Cseh, E.

Demeter, A.

Dombradi, V.

Farkas, I.

Gergely, P.

Koncz, C.

Kovacs, I.

Okresz, L.

Szabados, L.

English

MPG.PuRe

Identification of Arabidopsis mutants carrying T-DNA inserts in phosphoprotein phosphatase genes

the protein structure validated by in vivo assays could be a goodalternative for the time consuming crystallographic method.A2-017PHigh expression of recombinant humanPlatelet Factor 4 in Escherichia coli and itsbioactivitiesN. L. Cheng, J. Xie, J. F. Zhang, Y. H. Zhang, B. F. Yu,X. J. Chen, X. M. Xu and B. NiuDepartment of Biochemistry and Molecular Biology, ShanxiMedical University, Taiyuan, Shanxi, PR China.E-mail: xiejunty@yahoo.comIn order to improve the expression of human Platelet Factor 4(hPF4) in Escherichia coli, we have constructed a prokaryoticexpression vector pBV220-hPF4 by DNA polymerase chainreaction (PCR) and DNA recombinant technology, 3¢-UTR ofPF4 cDNA was deleted and TAG was mutated to TAATAA.The yield of recombinant hPF4 is 160 mg/L in shaking flaskculture. The expression level has been improved by 80-fold com-pared with that of PT7-7 hPF4 expression system. After wewashed, dissolved and renatured the inclusion bodies, inhibitionexperiment of blood vessel proliferation in chicken chorioallan-toic membrane was carried out to determine the bioactivities ofhPF4. The experimental result demonstrated that hPF4 pre-pared by our methods had the inhibitory activity against angio-genesis.A2-018PIdentification of Arabidopsis mutants carryingT-DNA inserts in phosphoprotein phosphatasegenesE´. Cseh1, A. Demeter1, L. O¨kre´sz2, I. Kova´cs2, C. Koncz3,L. Szabados2, P. Gergely1, V. Dombra´di1 and I. Farkas11Department of Medical Chemistry, University of Debrecen,Debrecen, Hungary, 2Institute of Plant Biology, BiologicalResearch Center of the Hungarian Academy of Sciences, Szeged,Hungary, 3Max Planck-Institut fu¨r Zu¨chtungsforschung, MaxPlanck-Institut fu¨r Zu¨chtungsforschung, Ko¨ln, Germany.E-mail: mumina@freemail.huAlthough members of the phosphoprotein phosphatase (PPP)enzyme family are known to be involved in numerous essential cel-lular processes, their functions are poorly characterized in plants.We have exploited an Arabidopsis T-DNA insertion mutant collec-tion to initiate functional analysis of PPP enzymes by reverse-gen-etics. Using a PCR screening technique described by Rı´os et al.(Plant J. (2004) 32, 243-253), we have identified T-DNA insertionsin genes encoding PP7, PP1 (TOPP-1), PP6At1, and a plant speci-fic phosphatase (the product of At2g27210). Analysis of homozy-gous insertion mutants revealed that none of the PPP genesstudied are essential for viability, as all PPP insertion mutant linesdisplay wild type phenotype under normal growth conditions.Study of various stress responses to osmotic and hormonal stres-ses, cadmium(II) salt, sugars, and photooxidative stress inducedby paraquat showed no significant difference between wild typeand most PPP mutants. However, the PP6 mutant displayedincreased sensitivity to abscisic acid inhibition of germination. Inresponse to blue light irradiation, the PP7 mutant exhibited a lossof hypocotyl growth inhibition. PP6 and At2g27210 plant specificphosphatase mutant plants showed a similar deficiency in bluelight-mediated inhibition of hypocotyl elongation. These resultssuggest that PP6, PP7 and At2g27210 may play important roles inthe regulation of growth responses by blue light.Acknowledgment: This work was supported by the grantOTKA T038324 from the Hungarian Scientific Research Fund.A2-019PMutational and crystallographic studies ofRNase HIII from Bacillus stearothermophilus:importance of N-terminal domain on substratebindingH. Chon1, K. Takano1,3, H. Matsumura2, Y. Koga1 andS. Kanaya11Department of Material and Life Science, Osaka University,Suita, Osaka, Japan, 2Department of Materials Chemistry, OsakaUniversity, Suita, Osaka, Japan, 3JST, PRESTO, Suita, Osaka,Japan. E-mail: hyongi@mls.eng.osaka-u.ac.jpMany organisms possess multiple RNase H genes in theirgenomes. For example, the Bacillus subtilis genome possessesthree RNase H-related genes. We have previously reported thattwo of them encode functional RNases H, RNases HII and HIII(Bsu RNases HII and HIII), while the other one, ypdQ, encodesan RNase HI homologue with no RNase H activity. Amino acidsequence of Bsu RNase HIII is similar to those of Bsu RNase HIIand Escherichia coli RNase HII, but is different from that ofE. coli RNase HI. However, the enzymatic properties of BsuRNase HIII are similar to those of E. coli RNase HI rather thanthose of Bsu RNase HII and E. coli RNase HII. Recently, wecloned the gene encoding RNase HIII from Bacillus stearothermo-philus (Bst RNase HIII) and characterized the enzymatic proper-ties of the recombinant protein. Bst RNase HIII highly resemblesto Bsu RNase HIII in enzymatic properties. Purpose of this studyis to understand the molecular mechanism of the enzymaticfunction of RNases HIII. Limited proteolysis of Bst RNase HIIIproduced two peptide fragments cleaved at Leu83-Ala84, suggest-ing that Bst RNase HIII consists of the N- and C-terminaldomains. The N-terminal domain alone and the C-terminaldomain alone of Bst RNase HIII were overproduced in E. coli,purified and characterized. Determination of the kinetic parame-ters for RNase H activity and the binding analyses of the proteinsto RNA/DNA hybrid using BIAcore indicated that the N-ter-minal domain of Bst RNase HIII is involved in substrate binding.To understand the structural basis for these results, Bst RNaseHIII was crystallized and the X-ray diffraction data sets of thenative crystal and the heavy-atom derivatives were collected.Structure determination by MIR method is in progress.A2-020PThe puzzling nature of cell wall anabolism:enzyme recruitment within and acrosspathwaysJ. J. Diaz-Mejia, E. Perez-Rueda and L. SegoviaLab 8, Cellular Enginery and Biocatalysis., National AutonomousUniversity of Mexico, Cuernavaca, Morelos, Mexico.E-mail: jdime@ibt.unam.mxEnzyme recruitment is one of the most important mechanisms tooriginate new reactions and metabolic pathways from the pre-existing ones. The two original models, the ‘‘patchwork’’ and‘‘stepwise’’, agree on the necessity of gene duplication as a prere-quisite for the recruitment, but have two main differences: (i) thedistance (number of reactions) among the recruited enzymes and,(ii) the chemical similarity of the reactions. Some major constitu-ents that distingue the bacterial cell wall from the eukaryotic andthe archaeal counterparts are synthesized by the peptidoglycan(PG), folate (FL) and formyl-tetrahydrofolate (FTHF) anabol-ism, thus the study of these routes is important to understandthe emergence of the three domains of life. In order to determinethe amount and nature of the enzyme recruitment that have per-formed the assemble of these and other pathways, we have com-pared the chemical properties of their reactions, from a networkAbstracts83

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Identification of Arabidopsis mutants carrying T-DNA inserts in phosphoprotein phosphatase genes

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