• International Journal of Industrial Entomology and Biomaterials
• /
• 제20권2호
• /
• pp.107-114
• /
• 2010

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5'-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• The Plant Pathology Journal
• /
• 제32권2호
• /
• pp.102-111
• /
• 2016

Olive culture is very important in the Mediterranean Basin. A severe outbreak of Olive Quick Decline Syndrome (OQDS) caused by Xylella fastidiosa infection was first noticed in 2013 on olive trees in the southern part of Apulia region (Lecce province, southern Italy). Studies were carried out for detection and diversity evaluation of the Apulian strain of Xylella fastidiosa. The presence of the pathogen in olive samples was detected by PCR amplifying the 16S rDNA, gyrase B subunit (gyrB) and HL hypothetical protein genes and single nucleotide polymorphisms (SNPs) assessment was performed to genotype X. fastidiosa. Twelve SNPs were recorded over gyrB and six SNPs were found for HL gene. Less variations were detected on 16S rDNA gene. Only gyrB and HL provided sufficient information for dividing the Apulian X. fastidiosa olive strains into subspecies. Using HL nucleotide sequences was possible to separate X. fastidiosa into subspecies pauca and fastidiosa. Whereas, nucleotide variation present on gyrB gene allowed separation of X. fastidiosa subsp. pauca from the other subspecies multiplex and fastidiosa. The X. fastidiosa strain from Apulia region was included into the subspecies pauca based on three genes phylogenetic analyses.

• Journal of Microbiology and Biotechnology
• /
• 제33권2호
• /
• pp.211-218
• /
• 2023

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Em^r), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

• 한국환경농학회지
• /
• 제22권3호
• /
• pp.203-209
• /
• 2003

항진균 활성균주를 선발하는 과정에서 DM3 균주를 대천 바닷가에서 수집된 진흙 시료로부터 분리하였으며 API 50CHB kit를 이용하여 동정한 결과 Bacillus licheniformis로 동정되었다. 이 균주는 12종의 식물병원성 진균에 대해 항균활성을 나타내었다. 감마선($^{60}Co$)을 조사하여 항진균 활성 결핍 돌연변이체(mDMB)를 유도한 후, 이차원전기영동으로 단백질을 분석한 결과 DM3와 mDM3에만 존재하는 각각 4종과 3종의 단 단백질을 확인할 수 있었다. 2-DE 결과 B. licheniformis DM3에서 spot 1은 serine hydroxymethyltransferase(45.0kDa), spot 2는 hypothetical protein(40.7 kDa), spot 3는 NifU protein homolog(15.4 kDa), 그리고 spot 4는 resolvase(12.5 kDa)와 상동성을 지닌 단백질로 동정되었고 B. licheniformis mDM3에서만 발현된 spot 5는 lysozyme(18.1 kDa)과 spot 6, 7은 alkyl hydroperoxide reductase(15.6 kDa)으로 동정되었다. B. licheniformis DM3에서 이들 단백질들의 항진균 활성 관련 여부를 규명하기 위해서 더 연구가 진행되어야 할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
• /
• 제17권2호
• /
• pp.207-217
• /
• 2007

The Saccharomyces0 cerevisiae KNU5377 strain, which was isolated from spoilage in nature, has the ability to convert biomass to alcohol at high temperatures and it can resist against various stresses [18, 19]. In order to understand the defense mechanisms of the KNU5377 strain under menadione (MD) as oxidative stress, we used several techniques for study: peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) followed by two-dimensional (2D) gel electrophoresis, liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) technology. Among the 35 proteins identified by MALDI-TOF MS, 19 proteins including Sod1p, Sod2p, Tsa1p, and Ahp1p were induced under stress condition, while 16 proteins were augmented under normal condition. In particular, five proteins, Sod1p, Sod2p, Ahp1p, Rib3p, Yaf9p, and Mnt1p, were induced in only stressed cells. By LC-ESI-MS/MS analysis, 37 proteins were identified in normal cells and 49 proteins were confirmed in the stressed cells. Among the identified proteins, 32 proteins were found in both cells. Five proteins including Yel047cp and Met6p were only upregulated in the normal cells, whereas 17 proteins including Abp1P and Sam1p were elevated in the stressed cells. It was interesting that highly hypothetical proteins such as Ynl281wp, Ygr279cp, Ypl273wp, Ykl133cp, and Ykr074wp were only expressed in the stressed cells. SELDI-TOF analysis using the SAX2 and WCX2 chips showed that highly multiple-specific protein patterns were reproducibly detected in ranges from 2.9 to 27.0 kDa both under normal and stress conditions. Therefore, induction of antioxidant proteins, hypothetical proteins, and low molecular weight proteins were revealed by different proteomic techniques. These results suggest that comparative analyses using proteomics might contribute to elucidate the defense mechanisms of KNU5377 under MD stress.

• Journal of Microbiology and Biotechnology
• /
• 제32권9호
• /
• pp.1195-1208
• /
• 2022

Silver nanoparticles (AgNPs) have potential applications in medicine, photocatalysis, agriculture, and cosmetic fields due to their unique physicochemical properties and strong antimicrobial activity. Here, AgNPs were synthesized using actinobacterial SL19 strain, isolated from acidic forest soil in Poland, and confirmed by UV-vis and FTIR spectroscopy, TEM, and zeta potential analysis. The AgNPs were polydispersed, stable, spherical, and small, with an average size of 23 nm. The FTIR study revealed the presence of bonds characteristic of proteins that cover nanoparticles. These proteins were then studied by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and identified with the highest similarity to hypothetical protein and porin with molecular masses equal to 41 and 38 kDa, respectively. Our AgNPs exhibited remarkable antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. The combined, synergistic action of these synthesized AgNPs with commercial antibiotics (ampicillin, kanamycin, streptomycin, and tetracycline) enabled dose reductions in both components and increased their antimicrobial efficacy, especially in the case of streptomycin and tetracycline. Furthermore, the in vitro activity of the AgNPs on human cancer cell lines (MCF-7, A375, A549, and HepG2) showed cancer-specific sensitivity, while the genotoxic activity was evaluated by Ames assay, which revealed a lack of mutagenicity on the part of nanoparticles in Salmonella Typhimurium TA98 strain. We also studied the impact of the AgNPs on the catalytic and photocatalytic degradation of methyl orange (MO). The decomposition of MO was observed by a decrease in intensity of absorbance within time. The results of our study proved the easy, fast, and efficient synthesis of AgNPs using acidophilic actinomycete SL19 strain and demonstrated the remarkable potential of these AgNPs as anticancer and antibacterial agents. However, the properties and activity of such particles can vary by biosynthesized batch.

• Reproductive and Developmental Biology
• /
• 제32권4호
• /
• pp.223-227
• /
• 2008

Human embryonic stem (ES) cells retain the capacity for self-renewal, are pluripotent and differentiate into the three embryonic germ layer cells. The regulatory transcription factors Oct4, Nanog and Sox2 play an important role in maintaining the pluripotency of human ES cells. The aim of this research was to identify unknown genes upregulated in human ES cells along with Oct4, Nanog, and Sox2. This study characterizes an unknown gene, named chromosome 1 open reading frame 31 (C1orf31) mapping to chromosome 1q42.2. The product of C1orf31 is the hypothetical protein LOC388753 having a cytochrome c oxidase subunit VIb (COX6b) motif. In order to compare expression levels of C1orf31 in human ES cells, human embryoid body cells, vascular angiogenic progenitor cells (VAPCs), cord-blood endothelial progenitor cells (CB-EPCs) and somatic cell lines, we performed RT-PCR analysis. Interestingly, C1orf31 was highly expressed in human ES cells, cancer cell lines and SV40-immortalized cells. It has a similar expression pattern to the Oct4 gene in human ES cells and cancer cells. Also, the expression level of C1orf31 was shown to be upregulated in the S phase and early G2 phase of synchronized HeLa cells, leading us to purpose that it may be involved in the S/G2 transition process. For these reasons, we assume that C1orf31 may play a role in on differentiation of human ES cells and carcinogenesis.