Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

Anaerobic induction of the maize<Emphasis Type="Italic"> GapC4</Emphasis> promoter in poplar leaves requires light and high CO<Subscript>2</Subscript>

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 (GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula × P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO₂ atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO₂ and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO₂ concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.Abbreviations CaMV cauliflower mosaic virus - GapC4 glyceraldehyde-3-phosphate dehydrogenase gene 4 - GUS -glucuronidase - 4-MU methylumbelliferone - STLS-1 stem- and leaf-specific promoter 1     

Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.     

乳房链球菌GapC蛋白的表达及其B细胞抗原表位的预测与鉴定

乳房链球菌Streptococcus uberis的GapC蛋白是一种位于该菌表面的具有甘油醛-3-磷酸脱氢酶活性的蛋白,其参与细胞活动,表现出多种生物学活性,此外还具有良好的抗原性。文中旨在对乳房链球菌GapC蛋白可能的B细胞抗原表位进行预测,分析和验证候选表位肽的免疫原性。利用S. uberis分离株RF5-1克隆gapC基因,构建重组表达质粒pET-28a-GapC,诱导表达GapC重组蛋白,并以纯化蛋白免疫家兔,获得抗GapC多抗。利用生物信息学软件预测并分析GapCB细胞抗原表位的三维结构和空间位置及对GapC蛋白及表位的同源性比较。结果表明,表达纯化了44kDa的GapC蛋白具有良好的反应性。利用表位预测软件筛选并合成针对S.uberisGapC蛋白的6个线性和3个构象优势B细胞表位多肽,三维结构的分析显示,筛选的多肽具有良好的抗原表位形成条件。以纯化的S.uberis GapC蛋白免疫家兔制备多抗,通过间接ELISA对抗原表位进行鉴定。ELISA检测结果显示,9条抗原表位肽均可不同程度地与抗GapC多抗反应,其中表位266AANDSYGYTEDPIVSSD282与多抗反应...     

The maize GapC4 promoter confers anaerobic reporter gene expression and shows homology to the maize anthocyanin regulatory locus C1

The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) gene family of maize is differentially expressed in response to anaerobic stress. While GapC1 and GapC2 are downregulated, GapC3 and GapC4 are anaerobically induced. We have sequenced and analyzed a 3073 bp promoter fragment of GapC4. The promoter confers anaerobic induction of a reporter gene construct in a transient gene expression system in maize. Deletion analysis of the GapC4 promoter revealed a 270 bp long DNA region required for anaerobic induction. This region contains sequence motifs resembling the cis-acting sequences of the anaerobically induced maize Adh1 and Adh2 genes. Furthermore, the 3073 bp GapC4 promoter fragment displays homology to long terminal repeats of maize retrotransposons and to the 3 region of the maize anthocyanin regulatory locus C1.     

Intron-specific stimulation of anaerobic gene expression and splicing efficiency in maize cells

Most of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes characterized in plants and algae to date have one intron very close to the 5 end of the gene. To study the functional relevance of some of these introns for gene expression we have analysed the influence of three 5 introns on transient gene expression of the anaerobically inducible maizeGapC4 promoter in maize cells. Under aerobic conditions, reporter gene expression is increased in the presence of the first introns of theGapC4 andGapC1 genes, and the first intron of the nuclear encoded chloroplast-specificGapA1 gene. In contrast, theGapC4 intron increases anaerobic gene expression above the level obtained for the intronless construct, while anaerobic expression of constructs harboring theGapA1 andGapC1 introns was similar to the anaerobic expression level of the intronless construct. Splicing analysis revealed that theGapC4 intron is processed more efficiently under anaerobic conditions, while no change in splicing efficiency is observed for theGapC1 and theGapA1 introns when subjected to anaerobic conditions. These results suggest that an increase in splicing efficiency contributes to the anaerobic induction of the maizeGapC4 gene.     

Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro

Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliana, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cys(149). Catalytic Cys(149) is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (pK(a)=5.7). This property makes GapC1 sensitive to oxidation by H(2)O(2), which appears to inhibit enzyme activity by converting the thiolate of Cys(149) (-S-) into irreversible oxidized forms (-SO(2)(-) and -SO(3)(-)) via a labile sulfenate intermediate (-SO(-)). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys(149) sulfenates to give rise to a mixed disulfide (Cys(149)-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys(149).     

Light-dependent Anaerobic Induction of the Maize Glyceraldehyde-3-Phosphate Dehydrogenase 4 (GapC4) Promoter in Arabidopsis thaliana and Nicotiana tabacum

The maize glyceraldehyde-3-phosphate dehydrogenase 4 (GapC4)promoter confers strong and specific anaerobic gene expressionin tobacco (Nicotiana tabacum) and potato (Solanum tuberosum).Here we show that the promoter is also anaerobically inducedin Arabidopsis thaliana. Histochemical analysis demonstratesthat the promoter is anaerobically induced in roots, leaves,stems and flower organs. Surprisingly, the strong anaerobicinduction of the promoter is dependent on light and on the substitutionof oxygen with carbon dioxide. High carbon dioxide concentrationalone does not induce the promoter in the presence of oxygenand light. If anaerobic conditions are generated under completedarkness or if plants are submerged, no induction above backgroundis observed. When transgenic tobacco harbouring a GapC4 promoter–reportergene construct is analysed for light dependent anaerobic induction,the results are indistinguishable from those with arabidopsis.The implications for using the GapC4 promoter as an anaerobicreporter for monitoring alterations in the anaerobic signaltransduction pathway are discussed.     

GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity.

In this study we have determined gap sequences from nine different spirochetes. Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families. Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor. A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa. Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred. Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent.     

PCR-mediated direct gene disruption in Schizosaccharomyces pombe.

We have examined the feasibility and efficiency of PCR-mediated direct gene disruptions in the fission yeast Schizosaccharomyces pombe. In the present study, the S.pombe ura4+ gene was amplified by PCR with oligonucleotides that had short flanking regions ( approximately 40 bp) to the target gene. Using this purified PCR product we were able to disrupt genes in an S. pombe strain bearing aura4 deletion, with an efficiency ranging between 1 and 3% among selected transformants. The results indicated that despite S.pombe's preference for non-homologous or illegitimate recombination, even very short stretches of homologous regions could be used to target genes at a defined frequency in this organism. The successful disruption of four independent genes (sts1+, gcs1+, gsh2+and hmt1+) by this method further demonstrates that, despite the relatively low efficiency, the method is very feasible, and it's simplicity, especially when coupled to phenotype-based screening, should greatly facilitate disruption of genes in S.pombe.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.     

Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes

Utilizing yeast strains containing insertion mutations in each of the three glyceraldehyde-3-phosphate dehydrogenase structural genes, the level of expression of each gene was determined in logarithmically growing cells. The contribution of the TDH1, TDH2, and TDH3 gene products to the total glyceraldehyde-3-phosphate dehydrogenase activity in wild type cells is 10-15, 25-30, and 50-60%, respectively. The relative proportions of expression of each gene is the same in cells grown in the presence of glucose or ethanol as carbon source although the total glyceraldehyde-3-phosphate dehydrogenase activity in cells grown in the presence of glucose is 2-fold higher than in cells grown on ethanol. The polypeptides encoded by each of the structural genes were identified by two-dimensional polyacrylamide gel electrophoresis. The TDH3 structural gene encodes two resolvable forms of glyceraldehyde-3-phosphate dehydrogenase which differ by their net charge. The apparent specific activity of glyceraldehyde-3-phosphate dehydrogenase encoded by the TDH3 structural gene is severalfold lower than the enzymes encoded by TDH1 or TDH2. The polypeptides encoded by the TDH2 or TDH3 structural genes form catalytically active homotetramers. The apparent Vmax for the homotetramer encoded by TDH3 is 2-3-fold lower than the homotetramer encoded by TDH2. Evidence is presented that isozymes of glyceraldehyde-3-phosphate dehydrogenase exist in yeast cells, however, the number of different isozymes formed was not established. These data confirm that the three yeast glyceraldehyde-3-phosphate dehydrogenase genes encode catalytically active enzyme and that the genes are expressed at different levels during logarithmic cell growth.     

Nucleotide distribution in gymnosperm nuclear sequences suggests a model for GC-content change in land-plant nuclear genomes

Nuclear protein coding sequences from gymnosperms are currently scarce. We have determined 4 kb of nuclear protein coding sequences from gymnosperms and have collected and analyzed >60 kb of nuclear sequences from gymnosperms and nonspermatophytes in order to better understand processes influencing genome evolution in plants. We show that conifers possess both biased and nonbiased genes with respect to GC content, as found in monocots, suggesting that the common ancestor of conifers and monocots may have possessed both biased and nonbiased genes. The lack of biased genes in dicots is suggested to be a derived character for this lineage. We present a simple but speculative model of land-plant genome evolution which considers changes in GC bias and CpG frequency, respectively, as independent processes and which can account for several puzzling aspects of observed nucleotide frequencies in plant genes.Abbreviations GC guanosine plus cytosine - GapC glycolytic glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12 - GapA Calvin cycle glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.13 - O/E ratio of observed-to-expected dinucleotide frequencies Correspondence to: W. Martin     

多重实时荧光PCR相对定量法快速诊断唐氏综合征

为了建立一种基于多重实时荧光相对定量PCR技术并应用之于唐氏综合征分子诊断, 选择21号染色体上唐氏综合征特异区域基因片段(DSCR3)为目的基因, 以12号染色体上的磷酸甘油醛脱氢酶基因(GAPDH)为参照基因, 设计合成两对引物以及分别以不同荧光标记的TaqMan探针, 在同一个反应管中进行扩增。以相对定量指标△CT值区分唐氏综合征患者与正常人。采用EB 病毒转化技术, 把唐氏综合征患者外周血B 淋巴细胞转化成永生淋巴母细胞系作为标准品。通过优化反应条件, 使得目的基因和参照基因的扩增效率基本一致, 接近100%, 模板浓度在3～300 ng/μL范围内, △CT值的变异系数小于15%, 浓度在30 ng/μL时, 变异系数最小(＜10%), 以该浓度的DNA作为模板进行批内和批间实验的△CT值重复性好, 变异系数分别为9.8%和13.3%。运用建立的方法检测20例唐氏综合征患者的血标本和30例正常人的血标本, 正常人△CT值范围是-1.90～-1.30, 患者的△CT值范围是-2.95～-2.15, 两组之间无交叉重叠, 有明显差异(P＜0.001)。唐氏综合征患者永生细胞系建系成功 ,染色体核型和DNA 分析表明建系前后遗传是稳定的。因此, 实时荧光定量PCR比较△CT值的相对定量法快速诊断唐氏综合征是可行的。     

The mitochondrial genome of the legume Vigna radiata and the analysis of recombination across short mitochondrial repeats

The mitochondrial genomes of seed plants are exceptionally fluid in size, structure, and sequence content, with the accumulation and activity of repetitive sequences underlying much of this variation. We report the first fully sequenced mitochondrial genome of a legume, Vigna radiata (mung bean), and show that despite its unexceptional size (401,262 nt), the genome is unusually depauperate in repetitive DNA and "promiscuous" sequences from the chloroplast and nuclear genomes. Although Vigna lacks the large, recombinationally active repeats typical of most other seed plants, a PCR survey of its modest repertoire of short (38-297 nt) repeats nevertheless revealed evidence for recombination across all of them. A set of novel control assays showed, however, that these results could instead reflect, in part or entirely, artifacts of PCR-mediated recombination. Consequently, we recommend that other methods, especially high-depth genome sequencing, be used instead of PCR to infer patterns of plant mitochondrial recombination. The average-sized but repeat- and feature-poor mitochondrial genome of Vigna makes it ever more difficult to generalize about the factors shaping the size and sequence content of plant mitochondrial genomes.