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무포자 효모인 Candida속의 효모는 aikane계 화합물을 이용하며 cellubiose나 xylose를 동화하는 등 산업적으로 이용가능성이 높다. Candida tropicalis는 세포질 인자인 plasmid가 발견되지 않으므로 염색체 유래의 자율복제기점을 non-replicative plasmid인 YIp5에 삽입시켜 S. cevisiae YNN 27에서 분리하였다. 분리된 자율 복제 기점을 Hind III과 EcoR I제한 효소로 절단하고 각각 2.7kb와 2.3kb fragment를 회수한 후 동일 제한 효소로 절단한 YIp5와 YIp32에 ligation시켜 pIKS523, pIKS527및 pIKC27을 구축하였다. 특히, pIKC27은 2.7kb의 자율복제 기점을 가진 plasmid로서 S. cerevisiae DBY746과 C.maltosa J288에서 높은 형질 전환체를 나타내었다. 기존의 plasmid와 안정성을 비교하였으며, 여러 제한효소에 의해서 얻어진 결과로서 상세한 restrition map을 작성하였다. Candida species are special interest because of its ability to metabolize hydrocarbon, covet this material into single cell protein and assimilate cellobiose and xylose as substrate. Cadida tropicalis has no native plasmid similar to 2㎛ plasmid. Therefore, Candida tropicalis is regarded as a new yeast host. An 8-kb fragment was isolated from Sal I digest of Candida tropicalis IFO 0518 genomic DNA which conferred the property of autonomous replication in Saccharomyces cerevisiae YNN 27. The vectors for gene manipulation of Yeast, pIKS 523, pIKS 527 and pIKC 27, were constructed by combinding the ARS fragment and integration plasmid YIP 5 and YIP 32, respectively. One of the recombinant plasmids, pIKC 27 (9.4kb), was capable of autonomous replication in both S. cerevisiae DBY 746 to Leu+ at a frequency of 600 transformants per ㎍ DNA, and transformed Candida maltosa J288 to Leu+ at a frequency of 230 transformants per ㎍ DNA. These results indicate that the 2.7 kb ARS element was necessary for high frequency transformation and autonomous plasmid replication in both S. Cerevisiae and C. maltosa.
In the present, batch test was conducted to evaluate the neutralization and adsorption of heavy metals from wastewater using waste egg shells. Neutralization and removal rate of heavy metals were excellent in the increase of waste concrete amounts and a small size. It seemed that adsorption efficiencies of heavy metals were influenced by solubility. As a result on the experiments of Freundlich isotherm, the adsorption capacities(k) were Cr 3.11, Cu 2.61, Mn 3.02 and Pb 0.95, respectively and the adsorption intensities(1/n) were Cr 0.35, Cu 0.44, Mn 0.4 and Pb 0.41, respectively. In view of these results, it showed that wastes containing the similar compositions as waste egg shells could utilize the neutralization and adsorption of heavy metals in wastewater.
Choi, Won-Young,Hwang, Ji-Hyun,Cho, Ann-Na,Lee, Andrew J.,Jung, Inkyung,Cho, Seung-Woo,Kim, Lark Kyun,Kim, Young-Joon Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.12
Cell type specification is a delicate biological event in which every step is under tight regulation. From a molecular point of view, cell fate commitment begins with chromatin alteration, which kickstarts lineage-determining factors to initiate a series of genes required for cell specification. Several important neuronal differentiation factors have been identified from ectopic over-expression studies. However, there is scarce information on which DNA regions are modified during induced pluripotent stem cell (iPSC) to neuronal progenitor cell (NPC) differentiation, the cis regulatory factors that attach to these accessible regions, or the genes that are initially expressed. In this study, we identified the DNA accessible regions of iPSCs and NPCs via the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). We identified which chromatin regions were modified after neuronal differentiation and found that the enhancer regions had more active histone modification changes than the promoters. Through motif enrichment analysis, we found that NEUROD1 controls iPSC differentiation to NPC by binding to the accessible regions of enhancers in cooperation with other factors such as the Hox proteins. Finally, by using Hi-C data, we categorized the genes that directly interacted with the enhancers under the control of NEUROD1 during iPSC to NPC differentiation.
One of the cryptic plasmids from the oil degrading bacterium Klebsiella sp. KCL-2, the small plasmid pGD2, has been identified and characterized. This plasmid has a size of 3.6 kb with unknown functions. We constructed the recombinant plasmid pMGD2. The nucleotide sequences of the plasmid were determined and two open reading frames were detected. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology with the protein of ColE2 plasmid. The product encoded by ORF2 showed a high similarity with the transposase protein of IS5. IS5 is 1195 by long and contains an inverted terminal repetition of 16 bp with one mismatch. Stem-loop structures in the 5'untranslated region of the repA suggest that a putative gene, incA, is located in a complementary strand to the leader region of the repA mRNA.
Eighty-seven strains of indigenous rhizobia were isolated from the nodule of soybean cultivar, Danyup, cultivated in four different soils sampled from continuously soybean cultivated and newly reclaimed fields. The strains were groups into Bradyrhizobium (slow-grower; 55 strains) and Rhizobium japonicum(fast-grower; 32 strains). The both groups could be divided into two sub-groups according to the denitrification characteristics, that is, denitrifying fast-grower(F-I), nitrate respiring fast-grower (F-II), denitrifying slow-grower (S-I), and nitrate respiring slow-grower (S-II). Among the 87 isolates, F-I, F-II, S-I and S-II sub-groups were 10, 22, 48 and 7 strains, respectively. The one-and two-dimensional polyacrylamide gel electrophoretic pattern of the four sub-groups were compared and discernible difference was observed between fast and solw-grower, but the difference was not discernible between sub-group within the same growth rate group.
We have previously cloned a novel-type bacterial lipase gene which is classified into lipase family I.1 based on the amino acid sequence similarities but into lipase family I.2 based on molecular weight (33 kDa) and active-sitemotif (GHSQG). When this gene was expressed under T7 promoter using aexpression vector, pET29a(+) in Escherichia coli (BL21 DE3), recombinant lipase was overproduced in an insoluble form. To obtain an active enzyme, after this protein was solubillized in the presence of 8 M urea, it was purified in aurea-denatured form by ion-exchange chromatography on DEAE-Sepharose and refolded by removing urea in the presence of the Ca^(2+) ion. Gel filtration chromatography indicated that this refolded protein is monomeric. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed an molecular weight of 33 kDa for the purified protein. rPSL showed relatively broad substrate specificities. The optimum pH and temperature waspH 8.0 and 50℃, respectively. Th enzyme was stable in the range of pH 8-9and at below 50℃ in the presence of 5 mM CaCl₂. Among trhe p-nitrophenyl(PNP) esters tested the best substrate was PNP-myristate with Km and Vmax values of 44μM and 24 nM/min^(-1).
Lee Yeongun,Dho So Hee,Lee Jiyeon,Hwang Ji-Hyun,Kim Minjung,Choi Won-Young,Lee Jin-Young,Lee Jongwon,Chang Woochul,Lee Min Young,Choi Jungmin,Kim Tae-You,Kim Lark Kyun 생화학분자생물학회 2022 Experimental and molecular medicine Vol.54 No.-
Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.
The five components of leghemoglobin from alfalfa root nodules were purified by DEAE-cellulose(DE52) and Sephadex G-100 chromatography and antiserum against the purified alfalfa leghemoglobin was prepared. RNA was extracted from alfalfa root nodules and poly(A)-mRNA was purified by oligo(dT)-cellulose affinity chromatography. The leghemolgobin messages were observed to be 23 % of the poly(A)-mRNA. The poly(A)-mRNA was fractionated by sucrose density gradient centrifugation and the mRNA had 9, 18 and 28S value populations with 58, 21 and 36 % of the leghemoglobin messages.