Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants |
|Research Institution||Okayama University |
OHMORI Shinji Faculty of Pharmaceutical Sciences, Okayama University, 薬学部, 教授 (10032872)
SHIOZAWA Jud マックス, プランク生化学研究所, 研究員
LOTTSPEICH Friedrich Max-Planck-Institute of Biochemistry, プランク生化学研究所, 教授
OESTERHELT Dieter Max-Planck-Institute of Biochemistry, プランク生化学研究所・所長, 教授
MORITANI Chie Faculty of Pharmaceutical Sciences, Okayama University, 薬学部, 助手 (60253001)
OTSUKA Masato Faculty of Pharmaceutical Sciences, Okayama University, 薬学部, 助手 (30243489)
IKEDA Mikiko Faculty of Health and Welfare Science, Okayama Prefectural University, 保健福祉学部, 教授 (20112154)
SHIOZAWA Judith a. Max-Planck-Institute of Biochemistry
JUDITH A. Sh マックス, プランク生化学研究所・膜生化学部門, 研究員
FRIEDRICH Lo マックス, プランク生化学研究所・遺伝子センター, 教授
DIETER Oeste マックス, プランク生化学研究所・膜生化学部門, 教授
|Project Period (FY)
1992 – 1993
Completed (Fiscal Year 1993)
|Budget Amount *help
¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 1993: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1992: ¥2,700,000 (Direct Cost: ¥2,700,000)
|Keywords||Cl^--ATPase / cloning / F type ATPase / sulfate permease / cysA gene / sbp gene / ABC type ATPase / Acetabularia acetabulum / Cl^-ATPase / アニオン / 一次性能動輸送系 / 遺伝子クローニング|
1. Cl^--translocating ATPase
(1) Molecular cloning of the b (50 kDa) subunit : Overlapping 4 clones, pCLBl to 4 were cloned and sequenced by RT-PCR of total RNA and poly(A)^+ RNA isolated from Acetabularia acetabulum. Combined clones encoded the entire ORF of the Cl^--ATPase, b subunit which consisted of 478 amino acids and the molecular weight was calculated to be 51,513. The primary structure showed the highest similarity to Chlamydomonas CF_1-ATPase, beta subunit among cation-translocating ATPases, F, P and V(A) type.
(2) Molecular cloning of the a (54 kDa) subunit : A clone, pCLAl (803 bp) encoding the N-terminal half region of the a aubunit gene was obtained and sequenced. The deduced amino acid sequence was ca. 75% identical with that of spinach CF_1-ATPase, alpha subunit.
(3) Northern analysis : We have also obtained putative gene fragments coding for A. acetabulum CF_1- (273 bp) and MF_1-ATPases (332 bp), beta subunits. Northern analysis was performed for total RNA using the three
beta like gene fragments. Ca. over 10 kb, 7.5 and 3.8 kb of RNA were detected with the probes derived from the Cl^--b subunit. Ca.2.2 kb of RNA positively hybridized with the CF_1- and MF_1-beta specific probes, respectively.
A gene fragment (260 bp) encoding a putative CF_1-alpha subunit was also obtained. Northern analysis demonstrated the expression of different sizes of RNA (ca. 3.2 kb for the Cl^--a and 2.4 kb for the CF_1-alpha).
In conclusion, the Cl^--ATPase belongs to the F type ATPase family and the presence of a small multigene family for the F type ATPase was suggested in this organism.
2. Sulfate permease
(1) Sulfate uptake by sulfate-starved cells : About 3- to 4-fold increase in sulfate uptake was observed for sulfate-starved cells (3 to 4 cm in length).
(2) Molecular cloning : A putative cysA (359 bp) and sbp (552 bp) gene fragments were obtained from cDNA library and total RNA, respectively.
(3) Northern analysis : Ca. 2.4 kb for cysA and 1.55 kb for sbp were detected.
3. Maltose permease : A putative malk gene fragment (369 bp) was also cloned and sequenced. Ca. 1.65 kb of RNA was detected by Northern analysis.
The results of 2 and 3 supported the presence of ATP-binding cassette (ABC) type ATPase in A. acetabulum. This is the first report describing the ATPase specific for bacteria in plant materials. Less