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이명철,Rod A Wing,백형진,주석철,은무영 한국국제농업개발학회 2007 韓國國際農業開發學會誌 Vol.19 No.3
A large fraction of maize genome consists of LTR retrotransposons that are heavily methylated and estimated over 60% of genome. In contrast, functional genes exist in hypomethylated CpG islands and intermixed with large stretches of retrotransposon and repetitive DNA. It has been hypothesized that efficient exclusion of methylated retrotransposons and repeated DNA region is one of the rapid and cost-effective approaches for comprehensive gene discovery in large genome size of maize. Three kinds of methylation-sensitive restriction enzymes, HapII, MspI and McrBC, were used to identify the restriction frequency of cytosine methylation sites in maize genome. Roughly 60% of total maize genomic DNA restricted less than 500bp by McrBC restriction enzyme, and the most of restricted small size fraction was composed retrotransposon. In order to validate the efficient construction of gene-rich shotgun library, we compare two methylation filtering methods gene-rich methyl-filtration shotgun libraries of maize were maid and characterized in vivo and in vitro methyl-filtration system. The size selected DNA fraction by Sau3A-McrBC enzyme treated was very stable and has not appeared modification in E.coli, but most insert DNA size of partially digested with Sau3A were decrease less than 500bp by bacterial methylation-modification system. In compare of retroelements portion, Approximately 44.6% of the sequences were retroelement in unmethyl-filtered library, and the most of them was Copia type, such as Prem, Opie and Ji. The portion of retroelement was drastically decrease about 25% and 20% by in vivo and in vitro filtration system, respectively.
Lee, Myung-Chul,Wing, Rod A,Suh, Seok-Cheol,Eun, Moo-Young The Plant Resources Society of Korea 2007 한국자원식물학회지 Vol.20 No.6
It has been hypothesized that efficient exclusion of methylated retrotransposons and repeated DNA region is one of the rapid and cost-effective approaches for comprehensive gene discovery in large genome size of maize. Three kinds of methylation-sensitive restriction enzymes, HapII, MspI and McrBC, were used to identify the restriction frequency of cytosine methylation sites in maize genome. Roughly 60% of total maize genomic DNA was restricted less than 500bp by McrBC, and the most of restricted small size fraction was composed retrotransposon. In order to validate the efficient construction of gene-rich shotgun library, we compare two gene-rich methyl-filtration shotgun libraries using in vivo and in vitro methyl-filtration system. The size selected DNA fraction by Sau3A-McrBC enzyme treated was very stable and has not appeared modification in E. coli, but most insert DNA size of partially digested with Sau3A were decrease less than 500bp by bacterial methylation-modification system. In compare of retroelements portion, A 44.6% of the sequences were retroelement in unmethyl-filtered library, and the most of them was Copia type, such as Prem, Opie and Ji. The portion of retroelement was drastically decreased to 25% and 20% by in vivo and in vitro filtration system, respectively.
Construction of Various Copy Number Plasmid Vectors and Their Utility for Genome Sequencing
Yang, Tae-Jin,Yu, Yeisoo,Frisch, David A.,Lee, Seunghee,Kim, Hye-Ran,Kwon, Soo-Jin,Park, Beom-Suk,Wing, Rod A. Korea Genome Organization 2004 Genomics & informatics Vol.2 No.4
We developed various plasmid cloning vectors that are useful in the construction of genomic and shotgun libraries. Two medium copy vectors, pCUGlblu21 (pCb21) and pAGlblu21 (pAb21), which are resistant to kanamycin ($Km^R$) and chloramphenicol ($Cam^R$), respectively, are useful for cloning DNA inserts ranging from 5kb to 15kb. Two high copy vectors, pCUGlblu31 (pCb31) and pAGlblu31 (pAb31), containing $Km^R$ and $Cam^R$, respectively, are useful for DNA inserts less than 5kb. These vectors are well adapted for large-scale genome sequencing projects by providing choice of copy number and selectable marker. The small vector size is another advantage of these vectors. All vectors contain lacZa including multicloning sites that originated from pBluscriptllsk- for easy cloning and sequencing. Two medium copy vectors contain unique and rare cutting Swal (ATTTAAAT) restriction enzyme sites for easy determination of insert size. We developed two combined vectors, pC21A31 and pC31A21, which are combinations of (pCb21 + pAb31) and (pCb31 + pAb21), respectively. These two vectors provide four choices of vectors such as $Km^R$ and medium, $Cam^R$ and high, $Cam^R$ and medium, and $Km^R$ and high copy vectors by restriction enzyme cutting, dephosphorylation, and gel purification. These vectors were successfully applied to high throughput shotgun sequencing of rice, tomato, and brassica BAC clones. With an example of extremely biased hydro sheared 3 kb shotgun library of a tomato BAC clone, which is originated from cytogenetically defined peri-centromeric region, we suggest the utility of an additional 10 kb library for sequence assembly of the difficult-to-assemble BAC clone.