Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

High Affinity of the 5'-Upstream Region of a Winged Bean Chymotrypsin Inhibitor Gene for Nuclear Matrix

The 5'-upstream region of a winged bean chymotrypsin inhibitorgene (WCI-3b) was found to have a high affinity for nuclearmatrix. The region, named WCI-3b MAR (matrix attachment region),is highly A+T-rich and contains multiple sites interacting withnuclear matrix. A MAR was also found in the corresponding regionof the WCI-x gene, another active gene of the WCI family. SeveralMAR-binding proteins were detected in the wheat nuclear matrix. ⁴Present address: Friedrich Miescher Institute, P.O. Box 2543,CH-4002 Basel, Switzerland. ⁵Present address: Research Institute for Biological Sciences(RIBS), Kayo-cho, Jyobo-gun, Okayama, 716–1241 Japan.     

Tissue-Dependent Expression of the Rice wx+ Gene Promoter in Transgenic Rice and Petunia

The waxy (wx) locus, which controls the amylose synthesis, isknown to be expressed specifically in the endosperm and pollen.To study the tissue-specific regulation of the wx⁺ gene, weintroduced a fusion gene that consisted of the upstream sequenceof the wx⁺ gene and the gene for rß-glucuronidase(GUS) into cells of rice (Oryza sativa L.) and petunia (Petuniahybrida L.). GUS activity was examined in the regenerated transgenicrice and petunia plants. In transgenic rice, the upstream sequenceof the wx⁺ gene was sufficient to direct the tissue-specificexpression of GUS in the endosperm and pollen, and the controlof expression was quantitative. By contrast, in transgenic petunia,the same fusion gene was expressed in pollen but not in theendosperm. These results suggest that the putative cis-actingelements that direct pollen-specific expression are common toor similar in both monocotyledonous and dicotyledonous plants,whereas ciy-elements responsible for the endosperm-specificexpression of the rice wx⁺ gene do not function in petunia,in which development of the endosperm differs from that in rice. ⁴Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan     

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.     

Genes Encoding the Group I Intron-containing tRNALeu and Subunit L of NADH Dehydrogenase from the Cyanobacterium Synechococcus PCC 6301

A part of the tRNA^Leu (UAA) gene containing a 240-nucleotidegroup I intron was amplified by PCR from cyanobacterium SynechococcusPCC 6301 genomic DNA. The pre-tRNA synthesized from the clonedPCR product was efficiently self-spliced in vitro under physiologicalconditions. The gene encoding the tRNA^Leu (UAA), trnL-UAA, wasisolated from a Synechococcus PCC 6301 genomic library and thenucleotide sequence of a 2,167-bp portion was determined. ThetrnL-UAA consists of a 34-bp 5' exon, a 240-bp group I intronand a 50-bp 3' exon. In addition, three open reading frames(ORF1, ORF2 and ORF3) were found in the 5' and 3' flanking regionsof trnL-UAA. The predicted protein sequence of ORF3, which islocated 74-bp upstream from trnL-UAA on the opposite strand,shows 66.2% amino acid identity to that of the SynechocystisPCC 6803 gene encoding subunit L of NADH dehydrogenase (ndhL).     

Combined Effects of Multiple cis-Acting Elements in Elicitor-Mediated Activation of PSCHS1 Gene

Promoter of a gene encoding chalcone synthase 1 (PSCHS1), amember of the defense-related genes in pea, was analyzed bytransient transfection assay. The results demonstrated thatin addition to the previously identified AT-rich sequences,at least four distinct cis-acting elements were required formaximal fungal elicitor-mediated activation of PSCHS1. ²Present address: Hikone Reserch Laboratories, Maruho Co. Ltd.,2763, Takamiya-cho, Hikone, Shiga, 522-02 Japan.     

The Spirulina platensis Adenylate Cyclase Gene, cyaC, Encodes a Novel Signal Transduction Protein

A cyaC gene encoding an adenylate cyclase of the filamentouscyanobacterium Spirulina platensis was se-quenced. The predictedamino acid sequence of the C-ter-minal region of cyaC is similarto the catalytic domains of adenylate cyclases in other cyanobacteriaand eukaryotes. The sequences of other regions are similar tothose of proteins consisting of the bacterial two-componentsignal transduction system: the sensory kinase and the responseregulator. The predicted gene product of cyaC contains, fromthe N-terminal end, a receiver domain of the response regulatorprotein (Rl), a domain similar to the ETR1 of Arabi-dopsis thaliana,a transmitter domain of the sensory kinase protein, a receiverdomain of the response regulator protein (R2), and a catalyticdomain of adenylate cyclase. The cyaC gene was expressed asan affinity-tagged protein in Escherichia coli, and the recombinantprotein was purified. The purified protein had adenylate cyclaseactivity which was activated by Mn²+. The results of Westernblotting using an anti-CyaC antiserum and the S. platensis cellextract confirmed that cyaC gene is expressed in S. platensis (Received February 27, 1997; Accepted April 26, 1997)     

Cell Cycle Timing of Replication of the Nuclear Gene Encoding Chloroplastic NADP-Specific Glutamate Dehydrogenase Isoenzymes in Chlorella sorokiniana

In synchronized Chlorella sorokiniana cells, the NH₄⁺ inducibleNADP-specific glutamate dehydrogenase enzyme (NADP-GDH) accumulatedin a linear manner throughout the first cell cycle. Early inthe following second cell cycle, an increase in its rate ofaccumulation occurred that was proportional to the increasein total cellular DNA in the previous cell cycle. In synchronizedbacterial cells, increases in rate of linear accumulation ofinducible enzymes coincide with the time of replication of theirstructural genes. To determine whether the rate change in NADPGDHaccumulation resulted from a delay in replication of its nuclearstructural gene (gdhN) in fully induced C. sorokiniana cells,the cell cycle timing of replication of this gene was comparedto that of another nuclear gene, nitrate reductase (nia), andof a chloroplast gene, ribulose bisphosphate carboxylase large-subunit(rbcL), in synchronized cells cultured in NH₄⁺ or NO₃^–(uninduced) medium. The gdhN and nia genes replicated withinthe period of nDNA synthesis and rbcL within the period of ctDNAsynthesis in cells growing in either nitrogen source. Therefore,the delayed rate change in enzyme accumulation results froma process that regulates expression of the gdhN gene after itsreplication. (Received July 16, 1994; Accepted November 28, 1994)     

Expression in Yeast of a Fusion Gene Composed of the Promoter of a Heat-Shock Gene from Arabidopsis and a Bacterial Gene for {beta}-Glucuronidase

Production of a functional ß-glucuronidase (GUS) proteinwas induced by exposure of exponentially growing yeast cellsto heat shock after transformation of the GUS gene under thecontrol of the promoter of the heat-shock gene, HSP18.2, fromArabidopsis. Yeast cyr and bcy mutations appeared to have essentiallyno effect. ¹Present Address: Laboratory of Plant Molecular Biology, TheRockefeller University, 1230 York Avenue, New York, NY 10021-6399,U.S.A.     

Transient Gene Expression in Plant Cells Mediated by Agrobacterium tumefaciens: Application for the Analysis of Virulence Loci

A simple system is described for detection of the transfer ofT-DNA from Agrobacterium cells to suspension-cultured tobaccoBY-2 cells. A modified reporter gene for rß-glucuronidase(GUS) that contained an intron sequence was introduced intothe T-DNA region such that the GUS protein could be synthesizedin plant cells only after transfer of the T-DNA to plant nuclei.When BY-2 cells were co-cultured with Agrobacterium cells thatcontained the modified reporter gene, transient synthesis ofGUS protein was observed between 36 and 48 h after the onsetof co-culture. The level of GUS activity reached a plateau withinas little as 48 h. This temporal profile of GUS activation suggeststhat the transient activity might have been due to expressionof the GUS gene in the T-DNA that had been transferred to theplant nuclei but had not yet been integrated into the plantchromosomes. Levels of transient GUS activity were also examinedwith various vir mutants of Agrobacterium and in a mutant withan altered chromosomal acvB gene, the gene for a protein thathas been postulated to function outside bacterial cells. Duringco-culture with virB, virD2, virD4 and acvB mutants, GUS activityremained at background levels, and the GUS activity in the caseof the virE2 mutant was thirty-fold lower than with the wildtype. On the basis of these results, we discuss the roles ofthese genes during infection by Agrobacterium of plant cells. ⁴Present address: Biochemistry Laboratory, Kanebo Ltd., 5-3-28Kotobuki-cho, Odawara, Kanagawa, 250 Japan     

Carboxyl Terminal Segment of T4 Gene 32 Protein is Dispensable in Vivo

We obtained a mutant of bacteriophage T4 which overcame thedeficiency in gene 49 endonuclease. The new mutation occurredin gene 32 and the mutant, which was viable, produced an amberfragment under non-suppressed conditions, lacking about 30 aminoacid residues at the carboxyl terminus. Its growth, recombination,and resistance to UV irradiation were affected to various degreesby the particular suppressor tRNA present. Growth was increasedby Su2⁺ to nearly that of the wild type, but growth of all otherswas reduced in the presence and absence of suppressors, suggestingthat the terminal domain of gene 32 protein is not indispensablefor the function but modulates it. We discuss the mechanismby which the mutation overcomes the defect in gene 49 endonuclease. ¹ This paper is dedicated to the memory of the late Dr. JojiAshida. (Received November 22, 1982; Accepted February 21, 1983)     

The Gene for Pyruvate,Orthophosphate Dikinase in C4 Plants: Structure, Regulation and Evolution

Pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1 [EC] ) is a keyenzyme in photosynthesis in plants that exploit the C4 photosyntheticpathway for the fixation of CO₂. This review focuses on thestructure, regulation and evolution of the C4-type ppdk genein the maize genome. The C4-ppdk gene in maize consists of 19exons spanning about 12 kbp. The gene is transcribed from twodifferent initiation sites under the control of two promotersto produce two mRNAs of different sizes. The larger one containsthe exon 1 sequence that encodes the chloroplast transit peptideand its product acts as C4-PPDK in chloroplasts, while the smallerone does not contain the sequence and its product may functionas a C3-enzyme in the cytosol. This unusual dual promoter systemis not unique to the maize C4-type ppdk gene since the sameorganization is also observed in the rice (C3 plant) ppdk geneand in Flaveria. Thus, the two-promoter system is common toplant ppdk genes from C3 and C4, monocot and dicot plants. Adiscussion is also presented of the generation of a system forregulation of the expression of the C4-type ppdk gene. A chimericgene consisting of a reporter gene under the control of thepromoter of maize CA-ppdk is exclusively expressed in photosynthetictissues and not in roots or stems of transgenic rice. The expressionof the introduced gene is also regulated by light: it is lowin etiolated leaves and is enhanced by illumination. These resultsindicate that the regulatory system that controls ppdk expressionin maize is not unique to C4 plants. ¹Recipient of the JSPP Young Investigator Award, 1995.     

A Role for the acrA Gene in the Protection of Escherichia coli Cells against Excess Sodium

The acrA gene determines the sensitivity of Escherichia coliK-12 cells to acriflavine, which is one of the acridine dyesand which effectively eliminates certain plasmids from the bacterialcells. The acriflavine-sensitivity mutation leads to instabilityof plasmids, such as sex (F)- and drug-resistance (R)-factorsand to loss of a membrane protein with molecular weight about60 kDa (Nakamura 1974, 1976, Nakamura et al. 1975, 1981). Wehave found that cells with a mutant acrA gene were also moresensitive to an excess of sodium ions in the medium than werethe wild-type acrA⁺ cells (Nakamura 1977). The product of theacrA gene hindered the accumulation of sodium by the cells.Although mutations in theproA or proB genes, which determinethe synthesis of the enzymes y-glutamyl phosphate reductaseand y-glutamyl kinase, respectively, in the proline-biosyntheticpathway also led to sensitivity to and accumulation of excesssodium, the presence of the acrA⁺ allele decreased both theseparameters. (Received November 1, 1989; Accepted April 27, 1990)     

Different Sets of cis-Elements Contribute to the Expression of a Catalase Gene from Castor Bean during Seed Formation and Postembryonic Development in Transgenic Tobacco

Deletion analysis of the promoter region of a gene for catalase,cat2, from castor bean (Ricinus communis) was performed to identifythe cis-regulatory elements responsible for the expression ofa rß-glucuronidase (GUS) fusion gene during seed formationand postembryonic development in transgenic tobacco. The analysisshowed that multiple cis-elements contribute to the activityof the cat2 promoter during seed formation and postembryonicdevelopment. The 5'-upstream regions from –1,241 to –816bp, from –720 to –682 bp, and from –632 to–535 bp, relative to the site of initiation of translationof cat2, contributed positively to the activity of the cat2promoter during both stages. By contrast, the region from –816to –720 bp had a negative effect at both stages. The regionfrom –682 to –632 bp contributed positively to theactivity during seed formation but negatively during postembyonicdevelopment. Histochemical analysis revealed that the multiplecis-elements determined not only the level of expression ofthe chimeric gene but also the tissue-specificity of such expression.For example, the region from –1,241 to –816 bp allowedexpression of the chimeric gene in the axis of the embryo ofthe dry seed, as well as in the cortex of the middle part ofthe hypocotyl and at the base of epicotyl in the young seedling. ¹Present address: Department of Plant Molecular and Cell Biology,University of Florida, Gainesville, Florida 32611-0511, U.S.A. ²Present address: Center for Molecular Biology and Genetics,Mie University, 1515 Kamihama, Tsu, Mie, 519 Japan ³Present address: Faculty of Biotechnology, Fukui PrefecturalUniversity, 4-1-1 Kenjojima, Matsuoka-cho, Yoshida-gun, Fukui,910-11 Japan