• Research in Plant Disease
• /
• v.19 no.4
• /
• pp.254-258
• /
• 2013

Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is relatively low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been identified in F. oxysporum. In order to explore the genetic element(s) for FA biosynthesis, restriction enzyme mediated integration (REMI) procedure as an insertional mutagenesis was employed using FA producing-F. oxysporum strains. Genetic transformation of two F. oxysporum strains by REMI yielded more than 7,100 transformants with efficiency of average 3.2 transformants/${\mu}g$ DNA. To develop a screening system using phytotoxicity of FA, eleven various grains and vegetable seeds were tested for germination in cultures containing FA: Kimchi cabbage seed was selected as the most sensitive host. Screening for FA non-producer of F. oxysporum was done by growing each fungal REMI transformant in Czapek-Dox broth for 3 weeks at $25^{\circ}C$ then observing if the Kimchi cabbage seeds germinated in the culture filtrate. Of more than 5,000 REMI transformants screened, fifty-three made the seeds germinated, indicating that they produced little or fewer FA. Among them, twenty-six were analyzed for FA production by HPLC and two turned out to produce less than 1% of FA produced by a wild type strain. Sequencing of genomic DNA regions (252 bp) flanking the vector insertion site revealed an uncharacterized genomic region homologous (93%) to the F. fujikuroi genome. Further study is necessary to determine if the vector insertion sites in FA-deficient mutants are associated with FA production.

• The Korean Journal of Physiology
• /
• v.26 no.2
• /
• pp.159-166
• /
• 1992

The present study was designed to evaluate effects of intermittent electrical stimulation of the sciatic nerve on the atrophic response of antigravity muscles, such as the soleus (slow m.) and medial gastrocnemius (fast m.) muscles. Rats (Sprague-Dawley, 245-255g) were subjected to a hindlimb suspension and divided into three groups : one was with hindlimb suspension (MS) and another with hindlimb suspension plus intermittent electrical stimulation of the sciatic nerve (HS ES). Control group (CONT) was kept free without strain of the hindlimb. After 7 days of hindlimb suspension, the soleus and medial gastrocnemius muscles were cut at their insertion sites, and were then connected to the force transducer to observe their mechanical properties. Optimal pulse width and frequency of electrical stimulation were 0.2ms, 20Hz for the soleus muscle and 0.3ms, 40Hz for the medial gastrocnemius muscle under supramaximal stimulation. Body weight and circumference of the hindlimb were significantly decreased in HS and HS-ES groups compared with the control group. In HS-ES group, however, the weight of the soleus muscle was not different from that in the control group while the weight of the medial gastrocnemius muscle was lower than that in the control group. In HS group, mechanical properties of muscle contraction including contraction time, half relaxation time, twitch tension, tetanic tension, and fatigue index of both muscles were significantly decreased compared with the control group except for twitch tension and tetanic tension of medial gastrocnemius muscle. The degree of atrophy of the soleus muscle in HS group was more prominent than that of the medial gastrocnemius muscle. Twitch tension and fatigue index of the soleus muscle and fatigue index of the medial gastrocnemius muscle in HS-ES group were not different from those of the control group. While mechanical properties of the soleus muscle examined were all significantly increased in HS-ES group compared with HS group, only contraction time and fatigue index of the medial gastrocnemius muscle were significantly increased in HS-ES group. These data indicate that intermittent electrical stimulation may be useful in prevention of muscle atrophy.

• Journal of Microbiology
• /
• v.43 no.4
• /
• pp.337-344
• /
• 2005

Although pneumococcus is one of the most frequently encountered opportunistic pathogen in the world, the mechanisms responsible for its infectiveness have not yet been fully understood. In this paper, we have attempted to characterize the effects of pneumococcal transformation on the pathogenesis of the organism. We constructed three transformation-deficient pneumococcal strains, which were designated as Nos. 1d, 2d, and 22d. The construction of these altered strains was achieved via the insertion of the inactivated gene, comE, to strains 1, 2 and 22. We then conducted a comparison between the virulence of the transformation-deficient strains and that of the wild-type strains, via an evaluation of the ability of each strain to adhere to endothelial cells, and also assessed psaA mRNA expression, and the survival of hosts after bacterial challenge. Compared to what was observed with the wild-type strains, our results indicated that the ability of all of the transformation-deficient strains to adhere to the ECV304 cells had been significantly reduced (p < 0.05), the expression of psaA mRNA was reduced significantly (p < 0.05) in strains 2d and 22d, and the median survival time of mice infected with strains Id and 2d was increased significantly after intraperitoneal bacterial challenge (p < 0.05). The results of our study also clearly indicated that transformation exerts significant effects on the virulence characteristics of S. pneumoniae, although the degree to which this effect is noted appears to depend primarily on the genetic background of the bacteria.

• Korean Journal of Veterinary Service
• /
• v.44 no.2
• /
• pp.73-83
• /
• 2021

Although many swine farms continuously vaccinated to sow to prevent Porcine epidemic diarrhea(PED), PED has occurred annually in swine herds in Jeonbuk province, Korea. In the present study, the small intestine and feces samples from 17 farms where severe watery diarrhea and death of newborn piglets occurred in 2019 were collected, amplified by RT-PCR and determined the complete nucleotide sequences of the spike (S) glycoprotein genes of nine Jeonbuk PEDV isolates. The spike (S) glycoprotein is an important determinant for molecular characterization and genetic relationship of PEDV. These nine complete S gene isolates were compared with other PEDV reference strains to identify the molecular diversity, phylogenetic relationships and antigenicity analysis. 9 field strains share 98.5~100% homologies with each other at the nucleotide sequence level and 97.3~100% homologies with each other at the amino acid level. The nine Jeonbuk PEDV isolates were classified into G2b group including a genetic specific signal, S-indels (insertion and deletion of S gene). In addition, comparisons the neutralizing epitopes of S gene between 9 field strains and domestic vaccine strains of Korea mutated 12-15 amino acids with SM-98-1 (G1a group) and mutated 0-3 amino acids with QIAP1401 (G2b group). Therefore, the development of G2b-based live vaccines will have to be expedited to ensure effective prevention of endemic PED in Korea. In addition, we will need to be prepared with periodic updates of preventive vaccines based on the PEDV variants for the re-emergence of a virulent strain.

• Journal of Mushroom
• /
• v.20 no.3
• /
• pp.178-182
• /
• 2022

Despite the long history of mushroom use, studies examining the genetic function of mushrooms and the development of new varieties via bio-molecular methods are significantly lacking compared to those examining other organisms. However, owing to recent developments, attempts have been made to use a novel gene-editing technique involving CRISPR/Cas9 technology and genetic scissors in mushroom studies. In particular, research is actively being conducted to utilize ribonucleoprotein particles (RNPs) that can be genetically edited with high efficiency without foreign gene insertion for ease of selection. However, RNPs are too large for Cas9 protein to pass through the cell membrane of the protoplasmic reticulum. Furthermore, guide RNA is unstable and can be easily decomposed, which remarkably affects gene editing efficiency. In this study, nanoparticles were used to mitigate the shortcomings of RNP-based gene editing techniques and to obtain transformants stably. We used Lentinula edodes (shiitake mushroom) Sanjo705-13 monokaryon strain, which has been successfully used in previous genome editing experiments. To identify a suitable osmotic buffer for the isolation of protoplast, 0.6 M and 1.2 M sucrose, mannitol, sorbitol, and KCl were treated, respectively. In addition, with various nanoparticle-forming materials, experiments were conducted to confirm genome editing efficiency via the formation of nanoparticles with calcium phosphate (CaP), which can be bound to Cas9 protein without any additional amino acid modification. RNPs/NP complex was successfully formed and protected nuclease activity with nucleotide sequence specificity.

• Journal of the Korea Institute of Military Science and Technology
• /
• v.27 no.4
• /
• pp.507-515
• /
• 2024

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled in silico anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General's Mechanism(UNSGM)'s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were B. anthracis through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of B. anthracis were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of B. anthracis, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as B. anthracis strain A4564 which is associated with injectional anthrax isolates in heroin users.

• Tuberculosis and Respiratory Diseases
• /
• v.50 no.1
• /
• pp.94-105
• /
• 2001

Background : Examining the biological susceptibility of Mycobacterium tuberculosis to pyrazinamide (PZA) in vitro is very difficult as PZA is inactive under normal culture conditions. The biological susceptibility test, an enzyme assay for Pzase activity, and a genetic test for pncA gene mutations, were performed in order to predict PZA resistance. Methods : 28 cultured clinical isolates of Mycobacterium tuberculosis were tested. The biological susceptibility was performed by the absolute concentration method using Lowenstein-Jensen media. The PZase activity was tested by means of Wayne's method. A 710-bp region includes the entire open reading frame of pncA was amplified and sequenced. Results : All six strains with positive PZase activity exhibited no pncA mutations with one strain showing a false resistance in the biological susceptibility test. Among the 22 strains with no PZase activity, 21 exhibited showed pncA mutations. In the biological susceptibility test, 20 strains were resistant, and one was susceptible, and the other flied to test. The mutation types varied with ten missense, one silent and one nonsense mutation 1 slipped-strand mispairing, and 6 frameshift mutations. Three strains had an adenine to guanine mutation at position -11 upstream of the start codon. Conclusion : The mutation at the pncA promotor region is frequent at -11 upstream position. Automatic sequencing of pncA is a useful tool for rapid and accurate detection of PZA resistant M. tuberculosis, and for demonstrating the epidemiological relatedness of the PZA resistant M. tuberculosis strains.