• BMB Reports
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• 제43권7호
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• pp.474-479
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• 2010

Three assay methods for quantification of the two galactose-operon mRNAs that only differ by 5 bases in their 5'-end are presented. The 5' ends of each mRNA were extended by ligating the 3'-end of the abundant 5S rRNA. This ligation extends the 5' ends of the two gal mRNAs long enough to be distinguished by the specific PCR primers in the following quantification reactions. Quantification of the corresponding cDNAs was performed either by primer extension assay or real-time qPCR. To circumvent the problem of the RNA ligation reaction (i.e. very low ligation efficiency), we devised a new method that employs real-time qPCR directly for the quantification of the gal transcripts which differ by 5 bases in their 5'-ends.

• 한국토양동물학회지
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• 제8권1_2호
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• pp.1-6
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• 2003

Hox genes are pivotal in the control of morphogenesis along the anterior-posterior (AP) axis in various bilaterians. Several indications suggest their involvement in the control of cell growth and regeneration. For the labial full-length fragment, RACE-PCR was employed to obtain the 3' and 5' franking regions. Semi-quantitative RT-PCR analysis revealed that the labial expression began to increase at 12 hours after amputation. The peak expression was approximately 1.5-fold more than the unamputated controls. This result could give us information on the significance of Hox genes and the relationships between Hox genes during regeneration.

• 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.

• 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.

• Environmental Analysis Health and Toxicology
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• 제26권
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• pp.5.1-5.11
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• 2011

Objectives: In order to identify the possibility of slender bitterling (SB) (Acheilognathus yamatsutae) being used as a test species for estrogenic endocrine disrupting chemicals (EEDCs), we carried out the cloning and sequence characterization of the estrogen receptor (ER). Methods: The ER from a slender bitterling was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (5'-RACE and 3'-RACE) and T-vector cloning. The expression of ER mRNA was also analyzed in six tissues (brain, liver, kidney, gill, gonad, and intestines) by real-time PCR. Results: We obtained an ER from the slender bitterling. The SB ER cDNA was 2189 base pairs (bp) in length and contained a 1707 bp open reading frame that encoded 568 amino acid residues. The SB ER amino acid sequence clustered in a monophyletic group with the $ER{\alpha}$ of other fish, and was more closely related to zebrafish $ER{\alpha}$(88% identity) than to the $ER{\alpha}$ of other fish. The SB ER cDNA was divided into A/B, C, D, E and F domains. The SB ER has conserved important sequences for ER functions, such as the DNA binding domain (D domain), which are consistent with those of other teleosts. Conclusions: The ER of the slender bitterling could provide basic information in toxicological studies of EEDCs in the slender bitterling.

• 방사선산업학회지
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• 제2권3호
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• pp.97-104
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• 2008

Cinnamate-4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which leads a variety of secondary metabolites to participate in differentiation and protection of plant against environmental stresses. In this study, we isolated a full-length cDNA of the C4H gene from a black raspberry (Rubus occidentalis L.), using a reverse transcriptase-PCR and rapid amplification of the cDNA ends (RACE)-PCR. The full-length cDNA of the RocC4H gene contained a 1,515 bp open reading frame (ORF) encoding a 504 amino acid protein with a calculated molecular weight of about 57.9 kDa and an isoelectric point (pI) value of 9.1. The genomic DNA analysis revealed that RocC4H gene had three exons and two introns. By multiple sequence alignment, RocC4H protein was highly homologous with other plant C4Hs, and the cytochrome P450-featured motifs, such as the heme-binding domain, the T-containing binding pocket motif (AAIETT), the ERR triad, and the tetrapeptide (PPGP) hinge motif, were highly conserved. Southern blot analysis revealed that RocC4H is a single copy gene in R. occidentalis.

• 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.

• 대한수의학회지
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• 제52권1호
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• pp.61-64
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• 2012

This study evaluated the epizootiological characteristics of equine coital exanthema (ECE) in South Korea. A PCR test was used to determine the equine herpesvirus 3 (EHV-3) release period, excretion by suckling foals, morbidity rate, effect on fertility, and onset of breeding partner after treatment. The morbidity rate of ECE based on clinical symptoms was 8.3% (2/24) for stallions and 10.8% (45/416) for mares, and 29 of 45 (64.4%) animals were positive on the PCR test. Ten (22%) broodmares had symptoms before breeding, while 26 (58%) had symptoms after breeding. Nine (20%) mares had uncertain coverage periods and occurrence times. Suckling foals had no clinical findings and EHV-3 was not detected in their nostrils, although it was detected on teasers. No lesions were observed in the clitoral fossa on broodmares, although EHV-3 was detected by PCR. The period of EHV-3 emission was 22~23, 18~19, 6, and 58 days in stallions, broodmares, teasers, and mares with a mixed E. coli-like infection, respectively. ECE had no negative effects on the breeding capability of stallions and no symptoms were observed in broodmares after recovering from ECE.

• 한국수산과학회지
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• 제51권2호
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• pp.163-169
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• 2018

This study examined the expression of heat shock protein 70 (Hsp70) in red seabream Pagrus major infected by the, acanthocephalan parasites Longicollum pagrosomi. We cloned the full-length Hsp70 cDNA from the liver of the red seabream. The full-length cDNA had a 1,950 bp open reading frame (ORF) that encoded a protein of 650 amino acids. The deduced amino acid sequence of Hsp70 contained all of the conserved Hsp70 family signature sequences and an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding motif, including the EEVD (consensus sequence that terminates in Hsp70 family) consensus sequence. The expression of Hsp70 mRNA was upregulated int the fish head-kidney and liver, as determined by quantitative real-time PCR. We quantified the Hsp70 mRNA expression in normal red seabream and fish infected fish by L. pagrosomi. The expression of Hsp70 mRNA was significantly higher in the infected red seabream. These results suggest that Hsp70 play a role of protection against stress and inflammation caused by the parasite and may help maintain homeostasis.

• 한국버섯학회지
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• 제9권2호
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• pp.53-58
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• 2011

In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3'- and 5'-RACE PCR and RT-PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.