Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase)

Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme complexes located in the membranes of eukaryotic cells regulating cytoplasmic pH. So far, nothing is known about the genomic organization and chromosomal location of the various subunit genes in higher eukaryotes. Here we describe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porcine V-ATPase subunit alpha and beta isoforms. We have determined the genomic structure of the V-ATPase subunit gene spanning at least 62 kb on Chromosome (Chr) 4q14-q16. It consists of 14 exons with sizes ranging from 54 bp to 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms. The 5′ end of the V-ATPase cDNA was isolated by RACE-PCR. The V-ATPase alpha isoform mRNA, lacking the seventh exon, has an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pI of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa. Amino acid and DNA sequence comparison revealed that the porcine V-ATPase subunit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans. Received: 14 May 1998 / Accepted: 20 October 1998     

Structural features of a gene encoding the vacuolar H+-ATPase c subunit from a marine red alga, Porphyra yezoensis.

We report the nucleotide sequence of a gene encoding the c ('16 kDa') subunit of the vacuolar-type H+-ATPase (V-ATPase) from a marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and analyzed for the sequence. The genomic DNA sequence was directly determined by nested PCR. The structural gene contained four introns within a coding sequence of 483 base pairs which encodes a polypeptide of 161-amino acids with four hydrophobic transmembrane-spanning regions. Comparison of the deduced amino acid sequences showed higher similarity to the land plant Oryza sativa (69.1%) than to the Ulvophyceae Acetabularia acetabulum (64.1%). The mRNA was detected both in the leafy gametophytes and filamentous sporophytes.     

利用mRNA差异显示技术分离甜菜M14品系特异表达基因的cDNA片段

二倍体栽培甜菜与白花甜菜杂交、进一步回交而获得的单体附加系M14,其染色体组成中除了含有18条栽培甜菜染色体外,还附加有一条野生白花甜菜第9号染色体,该附加染色体通过母本的传递率为96.5%;单体附加系传递率如此高的原因是因为M14中有无融合生殖基因的存在。本实验采用mRNA差异展示技术对甜菜无融合生殖品系M14和正常有性生殖的二倍体栽培甜菜A2Y花蕾减数分裂时期的基因表达进行了差异分析。采用GT15A,GT15G,GT15C3种锚定引物,共筛选了20个随机引物,通过RT-PCR检测,获得了6个阳性差异表达的cDNA片段,应用NCBI的BLASTx软件对测序结果进行同源序列、相似序列检索,为进一步克隆无融合生殖基因提供侯选cDNA片段。     

The 14-3-3 gene expression specificity in response to stress is promoter-dependent

Genomic clone coding for the 16R isoform of 14-3-3 proteins from potato plants has recently been described. This paper reports on 20R-gene isolation and analysis, and compares two isoforms. The northern blot analysis of mRNA of the 20R 14-3-3 isoform suggests its similarity to 16R. Vascular tissue-specific expression and age-dependent synthesis in potato leaves has been detected in both promoters. Screening of the potato genomic library using 20R cDNA isoform resulted in identification and isolation of the corresponding gene. This gene contains four exons and three introns. Inspecting the promoter sequence of the 20R isoform revealed several boxes important for the regulation of gene expression. The strongest GUS expression in transgenic potato plants transformed with the uidA reporter gene under the 20R promoter has been found in young leaf and stem vascular tissue, root tips, pollen and ovules. Mature fragments exhibit a significant decrease in GUS staining, which suggests age-dependent promoter activity. The analysis of transgenic plants transformed with 20R-GUS in contrast to 16R-GUS has revealed strong activation of the 20R promoter by metal ions and NaCl. Instead the 16R promoter is strongly affected by virus and salicylic acid treatments. The only factor, which strongly induced both promoters, was abscisic acid. It is thus suggested that promoter domain composition is the main factor differentiating the appearance of 14-3-3 isoforms.     

水稻液泡ATPase B 亚基基因的克隆及其在低磷胁迫下的表达特征分析

利用抑制性扣除杂交(SSH)技术构建水稻(Oryza sativa L.)根系磷饥饿诱导cDNA文库,获得编码液泡ATPase (V-ATPase) B亚基的克隆,通过反转录PCR方法获得该基因的完整序列.该基因编码487个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量为54.06 kD,等电点为4.99.Southern印迹表明,V-ATPase B亚基基因在水稻基因组中以单拷贝形式存在.氨基酸同源性分析发现,V-ATPase B亚基是一个较为保守的蛋白亚基,其序列变化伴随生物的进化过程同步进行.Northern印迹表明,V-ATPase B亚基在水稻根系中受到磷饥饿诱导表达,磷饥饿6～12 h出现表达高峰,而在叶片中表达高峰有所滞后(24～48 h).在缺磷环境条件下,ATPase B亚基可能通过提高其表达量,进而提高质子转运活性,形成跨膜的电化学梯度,为体内储备磷跨液泡膜运输提供能量,从而提高植物体内磷的利用效率及其耐低磷的能力.     

Sequence analysis and characterization of vacuolar-type H+ -ATPase proteolipid transcript from Acanthus ebracteatus Vah1.

The vacuolar-type H+ -ATPase (V-ATPase) is a multimeric enzyme with diverse functions in plants such as nutrient transport, flowering, stress tolerance, guard cell movement and development. A partial sequence of V-ATPase proteolipid was identified among the expressed sequence tags (ESTs) generated from Acanthus ebracteatus, and selected for full-length sequencing. The 876-nucleotide cDNA consists of an open reading frame of 165 amino acids. The deduced amino acid sequence displays high similarity (81%) with its homologs from Arabidopsis thaliana, Avecinnia marina and Gossypium hirsutum with the four transmembrane domains characteristics of the 16 kDa proteolipid subunit c of V-ATPase well conserved in this protein. Southern analysis revealed the existence of several members of proteolipid subunit c of V-ATPase in A. ebracteatus. The mRNA of this gene was detected in leaf, floral, stem and root tissues, however, the expression level was lower in stem and root tissues.     

盐地碱蓬叶片液泡膜H+-ATPase B亚基的克隆及盐胁迫下表达分析

植物液泡膜H -ATPase在建立跨液泡膜质子梯度、促进液泡Na 区域化、提高植物耐盐性方面发挥着重要作用.本实验从盐生植物盐地碱蓬(Suaeda salsa L.)cDNA文库分离到碱蓬叶片液泡膜H -ATPase B亚基cDNA克隆.测序表明该基因长达1 974 bp,开放阅读框有1 470 bp编码489个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量约为54.29 kD.Northem及Western印迹表明盐地碱蓬液泡膜H -ATPase B亚基表达明显受NaCl胁迫诱导,并且在NaCl胁迫下,B亚基在转录及翻译水平上与液泡膜H -ATPase c亚基存在协同作用.盐胁迫下,盐地碱蓬液泡H -ATPase B亚基与c亚基的协同表达增加了液泡H -ATPase的数量,从而提高了液泡H -ATPase活性,为碱蓬叶片液泡Na 区域化提供了动力,最终提高了碱蓬植株的耐盐性.     

a4, a unique kidney-specific isoform of mouse vacuolar H+-ATPase subunit a

The vacuolar-type H+ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical alpha- and beta-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.     

过表达ThVHAc1对拟南芥V-ATPase各亚基表达的影响

V-ATPase是多亚基复合蛋白,其c亚基负责V-ATPase的组装及质子通道的形成。本研究拟分析盐胁迫下过表达ThVHAc1基因拟南芥V-ATPase各亚基的表达,探讨过表达外源c亚基对拟南芥V-ATPase全酶响应盐胁迫表达模式的影响。实时荧光定量PCR结果显示,盐胁迫下,过表达外源ThVHAc1拟南芥V-ATPase 28个亚基的表达发生了明显改变,且拟南芥5个c亚基的表达均不同程度的被抑制。表明外源ThVHAc1基因能影响拟南芥V-ATPase各亚基的表达以调节V-ATPase全酶的活性,但各亚基的表达模式与V-ATPase活性非简单对应关系,各亚基互相协调决定V-ATPase活性。