• Journal of Life Science
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• v.9 no.5
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• pp.531-536
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• 1999

As an attempt to preserve resources in marine biological organisms, we first constructed a cDNA library from the red alga Porphyra yezoensis. The library construction method from P. yezoensis consists of three steps; those include protoplast presparation, RNA isolation, and phage library construction. Protoplast was prepared in order to remove much of the carbohydrate compounds which are characteristics of algal cell walls. Carbohydrate contamination in the purified RNA may inhibit further enzyme reactions, those carbohydrates should be removed. RNA samples prepared from protoplast still seemed to contain residual amount of carbohydrate because mRNA isolation with conventional method failed. We therefore developed a method with Poly ATtract mRNA isolation system. The constructed phage library was tested by analyzing cDNA insert in phage vector from randomly picked ten independent white plagues. All of the phages contained cDNA inserts with sizes ranging 0.5kb and 2.0kb.

• Animal cells and systems
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• v.1 no.4
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• pp.629-635
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• 1997

Previously we reported the migration and rearrangement of a chloroplast gene cluster into mitochondria. The exact genomic locations of the clusters, modes of the gene rearrangement and mechanisms of the interorganellar migration of the clusters have yet to be understood. The detailed analysis needs to include a larger region of DNA surrounding each cluster. To study DNA rearrangement and migration in more detail a cosmid library was constructed using the total rice genomic DNA including nuclear, chloroplast and mitochondrial DNA. From this cosmid library, a sub-library was obtained by selecting the clones hybridized to various regions of chloroplast DNA. According to the hybridization pattern 136 clones from the sub-library were classified into 29 groups. Detailed analysis of these clones revealed that in addition to authentic chloroplast DNA, the clones contain its homologs resulted from rearrangement and mutation. We analyzed two clones in detail, which contain different rp12 homologs resulted from rearrangement and/or migration, respectively.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.359-363
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• 2004

I investigated an effective way to generate a metagenomic library from DNA prepared from environental samples. The sizes of DNA extracted from environmental samples were usually in the range of 10 to 100 kbp as estimated from $0.4\%$ agarose gel electrophoresis. Because of this small size, a fosmid, rather than BAC, was chosen as a vector. It was found that, for the successful generation of metagenomic library, the selection of DNA with the sized of about 40 kbp was critical and, therefore, a simple agarose gel electrophoresis system was developed to select this size of DNA. By the procedure described in this report, I obtained metagenomic libraries containing 25,000 fosmid clones, which corresponded to 1,000 Mb of metagenomic DNA.

• The Plant Pathology Journal
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• v.31 no.2
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• pp.108-114
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• 2015

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.13-18
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• 1996

To secure the genetic resources of silkworm, Bomyx mori, the cDNA library was constructed with mRNA isolated from fifth instar larvae. Titer of the cDNA library was about 1.3 X 106 plaques in total. We presumed that the titer covered all transcripts existed in Bombyx mori. Meanwhile, it is knowen that partial cDNA sequences, Expressed Sequence Tags(ESTs), have a good value for the discovery of novel genes and the elucidation of their structures. For this purpose, partial cDNA sequencing was carried out from randomly selected cDNA clones in the library. Partial cDNA sequences of 37 clones were determined and an average of 212 nucleotides of sequence can be read from the clone. The ESTs were searched in GenBAnk database and fifteen ESTs showed significant similarities to enlisted sequences. They included the genes of storage protein, heat shock protein, actin, catalase and so forth. We presumed that the 22 unmatched ESTs were novel genes.

• Entomological Research
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• v.48 no.5
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• pp.384-389
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• 2018

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site-specific recombination of bacteriophage ${\lambda}$. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of $8.4{\times}10^6$ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus-induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi-induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.43-48
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• 1993

These experiments were carried out to construct mouse testis cDNA library and to to seen H-Y Ag gene. Mouse testis was obtained from BALB/c inbreed mouse that was after-born 1 week. Isolation of mouse testis total RNA was carried out by guanidum/cesium choloride, poly(A+) mRNAs were purified by oligo d(T)-cellulose chromatography method. To investigate protein synthesis activity, in-vitro translation carried out by total RNA and poly(A+) mRNA. The products of in-vitro translation were identified in 12.5% PAGE. Single strand DNA and double strand DNA were synthesized from poly(A+) mRNA and purified using phenol/chloroform/isoamylalcohol. Synthesized cDNA was combined with cohesive Eco RI polylinker, its recombination efficiencies were identified by X-gal and IPTG. In the cDNA library, 1$\times$107 phagemids were screened with 32P labelled probe. Hybridization were carried on $65^{\circ}C$ for 16~20hours. And 1$\times$106 phagemids were screened with rabbit-anti-H-Y. In former, select 5 positive clones, and later, 1 positive clone. Its southern blot analysis showed various size of insert cDNA from 0.7kb to 3kb.

• Journal of Life Science
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• v.10 no.1
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• pp.10-13
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• 2000

To develope the genetic source of oak wild silkworm, Antheraea yamamai, the cDNA library was constructed with poly A+ mRNA isolated from posterial silk gland of fifth instar larvae. Titer of the cDNA library was about 5.1$\times$105 pfu in total. We presumed that the titer covered almost all transcripts existed in Antherea yamamai. From cDNA library of Antheraea yamamai, fibroin heavy chain gene, which is specifically expressed from posterial silk gland of Antheraea yamamai, was screened using oligonuclotide probe specific to alanine rich motif of fibrin heavy chain gene of Antheraea pernyi. As a result, fibroin clones isolated from 5$\times$104 plaques showed the highest homolgy (95%) with that of Antherea pernyi in nucleotide of Anthereaea yamamai and Bombyx mori shows that there is no homologous sequence in the 3+ partial 채야후 region Genomic southern hybridization suggested that one copy is present. Northern hybridization showed that fibroin transcript was approximateely 9 kb in length.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.547-553
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• 2015

The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.797-805
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• 1999

T sergenti cDNA library were constructed to get a more broad information about the structural, functional or antigenic properties of the proteins, and analyzes for their partial cDNA sequences and expression sequences tags(ESTg). The mRNA were purified from T sergenti isolates to identify the information of antigen gene, then first and second strand cDNA was synthesized. EcoR I adaptor ligation and Xho I enzyme restriction were used to the synthesized cDNA, and ligated into a Uni-ZAP XR vector. T sergenti cDNA library was constructed with packaging and amplification in vitro. Antibody screening was performed with constructed T sergenti cDNA library using antisera against T sergenti. Among those clones, eight phagemids were rescued from the recombinant in vivo excision with f1 helper phage. Using the analysis of endonuclease restriction and PCR, the recombinant cDNA were proved having a 0.5-3.0kb of inserts. The eight of partial cDNA clones' sequences were obtained and examined for their homology using BLASTN and BLASTX. The eight of sequenced clones were classified into three groups according to the basis of database searches. A total 3,045bp of partial cDNA sequence were determined from six clones. The putatively identified clones contain a cytochrome c gene, a heat shock protein gene, a cyclophilin gene, and a ribosomal protein gene. The unidentified clones have a homology to ATP-binding protein(mtrA) gene of S argillaceus, DNA-binding protein(DBP) gene of Pseudorabies virus 85kDa merozoite protein gene of B bovis, mRNA spm1 protein of T annulata and glycine-rich RNA-binding protein mRNA of O sativa etc.