• BMB Reports
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• v.35 no.3
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• pp.348-351
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• 2002

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed $\alpha$ and $\beta$) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the $\alpha$ and $\beta$ subunits of M.AquI into expression vectors. The overexpressed His-fusion $\alpha$ and $\beta$ subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The $\alpha$ and $\beta$ subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-L-methionine to DNA is reconstituted. We also showed that the $\beta$ subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the $\alpha$ subunit.

• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.407-414
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• 2009

A cDNA clone encoding phytocystatin was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (name as Brcpi1; GenBank accession no.: EF079953) had a total length of 881 bp with an open reading frame of 609 bp, and encoded predicted polypeptide of 203 amino acid (aa) residues including a putative N-terminal signal peptide. Other relevant regions found its sequence included the G and PW conserved aa motifs, and the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The BrCPI1 protein shared 95, 94, 81, 80 and 78% identity with other CPI proterins isolated from Brassica oleracea (BoCPI-1), Arabidopsis thaliana (AtCY SB), Glycine max (GmCPI), Oryza sativa (OsCYS-2) and Zea may (ZmCPI) at amino acid level, respectively. Southern blot analysis showed that Brcpi1 was a low copy gene. Expression pattern analysis revealed that Brcpi1 was a tissue-specific expressing gene during reproductive growth and strongly expressed at mature seedling stages. Furthermore, overexpression of Brcpi1 in transgenic Arabidopsis was enhanced tolerance to salt and cold stresses. Meanwhile the juvenile seedling of Brcpi1 transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that Brcpi1 functioned as a cysteine protease inhibitor and it exhibited a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner.

• The Plant Pathology Journal
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• v.32 no.4
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• pp.347-356
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• 2016

Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), Vf-CXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, Vf-CXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines.

• Molecules and Cells
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• v.20 no.2
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• pp.210-218
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• 2005

The rice genome contains at least 28 EXPA (${\alpha}$-expansin) genes. We have obtained near full-length cDNAs from the previously uncharacterized genes. Analysis of these newly identified clones together with the 12 identified earlier showed that the EXPA genes contain up to two introns and encode proteins of 240 to 291 amino acid residues. The EXPA proteins contain three conserved motifs: eight cysteine residues at the N-terminus, four tryptophan residues at the C-terminus, and a histidine-phenylalanine-aspartate motif in the central region. EXPA proteins could be divided into six groups based on their sequence similarity. Most were strongly induced in two-day-old seedlings and in the roots of one-week-old plants. However, only 14 genes were expressed in the aboveground organs, and their patterns were quite diverse. Transcript levels of EXPA7, 14, 15, 18, 21, and 29 were greater in stems, while EXPA2, 4, 5, 6, and 16 were highly expressed in both stem and sheath but not in leaf blade. EXPA1 is leaf blade-preferential, and EXP9 is leaf sheath-preferential. Most of the root-expressed genes were more strongly expressed in the dividing zone. However, the Group 2 EXPA genes were also strongly expressed in both mature and dividing zones, while EXPA9 was preferentially expressed in the elongation zone. Fourteen EXPA genes were expressed in developing panicles, with some being expressed during most developmental stages, others only as the panicles matured. These diverse expression patterns of EXPA genes suggest that in general they have distinct roles in plant growth and development.

• Journal of the Korean Chemical Society
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• v.46 no.2
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• pp.157-163
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• 2002

To identify RNA motifs interacting with $tRNA^{Val}$, a SELEX(Systematic Evolution of Ligands by Exponential Enrichment) was applied. Random DNA library which contains a region of ran-domized 48-mer oligonucleotide flanked by conserved sequ ence primers was transcribed into RNA pool using T7 RNA polymerase and RNA aptamers were selected with $tRNA^{Val}$ -immobilized affinity column through 14 rounds of SELEX. Some of the resulting aptamers contained a consensus sequence similar to the sequence in the loop regions of three rRNAs; C43GAAC47 sequence of 5S rRNA, G1491AAGU1495, G1379UUCC1383 sequence of 16S rRNA and C1064UUAG1068, G2110UGUA2114, C2480GACGG2485, A2600CAGU2604 sequence of 23S rRNA. These results suggest that $tRNA^{Val}$ can interact with 5S rRNA, 16S rRNA and 23S rRNA with variety in ribosome.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.133-144
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• 2000

Objective: Several water channels (aquaporins; AQP) that belong to the MIP (major intrinsic protein) family have identified. In the selected tissues including red blood cells or renal tubules, water movements are abundant and/or physiologically important. Unexpectedly, a high water permeability of human and ram sperm has been reported. Recent studies showed that AQP7 and AQP8 are present in testes, so that the high water permeability of human sperm suggested to be mediated by AQPs. Method: To identify the identity of aquaporins expressed in testes, RT-PCR was performed using degenerative primers, which were designed to correspond to highly conserved sequences surrounding the Asn-Pro-Ala (NPA) motifs in the aquaporins. New expressed AQP series were reconfirmed by immunohistochemical study using rabbit polyclonal antibodies. Results: DNA sequencing of PCR products revealed that AQP2 and AQP3 mRNA as well as AQP7 and AQP8 are expressed in human and rat testes. In human and rat testes, AQP2 are expressed in spermatozoa, interstitial cells and myofibroblasts and AQP3 are expressed in myofibroblasts of semineferous tubules on immunocytochemical stain. Conclusion: These results indicate that multiple aquaporins are expressed in testes, and that they may have important roles in the spermatogenesis and the germ cell function of testis.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.889-893
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• 2012

Appressorium is a specialized infection structure of filamentous pathogenic fungi and plays an important role in establishing a pathogenic relationship with the host. The Egh16/Egh16H family members are involved in appressorium formation and pathogenesis in pathogenic filamentous fungi. In this study, a homolog of Egh16H, Magas1, was identified from an entomopathogenic fungus, Metarhizium acridum. The Magas1 protein shared a number of conserved motifs with other Egh16/Egh16H family members and specifically expressed during the appressorium development period. Magas1-EGFP fusion expression showed that Magas1 protein was not localized inside the cell. Deletion of the Magas1 gene had no impact on vegetative growth, conidiation and appressorium formation, but resulted in a decreased mortality of host insect when topically inoculated. However, the mortality was not significant between the Magas1 deletion mutant and wild-type treatment when the cuticle was bypassed by injecting conidia directly into the hemocoel. Our results suggested that Magas1 may influence virulence by affecting the penetration of the insects' cuticle.

• Journal of Radiation Industry
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• v.2 no.3
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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.

• Animal Bioscience
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• v.35 no.4
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• pp.517-526
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• 2022

Objective: The growth differentiation factor 8 (GDF8) gene plays a key role in bone formation, resorption, and skeletal muscle development in mammals. Here, we studied the genetic variants of GDF8 and their contribution to body conformation traits in Chinese Dabieshan cattle. Methods: Single nucleotide polymorphisms (SNPs) were identified in the bovine GDF8 gene by DNA sequencing. Phylogenetic analysis, motif analysis, and genetic diversity analysis were conducted using bioinformatics software. Association analysis between five SNPs, haplotype combinations, and body conformation traits was conducted in 380 individuals. Results: The GDF8 was highly conserved in seven species, and the GDF8 sequence of cattle was most similar to the sequences of sheep and goat based on the phylogenetic analysis. The motif analysis showed that there were 12 significant motifs in GDF8. Genetic diversity analysis indicated that the polymorphism information content of the five studied SNPs was within 0.25 to 0.5. Haplotype analysis revealed a total of 12 different haplotypes and those with a frequency of <0.05 were excluded. Linkage disequilibrium analysis showed a strong linkage (r²>0.330) between the following SNPs: g.5070C>A, g.5076T>C, and g.5148A>C. Association analysis indicated these five SNPs were associated with some of the body conformation traits (p<0.05), and the animals with haplotype combination H1H1 (-GGGG CCTTAA-) had greater wither height, hip height, heart girth, abdominal girth, and pin bone width than the other (p<0.05) Dabieshan cattle. Conclusion: Overall, our results indicate that the genetic variants of GDF8 affected the body conformation traits of Chinese Dabieshan cattle, and the GDF8 gene could make a strong candidate gene in Dabieshan cattle breeding programs.

• Fisheries and Aquatic Sciences
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• v.25 no.11
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• pp.559-571
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• 2022

Galectins, a family of ß-galactoside-binding lectins, have emerged as soluble mediators in infected cells and pattern recognition receptors (PRRs) responsible for evoking and regulating innate immunity. The present study aimed to evaluate the role of galectin-1 in the host immune response of redlip mullet (Liza haematocheilia). We established a cDNA database for redlip mullet, and the cDNA sequence of galectin-1 (LhGal-1) was characterized. In silico analysis was performed, and the spatial and temporal expression patterns in gills and blood in response to lipopolysaccharide polyinosinic:polycytidylic acid, and Lactococcus garvieae were estimated via quantitative real-time PCR. Functional assays were conducted using recombinant protein to investigate carbohydrate binding, bacterial binding, and bacterial agglutination activity. LhGal-1 was composed of 135 amino acids. Conserved motifs (H-NPR, -N- and -W-E-R) within the carbohydrate recognition domain were found in LhGal-1. The tissue distribution revealed that the healthy stomach expressed high levels of LhGal-1. The temporal monitoring of LhGal-1 mRNA expression in the gill and blood showed its significant upregulation in response to immune challenges with different stimulants. rLhGal-1 exhibited binding activity in response to carbohydrates and bacteria. Moreover, the agglutination of rLhGal-1 against Escherichia coli was observed. Collectively, our findings suggest that LhGal-1 may function as a PRR in redlip mullet. Furthermore, LhGal-1 can be considered a significant gene to play a protective role in redlip mullet immune system.