The nifH-Like (frxC) Gene Is Involved in the Biosynthesis of Chlorophyll in the Filamentous Cyanobacterium Plectonema boryanum

The frxC gene found in the DNA of the liverwort chloroplastencodes a protein of unknown function. The deduced amino acidsequence shows significant homology to that of the nitrogenaseFe-protein encoded by the nifH gene. We previously identifiedthe frxC and nifH genes in the filamentous cyanobacterium Plectonemaboryanum. We describe here the isolation of targeted mutantsof frxC (YFC1004) and nifH (YFH201) which were generated byinsertion of a kanamycin-resistance gene into the structuralportion of the respective genes. As expected, YFH201 cannotgrow under nitrogen-fixing conditions. However, YFC1004 growsas well as the wild type does under nitrogen-fixing, photoautotrophicand chemoheterotrophic conditions, indicating that the FrxCprotein is essential neither for nitrogen fixation nor for majorenergytransduction systems, such as photosynthesis and respiration.YFC1004 synthesizes chlorophyll (Chi) normally in the lightbut not in the dark, and it accumulates a precursor to Chi,protochlorophyllide (Pchlide) in the dark. These phenotypiccharacteristics of YFC1004 suggest that the cyanobacterium hastwo pathways for the reduction of Pchlide: a light-dependentand a lightindependent system. The FrxC protein appears to beinvolved in the light-independent reduction of Pchlide. (Received September 24, 1991; Accepted November 11, 1991)     

A frxC Homolog Exists in the Chloroplast DNAs from Various Pteridophytes and in Gymnosperms

Chloroplast DNAs (ctDNAs) from several pteridophytes were analysedby cross-hybridization with the liverwort frxC gene as probe.Hybridization signals were observed without exception, suggestingthe presence of the frxC gene in ctDNAs of non-flowering vascularplants as in liverwort. A frxC homolog was also identified intwo gymnosperms. (Received November 7, 1991; Accepted January 13, 1992)     

The Effect of Oxygen Radicals on Proteolysis in Isolated Oat Chloroplasts

Chloroplast DNAs (ctDNAs) from several pteridophytes were analysedby cross-hybridization with the liverwort frxC gene as probe.Hybridization signals were observed without exception, suggestingthe presence of the frxC gene in ctDNAs of non-flowering vascularplants as in liverwort. A frxC homolog was also identified intwo gymnosperms. (Received July 5, 1991; Accepted January 28, 1992)     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Identification of a nifDK-Like Gene (ORF467) Involved in the Biosynthesis of Chlorophyll in the Cyanobacterium Plectonema boryanum

The frxC gene, which is found in chloroplast DNA (ctDNA) andin cyanobacteria, encodes a protein that is required for thelight-independent reduction of protochlorophyllide (Pchlide)to chlorophyllide a (Chlide). A DNA fragment downstream of frxCin the filamentous cyanobacterium Plectonema boryanum was clonedand analyzed. Sequencing of the DNA fragment revealed an openreading frame (ORF) that encoded a protein of 467 amino acidresidues (designated ORF467), which showed extensive homologyto the proteins encoded by genes on ctDNAs (ORF465 in liverwort,gidA in pine and chlN in Chlamydomonas reinhardtii) and to ORF469protein of the cyanobacterium Synechocystis sp. strain PCC 6803.We isolated a targeted mutant YFM6D-3 in which ORF467 was inactivatedby the insertion of a kanamycin-resistance gene into the codingregion. YFM6D-3 exhibited a phenotype similar to that of YFC1004,an frxC-disrupted mutant, which did not synthesize chlorophyll(Chl) and accumulated Pchlide, a precursor to Chl, in the dark.These phenotypic characteristics of YFM6D-3 indicate that thelight-independent reduction of Pchlide requires not only theFrxC protein but also the ORF467 protein. The amino acid sequencesof the homologues of ORF467 exhibit low but significant similarityto those of the and ß subunits of nitrogenase MoFe-protein,suggesting a phylogenetic relationship between the light-independentPchlide reductase and nitrogenase, as is observed between theFrxC protein and the Fe-protein of nitrogenase. ¹Institute for Protein Research, Osaka University, Suita, Osaka,565 Japan     

Stabilizing Effect of Lipopolysaccharide on the l-Glycerol 3-Phosphate Acyltransferase of Escherichia coli Membrane

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469–0RF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifHl from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the β subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.     

Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers

Strong hybridization signals were obtained from total DNA of two conifers, lodgepole pine (Pinus contorta) and Norway spruce (Picea abies), in a Southern blot analysis using a probe derived from the chloroplast gidA gene of the green alga Chlamydomonas reinhardtii. The pine fragments detected by the probe were found to originate from the chloroplast genome and, as judged by the signal intensity, this was also true for the spruce fragments. Sequence analysis of the hybridizing pine chloroplast DNA region revealed an open reading frame potentially encoding a 459 amino acid polypeptide, highly homologous to that deduced from the algal gene and to ORF465 of liverwort chloroplast DNA. Upstream of the gidA sequence, we found a trnN(GUU) gene and an open reading frame of 291 codons which was 78% identical to the frxC gene of liverwort. Since ORF465 is located immediately downstream of trnN and frxC in liverwort, the genetic organization of this region is very similar in the two plants. In contrast, neither the gidA nor the frxC gene is present in the chloroplast DNA of tobacco or rice. It was recently reported that deletions in the gidA region of the chloroplast genome of Chlamydomonas reinhardtii abolish the light-independent pathway of chlorophyll synthesis which exists in many algae and lower plants. The presence of the gidA gene on the chloroplast genomes of conifers may therefore be of significance with respect to the ability of these plants to synthesize chlorophyll in the dark.     

甜菜夜蛾泛素延伸蛋白基因cDNA的3′RACE-PCR扩增及其克隆和表达

根据已知的草地夜蛾Spodoptera frugiperda的泛素延伸基因 5'端核苷酸序列设计引物,应用3'RACE-PCR技术,从甜菜夜蛾S. exigua脂肪体组织总RNA中反转录扩增泛素基因的cDNA片段。扩增得到的片段全长513 bp,3'末端有123 bp的非翻译区,翻译区编码一个长为129个氨基酸残基的蛋白质,预测分子量为14.8 kD。同源分析表明,此cDNA序列为ubiquitin-53aa extension protein(ubi-53) 基因,在泛素蛋白后融合了一个核糖体L40蛋白（ribosomal L40 protein）。用MagAlign和Genedoc软件对cDNA编码的氨基酸序列进行了同源性分析,结果表明： 甜菜夜蛾的ubi-53基因与真核生物家蚕Bombyx mori、草地夜蛾、果蝇Drosophila melanogaster和人Homo sapienes泛素的同源性分别为96.9%、98.5%、95.3%和93.0%,与甜菜夜蛾核型多角体病毒(SeNPV)泛素的同源性为78.8%,说明真核生物的泛素基因与核型多角体病毒的泛素基因可能存在不同的分子进化途径。将甜菜夜蛾的ubI-53基因克隆到原核表达载体pET-28a上,转化至BL21（DE3）中,用IPTG进行诱导表达,用异源泛素单克隆抗体进行Western blot检测,证明原核表达蛋白是目的蛋白。     

Identification of the chlB Gene and the Gene Product Essential for the Light-Independent Chlorophyll Biosynthesis in the Cyanobacterium Plectonema boryanum

We cloned a 6.0-kb HindIII fragment from the cyanobacteriumPlectonema boryanum using the chloroplast chlB (ORF513) geneof the liverwort (Marchantia polymorpha) as a probe. An openreading frame (ORF508) encoding a polypeptide of 508 amino acidresidues was found within the nucleotide sequence of the 4,437-bpHindIII-EcoRV subfragment. The deduced amino acid sequence ofORF508 shows very high similarity to that encoded by the liverwortchlB gene (72.7%). A mutant, YFB14, in which ORF508 was inactivatedby the insertion of a kanamycin-resistance cartridge, was unableto synthesize chlorophyll, accumulating protochlorophyllidein darkness while synthesizing chlorophyll normally in the light.Thus, the chlB gene is the third gene that is essential forthe light-independent reduction of protochlorophyllide. Theother two genes are chlL and chlN, and the results suggest thatthe light-independent protochlorophyllide reductase consistsof at least three subunits, which are encoded by chlL, chlNand chlB. Using an antiserum prepared against a ChlB-6xHis fusionprotein expressed in Escherichia coli, we detected a proteinwith an apparent molecular weight of 58,000 in the membranefraction of the cyanobacterium. These results indicate thateither the cytoplasmic or thylakoid membranes could be the siteof the light-independent reduction of protochlorophyllide. (Received November 16, 1995; Accepted February 7, 1996)     

A Novel Synthesis of (±)-Serricornin 4,6-Dimethyl-7-hydroxy-nonan-3-one The Sex Pheromone of Cigarette Beetle (Lasioderma serricorne F.)

A 5.5-kb HindIII fragment of Synechocystis PCC6803 containing a liverwort (ORF316) homolog encoding a putative zinc finger protein was cloned. Nucleotide sequence analysis showed that the homology of the amino acid sequence deduced from the ORF326 of Synechocystis PCC6803 with the counterparts of a liverwort and tobacco was 50% and 46%, respectively. Synechocystis ORF326 also showed 38% homology with the dedB gene in Escherichia coli. The gene organization of the region in these species of organisms was quite different. This suggests that the Synechocystis ORF326 and liverwort ORF316 genes may be related to a common regulatory gene, but not photosynthetic gene characteristic to chloroplasts.     

Characterization of the gene for the Fe-protein of the vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tn5 mutant

Summary A sequence homologous to the conventional nifH gene has been cloned from a different region of the Azotobacter vinelandii genome. Tn5 insertions were obtained in this clone and the mutagenized plasmid was used for marker exchange with A. vinelandii strain CA12 (nifHDK) to obtain Tn5 mutants. These mutants exhibited a Nif^- phenotype in the presence of vanadium, unlike CA12 which was Nif⁺ on vanadium-containing medium. The gene in the cloned nifH-like region is therefore apparently involved in the vanadium dependent alternative pathway of nitrogen fixation. This gene, nifH2, has been sequenced and encodes a protein of 289 amino acids that is similar to nifH in nucleotide sequence, deduced amino acid sequence, predicted secondary structure and hydrophobicity profile. A second open reading frame downstream of nifH2 codes for a protein of 64 amino acids, similar to the ferredoxin (Fd)-like protein encoded downstream of nifH ^* in A. chroococcum. Sequence analysis suggests that the nifH2 and Fd-like genes are in a single operon.     

The Amino Acid Sequence of Ascorbate Peroxidase from Tea Has a High Degree of Homology to That of Cytochrome c Peroxidase from Yeast

The chloroplast isozyme of ascorbate peroxidase from tea leaveswas digested with lysyl endopeptidase, and the amino acid sequencesof the peptide fragments were determined. These sequences accountedfor 64% of the amino acids in the entire protein. The sequenceof one of the peptides can be aligned with the region whichincludes the proximal histidine that serves as the fifth ligandof the heme iron in guaiacol peroxidases and cytochrome c peroxidase.The sequences of the peptides from ascorbate peroxidase exhibita higher degree of homology to the sequence of cytochrome cperoxidase from yeast than to those of guaiacol peroxidasesfrom plants. In addition, three of the peptides from ascorbateperoxidase show a high degree of homology to triose-phosphateisomerase from maize. From the available amino acid sequencesand the enzymatic and molecular properties of ascorbate andcytochrome c peroxidases, we propose that these hydrogen peroxide-scavengingperoxidases that use either cytochrome c or ascorbate as theelectron donor originated from the same ancestral protein. (Received July 5, 1991; Accepted December 6, 1991)     

A Novel 3.5 kDa Protein Component of Cyanobacterial Photosystem I Complexes

A novel protein component of 3.5 kDa was detected in photosystemI complexes prepared from several cyanobacteria, viz. Synechococcusvulcanus, Synechococcus elongotus BP-1, Synechococcus sp. FCC7002 and Synechocystis sp. FCC 6803. The complete amino acidsequence of this component was determined by direct proteinsequencing. The sequences of the 3.5 kDa proteins from thesefour organisms were highly homologous to each other, featuringa hydrophobic domain in the middle. The cyanobacterial consensussequence exhibits significant homology to the presumed productof ORF32 in the chloroplast DNA of liverwort (Marchantia polymorpha),but no homologous ORF is present in the chloroplast DNA of tobaccoor rice. Since this protein appears to interact strongly withthe PS I reaction center complex, it may play some role in thefunction and maintenance of the structure of PS I. (Received May 25, 1992; Accepted August 18, 1992)     

Using bacteria to analyze sequences involved in chloroplast gene expression

The complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. Other genes involved in photosynthesis, respiratory electron transport, and membrane-associated transport in chloroplasts were predicted by the amino acid sequence homology and secondary structure of gene products. Thirty-three open reading frames in the liverwort chloroplast genome remain unidentified. However, most of these open reading frames are also conserved in the chloroplast genomes of two species, a liverwort, Marchantia polymorpha, and tobacco, Nicotiana tabacum, indicating their active functions in chloroplasts.Abbreviations bp base pair - kDa kilodalton - IR inverted repeat - ORF open reading frame - DALA -aminolevulinate     

蓖麻蚕线粒体基因组细胞色素氧化酶亚基Ⅲ的序列及其分子进化分析

测定了蓖麻蚕Samia cynthia ricini线粒体基因组(mtDNA)含完整的细胞色素氧化酶亚基Ⅲ(COX3)、tRNA-Gly和部分的NADH亚基Ⅲ(ND3)基因的DNA片段序列。COX3基因编码框包含789个核苷酸,编码262个氨基酸的蛋白质。通过同源性比较,发现COX3基因的3′端比5′端要保守,其编码的蛋白在C端有两个保守序列存在。COX3的下游为66 bp的tRNA-Gly基因。蓖麻蚕的COX3与家蚕COX3同源性最高,核苷酸和氨基酸序列同源性分别是80.2%和85.6%。根据COX3氨基酸序列进行了12种无脊椎动物的分子进化树分析,认为在采用线粒体基因序列进行分子进化分析时,应该综合考虑物种的繁殖模式及生态特点。