• Molecules and Cells
• /
• v.27 no.1
• /
• pp.47-54
• /
• 2009

A cDNA clone for a transcript preferentially expressed during an early phase of flooding was isolated from Nicotiana tabacum. Nucleotide sequencing of the cDNA clone identified an open reading frame that has high homology to the previously reported glycine-rich RNA-binding proteins. The open reading frame consists of 157 amino acids with an N-terminal RNA-recognition motif and a C-terminal glycine-rich domain, and thus the cDNA clone was designated as Nicotiana tabaccum glycine-rich RNA-binding protein-1 (NtGRP1). Expression of NtGRP1 was upregulated under flooding stress and also increased, but at much lower levels, under conditions of cold, drought, heat, high salt content, and abscisic acid treatment. RNA homopolymer-binding assay showed that NtGRP1 binds to all the RNA homopolymers tested with a higher affinity to poly r(G) and poly r(A) than to poly r(U) and poly r(C). Nucleic acid-binding assays showed that NtGRP1 binds to ssDNA, dsDNA, and mRNA. NtGRP1 suppressed expression of the fire luciferase gene in vitro, and the suppression of luciferase gene expression could be rescued by addition of oligonucleotides. Collectively, the data suggest NtGRP1 as a negative modulator of gene expression by binding to DNA or RNA in bulk that could be advantageous for plants in a stress condition like flooding.

• Microbiology and Biotechnology Letters
• /
• v.49 no.3
• /
• pp.440-448
• /
• 2021

Helicobacter pylori (H. pylori) establishes long-term infections associated with severe gastric diseases such as peptic ulceration and gastric cancer. Exposure to an antibacterial agent can help regulate the expression levels of its pathogenic genes. In this study, we analyzed the transcriptional changes in H. pylori genes induced by β-caryophyllene. We used next-generation sequencing (NGS) to analyze RNA expression changes, and reverse transcription-polymerase chain reaction (RT-PCR) was performed as required to verify the results. The NGS results showed that 30 out of 1,632 genes were expressed differentially by β-caryophyllene treatment. Eleven genes associated with DNA replication, virulence factors, and T4SS components were significantly downregulated. RT-PCR confirmed that treatment reduced the expression levels of 11 genes. RT-PCR showed the reduced expression of 11 genes (dnaE, dnaN, holB, gyrA, cagA, vacA, secA, flgE, virB2, virB4, and virB8) following β-caryophyllene treatment. These results suggest that β-caryophyllene can modulate various H. pylori pathogenic determinants and be a potential therapeutic agent for H. pylori infection.

• Asian-Australasian Journal of Animal Sciences
• /
• v.28 no.1
• /
• pp.50-57
• /
• 2015

This study was conducted to investigate the effect of soybean meal (SM) and soluble starch (SS) on biogenic amine production and microbial diversity using in vitro ruminal fermentation. Treatments comprised of incubation of 2 g of mixture (expressed as 10 parts) containing different ratios of SM to SS as: 0:0, 10:0, 7:3, 5:5, 3:7, or 0:10. In vitro ruminal fermentation parameters were determined at 0, 12, 24, and 48 h of incubation while the biogenic amine and microbial diversity were determined at 48 h of incubation. Treatment with highest proportion of SM had higher (p<0.05) gas production than those with higher proportions of SS. Samples with higher proportion of SS resulted in lower pH than those with higher proportion of SM after 48 h of incubation. The largest change in $NH_3$-N concentration from 0 to 48 h was observed on all SM while the smallest was observed on exclusive SS. Similarly, exclusive SS had the lowest $NH_3$-N concentration among all groups after 24 h of incubation. Increasing methane ($CH_4$) concentrations were observed with time, and $CH_4$ concentrations were higher (p<0.05) with greater proportions of SM than SS. Balanced proportion of SM and SS had the highest (p<0.05) total volatile fatty acid (TVFA) while propionate was found highest in higher proportion of SS. Moreover, biogenic amine (BA) was higher (p<0.05) in samples containing greater proportions of SM. Histamines, amine index and total amines were highest in exclusive SM followed in sequence mixtures with increasing proportion of SS (and lowered proportion of SM) at 48 h of incubation. Nine dominant bands were identified by denaturing gradient gel electrophoresis (DGGE) and their identity ranged from 87% to 100% which were mostly isolated from rumen and feces. Bands R2 (uncultured bacterium clone RB-5E1) and R4 (uncultured rumen bacterium clone L7A_C10) bands were found in samples with higher proportions of SM while R3 (uncultured Firmicutes bacterium clone NI_52), R7 (Selenomonas sp. MCB2), R8 (Selenomonas ruminantium gene) and R9 (Selenomonas ruminantium strain LongY6) were found in samples with higher proportions of SS. Different feed ratios affect rumen fermentation in terms of pH, $NH_3$-N, $CH_4$, BA, volatile fatty acid and other metabolite concentrations and microbial diversity. Balanced protein and carbohydrate ratios are needed for rumen fermentation.

• Journal of Life Science
• /
• v.30 no.3
• /
• pp.313-319
• /
• 2020

Recently, cattle epidemic diseases are caused by a pathogen such as a virus or bacterium. Such diseases can spread through various pathways, such as feed intake, respiration, and contact between livestock. Diagnosis based on the ELISA (Enzyme-linked immunosorbent assay) and PCR (Polymerase chain reaction) methods has limitations because these traditional diagnostic methods are time consuming assays that require multiple steps and dedicated equipment. In this review, we propose the use of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas system based on DNA and RNA levels for early point-of-care diagnosis in cattle. In the CRISPR/Cas system, Cas effectors are classified into two classes and six subtypes. The Cas effectors included in class 2 are typically Cas9 in type II, Cas12 in type V (Cas12a and Cas12b) and Cas13 in type VI (Cas13a and Cas13b). The CRISPR/Cas system uses reporter molecules that are attached to the ssDNA strands. When the Cas enzyme cuts the ssDNA, these reporters either fluoresce or change color, indicating the presence of a specific disease marker. There are several steps in the development of a CRISPR/Cas system. The first is to select the Cas enzyme depending on DNA or RNA from pathogens (viruses or bacteria). Based on that, the next step is to integrate the optimal amplification, transducing method, and signal reporter. The CRISPR/Cas system is a powerful diagnostic tool using a gene-editing method, which is faster, better, and cheaper than traditional methods. This system could be used for early diagnosis of epidemic cattle diseases and help to control their spread.

• Journal of Crop Science and Biotechnology
• /
• v.10 no.3
• /
• pp.147-158
• /
• 2007

Xanthomonas axonopodis pv. glycines(Xag) is a pathogen that causes bacterial leaf pustule(BLP) disease in soybeans grown in Korea and the southern United States. Typical and early symptoms of the disease are small, yellow to brown lesions with raised pustules that develop into large necrotic lesions leading to a substantial loss in yield due to premature defoliation. After Xag infects PI 96188, only pustules without chlorotic haloes were observed, indicating the different response to Xag. To identify differentially expressed genes prior to and 24 hr after Xag inoculation to PI 96188 and BLP-resistant SS2-2, an oligonucleotide macroarray was constructed with 100 genes related to disease resistance and metabolism from soybean and Arabidopsis. After cDNAs from each genotype were applied on the oligonucleotide macroarrays with three replicates and dye swapping, 36 and 81 genes were expressed as significantly different between 0 hr and 24 hr in PI 96188 and SS2-2, respectively. Six UniGenes, such as the leucine-rich repeat protein precursor or 14-3-3-like protein, were selected because they down-regulated in PI 96188 and up-regulated in SS2-2 after Xag infection, simultaneously. Using tubulin and cDNA of Jangyeobkong(BLP-susceptible) as controls, the oligonucleotide macroarray data concurred with quantitative real-time RT-PCR(QRT RT-PCR) results in most cases, supporting the accuracy of the oligonucleotide macroarray experiments. Also, QRT RT-PCR data suggested six candidate genes that might be involved in a necrotic response to Xag in PI 96188.

• Biomedical Science Letters
• /
• v.26 no.2
• /
• pp.114-119
• /
• 2020

Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.

• BMB Reports
• /
• v.31 no.2
• /
• pp.149-155
• /
• 1998

The N199H variant of the EcoRI endonuclease has about twice the catalytic activity of the wild-type. A comparison of their biochemical characteristics, using synthetic oligonucleotides 5'-dAAAACTTAAGAAAAAAAAAAA-3' (KA) and 5'-dTTTTTGAATTCTTTTTTTTTT-3' (KT), helps to define the cleavage reaction pathway of these enzymes. Both EcoRI and EcoRI variant N199H were found to cleave singlestranded KA or KT about three times faster than the double-stranded forms, although the KT oligonucleotide was more susceptible. Using the ssDNA substrate in kinetic analyses, lower $K_m$ values were obtained for the N199H variant than for the wild-type at low (50 mM), as well as high (200 mM), sodium chloride concentrations. This difference between the endonucleases is attributed to a grealter accessibility for tbe substrate by the variant, and also a higher affinity for the DNA backbone. It also appears that the relative activities of the two enzymes, particularly at high ionic strength, are proportional to their populations in the monomeric enzyme form. That is, according to gel filtration data, half of the N199H molecules exist as monomers in 200 mM NaCl, whereas those of the wild-type are mainly dimeric. Consequently, the Asp199 residue of the EcoRI endonuclease may be implicated in the protein-protein interaction leading to dimerization, as well as in coupling to DNA substrates. In summary, it is proposed that active monomeric endonuclease molecules, derived from the dimeric enzyme, recognize and form a complex with a single stranded form of the DNA substrate, which then undergoes nucleophilic substitution and cleavage.

• Journal of Microbiology
• /
• v.33 no.2
• /
• pp.136-141
• /
• 1995

From a cluster of structural rRNA genes which has previsouly been cloned (Hwang and Kim, in submission; J. Microbiol. Biotechnol.), a 1.0-kb Eco RI fragment of DNA which shows significant homology to the 25S and rRNA s of Tricholoma matsutake was used for sequence analysis. Nucleotide sequence was bidirectionally determined using delection series of the DNA fragment. Comparing the resultant 1016-base sequence with sequences in the database, both the 3'end of 25S-rRNA gene and 5S rRNA gene were searched. The 5S rRNA gene is 118-bp in length and is located 158-bp downstream of 3'end of the 25S rRNA gene. IGSI and IGS2 (partial) sequences are also contained in the fragment. Multiple alignment of the 5S rRNA sequences was carried out with 5S rRNA sequences from some members of the subdivision Basidiomycotina obtained from the database. Polygenetic analysis with distance matrix established by Kimura's 2-parameter method and phylogenetic tree by UPGMA method proposed that T. matsutake is closely related to efibulobasidium allbescens. Secondary structure of 5S rRNA was also hypothesized to show similar topology with its generally accepted eukaryotic counterpart.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.18 no.2
• /
• pp.534-537
• /
• 2004

We produced twelve monoclonal antibodies(mAbs) against thyroglobulin and characterized the bindig profiles. Among them, three mAbs(TN-1, TN-2 and TN-3) were further characterized their binding specificities. TN-2 had a potent lupus anticoagulant activity and potentiated the anticoagulant effect of venom phospholipase A₂. he anticoagulant mechanism of TN-2 was elongation of the partial thromboplastin time and binding to phosphatidylserine which may have a pivot role in blood coagulation. And TN-2 was cross-reacted with ss-DNA and ds-DNA and had a characteristic of autoantibody. These results suggest that TN-2 may provide a useful tool for studying the correlation between autoimmune thyroiditis and its therapeutic effect.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.3
• /
• pp.457-462
• /
• 2003

The complete nucleotide sequence of a plasmid, pMG1, isolated from Bifidobacterium longum MG1 has been determined. This plasmid, composed of 3,862 base pairs with 65.1% of G+C content. harbors two major open reading frames (ORF) encoding putative proteins of 29 kDa (ORF I) and 71 kDa (ORF II). ORF I showed relatively high amino acid sequence homology with replication proteins of other plasmids from Gr Im-positive and -negative bacteria. Upstream of ORF I, four sets of tandem repeat sequences resembling the iteron structure of related plasmids were found. S1 endonuclease treatment and Southern blot analysis revealed that pMG1 accumulates single-stranded DNA (ssDNA) intermediate, which indicate i the rolling circle replication (RCR) mechanism of this plasmid. Homology search indicated that ORF II encodes plasmid mobilization protein, and the presence of highly conserved oriT sequence in the upstream of this gene supported this assumption. RT-PCR showed that only ORF I is expressed in vivo. Based on these results, pMG 1 was exploited to construct a shuttle vector, pBES2. It was successfully transformed into Bifidobacterium and maintained stably.