Studies on active anion transport systems in a biological membrane

1.（1）B（50 kDa）亚基的分子克隆：从髋臼菌中分离总RNA和poly（A）^+ RNA，通过RT-PCR方法克隆并测序了4个重叠克隆pCLB 1 - 4。合并的克隆编码Cl^--ATP酶，B亚基的完整ORF，其由478个氨基酸组成，计算的分子量为51，513。一级结构与衣原体CF_1-ATP酶β亚基的相似性最高。(2)a（54 kDa）亚基的分子克隆：获得编码a亚基基因的N-末端半区的克隆pCLA 1（803 bp）并测序。推导的氨基酸序列为ca。与菠菜CF_1-ATP酶α亚基的一致性为75%。(3)北方分析：我们还获得了编码A.髋臼CF_1-（273 bp）和MF_1-ATP酶（332 bp）为β亚基。使用三种方法对总RNA进行北方分析。 ...更多信息 β类基因片段约用来自Cl^--B亚基的探针检测到超过10 kb、7.5和3.8 kb的RNA。结果表明，CF_1-β和MF_1-β特异性探针分别与约2.2 kb的RNA杂交，获得了一个编码CF_1-α亚基的基因片段（260 bp）。北方分析表明，不同大小的RNA（约。3.2 Cl^--α和CF_1-α分别为2.4kb和2.4kb），因此，Cl^--ATP酶属于F型ATP酶家族，并且在该生物中存在一个小的多基因家族.硫酸盐通透酶（1）硫酸盐饥饿细胞的硫酸盐摄取：对于硫酸盐饥饿细胞（3至4cm长），观察到硫酸盐摄取增加约3至4倍。(2)分子克隆：从cDNA文库和总RNA中分别获得cysA和sbp基因片段，大小分别为359 bp和552 bp。(3)北方分析：Ca. 2.4 cysA和sbp分别为1.55kb和1.55kb.麦芽糖通透酶：一个假定的malk基因片段（369 bp）也被克隆和测序。约1.65通过北方分析，发现2和3的RNA分子量约为100 kb，支持A.髋臼这是首次报道植物材料中细菌特异性ATP酶。少

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 … More 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