Differential Expression of an Auxin-Regulated Gene, parC, and a Novel Related Gene, C-7, from Tobacco Mesophyll Protoplasts in Response to External Stimuli and in Plant Tissues

As part of an extended search for auxin-regulated genes otherthan parA and parB in the cDNA library from cultured tobaccomesophyll protoplasts, we have isolated the cDNA for a genedesignated parC. This gene is expressed during the transitionfrom the G₀ to S phase of the cell cycle. The nucleotide sequenceof parC cDNA was similar to that of parA. Using the parC cDNAas a probe we have isolated cDNA for a gene designated C-7 byhybridization at reduced stringency. Even though C-7 is relatedto parC, as is parA, its mode of expression was, to our surprise,completely different from that of parC. The C-7 gene is predominantlyexpressed in mature leaves and in freshly prepared protoplasts,but its level of expression did not change in response to auxintreatment. Although parC and C-7 are related to one anotherand exhibit homology to the parA gene, the two former genesdemonstrated conspicuous differences in their responses to externalstimuli, which included auxin, as well as their tissue-specificexpression. These results provide us an interesting system forthe analysis of the differential expression of closely relatedgenes. The significance of our observations is discussed withreference to the other members of the family of parA genes. (Received May 8, 1992; Accepted June 17, 1992)     

Isolation of a Tobacco cDNA Encoding Sar1 GTPase and Analysis of Its Dominant Mutations in Vesicular Traffic Using a Yeast Complementation System

The cDNA clone of NtSARl, a gene encoding the small GTPase Sar1pwhich is essential for vesicle formation from the endoplasmicreticulum (ER) membrane in yeast, has been isolated from Nicotianatabacum BY-2 cells. NtSAR1 as well as AtSAR1 cDNA isolated fromArabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11: 4205]could complement the lethality of the disruption of SARI inyeast cells in a temperature-sensitive fashion. They also suppressedyeast sec12 and sec16 temperature-sensitive mutations as yeastSARI does. Using this complementation system, we analyzed thephenotypes of several mutations in plant SAR1 cDNAs in yeastcells. The expression of NtSAR1 H74L and AtSAR1 N129I showeddominant negative effect in growth over the wild-type SARI,which was accompanied by the arrest of ER-to-Golgi transport.Such dominant mutations will be useful to analyze the role ofmembrane trafficking in plant cells, if their expression canbe regulated conditionally. (Received October 29, 1997; Accepted March 17, 1998)     

A Protein Encoded by din1, a Dark-Inducible and Senescence-Associated Gene of Radish, Can Be Imported by Isolated Chloroplasts and Has Sequence Similarity to Sulfide Dehydrogenase and Other Small Stress Proteins

In an attempt to isolate cDNA clones for dark-inducible chloroplastproteins, we screened a cDNA library which was prepared fromradish cotyledons by a two-step method. The source plants weregrown under continuous light for 14 d and kept in darkness for24 h. One of the selected clones, S2D12, corresponded to thedin1 gene which we previously reported as a dark-inducible,senescence-associated gene [Azumi and Watanabe (1991) PlantPhysiol. 95: 577]. A 22 kDa polypeptide was produced from thecDNA in an in vitro expression system in the presence of [³⁵S]methionine.This polypeptide was capable of being imported by isolated chloroplasts,processed to a smaller mature form and localized in the stromalfraction. As the amino acid sequence of the putative matureprotein has no homology to any known chloroplast protein, din1was suggested to be the first gene for a chloroplast proteinwhich is negatively controlled by light. The putative matureprotein has similarity to sulfide dehydrogenase from Wolinellasuccinogenes and other small stress proteins; glpE and pspEfrom Escherichia coli and hsp67B2 from Drosophila melanogaster. ¹ The nucleotide sequence data in this paper has been submittedto EMBL, GenBank and DDBJ Data Libraries under the acces sionnumber AB004242 ² Present address: The Institute of Physical and Chemical Research(RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-01 Japan     

Cloning and Characterization of HumanSep1 (hSEP1) Gene and Cytoplasmic Localization of its Product

We isolatedand sequenced a human cDNA (designated as hSEP1)encoding both a homologue of mouse Dhm2 and budding yeast SEP1.The gene was shown to be locatedon the long arm of chromosome3 (3q25-26.1). The putative hSEP1 product (hSEP1p) consistedof 1694 amino acid residues with a molecular mass of about 190kDa. Northern blot analysis showeda major 10-kb mRNA expressedubiquitously in variousorgans as well as a minor 5.5-kb mRNAexpressed relatively highly in the testis and placenta. hSEP1pis localizedin the cytoplasm as examinedb y cytochemical andWestern blot analyses of fractionated cellular extracts. Thebiological function of hSEP1p was discussed in correlation withits cytoplasmic localization.     

Isolation and Characterization of cDNA Clones with Enhanced Expression in Tobacco Plants Expressing the Rice Homeobox Gene, OSH1

We have used subtractive hybridization to isolate cDNA cloneswhose expression were up-regulated in transgenic tobacco ectopicallyexpressing the rice homeobox gene, OSH1. Thirty-nine distinctcDNA clones, which we term HRGs (Homeobox Regulated Genes),were identified. Some of them were specifically expressed intransformants, indicating that their expression was possiblyregulated by transgene. (Received January 9, 1997; Accepted March 8, 1997)     

Expression of a Gene for a Protein Similar to HIV-1 Tat Binding Protein 1 (TBP1) in Floral Organs of Brassica rapa

A cDNA for a protein similar to human immunodeficiency virusTat binding protein was isolated from an anther cDNA libraryof Brassica rapa. RNA in situ analysis in flower buds showedthat the gene for this cDNA was specifically expressed in thetapetum and middle layer of anthers and pollen. (Received February 15, 1997; Accepted May 28, 1997)     

氟虫脲对东亚飞蝗和中华稻蝗几丁质合成酶基因表达的影响

为了探讨氟虫脲可能的作用靶标及毒性机制, 本研究以重要农业害虫东亚飞蝗Locusta migratoria manilensis (Meyen)和中华稻蝗Oxya chinensis (Thunberg)为材料, 采用简并引物扩增中华稻蝗几丁质合成酶1基因（OcCHS1）的部分cDNA序列; 以氟虫脲浸渍法处理2龄中期中华稻蝗及1, 2和3龄东亚飞蝗若虫为处理组, 丙酮处理为对照组, 使用RT-PCR和实时荧光定量PCR方法分析氟虫脲对蝗虫几丁质合成酶基因mRNA表达的影响。结果获得的OcCHS1部分cDNA序列, 其长度为312 bp, 编码104个氨基酸, GenBank登录号为HM214491, 与东亚飞蝗几丁质合成酶1基因（LmCHS1）在氨基酸水平上相似度达95％。RT-PCR结果显示, 处理组几丁质合成酶1扩增带均强于对照组。实时荧光定量PCR结果表明: 与对照组相比, 处理组中华稻蝗2龄中期若虫OcCHS1 mRNA表达提高了1.02倍, 东亚飞蝗1, 2, 3龄若虫LmCHS1 mRNA表达分别提高了34%, 82%和89%, 差异显著（P<0.05）。分析基因表达提高的原因是几丁质合成受阻后基因表达水平的一种代偿性增加, 由此推测几丁质合成酶可能是氟虫脲作用的靶标之一。     

Primary Structure and Properties of Glutathione Reductase from Arabidopsis thaliana

A cDNA encoding glutathione reductase (EC 1.6.4.2 [EC] ) from Arabidopsisthaliana was cloned by immunoscreening. The amino acid sequencededuced from the nucleotide sequence agrees with the N-terminalamino acid sequence of the major isozyme (GR II) purified fromleaves of A. thaliana. The predicted polypeptide comprises anN-terminal leader sequence of 74 amino acids, which has featuresof chloroplast-targeting peptides, and a mature polypeptideof 491 residues with a molecular mass of 52.7 kDa, which showshomology with glutathione reductases from other species. TheK_m for GSSG was 44 µM and that for NADPH was 5.0 µMfor GR II at 25C. The pH optimum for GR II was 7.5 to 8.0.The native molecular mass of GR II was 110 kDa, indicating thatGR II is a homodimer. GR II had an isoelectric point of 4.8.The cDNA hybridizes with a 2.1-kb poly(A)⁺RNA from leaves ofA. thaliana. Genomic Southern analysis indicates that the genecorresponding to the cDNA is likely a single-copy gene. (Received July 1, 1993; Accepted September 8, 1993)     

Molecular Cloning of Plant Spermidine Synthases

Four cDNAs for spermidine synthase (SPDS), which converts thediamine putrescine to the higher polyamine spermidine usingdecarboxylated S-adenosylmethionine as the co-factor, were isolatedfrom Nicotiana sylvestris, Hyoscyamus niger, and Arabidopsisthaliana. When the N. sylvestris SPDS cDNA was expressed ina SPDS-deficient E. coli mutant, the recombinant protein showedhigh SPDS activity, but did not have any spermine synthase activity.The plant SPDSs have molecular masses of about 34 kDa, possessthe co-factor binding motifs which have been proposed for S-adenosylmethionine,and are more homologous in amino acid sequence to tobacco putrescineN-methyltransferase (PMT) than to SPDSs from mammals and E.coli. The SPDS gene is expressed in root, stem, and leaf inN. sylvestris, whereas the PMT gene is expressed only in root.The potential evolution of plant SPDS and PMT, and their evolutionaryrelationships with animal SPDS are discussed. (Received September 3, 1997; Accepted November 5, 1997)     

Isolation and Mapping of a Family of Putative Zinc-finger Protein cDNAs from Rice

To understand the functions of rice homologues of the Arabidopsisflowering-time gene CONSTANS (CO) and salt-tolerance gene STO,we performed a similarity search of the single-run sequencedata of cDNA clones accumulated by the Rice Genome ResearchProgram, and isolated seven rice cDNA clones (S3574, C60910,S12569, R2931, R1479, R1577, and E10707) coding for proteinscontaining one or two zinc-finger-like motifs. Comparison ofthe deduced amino acid sequences between these cDNAs and theCO gene revealed significant similarities (46%-;61%) in theregion of zinc-finger motifs. A domain having a high contentof basic amino acids at the C-terminus of the CO protein wasfound in the corresponding region of proteins predicted fromcDNAs S3574, C60910, and S12569. Two amino acid sequences, "CCADEAAL"and "FCV(L)EDRA," which were present inside each zinc-fingerin the Arabidopsis regulatory protein STO, were also found ineach of the two zinc-finger regions of proteins predicted fromcDNAs R2931, R1479, R1577, and E10707. Using restriction fragmentlength polymorphism (RFLP) linkage analysis, we determined thechromosomal location of the seven cDNA clones. The positionof R2931 on the RFLP linkage map was closely linked to Hd-3,one of the putative quantitative trait loci (QTL) controllingheading date in rice.     

Intergenic transcripts containing a novel human cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene

The cytochrome P450 2C (CYP2C) gene locus was found to includea novel exon 1 sequence with high similarity to the canonicalexon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) andPCR amplifications of human liver cDNA revealed the presenceof several intergenic species containing the CYP2C18 exon 1–likesequence spliced to different combinations of exonic and intronicsequences from the CYP2C9 gene. One splice variant was foundto have an open reading frame starting at the canonical translationinitiation codon of the CYP2C18 exon 1–like sequence.Another variant consisted of the nine typical CYP2C9 exons splicedafter the CYP2C18 exon 1–like sequence through a segmentof CYP2C9 5' flanking sequences. Moreover, analysis of bacterialartificial chromosome (BAC) clones revealed that the CYP2C18exon 1–like sequence was located in the intergenic regionbetween the CYP2C19 and CYP2C9 genes. The finding that a solitaryexon is spliced with sequences from a neighboring gene may beinterpreted as representing a general evolutionary mechanismaimed at using the full expression potential of a cell's genomicinformational content.     

Identification of a Novel Human Gene Containing the Tetratricopeptide Repeat Domain from the Down Syndrome Region of Chromosome 21

The Down syndrome (DS) region on chromosome 21, which is responsiblefor the DS main features, has been defined by analysis of DSpatients with partial trisomy 21. Within the DS region, we constructeda 1.6-Mb P1 contig map previously. To isolate gene fragmentsfrom the 1.6-Mb region, we performed direct cDNA library screeningand exon trapping using the P1 clones and a human fetal braincDNA library, and obtained 67 cDNA fragments and 52 possibleexons. Among them, 23 cDNA fragments and 4 exons were interpretedto be derived from a single gene by localization on P1 clonesand by Northern analysis. To obtain the full-length cDNA sequence,longer cDNA clones were further screened from another humancDNA library which was enriched with longer cDNA species. Theseclones were sequenced and assembled to a sequence of 9045 bp.This transcribed sequence encodes a novel 2025 amino-acid proteincontaining tetratricopeptide repeat (TPR) motifs and thereforethe gene was designated as TPRD (a gene containing the TPR motifson the Down syndrome region). The TPR domain has been foundin a certain protein phosphatase and in other proteins involvedin the regulation of RNA synthesis or mitosis. The TPRD gene,the novel gene which was proved to be in the 1.6-Mb region andto have the interesting features described above, is a candidatefor genes responsible for the DS phenotypes.     

C-ABI3, the Carrot Homologue of the Arabidopsis ABI3, is Expressed during Both Zygotic and Somatic Embryogenesis and Functions in the Regulation of Embryo-Specific ABA-Inducible Genes

A carrot gene homologous to the ABI3 gene of Arabidopsis wasisolated from a carrot somatic embryo cDNA library and designatedC-ABI3. The sequence of C-ABI3 was very similar to those ofABI3 of Arabidopsis and VP1 of maize in certain conserved regions.The expression of C-ABI3 was detected specifically in embryogeniccells, somatic embryos and developing seeds. Thus, expressionof C-ABI3 was limited to tissues that acquired desiccation tolerancein response to endogenous or exogenous abscisic acid (ABA).Endogenous levels of ABA in seeds increased transiently andthen desiccation of seeds started. The expression of C-ABI3in developing seeds was observed prior to the increase in levelsof endogenous ABA that was followed by desiccation of seeds.In transgenic mature leaves in which C-ABI3 was ectopicallyexpressed, expression of ECP31, ECP63 and ECP40 was inducedby treatment with ABA, which indicates that the expression ofECP genes was controlled by the pathway(s) that involved C-ABI3and ABA. This suggests that C-ABI3 has the same function asVP1/ABI3 factor in carrot somatic embryos. (Received March 4, 1998; Accepted September 4, 1998)