• Parasites, Hosts and Diseases
• /
• 제37권4호
• /
• pp.257-263
• /
• 1999

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC ＃289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8％) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.

• Journal of Microbiology and Biotechnology
• /
• 제31권6호
• /
• pp.882-889
• /
• 2021

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→ C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.

• IMMUNE NETWORK
• /
• 제14권3호
• /
• pp.138-148
• /
• 2014

MicroRNAs (miRNAs) are endogenous small RNA molecules best known for their function in post-transcriptional gene regulation. Immunologically, miRNA regulates the differentiation and function of immune cells and its malfunction contributes to the development of various autoimmune diseases including systemic lupus erythematosus (SLE). Over the last decade, accumulating researches provide evidence for the connection between dysregulated miRNA network and autoimmunity. Interruption of miRNA biogenesis machinery contributes to the abnormal T and B cell development and particularly a reduced suppressive function of regulatory T cells, leading to systemic autoimmune diseases. Additionally, multiple factors under autoimmune conditions interfere with miRNA generation via key miRNA processing enzymes, thus further skewing the miRNA expression profile. Indeed, several independent miRNA profiling studies reported significant differences between SLE patients and healthy controls. Despite the lack of a consistent expression pattern on individual dysregulated miRNAs in SLE among these studies, the aberrant expression of distinct groups of miRNAs causes overlapping functional outcomes including perturbed type I interferon signalling cascade, DNA hypomethylation and hyperactivation of T and B cells. The impact of specific miRNA-mediated regulation on function of major immune cells in lupus is also discussed. Although research on the clinical application of miRNAs is still immature, through an integrated approach with advances in next generation sequencing, novel tools in bioinformatics database analysis and new in vitro and in vivo models for functional evaluation, the diagnostic and therapeutic potentials of miRNAs may bring to fruition in the future.

• 식물조직배양학회지
• /
• 제24권3호
• /
• pp.153-160
• /
• 1997

오이 모자이크바이러스(CMV, cucumber mosaic virus)는 작물의 생산량과 품질에 심각한 피해를 주기 때문에 외피단백질 유전자(CP, coat protein gene)를 도입하여 저항성 작물를 개발하고자 하였다. CMV CP유전자가 도입된 형질전환 담배 39 계통을 대상으로 오이모자이크바이러스 저항성을 검정하였다. 바이러스 저항성은 바이러스 감염으로 인한 생장 억제정도, 병징발현에 따른 잎모양의 변화로서 고도저항성, 저항성, 중간성, 감수성 등으로 판정하였고 39개 계통중 16 계통이 뚜렷한 바이러스 저항성을 보였다. 특히, 저항성 계통중 2 계통은 생장량과 잎모양에서 다른 저항성 계통보다 우수하여 고도저항성으로 세분하였다. 각 형질전환계통에서 CP단백질과 CP RNA 생성량을 조사하였는바, CP단백질 생합성은 대부분의 저항성과 감수성계통에서 검출되어 저항성과 특별한 관련을 인정할 수 없었으나 CP RNA는 대부분의 저항성 및 중간성 계통에서 다량 축적되는 경향을 보여 CP RNA가 저항성에 좀더 밀접함을 알수 있었다. 그러나 고도저항성 계통에서는 CP RNA가 검출되지 않아 저항성의 근원을 파악하기 위해서는 계속적인 연구가 요구된다.

• 생명과학회지
• /
• 제25권9호
• /
• pp.1007-1013
• /
• 2015

원핵생물체의 생명유지에 중요한 역할을 담당하는 유전자들을 밝히기 위해 미생물 유전체들 사이의 공통적 유전자를 파악하는 COG 알고리즘을 이용하였다. 원핵생물 711종 모두에 보존적인 것은 COG0080 (Ribosomal protein L11) 1개였다. 708종 이상의 원핵생물에 보존적인 22개의 ortholog 중 전사관련 2개, tRNA synthetase 관련4개, ribosamal large subunit 8개, ribosomal small subunit 7개였다. 700종 이상의 원핵생물에 보존적인 COG는 58개였다. 이중 리보좀을 구성하는 소단위체 등 번역 관련 COG가 50개(86.2%), 전사관련 COG가 4개(6.9%)로 나타나 생명현상에서의 단백질의 중요성을 알 수 있었다. 58개의 COG 중 보존성은 COG0060 (Isoleucyl tRNA synthetase)이 가장 높았고 COG0143 (Methionyl tRNA synthetase)이 가장 낮았다. 문(phylum)과 강(class) 수준에서 보존적 유전자들의 평균과 분산으로 유전체 분석을 수행한 결과 변이가 큰 고세균은 진정세균과 구분되었으며 편차는 일부 진정세균이 고세균보다 컸다. 보존적 유전자를 탐색하는 본 연구의 기법은 기초과학 연구와 함께 항균제 개발과 항암요법 개발 등에도 유용할 것이다.

• Molecules and Cells
• /
• 제22권3호
• /
• pp.314-322
• /
• 2006

The whole mitochondrial genome (14,915 nt) of Pollicipes mitella (Crustacea, Maxillopoda, Cirripedia, Thoracica) was sequenced and characterized. It is the shortest of the 31 completely sequenced crustacean mitochondrial genomes, with the exception of a copepod Tigriopus japonicus (14,628 nt). It consists of the usual 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 relatively short non-coding region (294 nt). The thoracican cirripeds apart from Megabalanus volcano have the same arrangement of protein-coding genes as Limulus polypemus, but there are frequent tRNA gene translocations (at least 8). Some interesting translocation features that may be specific to the thoracican cirriped lineage are as follows: 1) trnK-trnQ lies between the control region and trnI, 2) trnA-trnE lies between trnN and trnS1, 3) trnP lies between ND4L and trnT, and 4) trnY-trnC lies between trnS2 and ND1. In P. mitella there are two trnL genes (L1 and L2) in the typical crustacean positions (ND1-L1-LrRNA and CO1-L2-CO2). The present result is compared and discussed with the other three cirriped mitochondrial genomes from one pedunculate (Pollicipes polymerus) and two sessiles (Tetraclita japonica and M. volcano) published so far. Mitochondrial protein phylogenies reconstructed by the BI and ML algorithms show that the thoracican Cirripedia is monophyletic (BPP 100/BP 100) and associated with Remipedia (BPP 98/BP 35). In addition, Oligostraca, including Ostracoda, Branchiura, and Pentastomida, is a monophyletic group (BPP 99/BP 68), and is basal to all the other examined arthropods. Remipedia + Cirripedia appears as an independent lineage within Arthropoda, apart from Thoracopoda (Malacostraca, Branchiopda, and Cephalocarida). The Thoracopoda is paraphyletic to Hexapoda. The present result suggests that the monophylies of Crustacea and Maxillopoda should be reconsidered.

• 대한수의학회지
• /
• 제51권2호
• /
• pp.107-115
• /
• 2011

Theileria (T.) buffeli (formerly T. sergenti/T. orientalis) is the major hemo-protozoan distributed in the Far East Asian countries such as Korea, China and Japan. It is responsible for the clinical symptoms of anorexia, ateliosis, anemia, fever and icterus. It also causes abortion and sudden death under severe cases, resulting in economic losses for many livestock farms. The objective of this study was to analyze the genetic diversity of the major surface protein (Msp) gene in T. buffeli in Holstein in Korea, and we characterized the association of the diversification of the Msp gene and its relationship with the pathogenicity of Theileria. For this, complete blood counts and Theileria PCR sequence analysis were performed from 57 Holstein in Jeju Island. A total of 26 PCR positive Holstein (16 anemic and 10 non-anemic) were then randomly selected based on 18s rRNA sequence typing of the Theileria Msp gene. The DNA sequence of the T. buffeli Msp gene in Holstein showed 99.0%, 99.2%, 99.9%, 99.5%, 98.7%, 98.4% and 98.4% homology with T. sergenti, Theileria spp., T. sergenti, Theileria spp., Theileria spp., Theileria spp. and Theileria spp., respectively. The result showed a genetic variation of 57.7% (type I), 3.8% (type II), 15.4% (type III), 7.7% (type IV), 13.5% (type V) and 1.9% (type VI). Type I is the most frequent type in both anemic and non-anemic Holstein while type II was found in only non-anemic Holstein. This results of our study help confirm the diversity of Msp gene types and demonstrate that the gene type distribution of Msp genes varies among Theileria-infected Holstein in Jeju Island.

• 미생물학회지
• /
• 제55권3호
• /
• pp.283-285
• /
• 2019

이 연구에서는 Illumina Hiseq platform을 사용하여 동해 심층 해양수로부터 분리된 Pelagicola sp. DSW4-44 (= KCTC 62762 = KCCM 43261)의 초안 유전체 염기서열 해독을 수행하였다. 그 결과, 유전체는 대략 4.85 Mbp의 길이 및 54.3%의 G + C 함량으로 구성되었고, 전체 4,566개의 단백질 암호 유전자, 3개의 rRNA 유전자, 48개의 tRNA 유전자, 3개의 non-coding RNA 유전자 및 67개의 위유전자(pseudo gene)가 확인되었다. 초안 유전체에서 균주 DSW4-44는 Pelagicola 속의 다른 균주에서 발견되지 않는 이화적 질산염의 암모늄 환원과 탈질화의 질소대사 유전자를 가지고 있었다.

• BMB Reports
• /
• 제50권4호
• /
• pp.158-159
• /
• 2017

MicroRNAs (miRNAs) regulate gene expression by guiding the Argonaute (Ago)-containing RNA-induced silencing complex (RISC) to specific target mRNA molecules. It is well established that miRNAs are stabilized by Ago proteins, but the molecular features that trigger miRNA destabilization from Ago proteins remain largely unknown. To explore the molecular mechanisms of how targets affect the stability of miRNAs in human Ago (hAgo) proteins, we employed an in vitro system that consisted of a minimal hAgo2-RISC in HEK293T cell lysates. Surprisingly, we found that miRNAs are drastically destabilized by binding to seedless, non-canonical targets. We showed that miRNAs are destabilized at their 3' ends during this process, which is largely attributed to the conformational flexibility of the L1-PAZ domain. Based on these results, we propose that non-canonical targets may play an important regulatory role in controlling the stability of miRNAs, instead of being regulated by miRNAs.

• Journal of Microbiology and Biotechnology
• /
• 제29권8호
• /
• pp.1266-1272
• /
• 2019

A bacterial strain, labeled $UR11^T$, was isolated from green alga Ulva pertusa collected from Jeju Island, Korea. $UR11^T$ was identified as a gram-negative, rod-shaped, motile by gliding and aerobic bacterial strain with yellow colonies on R2A plates. The strain $UR11^T$ grew over at a temperature range of $10^{\circ}C$ to $30^{\circ}C$ (optimally at $25^{\circ}C$), a pH range of 6.0-11 (optimally at pH 7.0) and a Nacl range of 0.5-5% Nacl (w/v). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain $UR11^T$ was a member of the genus Flavobacterium. Strain $UR11^T$ shared close similarity with F. jejuensis $EC11^T$ (98.0%) F. jumunjinense $HME7102^T$ (96.1%), F. haoranii $LQY-7^T$ (95.3%), F. dongtanense $LW30^T$ (95.1%), and F. ahnfeltiae 10Alg $130^T$(94.9%). The major fatty acids (>5%) were $iso-C_{15:0}$ (33.9%), $iso-C_{15:1}$ G (12.4%), $iso-C_{17:0}$ 3-OH (9.0%), $isoC_{16:0}$ (7.0%) and $iso-C_{15:0}$ 3-OH (6.3%). The major polar lipids were phosphatidylethanolamine, seven unknown aminolipids, two unknown aminopolarlipids and two unknown lipids. DNA-DNA hybridization value was 58% at strain $UR11^T$ with F. jejuensis $EC11^T$. Based on phenotypic, chemotaxonomic and phylogenetic evidence, strain $UR11^T$ represents a novel species of the genus Flavobacterium, for which the name Flavobacterium jocheonensis sp. nov. is proposed. The type strain is Flavobacterium jocheonensis is $UR11^T$ (=KCTC $52377^T$ =JCM $31512^T$).