• Title/Summary/Keyword: conserved

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Site-directed Mutagenesis of the Evolutionarily Conserved Tyr8 Residue in Rice Phi-class Glutathione S-transferase F3

  • Jo, Hyun-Joo;Pack, Mi-Jin;Seo, Jin-Young;Lim, Jin-Kyung;Kong, Kwang-Hoon
    • Bulletin of the Korean Chemical Society
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    • v.34 no.9
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    • pp.2671-2674
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    • 2013
  • To elucidate the role of the evolutionarily conserved Tyr8 residue in rice Phi-class GSTF3, this amino acid was replaced with alanine and phenylalanine by site-directed mutagenesis, respectively. The replacement of Tyr8 with Ala significantly affected the catalytic activity and the kinetic parameters, whereas the substitutions of Tyr8 with Phe had almost no effect. The Y8A mutant resulted in approximately 90-100% decrease of the specific activity. Moreover, the Y8A mutant resulted approximately in 2-fold increase of $K_m$, approximately 60-80% decrease of $k_{cat}$, and approximately 6.5-fold decrease in $k_{cat}/K_m$. From the pH/log $k_{cat}/K_m$ plot, $pK_a$ values of the GSH in the wild-type enzyme-GSH complex, Y8A-GSH complex and Y8F-GSH complex were estimated to be approximately 6.8, 8.5 and 6.9, respectively. From these results, we suggest that the evolutionarily conserved Tyr8 residue in OsGSTF3 seems to influence the structural stability of the active site of OsGSTF3 rather than directly its catalytic activity.

New Approach to Predict microRNA Gene by using data Compression technique

  • Kim, Dae-Won;Yang, Joshua SungWoo;Kim, Pan-Jun;Chu, In-Sun;Jeong, Ha-Woong;Park, Hong-Seog
    • Proceedings of the Korean Society for Bioinformatics Conference
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    • 2005.09a
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    • pp.361-365
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    • 2005
  • Over the past few years, the complex and subtle roles of microRNA (miRNA) in gene regulation have been increasingly appreciated. Computational approaches have played one of important roles in identifying miRNAs from plant and animals, as well as in predicting their putative gene target. We present a new approach of comprehensive analysis of the evolutionarily conserved element scores and applied data compression technique to detect putative miRNA genes. We used the evolutionarily conserved elements [19] (see more detail on method and material) to calculate for base-by-base along the candidate pre-miRNA gene region by detecting common conserved pattern from target sequence. We applied the data compression technique [20] to detect unknown miRNA genes. This zipping method devises, without loss of generality with respect to the nature of the character strings, a method to measure the similarity between the strings under consideration [20]. Our experience to using our new computational method for detecting miRNA gene identification (or miRNA gene prediction) has been stratified and we were able to find 28 putative miRNA genes.

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Probing Starch Biosynthesis Enzyme Isoforms by Visualization of Conserved Secondary Structure Patterns

  • Vorapreeda, Tayvich;Kittichotirat, Weerayuth;Meechai, Asawin;Bhumiratana, Sakarindr;Cheevadhanarak, Supapon
    • Proceedings of the Korean Society for Bioinformatics Conference
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    • 2005.09a
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    • pp.215-220
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    • 2005
  • Generally, enzymes in the starch biosynthesis pathway exist in many isoforms, contributing to the difficulties in the dissection of their specific roles in controlling starch properties. In this study, we present an algorithm as an alternative method to classify isoforms of starch biosynthesis enzymes based on their conserved secondary structures. Analysis of the predicted secondary structure of plant soluble starch synthase I (SSI) and soluble starch synthase II (SSII) demonstrates that these two classes of isoform can be reclassified into three subsets, SS-A, SS-B and SS-C, according to the differences in the secondary structure of the protein at C-terminus. SS-A reveals unique structural features that are conserved only in cereal plants, while those of SS-B are found in all plants and SS-C is restricted to barley. These findings enable us to increase the accuracy in the estimation of evolutionary distance between isoforms of starch synthases. Moreover, it facilitates the elucidation of correlations between the functions of each enzyme isoforms and the properties of starches. Our secondary structure analysis tool can be applicable to study the functions of other plant enzyme isoforms of economical importance.

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성장중인 생쥐와 돼지난자의 성숙억제요인에 관하여

  • 이원교;권혁방
    • The Korean Journal of Zoology
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    • v.31 no.4
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    • pp.265-272
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    • 1988
  • 세포융합방법을 사용하여 성장증인 포유동물의 난자에 들어있는 성숙억제요인(maturation inhibiting activity, 1연Al에 대해 조사하였다. 성장중인 생쥐난자와 성장한 미성숙난자를 1:1로 융합하여 배양했을 서 (14-17시간)에는 거의 모두 핵붕괴를 일으키었으나(90oyo), 2:1로 융합했을 때는 대부분(약 64%) 3개의 핵을 모두 간직하고 있었다. 돼지난자의 경우는 성장중인 것깎 성장한 것을 1:1로 융합하여 배양했을 때에도 융합체들은 모두 핵을 간직하고 있었으며 돼지의 성장중인 난자와 생쥐의 성장한 난자를 융합했을 때에도 모두 핵을 보존하고 있었다. 이에 반하여 돼지와 생쥐 모두에서 성장한 난자끼리 융합했을 때에는 예외없이 핵붕괴가 일어났다. 이러한 결과는 성장중인 생쥐나 돼지의 난자에 각IA가 존재한다는 열과 이종간에도 효과가 있다는 것을 보여주고 있다. 또한 이는 MIA와 성숙촉진요인(maturation promoting factor, MPH의 상대적인 양의 변화가 난자의 성숙조절에 증요한 9f할을 한다는 것을 시사해주고 있다.In an attempt to elucidate the nature of maturation inhibiting activity (MIA) in growing mamma-lian oocvtes, growing mouse and pig oocytes incompetent to resume meiosis were fused with fully grown immature oocvtes in various combinations and cultured for 14-17 hours. In slant cells composed of two mouse growing ooh임es and one large immature oocyte (2:기, their GVs remained well conserved (about 64%) after culture, but not in the ceils composed of one by one pairs. In giant cells of pig composed of one growing and onto large immature oocytes, both GVs remained conserved. In the cells composed of one pig growing and one mouse large oocytes, both GVs were also conserved. In contrast to this, pairs of large mouse oocvtes or those of large pig oocvtes had no CVs after culture. Thus, we could acertain the existEnce of MIA and none-pecificty of it in the mouse and pig growing oocvtes. The results also suggest that the relative amount of substances showlns MfA or MPF activity may be important in the regulation of oocyte amount of substances showing MIA or MPF activity may be important in the regulation of oocyte maturation.

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Identification of the spk Gene Encoding Sphingosine Kinase in Sphingomonas chungbukensis DJ77 and Its Expression in Escherichia coli (Sphingomonas chungbukensis DJ77에서 Sphingosine Kinase를 암호화하는 spk 유전자의 동정과 대장균에서의 발현)

  • Lee Su-Ri;Um Hyun-Ju;Kim Young-Chang
    • Korean Journal of Microbiology
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    • v.41 no.2
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    • pp.93-98
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    • 2005
  • The sphingosine kinase gene, which is 969-nucleotide long, was identified during the whole genome sequencing of Sphingomonas chungbukensis DJ77. The amino acid sequence showed the identity of $55\%$ with that of Zymomonas mobilis subsp. mobilis ZM4. C2, C3, and C5 domains of eukaryotic sphingosine kinase were found in sphingosine kinase from Sphingomonas chungbukensis DI77. One of these three conserved sites, GGDG, was predicted as a ATP-binding site, and the functions of the others were unknown currently. The phylogenetic tree constructed by ClustalX indicated that the sphingosine kinase of S. chungbukensis DJ77 was near the phylogenetic group COG1597, and did not belong to the group of diacylglycerol kinase of the same strain. The recombinant sphingosine kinase was expressed in Escherichia coli, but it was made in form of inclusion body.

Marker Production by PCR Amplification with Primer Pairs from Conserved Sequences of WRKY Genes in Chili Pepper

  • Kim, Hyoun-Joung;Lee, Heung-Ryul;Han, Jung-Heon;Yeom, Seon-In;Harn, Chee-Hark;Kim, Byung-Dong
    • Molecules and Cells
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    • v.25 no.2
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    • pp.196-204
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    • 2008
  • Despite increasing awareness of the importance of WRKY genes in plant defense signaling, the locations of these genes in the Capsicum genome have not been established. To develop WRKY-based markers, primer sequences were deduced from the conserved sequences of the DNA binding motif within the WRKY domains of tomato and pepper genes. These primers were derived from upstream and downstream parts of the conserved sequences of the three WRKY groups. Six primer combinations of each WRKY group were tested for polymorphisms between the mapping parents, C. annuum 'CM334' and C. annuum 'Chilsung-cho'. DNA fragments amplified by primer pairs deduced from WRKY Group II genes revealed high levels of polymorphism. Using 32 primer pairs to amplify upstream and downstream parts of the WRKY domain of WRKY group II genes, 60 polymorphic bands were detected. Polymorphisms were not detected with primer pairs from downstream parts of WRKY group II genes. Half of these primers were subjected to $F_2$ genotyping to construct a linkage map. Thirty of 41 markers were located evenly spaced on 20 of the 28 linkage groups, without clustering. This linkage map also consisted of 199 AFLP and 26 SSR markers. This WRKY-based marker system is a rapid and simple method for generating sequence-specific markers for plant gene families.

Development of Detection Method for Cyclomaltodextrinase Family Genes using Degenerate PCR Primers

  • Oh, Su-Won;Jang, Myoung-Uoon;Jeong, Chang-Ku;Yuk, Jeong-Bin;Park, Jung-Mi;Park, Kwan-Hwa;Kim, Tae-Jip
    • Food Science and Biotechnology
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    • v.15 no.6
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    • pp.967-974
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    • 2006
  • Cyclomaltodextrinases (CDases), maitogenic amylases, and neopullulanases share highly conserved primary structures and similar characteristics, and are thus classified into the same family. BLAST search has showed that a variety of bacterial strains harbor putative CDase family genes with several well-conserved motif amino acid sequences. In this study, four degenerate polymerase chain reaction (PCR) primer sets were designed for the detection of CDase genes, on the basis of their highly conserved amino acid blocks (WYQIFP, DGWRLD, LGSHDT, and KCMVW). The PCR detection conditions were optimized and the detection specificity of each for the primer sets was tested against the genomic DNAs isolated from 23 different Bacillus-associated species. Consequently, all tested primer sets evidenced successful amplification of specific PCR products in length, which share 55-98% amino acid sequence identity with known and putative CDases. The primers developed herein, therefore, can be applied for the easy and efficient detection and isolation of CDase family genes for the modification of functional food carbohydrates.