Studies on active anion transport systems in a biological membrane

1. (1) b （50 kDa）亚基的分子克隆：克隆了4个重叠克隆，pCLBl ~ 4，并采用RT-PCR方法对从髋臼分离的总RNA和poly(A)^+ RNA进行了测序。组合克隆编码了Cl^- ATPase的整个ORF， b亚基由478个氨基酸组成，计算分子量为51,513。初级结构与衣藻CF_1-ATPase（阳离子易位atpase中的β亚基）、F型、P型和V(A)型的相似性最高。(2) a （54 kDa）亚基的分子克隆：获得a基因n端半区编码的克隆pCLAl （803 bp）并测序。推导出的氨基酸序列与菠菜cf_1 - atp酶α亚基的序列一致性约75%。(3) Northern分析：我们还获得了A. acetabulum CF_1- (273 bp)和MF_1-ATPases (332 bp) β亚基的推定基因片段。Northern对总RNA进行了分析，使用了三个更像β的基因片段。由Cl^- b亚基衍生的探针可检测到超过10kb、7.5和3.8 kb的RNA。ca2.2 kb的RNA分别与CF_1-和mf_1 - β特异性探针正杂交。还获得了一个编码cf_1 - α亚基的基因片段（260 bp）。Northern分析显示不同大小的RNA表达（Cl^- a约3.2 kb， CF_1-alpha约2.4 kb）。综上所述，Cl^- ATPase属于F型ATPase家族，并且在该生物中存在一个小的多基因ATPase家族。硫酸盐渗透酶(1)硫酸盐缺乏细胞对硫酸盐的摄取：在硫酸盐缺乏细胞（3 - 4厘米长）中，硫酸盐摄取增加了约3- 4倍。(2)分子克隆：从cDNA文库和总RNA中分别获得一个推测的cysA （359 bp）和sbp （552 bp）基因片段。(3)北部分析：cysA基因约为2.4 kb， sbp基因约为1.55 kb。麦芽糖渗透酶：一个推测的麦芽糖基因片段（369 bp）也被克隆和测序。Northern分析法检测到约1.65 kb的RNA。结果2和3支持atp结合盒（ABC）型atp酶在A.髋臼的存在。这是对植物材料中细菌特异性atp酶的首次报道。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