, every BAC fragment has been matched to its appropriate chromosomal location. We have placed 86 (156 BAC clones) cytogenetically defined landmarks to help with the characterization of known cancer genes. Microarray techniques would be applied in CGH by replacement of metaphase chromosome to arrayed BAC confirming in oncogene and tumor suppressor gene: and an array BAC clones from the collection is used to perform a genome-wide scan for segmental aneuploidy by array-CGH. Therefore, the genomic DNA chip (arrayed BAC) will be undoubtedly provide accurate diagnosis of deletions, duplication, insertions and rearrangements of genomic material related to various human phenotypes, including neoplasias. And our tumor markers based on genetic abnormalities of cancer would be identified and contribute to the screening of the stage of cancers and/or hereditary diseases

• 대한바이러스학회지
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• 제28권3호
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• pp.215-224
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• 1998

Cloning, sequencing and expressing in E. coli of the thymidine kinase (TK) gene of Herpes simplex virus type-1 (HSV-1) strain F was investigated. The TK gene, located in the BamHI 3.74 kb DNA fragment of the plasmid pHLA-12, was amplified by polymerase chain reaction (PCR). The 1,131 kb PCR product was cloned into the BamHI and EcoRI sites of pBacPAK9 plasmid and then named pBac-TK recombinant. The TK gene was subcloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The nucleotide sequence of the 1,131 kb TK gene was determined, and the GC content was 65.13%. There were deduced 367 amino acid residues with a total molecular weight of 43 kDa. The weight was confirmed by the protein produced by E. coli M15/pQE-TK on the SDS-PAGE and Western blot. The production of the TK protein in the IPTG induced cells was measured over 4 h. At the end of 1, 2 and 3 h the level increased by 146, 204 and 242%, respectively. The amount of the protein at the highest fraction purified with Ni-NTA resin chromatography was $0.68\;{\mu}g$ per ml. The soluble state TK protein was present in the cytoplasm. In these results the F strain was different in base sequence and amino acid sequence from that of the CL101 strain, which caused difference in their strains.