Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.     

信号肽捕获系统的建立

细胞分泌蛋白的分泌有赖于蛋白质N端的信号肽的存在,利用酵母建立了从cDNA文库中筛选编码信号肽的基因片段的遗传系统,为此,用一步基因破坏法对酿酒酵母EGY48基因组中的suc2基因（编码酵母蔗糖转换酶）进行了定位突变,获得了无蔗糖转换酶表达的酵母株EGY48－suc。将无信号肽的suc 2成熟肽基因克隆于酵母乙 氢酶（ADHI）基因启动子下游,得到用于文库筛选的酵母真核表达工体,启动子与成熟肽基因之间为多克隆位 ,用于插入待筛选的CDNA文库,用此载体转化酵母EGY48－suc,所得克隆可以在葡萄糖为碳源的培养基上生长,但不能在以棉子糖为碳源的培养基上生长,在suc 2成熟肽基因前分别插入suc 2信号肽基因片段或人IL－2受体α链信号肽基因片段,然后转染EGY48－suc,所得克隆既能在以葡萄糖为碳源的培养基上生长,也能在以棉子糖为碳源的培养基上生长,表明构建的系统可用于筛选插篱多克隆位点cDNA片段是否具有编码信号肽的功能。     

Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant

Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine-5′-phosphosulfate (APS). Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)⁺ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3-1 and StMet3-2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers. To the best of the authors' knowledge this is the first cloning and identification of genes involved in the reductive sulfate assimilation pathway from higher plants.     

The expression of the open reading frame of Arabidopsis CAX1, but not its cDNA, confers metal tolerance in yeast

The biochemical properties and regulation of several plant CAX (CAtion eXchanger)-type vacuolar Ca2+/H+ exchangers have been extensively analysed in yeast expression assays. In the present study, we compare and contrast the phenotypes of yeast cells expressing the CAX1 cDNA and open reading frame (ORF). We report that the CAX1 ORF, but not the cDNA containing the 3′-untranslated region (UTR), was able to confer Ca2+ tolerance when expressed in a Ca2+-sensitive yeast mutant. Additionally, only yeasts expressing the N-terminal truncated CAX1 ORF were able to grow on high Mn2+ media, suggesting that removal of the 3′-UTR altered activity. However, removal of the 3′-UTR from another CAX did not alter the yeast phenotypes. Expression studies demonstrated that expressing the CAX1 ORF in yeast elevates CAX1 RNA and protein levels. Our results suggest that the 3′-UTR modulates expression of CAX1 in yeast.     

Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.     

植物液泡膜阳离子/H+反向转运蛋白结构和功能研究进展

阳离子转运蛋白在调节细胞质阳离子浓度过程中发挥关键作用。液泡是一个储存多种离子的重要细胞器,阳离子 (Ca2+)/H+反向转运蛋白CAXs定位在液泡膜上,主要参与Ca2+向液泡的转运,也参与其他阳离子的转运。近年来,植物中分离鉴定了多个CAX基因,植物CAXs主要有4个功能域：NRR通过自抑制机制调节Ca2+转运活性,CaD和C功能域分别赋予CAXs的Ca2+和Mn2+专一性转运活性,D功能域可调节细胞质pH。拟南芥AtCAXs参与植物的生长发育和胁迫适应过程,AtCAX3主要在盐胁迫下转运Ca2+,At     

The FOX hunting system: an alternative gain-of-function gene hunting technique

We have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T(1) mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T(2) progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.     

Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.

cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.     

Enhancing tonoplast Cd/H antiport activity increases Cd,Zn, and Mn tolerance,and impacts root/shoot Cd partitioning in <Emphasis Type="Italic">Nicotiana tabacum </Emphasis>L.

Sequestration mechanisms that prevent high concentrations of free metal ions from persisting in metabolically active compartments of cells are thought to be central in tolerance of plants to high levels of divalent cation metals. Expression of AtCAX2 or AtCAX4, which encode divalent cation/proton antiporters, in Nicotiana tabacum cv. KY14 results in enhanced Cd- and Zn-selective transport into root tonoplast vesicles, and enhanced Cd accumulation in roots of plants exposed to moderate, 0.02 μM Cd in solution culture (Korenkov et al. in Planta 225:403–411, 2007). Here we investigated effects of expressing AtCAX2 and AtCAX4 in the same lines on tolerance to growth with near-incipient toxicity levels of Cd, Zn and Mn. Less growth inhibition (higher tolerance) to all three metals was observed in 35S::AtCAX2 and FS3::AtCAX4 expressing plants. Consistent with the tolerance observed for Cd was the finding that while root tonoplast vesicle proton pump activities of control and FS3AtCAX4 expressing plants grown in 3 μM Cd were similarly reduced, and vesicle proton leak was enhanced, root tonoplast vesicle antiporter activity of these plants remained elevated above that in controls. We suggest that CAX antiporters, unlike tonoplast proton pump and membrane integrity, are not negatively impacted by high Cd, and that supplementation of tonoplast with AtCAX compensates somewhat for reduced tonoplast proton pump and proton leak, and thereby results in sufficient vacuolar Cd sequestration to provide higher tolerance. Results are consistent with the view that CAX2 and CAX4 antiporters of tonoplast play a role in tolerance to high, toxic levels of Cd, Zn, and Mn in tobacco.     

Changes of cationic transport in <Emphasis Type="Italic">AtCAX5</Emphasis> transformant yeast by electromagnetic field environments

The electromagnetic field (EMF) is newly considered as an exogenous environmental stimulus that is closely related to ion transportation on the cellular membrane, maintaining the internal ionic homeostasis. Cation transports of Ca²⁺ and other metal ions, Cd²⁺, Zn²⁺, and Mn²⁺were studied in terms of the external Ca²⁺ stress, [Ca²⁺]_ext, and exposure to the physical EMF. A specific yeast strain K667 was used for controlling CAX5 (cation/H⁺ exchanger) expression. Culture samples were exposed to 60 Hz, 0.1 mT sinusoidal or square magnetics waves, and intracellular cations of each sample were measured and analyzed. AtCAX5 transformant yeast grew normally under the metallic stress. However, the growth of the control group was significantly inhibited under the same cation concentration; 60 Hz and 0.1 mT magnetic field enhanced intracellular cation concentrations significantly as exposure time increased both in the AtCAX5 transformed yeast and in the control group. However, the AtCAX5-transformed yeast showed higher concentration of the intracellular cations than the control group under the same exposure EMF. AtCAX5-transformed yeasts displayed an increment in [Ca²⁺]_int, [K⁺]_int, [Na⁺]_int, and [Zn²⁺]_int concentration under the presence of both sinusoidal and square-waved EMF stresses compared to the control group, which shows that AtCAX5 expressed in the vacuole play an important role in maintaining the homeostasis of intracellular cations. These findings could be utilized in the cultivation of the crops which were resistant to excessive exogenous ions or in the production of biomass containing a large proportion of ions for nutritional food or in the bioremediation process in metal-polluted environments.     

Molecular cloning and expression in yeast of caprine prochymosin

We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats. This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level. The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors. The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively. Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH. The FLAG-prochymosin fusion was purified from S. cerevisiae culture supernatants by affinity chromatography. Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed kappa-casein.     

Production and Purification of Human Fibroblast Collagenase (MMP-1) Expressed in the Methylotrophic YeastPichia pastoris

The cDNA that encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was expressed in the methylotrophic yeastPichia pastoris.The proMMP-1 encoding DNA was fused to theSaccharomyces cerevisiaepre-pro α-mating factor secretion signal in theP. pastorispPIC9 expression plasmid, transformed into strain GS115 (His⁻), and His⁺Mut^s(slow methanol utilization) transformants were selected. Full-length proenzyme and processed forms of the protein could be detected in yeast culture supernatants following shake flask and 10-liter fermentations. The protein was purified to greater than 95% homogeneity. The recombinant proMMP-1 was comparable to the native fibroblast material based on (i) migration of the full-length molecule as a 52-kDa protein on reducing SDS–PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuric acetate to yield processed protein species, (iv) degradation of gelatin as monitored by zymogram gels, and (v) enzymatic activity. These data suggest that theP. pastorisexpression system offers a convenient and efficient means to produce and purify MMP-1.     

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.     

Identification of the N-terminal region of TjZNT2, a Zrt/Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity

The Zrt/Irt-like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N-terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn(2+) and Mn(2+) transporters, respectively. To identify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn(2+) uptake of yeast cells expressing them. As a result, the extracellular N-terminal ends were identified as regions involved in Zn(2+) selectivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N-terminal extension was shown to possess Zn(2+) uptake activity. This suggests that the extended N-terminal region inhibits Zn(2+) transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N-terminus that is important for the inhibition of Zn(2+) transport. Furthermore, the N-terminal truncated TjZNT2 lacked Mn(2+) uptake activity. These findings suggest that the N-terminal region is a novel substrate selector in the ZIP family of transporters.     

Differential signaling pathways following oxidative stress in mutant myotonin protein kinase cDNA-transfected C2C12 cell lines

We investigated the response to oxidative stress in a model system established in C2C12 cells stably transfected with myotonin protein kinase (MtPK) cDNAs having 5, 46, 60, or 160 CTG repeats. The transformants showed CTG repeat number-dependent susceptibility to oxidative stress. Mutant MtPK cDNA transformants containing 160 CTG repeats showed apoptotic cell death by the exposure to an oxidant, a very low level of methylmercury. The addition of the antioxidant Trolox protected transformants against apoptosis. Oxidative stress activated the extracellular signal-regulated kinases (ERKs) pathway leading to cell survival in wild-type MtPK cDNA transformants, whereas mutant MtPK cDNA transformants having 160 CTG repeats were defective in the induction of the ERK pathway, although the activation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) was strong and sustained. These results suggest that the susceptibility to oxidative stress in mutant MtPK cDNA transformants involves differential signaling pathways evoked following oxidative stress.     

Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.

Active loading of the phloem with sucrose in leaves is an essential part of the process of supplying non-photosynthetic tissues with carbon and energy. The transport is protein mediated and coupled to proton-symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression. Yeast strains that allow the phenotypic recognition of a sucrose carrier activity were constructed by expressing a cytoplasmic invertase from yeast, or the potato sucrose synthase gene, in a strain unable to transport or grow on sucrose due to a deletion in the SUC2 gene. A spinach cDNA expression library established from the poly(A)+ RNA from source leaves of spinach and cloned in a yeast expression vector yielded transformed yeast clones which were able to grow on media containing sucrose as the sole carbon source. This ability was strictly linked to the presence of the spinach cDNA clone pS21. Analysis of the sucrose uptake process in yeast strains transformed with this plasmid show a pH-dependent uptake of sucrose with a Km of 1.5 mM, which can be inhibited by maltose, alpha-phenylglucoside, carbonyl cyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid. These data are in accordance with measurements using both leaf discs and plasma membrane vesicles from leaves of higher plants. DNA sequence analysis of the pS21 clone reveals the presence of an open reading frame encoding a protein with a molecular mass of 55 kDa. The predicted protein contains several hydrophobic regions which could be assigned to 12 membrane-spanning regions.(ABSTRACT TRUNCATED AT 250 WORDS)