• Asian-Australasian Journal of Animal Sciences
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• v.21 no.1
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• pp.11-18
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• 2008

We created a cDNA microarray representing approximately 3,500 pig genes for functional genomic studies. The array elements were selected from 6,494 cDNA clones identified in a large-scale expressed sequence tag (EST) project. These cDNA clones came from normalized and subtracted porcine adipose tissue cDNA libraries. Sequence similarity searches of the 3,426 ESTs represented on the array using BLASTN identified 2,790 (81.4%) as putative human orthologs, with the remainder consisting of "novel" genes or highly divergent orthologs. We used the gene microarray to profile transcripts expressed by adipose tissue of fatty Chinese Xiang pig (XP) and muscley Large White (LW). Microarray analysis of RNA extracted from adipose tissue of fatty XP and muscley LW identified 81 genes that were differently expressed two fold or more. Transcriptional differences of four of these genes, adipocyte fatty acid binding protein (aP2), stearyl-CoA desaturase (SCD), sterol regulatory element binding transcription factor 1 (SREBF1) and lipoprotein lipase (LPL) were confirmed using SYBR Green quantitative RT-PCR technology. Our results showed that high expression of SCD and SREBF1 may be one of the reasons that larger fat deposits are observed in the XP. In addition, our findings also illustrate the potential power of microarrays for understanding the molecular mechanisms of porcine development, disease resistance, nutrition, fertility and production traits.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.3
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• pp.421-426
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• 2002

The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• The Korean Journal of Malacology
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• v.13 no.2
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• pp.91-96
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• 1997

한국산 논우렁이(CIpangopaludina chinensis malleata)와 큰논우렁이 (C. japomica)는 형태학적으로 유사하여 그 감별이 용이치 않다. 본 연구는 이 두 종을 대상으로 28S rDNA DI유전자를 7종의 제한효소로 처리하여 PCR-RDLP기법으로 그 절편을 비교하였다. 절편 상호간에는 차이점을 관찰할 수 없었으나, 두 종으로부터 분석된 28S rDNA DI 유전자의 염기서열에서는 4 부위에서 종간 차이를 보였다.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.90-95
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• 1999

A species-specific 760 base pair(bp) BamHI to EcoRI DNA fragment(fMG-2) of lipoprotein gene was isolated from a Mycoplasma gallisepticum(M gallisepticum) genomic library. Based on the DNA sequence data of fMG-2, a pair of 25bp primers was synthesized. When used in the polymerase chain reaction(PCR), 732bp DNA products were amplified from 6 standard strains and 10 field isolates of M gallisepticum, but not from 2 Mycoplasma synoviae and 7 other Mycoplasma species. The lower detection limit was 100fg of the genomic DNA. Identity of the PCR products was confirmed by comparison of patterns of restriction endonuclease analysis with AseI, DraI, EcoRV and SspI.

• Journal of Sericultural and Entomological Science
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• v.42 no.2
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• pp.126-141
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• 2000

A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in target genome. The nucleotide sequence encoded in the genome contains the information that specifies the amino acid sequence of every protein and functional RNA molecule. In principle, it will be possible to identify every protein resposible for the structure and function of the body of the target organism. The pattern of expression in different cell types will specify where and when each protein is used. The amino acid sequence of the proteins encoded by each gene will be derived from the conceptional translation of the nucleotide sequence. Comparison of these sequences with those of known proteins, whose sequences are sorted in database, will suggest an approximate function for many proteins. This mini review describes the development of new sequencing methods and the optimization of sequencing strategies for whole genome, various cDNA and genomic analysis.

• YAKHAK HOEJI
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• v.31 no.2
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• pp.116-125
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• 1987

It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of MRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5~4.0 kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4$\times$$10^5$~$10^{6}$ per $\mu\textrm{g}$ of rat liver poly (A$^+$)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.312-320
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• 2000

Abstract The full sequence of the plasmid pKJ36, which was derived from Bifidobacterium longum KJ, was determined and analyzed to construct shuttle vectors between E. coli and Bifidobacterium. The plasmid pKJ36 was composed of 3,625 base pairs with a 65.1% G+C content. The structural organization of pKJ36 was highly similar to that of pKJ50, and the three major ORFs on pKJ36 showed high amino acid sequence homologies with those of pKJ50. The putative proteins coded by these three ORFs were designated as RepB (32.0 kDa, pI=9.25), MembB (29.0 kDa, pI=12.25), and MobB (39.0 kDa, pI=IO.66), respectively. The amino acid sequence of RepB showed a 57% identity and 70% similarity with that of the RepA protein of pKJ50. Upstream of the repB gene, the so-called iteron sequence was directly repeated four-and-ahalf times and a conserved dnaA box was identified. An amino acid sequence comparison between the MobB and MobA of pKJ50 revealed a 48% identity and 61 % similarity. A conserved oriT sequence with an inverted repeat identical to that of pKJ50 was also found upstream of the mobB gene. A hydropathy analysis of MembB revealed four possible transmembrane regions. The expressions of the repB and membB genes were confirmed by RT-PCR. The in vitro translation reaction of pKJ36 showed protein bands with anticipated sizes with respect to each putative gene product. S 1 endonuclease treatment and Southern hybridization suggested that pKJ36 replicates by a rolling circle mechanism via a single-stranded DNA (ssDNA) intermediate. A shuttle vector between E. coli and Bifidobacterium sp. was constructed using the pKJ36, pBR322, and staphylococcal chloramphenicol acetyl transferase (CAT) gene. The successful transformation of the Bifidobacterium strains was shown by Southern hybridization and PCR. The transformation efficiency differed from strain to strain and, depending on the electroporation conditions, with a range between $1.2{\times}10^1-2.6{\times}10^2{\;}cfu/\mu\textrm{g}$ DNA.X> DNA.

• Journal of Microbiology
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• v.35 no.3
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• pp.222-227
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• 1997

DNA fragments homologous to chitin synthase gene were amplified from the genomic DNA of Beauveria brongniartii by PCR using degenerate primers. Cloning and sequencing of the PCR-amplified fragments led to the identification of a gene, designated BbCHSl. Comparison of the deduced amino acid sequence of BbCHSl with those of other Euascomycetes revealed that BbCHSl is a gene for class II chitin synthase. The Blastp search of the deduced amino acid sequence of BbCHSl displayed the highest rate of similarity, 95.8%, with CHS2 of Metarhizium unisopliae. Phylogenetic analysis of the amino acid sequences confirmed the taxonomic and evolutionary position of B. brongniartii, which was previously derived by traditional fungal classification based on morphological features.

• Animal Systematics, Evolution and Diversity
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• v.37 no.3
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• pp.235-241
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• 2021

To provide better taxonomic information of the genus Nectoneanthes, the two DNA barcode regions of mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal DNA (rDNA) sequences of Nectoneanthes oxypoda and N. uchiwa were determined. In addition, the respective sequences of four nereidid species closely related to Nectoneanthes were retrieved from GenBank for comparison and to estimate intra- and inter-specific genetic distances. The aligned sequence lengths of COI and 16S rDNA were 570 bp and 419 bp long, respectively. The mean intraspecific variation in both markers was less than 1% in all species except for that in COI of H. diadroma (1.87%). The mean interspecific variation between N. oxypoda and N. uchiwa was 12.02% regarding COI and 1.85% regarding 16S rDNA. In contrast, the mean interspecific variation between species of other genera was comparably higher(i.e., genus Perinereis: 20.5% in COI and 8.3% in 16S rDNA; genus Hediste: 13.18% in COI and 2.64% in 16S rDNA), compared with that between the two Nectoneanthes species. This result indicated that these Nectoneanthes species are genetically more closely related than other congeneric species of different genera. The DNA barcoding information on Nectoneanthes species generated in this study provides valuable insights for further biodiversity studies on nereidid species.

• Journal of Plant Biotechnology
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• v.48 no.1
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• pp.18-25
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• 2021

Solanum demissum is one of the wild Solanum species originating from Mexico. It has wildly been used for potato breeding due to its resistance to Phytophthora infestans. S. demissum has an EBN value of four, which is same as that of S. tuberosum, so that it is directly crossable for breeding purposes with the cultivated tetraploid potato (S. tuberosum). In this study, the chloroplast genome sequence of S. demissum obtained by next-generation sequencing technology was described and compared with those of seven other Solanum species to develop S. demissum-specific markers. Thetotal sequence length of the chloroplast genome is 155,558 bp, and its structural organization is similar to those of other Solanum species. Phylogenetic analysis with ten other Solanaceae species revealed that S. demissum is most closely grouped with S. hougasii and S. stoloniferum followed by S. berthaultii and S. tuberosum. Additional comparison of the chloroplast genome sequence with those of seven other Solanum species revealed two InDels specific to S. demissum. Based on these InDels, two PCR-based markers for discriminating S. demissum from other Solanum species were developed. The results obtained in this study will provide an opportunity to investigate more detailed evolutionary and breeding aspects in Solanum species.