Sequencing and Expression of the Gene that Encodes a 20-kDa Polypeptide of the PSI Complex from Cucumber Cotyledon

A cDNA clone encoding the polypeptide from cucumber PS I thatmigrates with an apparent molecular weight of 20 kDa on SDS-polyacrylamidegels has been isolated. The 907-bp sequence of this clone hasbeen determined and contains one large open reading frame thatencodes a 22,720-Da precursor polypeptide (207 amino acid residues).The molecular weight of the mature polypeptide was predictedto be 17,037-Da (153 amino acid residues). The deduced aminoacid sequence of this protein indicates that it is routed towardsthe stromal side of the thylakoid membrane and has no membrane-spanningregions. The sequence also confirmed the identity of the proteinas the product of the psa D gene. Chemical cross-linking offerredoxin to the PS I complex identified the 20-kDa subunitas the ferredoxin-binding protein. Northern hybridization experimentsrevealed that the mRNA of approximately 1,100 nucleotides forthe 20-kDa polypeptide was present in etiolated cucumber cotyledons,and its level increased about 5-fold during greening. The 20-kDapolypeptide was not detected by immunoblotting in etiolatedcotyledons, and it accumulated only after illumination. Labelingexperiments in vivo showed the absence of incorporation of [³⁵S]Metinto the polypeptide in etiolated cotyledons. These resultssuggest that the expression of the psa D gene is controlledat the translational level. (Received April 5, 1990; Accepted June 28, 1990)     

Hydroxypyruvate Reductase with a Carboxy-Terminal Targeting Signal to Microbodies is Expressed in Arabidopsis

Five Arabidopsis EST cDNA clones of hydroxypyruvate reductase(HPR), a photorespiratory enzyme in leaf peroxisomes, were sequenced.Deduced amino acid sequences revealed that HPR in Arabidopsiscontained the carboxy-terminal targeting signal to microbodies.Nucle-otide sequence analysis showed that the cDNA with thelongest insert contained an open reading frame of 1,158 bp whichencoded a polypeptide with 386 amino acids with a calculatedmolecular mass of 42,251 Da. A Southern blot analysis suggestedthat the Arabidopsis HPR gene, like that of the pumpkin HPRgene, exists as a single copy. Two kinds of pumpkin HPR mRNAmight be produced from a single gene by alternative splicing,but the structure of the genomic DNA indicated that the ArabidopsisHPR gene did not undergo alternative splicing. We detected apolypeptide with a molecular mass of 42 kDa in green leavesof Arabidopsis using an HPR-specific antibody. Immunoelectronmicroscopy revealed that Arabidopsis HPR protein was exclusivelylocalized in leaf peroxisomes in green leaves. These resultsindicate that HPR is expressed in a form with a carboxy-terminaltargeting signal to microbodies and is localized in microbodiesin Arabidopsis, suggesting that the differences in the genestructure and the regulation of gene expression of HPR are probablydue to species-specific differences in plants. (Received November 11, 1996; Accepted February 1, 1997)     

Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria

A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotrans-ferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hyper-sensitive reaction, as compared to virulent pathogens, which lead to disease.     

The Effect of NADP+ on Electrophoretic Behavior of Glucose 6-Phosphate Dehydrogenase from Sweet Potato

The gene from Bacillus brevis TT02–8 encoding arginase was cloned into Escherichia coli, and its nucleotide sequence was identified. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 298 amino acid residues with a predicted molecular weight of 31,891, which was consistent with that previously calculated for arginase purified from this bacterium. Comparison of the deduced amino acid sequence of the B. brevis TT02–8 arginase with that of the prokaryotic and eukaryotic arginases of Bacillus caldovelox, Bacillus subtilis, Agrobacterium Ti plasmid C58, Saccharomyces cerevisiae, Coccidioides immitis, Xenopus laevis, Rana catesbeiana, rat liver, and human liver, showed 33–66% of the sequences to be similar; there were several highly conserved regions. Arginase activity was detected in Escherichia coli cells transformed with an expression plasmid of the cloned arginase gene.     

Structural Analysis of a recA-like Gene in the Genome of Arabidopsis thaliana

A recA-like gene was identified in the genome of Arabidopsisthaliana by means of PCR using primers designed on the basisof previously reported amino acid sequences of eukaryotic RecA-likeproteins. The structure of the gene, termed ArLIM15, was investigatedby comparing the primary structure of the genomic DNA with thatof the corresponding cDNA. The open reading frame, which wassplit into 15 exons, was established to have the capacity forencoding a 37.3-kDa polypeptide. The amino acid sequence ofthe putative product of ArLIM15 showed a high degree of similaritytothat of LIM15 in the monocotyledonous plant Lilium, includinga 93% identity, and to those of other recA-like genes in yeastsand vertebrates with identities of 69–71%. Phylogeneticanalysis indicated ArLIM15 to be much closer to meiosis-specificLIM15 and DMC1 in Saccharomyces cerevisiae than to RAD51 inS. cerevisiae and its homologues on an evolutionary scale.     

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

家蝇金属硫蛋白基因的克隆、原核表达及活性检测

金属硫蛋白是一类普遍存在于生物体内、富含半胱氨酸的小分子蛋白,能螯合多种金属离子。本研究根据EST序列信息,利用RACE技术克隆到1条家蝇Musca domestica金属硫蛋白基因MdMtn（GenBank登录号为GU289398）。序列分析表明,MdMtn cDNA全长408 bp,包含1个123 bp的开放阅读框,编码40个氨基酸残基,其中半胱氨酸残基10个,呈-C-X-C-方式排列。此蛋白理论分子量为3.8 kD,等电点为878。为了解家蝇金属硫蛋白对重金属的结合活性,构建了pET-DsbA-MT表达载体,并转化Escherichia coli BL21（DE3）宿主菌进行融合表达。研究发现MT重组菌对重金属镉的耐受性得到了明显加强,提示MdMtn基因可能在家蝇适应重金属环境中起到积极作用。     

A Serine/Threonine Protein Kinase Gene Isolated by an in vivo Binding Procedure Using the Arabidopsis Floral Homeotic Gene Product, AGAMOUS

During the course of characterizing fragments bound to an Arabidopsisfloral homeotic protein AGAMOUS in vivo, a gene encoding a putativeserine/threonine protein kinase was found on one of the fragments.The deduced 426 amino acid residues of the gene, named APK2a,are 65% identical to a previously reported Arabidopsisserine/threonineprotein kinase, APKla. The gene is composed of 6 exons and mapsat 10 cM from the upper end of chromosome 1. Northern hybridizationexperiments indicated that the gene is strongly expressed inleaves, moderately in roots, and very weakly in flowers. Furtherin situ analysis of the expression in floral buds showed thatthe APK2a gene is expressed at pedicels, is not expressed atthe floral organ primordia of wild type floral buds, but ismoderately expressed in the floral organ primordia of the agamousmutant. In vitro binding assay suggests that the AGAMOUS proteinbinds to a sequence similar to, but different from, the knownMADS-binding consensus sequences, the CArG box, located 3' downstreamof the APK2a gene. These results suggest that APK2a gene expressionis negatively regulated by the AG protein. A close homologue of the APK2a gene, named APK2b, was also isolatedfrom the Arabidopsis cDNA library. The expression pattern ofthe APK2b gene differs from that of APK2a. It is strongly expressedin leaves, moderately in flowers, and weakly in roots. ⁴Present address: Biomolecular Engineering Research Institute,6-2-3, Fruedai, Suita, Osaka, 565 Japan.