• 한국초지조사료학회지
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• 제40권4호
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• pp.285-290
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• 2020

To understand the role of small heat shock protein (sHSPs) in rice plant response to various stresses such as the heat and oxidative stresses, a cDNA encoding a 24.1 kDa mitochondrial small HSP (Oshsp24.1) was isolated from rice by rapid amplification of cDNA ends (RACE) PCR. The deduced amino acid sequence shows very high similarity with other plant small HSPs. DNA gel blot analysis suggests that the rice genome contains more than one copy of Oshsp24.1. High level of expression of Oshsp24.1 transcript was observed in rice seedlings in response to heat, methyl viologen, hydrogen peroxide, ozone, salt and heavy metal stresses. Recombinant OsHSP24.1 protein was produced in E. coli cells for biochemical assay. The protein formed oligomeric complex when incubated with Sulfo-EGS (ethylene glycol bis (succinimidyl succinate)). Our results shows that Oshsp24.1 has an important role in abiotic stress response and have potential for developing stress-tolerant plants.

• Asian-Australasian Journal of Animal Sciences
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• 제20권10호
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• pp.1473-1477
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• 2007

Four-and-a-half LIM-only protein 3 (FHL3) is a member of the LIM protein superfamily and can participate in mediating protein-protein interaction by binding one another through their LIM domains. In this study, the 5'- and 3'- cDNA ends were characterized by RACE (Rapid Amplification of the cDNA Ends) methodology in combination with in silico cloning based on the partial cDNA sequence obtained. Bioinformatics analysis showed FHL3 protein contained four LIM domains and four LIM zinc-binding domains. In silico mapping assigned this gene to the gene cluster MTF1-INPP5B-SF3A3-FHL3-CGI-94 on pig chromosome 6 where several QTL affecting intramuscular fat and eye muscle area had previously been identified. Transcription of the FHL3 gene was detected in spleen, liver, kidney, small intestine, skeletal muscle, fat and stomach, with the greatest expression in skeletal muscle. The A/G polymorphism in exon II was significantly associated with birth weight, average daily gain before weaning, drip loss rate, water holding capacity and intramuscular fat in a Landrace-derived pig population. Together, the present study provided the useful information for further studies to determine the roles of FHL3 gene in the regulation of skeletal muscle cell growth and differentiation in pigs.

• 방사선산업학회지
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• 제2권3호
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• pp.121-128
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• 2008

Flavanone-3-hydroxylase (F3H) is one of the key enzymes for the biosynthesis of flavonals, anthocyanins, catechins and proanthocyanins. F3H catalyzes the $3{\beta}$-hydroxylation of (2S)-flavonones to form (2R, 3R)-dihydroflavonols. In this report, we isolated a full-length cDNA of RocF3H from black raspberry (Rubus occidentalis L.) using a reverse transcriptase-PCR and rapid amplification of the cDNA ends (RACE)-PCR. The full-length cDNA of RocF3H contains a 1,098 bp open reading frame (ORF) encoding a 365 amino acid protein with a calculated molecular weight of about 41.1 kDa and isoelectric point (pI) of 5.45. The genomic DNA analysis revealed that the RocF3H gene had three exons and two introns. Comparison of the deduced amino acid sequence of the RocF3H with other F3Hs revealed that the protein is highly homologous with various plant species. The conserved amino acids ligating the ferrous iron and the residues participating in the 2-oxoglutarate binding (R-X-S) were found in RocF3H at the similar positions to other F3Hs. Southern blot analysis indicated that RocF3H exist a multi-gene family. The isolation of RocF3H gene will be helpful to further study the role of F3H gene in the biosynthesis of flavonoids in R. occidnetalis.

• BMB Reports
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• 제33권2호
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• pp.184-187
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• 2000

Prostaglandin $E_2$ ($PGE_2$) plays an important role in the regulation of various gastric functions, and the growth-inhibitory activities on tumor cells are studied in vitro and in vivo. Although the mechanisms have attracted many researchers in the past decade, the molecular mechanisms of cell cycle arrest, or induction of apoptosis by $PGE_2$, is unclear. We investigated the effects of $PGE_2$ on the growth of the human gastric carcinoma cell line SNU1 and genes that are regulated by $PGE_2$ and isolated them using differential display RT-PCR (DD RT-PCR). FACS analysis suggested that SNU1 cells were arrested at the G1 phase by $PGE_2$ treatment. This growth inhibitory effect was in a time- and dose-dependent manner. Treatment of SNU1 cells with $10\;{\mu}g/ml$ $PGE_2$, followed by DD RT-PCR analysis, revealed differently expressed bands patterns from the control. Among the differently expressed clones, we found an unidentified cDNA clone (HGP-27) overexpressed in $PGE_2$-treated cells. The full-length cDNA of HGP-27 was isolated using RACE, which consisted of a 30-nt 5'-noncoding region, a 891-nt ORF encoding the 296 amino acid protein, and a 738-nt 3'-noncoding region including a poly(a) signal. This gene was localized on the short arm of chromosome number 11. Using the Motif Finder program, a myb-DNA binding repeat signature was detected on the ORF region. The COOH-terminal half was shown to have similarity with the $NH_3$-terminal domain of thioredoxin (Trx). This relation between HGP-27 and Trx implied a potential role for HGP-27 in modulating the DNA binding function of a transcription factor, myb.

• 한국자원식물학회지
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• 제22권3호
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• pp.273-280
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• 2009

A cDNA clone encoding a MDR-like ABC transporter protein was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (named as Brmdr 1; GenBank accession no.: DQ296184 ) had a total length of 4222 bp with an open reading frame of 3900 bp, and encoded a predicted polypeptide of 1300 amino acids with a molecular weight of 143.1 kDa. The BrMDR1 protein shared 71.0, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAN28720), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Brmdr1 was a low-copy gene. Expression pattern analysis revealed that Brmdr1 constitutively expressed in the root, stem petals and stamens, but with lower expression in leaves and open flowers. The domains analysis showed that BrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that BrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.

• 한국산학기술학회논문지
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• 제21권5호
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• pp.216-222
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• 2020

Tumor necrosis factor receptor associated factor 4 (TRAF4)는 사람의 유방암에서 과발현 되며, 암세포전이, ROS 및 세포 극성 형성 등에 관여하는 것으로 알려져 있다. 그러나 돼지에서는 아직까지 그 기능과 특성에 대한 연구가 보고 된 바 없다. 따라서 본 연구에서는 돼지 TRAF4의 mRNA 전장서열을 분석하고, 그 기능과 특성을 알아보고자 수행되었다. TRAF4의 전장서열을 밝히기 위해 돼지 신장유래세포(pK15)에서 total RNA 추출하여 RACE (Rapid Amplification of cDNA ends) PCR을 수행하였다. 2,030 염기쌍의 mRNA 전장서열을 분석하였고, 470개의 아미노산으로 구성 되어 있는 것을 확인하였다. 사람과 쥐의 Homology를 분석한 결과 각각 93 % 그리고 90 %의 유사도를 가지며, 사람과는 8개, 쥐와는 12개의 아미노산 차이가 있음을 확인하였다. qPCR을 통하여 TRAF4, CLDN4, OCLN 그리고 TJP1의 발현을 분석한 결과 세포의 confluency 정도에 따라 발현이 다르게 나타남을 확인하였고, 세포가 40% 증식한 그룹 보다 60 %와 80 % 이상 증식 한 그룹에서 유의적으로 높게 나타났다. 또한 TRAF4의 기능을 확인하기 위하여 TRAF4 siRNA 처리 한 결과 TRAF4와 tight junction 관련 유전자가 낮게 발현됨을 관찰하였다. 따라서 사람과 마우스와 같이 돼지에서도 TRAF4가 발현되며, 세포-세포 간 중요한 역할을 하는 tight junction에 관여하는 것으로 사료된다.

• BMB Reports
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• 제40권2호
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• pp.270-276
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• 2007

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a $C_3HC_4$ ring finger domain and three $C_2H_2$ domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

• Asian Pacific Journal of Cancer Prevention
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• 제15권12호
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• pp.5055-5061
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• 2014

In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.

• 원예과학기술지
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• 제29권2호
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• pp.135-143
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• 2011

In the Doritaenopsis hybrid, like most of the orchid species and hybrids, temperature is crucial for the vegetative-to-reproductive transition, and low temperature is required for bud differentiation. To understand the molecular mechanism of this process, an orchid GIGANTEA (GI) gene, DhGI1, was isolated and characterized by using the rapid amplification of cDNA ends (RACE) PCR technique. Sequence analysis showed that the full-length cDNA is 4,022 bp with a major open reading frame of 3,483 bp, and the amino acid sequence showed high similarity to GI proteins in Zea mays, Oryza sativa, Arabidopsis thaliana and other plants. Semi-quantitative RT-PCR revealed that DhGI1 was expressed throughout development and could be detected in roots, stems, leaves, peduncles and flower buds. The expression level of DhGI1 was higher when the plants were flowering at low temperature (22/$18^{\circ}C$ day/night) than the other growth stages. Further analysis indicated that the accumulation of DhGI1 transcripts was significantly increased at low temperature, and concomitantly, initiation of the peduncle was observed. However, DhGI1 levels were low under high temperature (30/$25^{\circ}C$) conditions, and flower initiation was inhibited. These results indicate that the expression of DhGI1 is regulated by low temperature and that DhGI1 may play an important role in inflorescence initiation in this Doritaenopsis hybrid at low temperatures.

• Asian-Australasian Journal of Animal Sciences
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• 제17권7호
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• pp.897-902
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• 2004

PSMC5 subunit, which belongs to the 26S proteasomal subunit family, plays an important role in the antigen presentation mediated by MHC class I molecular. Full-length cDNA of porcine PSMC5 was isolated using the in silico cloning and rapid amplification of cDNA ends (RACE). Amino acid was deduced and the primary structure was analyzed. Results revealed that the porcine PSMC5 gene shares the high degree of sequence similarity with its mammalian counterparts at both the nucleotide level and the amino acid level. The RT-PCR was performed to detect the porcine PSMC5 expression pattern in seven tissues and the result showed that high express level was observed in spleen, lung, marrow and liver while the low express level was in muscle. The full-length genomic DNA sequence of porcine PSMC5 gene was amplified by PCR and the genomic structure revealed that this gene was comprised by 12 exons and 11 introns. Best alignment of the cDNA and genomic exon DNA sequence presents 4 mismatches and this information potentially bears further study in gene polymorphisms.