• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2005.10a
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• pp.119-122
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• 2005

본 연구는 한우 근내 지방 축적 기작을 구명하고 고급육과 저급육에서 차등 발현되는 유전자를 발굴 동정하여 한우 육질 진단을 위한 분자 표지 마커로 활용하기 위해 GeneFishing PCR 기법을 이용하여 한우 육질등급에 따른 등심조직에서 차등적으로 발현되는 유전자를 분석하였다. 한우 육질 등급($1^+$ 등급 vs 3 등급)간에 총 10개의 차등 발현 유전자가 확인되었고 이 가운데 고급육 한우 등심에서 발현량이 높은 유전자가 4개 그리고 저급육 등심에서 발현량이 높은 유전자 6개가 각각 검출되었다. 발현량 차이 유전자를 cloning하여 염기서열을 분석하고 상동성 검색을 실시한 결과 고급육에서 발현량이 높은 DEG는 주로 EST(expressed sequence tag) 유전자들로 밝혀졌고 저급육에서 발현량이 높은 DEG는 malate dehydrogenase 2(MDH2), myosin heavy chain 2a, triosephosphate isomerase 1(TPI 1), actin, alpha 1, skeletal muscle(ACTA1 ) 유전자들로 동정 되었다. 본 연구를 통해 한우 육질간 차등 발현되는 유전자들은 한우 육질 및 등급판정을 위한 표지인자(marker)로 활용할 수 있어 유전자 마커를 이용한 고급육 생산 한우의 육질 조기진단이 가능할 것이다.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.2
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• pp.108-112
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• 2007

A cellulase (endo-${\beta}$-1,4-D-glucanase(E.C.3.2.1.4)) was isolated from the hepatopancreas of abalone Haliotis discus hannai by EST analysis. The abalone cellulase named HdEG compassed 1977 bp, including 195 bp in the 5'untranslated region, 1680 bp in the open reading frame which encodes 560 amino acid residues, and 92 bp in the 3'-untranslated region. The C-terminal region of the HdEG showed 44-52% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and bacteria. The recombinant cellulase, pEHdEG was produced in E. coli with being fused with C-terminal His-tag. The expressed protein showed a single band (~62 kDa) on Western blotting which was consistent with the value (61,878 Da) calculated from the DNA sequence.

• Molecules and Cells
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• v.40 no.4
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• pp.262-270
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• 2017

MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and located in either intergenic regions or introns. The MER5A1, MER53, and MER91C sequences obtained from RepeatMasker were matched with the eca-miR-544b, eca-miR-1302, and eca-miR-652 precursor sequences derived from Ensembl transcript database, respectively. Each precursor form was anticipated to yield two mature forms, and we confirmed miRNA expression in six different tissues (cerebrum, cerebellum, lung, spleen, adrenal gland, and duodenum) of one thoroughbred horse. MER5A1-derived miRNAs generally showed significantly higher expression in the lung than in other tissues. MER91C-derived miRNA-5p also showed significantly higher expression in the duodenum than in other tissues (cerebellum, lung, spleen, and adrenal gland). The MER117-overlapped expressed sequence tag generated polycistronic miRNAs, which showed higher expression in the duodenum than other tissues. These data indicate that horse MER transposons encode miRNAs that are expressed in several tissues and are thought to have biological functions.

• The Plant Pathology Journal
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• v.30 no.4
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• pp.367-374
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• 2014

Genomes contain a large number of unique genes which have not been found in other species. Although the origin of such "orphan" genes remains unclear, they are thought to be involved in species-specific adaptive processes. Here, we analyzed seven orphan genes (MoSPC1 to MoSPC7) prioritized based on in planta expressed sequence tag data in the rice blast fungus, Magnaporthe oryzae. Expression analysis using qRT-PCR confirmed the expression of four genes (MoSPC1, MoSPC2, MoSPC3 and MoSPC7) during plant infection. However, individual deletion mutants of these four genes did not differ from the wild-type strain for all phenotypes examined, including pathogenicity. The length, GC contents, codon adaptation index and expression during mycelial growth of the four genes suggest that these genes formed during the evolutionary history of M. oryzae. Synteny analyses using closely related fungal species corroborated the notion that these genes evolved de novo in the M. oryzae genome. In this report, we discuss our inability to detect phenotypic changes in the four deletion mutants. Based on these results, the four orphan genes may be products of de novo gene birth processes, and their adaptive potential is in the course of being tested for retention or extinction through natural selection.

• Journal of Microbiology
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• v.40 no.1
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• pp.77-81
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• 2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematspoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator, In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His. Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-so1ub1e form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.

• Toxicological Research
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• v.26 no.1
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• pp.47-52
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• 2010

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.

• Development and Reproduction
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• v.19 no.3
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• pp.135-144
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• 2015

Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.77-87
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• 2009

To understand molecular and cellular mechanisms of many gene products in the female reproductive organs including the ovary and uterine endometrium as well as during embryo development, researchers have developed and utilized many effective methodologies to analyze gene expression in cells, tissues and animals over the last several decades. For example, blotting techniques have helped to understand molecular functions at DNA, RNA and protein levels, and the reverse transcription-polymerase chain reaction (RT-PCR) method has been widely used in gene expression analysis. However, some conventional methods are not sufficient to understand regulation and function of genes expressed in very complex patterns in many organs. Thus, it is required to adopt more high-throughput and reliable techniques. Here, we describe several techniques used widely recently to analyze gene expression, including annealing control based-PCR, differential display-PCR, expressed sequence tag, suppression subtractive hybridization and microarray techniques. Use of these techniques will help to analyze expression pattern of many genes from small scale to large scale and to compare expression patterns of genes in one sample to another. In this review, we described principles of these methodologies and summarized examples of comparative analysis of gene expression in female reproductive organs with help of those methodologies.

• Development and Reproduction
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• v.17 no.3
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• pp.221-229
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• 2013

Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87-98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder.

• BMB Reports
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• v.36 no.6
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• pp.572-579
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• 2003

An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts >500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of $\alpha$-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.