Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process but also to quantify interactions, for instance within molecular complexes. We combined fluorescence correlation spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (approximately = 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other. We also performed complementary continuous photobleaching experiments and found that the photobleaching decay rate of Alexa Fluor 647 in the excited state decreases by about 30% when incorporated into cDNA, while its nonradiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25% compared to free Alexa dyes.

Systems biology is the study of an organism, viewed as an integrated and interacting network of genes, proteins and biochemical reactions which give rise to life. Instead of analyzing individual components or aspects of the organism, systems biologists focus on all the components and the interactions among them, all as part of one system using computer-based or special algorithms-based analysis. Here we introduce a novel method for predicting genome-wide condition-specific subcellular locations of human proteins using only limited and condition-unspecified known locations. With systems biological analysis of glioma, the key target genes which involved in the control of the tumoregenesis process can be estimated. After systemic molecular biological experiments for target genes with immunohistochemical staining, FCCS, Olink system, western blotting, siRNA and growth/proliferation inhibition assay technique to determine protein binding sites on DNA and molecular imaging, the gene regulatory mechanism can be investigated and validated functionally based on key mechanisms. We discover that the relocated but still interacting PSPN and GFRa4 in endoplasmic reticulum do not interact with ret proto-oncogene (RET), whereas the three proteins are highly interact to each other in plasma membrane in normal brain tissues. The novel relocation of each gene discovered here can be a biomarker in glioma. FLUORESCENCE CORRELATION SPECTROSCOPY ON NANO-FAKIR SURFACES WITH TWO-PHOTON EXCITATION J. Delahaye *, S. Grésillon*, E. Fort*, N. Sojic** and S. Lévêq ue-Fort*** * Centre d’Imageries Plasmoniques Appliquées, Instit ut Langevin, ESPCI ParisTech, CNRS UMR 7587, 10 rue Vauquelin, 75 231 Paris Cedex 05, France **Institut des Sciences Moléculaires, UMR 5255 CNRS , Université Bordeaux 1 – ENSCPB, 16 Avenue Pey-Berland, 33607 Pessac Cedex, France ***Laboratoire de Photophysique Moléculaire, Univer sité Paris sud, bat 210, 91405 Orsay, France julie.delahaye@espci.fr Single biomolecule behaviour can reveal crucial inf ormation about processes not accessible by ensemble measurements. It thus repres ents a real biotechnological challenge. Common optical microscopy approaches require picoto nano-molar concentrations in order to isolate an individual molecule in the observatio n volume. However, biologically relevant conditions often involve micromolar concentrations. These impose a drastic reduction of the conventional observation volume by at least three o rders of magnitude. This confinement is also crucial for mapping subwavelength heterogeneit ies n cells which play an important role in many biological processes. We propose an original approach that couples Fluore scence Correlation Spectroscopy (FCS), powerful tool to retrieve essential informat ion on single molecular behaviour, and nanostructured plasmonic substrates with strong fie ld enhancements and confinements at their surface. These electromagnetic singularities at nan ometer scale, called “hotspots”, are the result of the unique optical properties of surface plasmons. They provide an elegant way for studying single-molecule dynamics at high concentra tions by reducing dramatically the excitation volume and enhancing the fluorophore sig nal by several orders of magnitude. Fig. 1 Scanning electron micrographs of a part of the nan otip array The nanostructured substrates are obtained by elect rochemistry of an optical fibre bundle covered by a metallic thin film (see Figure 1) [1]. This technique is versatile and highly reproducible. It permits to create “nano-fakir surf aces” which cover a wide range of surface topographies to tune and tailor the hotspots densit y, localisation and size. We present two-photon FCS results on these nano-fak ir substrates using fluorescent nanobeads. We show i) a dramatic reduction of the observation volume an d ii ) the ability to perform at high concentration measurements, which i s promising for biological applications. [1] V. Guieu, F. Lagugné-Labarthet, L. Servant et al., “Ultrasharp Optical-Fiber Nanoprobe Array for Raman Local-Enhancement Imaging” Small, 4, 96-99 (2008). Full correlation FCS (fcFCS) to investigate the bul k polymerization of styrene Maren Dorfschmid, Andreas Zumbusch, Dominik Wöll Zukunftskolleg/Department of Chemistry, Universität Konstanz, 78457 Konstanz, Germany E-mail: dominik.woell@uni-konstanz.de Radical polymerization represents a challenging top ic for basic kinetic research. Proceeding from dilute to semi-dilute and finally concentrated polymer solutions, the mobility of monomers, polymer chains and other solutes becomes more and m ore restricted. This restriction starts to dominate polymerization kinetics as soon as polymer ization or termination rates become diffusioncontrolled. In previous work, we have concentrated on studying the decrease of translational diffusion coefficients of single perylendiimide (PD I) molecules by fluorescence correlation spectroscopy (FCS) and wide-field fluorescence micr os opy (WFM).[1] Here, we present our latest results on rotational a d translational motion of different sized PDIs during bulk polymerization of styrene. Rotatio nal diffusion coefficients in dilute solutions were determined by fluorescence anisotropy measurem ents. When rotational diffusion times clearly exceed the fluorescence lifetime (ca. 5 ns for the PDI used), a situation which is reached at some point during increasing monomer-to-polymer conversi on, this method cannot be applied anymore. However, in this case, rotational diffusion can be observed by fcFCS with correlations down to nanoseconds.[2,3] The crosscorrelation curves show antibunching, rotational and translational motion. Triplet contributions do not appear due to the low ISC rate of the PDI. With fcFCS we were able to follow rotational motion of the probing dye over an extended range of monomer-to-polymer conversion. Thus, a direct comparison between rotat ion l and translational motion, both observed by the same fcFCS experiment, is possible. This kno wledge will be used to gain insight into changes in mobility during radical polymerization a d how they depend on the polymerization conditions. References [1] D. Wöll, H. Uji-i, T. Schnitzler, J. Hotta, P . Dedecker, A. Herrmann, F. C. De Schryver, K. Müllen, J. Hofkens, Angew. Chem. Int. Ed. 2008, 47, 783. [2] M. Wahl, H.-J. Rahn, I. Gregor, R. Erdmann, J . Enderlein, Rev. Sci. Instr. 2007, 78, 033106. [3] S. Felekyan, R. Kühnemuth, V. Kudryavtsev, C. S andhagen, W. Becker, C. A. M. Seidel, Rev. Sci. Instr. 2005, 76, 083104.

The plastid genome of higher plants is transcribed by two different types of RNA polymerases named nucleus encoded RNA polymerase (NEP) and plastid encoded RNA polymerase. Plastid encoded RNA polymerase is a multimeric enzyme comparable to eubacterial RNA polymerases. NEP enzymes represent a small family of monomeric phage-type RNA polymerases. Dicotyledonous plants harbor three different phage-type enzymes, named RPOTm, RPOTp, and RPOTmp. RPOTm is exclusively targeted to mitochondria, RPOTp is exclusively targeted to plastids, and RPOTmp is targeted to plastids as well as to mitochondria. In this article, we have made use of RPOTp and RPOTmp T-DNA insertion mutants to answer the question of whether both plastid-located phage-type RNA polymerases have overlapping or specific functions in plastid transcription. To this aim, we have analyzed accD and rpoB messenger RNAs (mRNA; transcribed from type I NEP promoters), clpP mRNA (transcribed from the −59 type II NEP promoter), and the 16S rRNA (transcribed from the exceptional PC NEP promoter) by primer extension. Results suggest that RPOTp represents the principal RNA polymerase for transcribing NEP-controlled mRNA genes during early plant development, while RPOTmp transcribes specifically the rrn operon from the PC promoter during seed imbibition.

We have investigated the function of one of the six plastid sigma-like transcription factors, sigma 3 (SIG3), by analysing two different Arabidopsis T-DNA insertion lines having disrupted SIG3 genes. Hybridization of wild-type and sig3 plant RNA to a plastid specific microarray revealed a strong reduction of the plastid psbN mRNA. The microarray result has been confirmed by northern blot analysis. The SIG3-specific promoter region has been localized on the DNA by primer extension and mRNA capping experiments. Results suggest tight regulation of psbN gene expression by a SIG3-PEP holoenzyme. The psbN gene is localized on the opposite strand of the psbB operon, between the psbT and psbH genes, and the SIG3-dependent psbN transcription produces antisense RNA to the psbT-psbH intergenic region. We show that this antisense RNA is not limited to the intergenic region, i.e. it does not terminate at the end of the psbN gene but extends as antisense transcript to cover the whole psbT coding region. Thus, by specific transcription initiation at the psbN gene promoter, SIG3-PEP holoenzyme could also influence the expression of the psbB operon by producing psbT antisense RNA.

We used quantitative phase tomography with synchrotron radiation to elucidate the 3D structure of Arabidopsis seeds in their native state. The cells are clearly distinguished, and their internal structure is revealed through local variations in electron density. We visualized a 3D network of intercellular air space that might allow immediate gas exchange for energy supply during germination and/or serve for rapid water uptake and distribution during imbibition.

Xin Zhang Xishuu Deng Yianni Deng Len Zhaohong Langping Yang Xiaili Fang Guirong Hou Lu Shan Jun Ma Hongjun Qing Peike Shi JIxiang Xie Cai Hao Yingjian Wang Songshu Pen Zeng Daqin Luo An Hengba Lei Dongshen Huang Jun Song JIxuan Wei Lou Ping Qiaosheng Xu Bo Liang Han Tian Liu Shaojun Zhixiang Hou Zhou Yucai Wu Yihu Shen Quntai Chen Hui Dou Ronhua An Li Wang Junlian C.l. Tang Caiming Fu Chen Ping Cheng Gong David J. Cooper Yu Dan Songjun Wuliang Chenqihua Shihaiyan Hou Feng Hanxiong Li Xiaoyin Li Zhangxin Liu Xichun Weng Xiaoxiong Xinghua Xiao Rong Jianhua Junqin Fu Chenxingquan Weiwu Qing Tan Fenglin Li Qingui Su Yuping Zhang Yuelin Li Songgui Yuan Xiuqun Li Zhiping Wu Long Yonghong Li Heqing Xiancheng Hou Xiao Mei Xiaolin Xu Yan Li Yang Yang Yangyong Sun Yi Deng Yingchun Liu Youcheng Xie Yuxiong Luo Zhaosheng Fan Zhang Xin Zhibin Qing ZhiQiang Liu Zhongwei Liu Zongli Shen H.X. Lii Heqing Li Liu Hui Li Xuyu Hongbing Tang Zhiyong He Kangxiong Wu B.Q. Li Jijun Yi X.Y. He Wangming Song Yuan Xiang Ail Zhou Zengzi Wei Richen Lou Xiao mei Run Qing Renjie Longkeju Wanglaowu Jian Ma Zhian Wang Xu Jia Desun Liu Juan Liu Xiangdong Zeng B.H. Xu Yideng Qinguang Zen Lian Wei Mingshun Li An He Qihua Chen

We report here on the genomic organization and expression of a nuclear gene coding for a plastid ribosomal protein. The gene encodes the plastid-specific ribosomal protein S22 (formerly named CS-S5). Southern blot analysis suggests that the gene is present in one copy in the spinach genome. The gene consists of 5 exons of sizes ranging from 108 to 273 bp and of 4 introns of 1410, 92, 386 and 82 bp. The exon-intron splice junctions and intron branch sites fit well the consensus sequences for plant introns. The major transcription start site has been determined 29 bp upstream of the AUG initiation codon by primer extension and S1 nuclease mapping. No canonical TATA box is found but some other possible promoter motifs are observed. Transcripts are detected in leaves, etiolated leaves, roots and seeds suggesting that the rps22 gene is expressed constitutively. During germination a marked increase in the relative steady-state level of the mRNA can be seen as soon as 24 h after imbibition of the seeds.

Plastid lipid-associated proteins of the fibrillin type have been found in a number of plants, but beyond their structural role in carotenoid-storing fibrils in chromoplasts, their functions remain poorly understood. We have identified 13 genes encoding members of this family in Arabidopsis. Some genes can be grouped in subfamilies of closer sequence similarity. Fibrillin polypeptides share typical signa- tures and potential functional domains, but appear quite diverse when other parameters are considered, namely their molecular weight, pI, or hydrophobic profiles. Most of these genes show differential expres- sion when stress- or organ-related expression is examined. An evolutionary scheme for fibrillin-related genes can be proposed. Three Arabidopsis fibrillin genes originate from relatively recent duplications, and are not found in the rice genome, which has ten orthologues. Four genes are found in a red alga. A diverse situation is found in cyanobacteria. ! -cyanobacteria have one orthologue of one plant gene and, in some species, a second gene (without plant orthologue). -Cyanobacteria have either one or two members of a third gene subfamily (without plant orthologue) or no such gene at all (in species adapted to low light). This suggests that plants multiplied their fibrillin genes from an ancestral gene that still has a relative in some cyanobacteria.

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5′-end and the long 3′-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.

The chloroplast chromosome of spinach (Spinacia oleracea) is a double-stranded circular DNA molecule of 150,725 nucleotide pairs. A comparison of this chromosome with those of the three other autotrophic dicotyledons for which complete DNA sequences of plastid chromosomes are available confirms a conserved overall structure. Three classes of open reading frames were distinguished: (1) genes of known function which include 108 unique loci, (2) three hypothetical chloroplast reading frames (ycfs) that are highly conserved interspecifically, and (3) species-specific or rapidly diverging 'open reading frames'. A detailed transcript study of one of the latter (ycf15) shows that these loci may be transcribed, but do not constitute protein-coding genes.

In order to study the regulation of nuclear genes coding for plastid ribosomal proteins, we have analysed the promoter region of spinach rps22 using both in vitro and in vivo approaches. By footprinting analyses, we have identified eight DNA elements interacting with spinach leaf nuclear factors in the 300 bp promoter region upstream of the transcription start site. Among these elements, four are short AT-rich sequences and one is identical to the Hex motif characterized initially in wheat histone genes. In transgenic tobacco plants, the reporter gene coding for the beta-glucuronidase (GUS) directed by a 1.2 kb upstream region of rps22 was expressed in several plant organs, with high levels in leaf mesophyll, embryo cotyledons and root meristematic cells and very low levels in other cell types. Interestingly, when deleted to -295, the promoter, which contained all the foot-printed elements, was still able to confer the same expression pattern, although the activity was relatively lower than with the 1.2 kb promoter. When deleted further to -154, the promoter, from which the AT-rich elements were eliminated, loses its activity almost completely, suggesting that these AT-rich elements are important for the rps22 promoter activity. Altogether, our results show that rps22 gene expression is controlled by specific cis elements not present in other nuclear-encoded plastid ribosomal protein genes studied so far.

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 bp to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 bp upstream of the ATG initiation codon to 9 bp downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes form a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

A clone containing a gene coding for a novel glycine-rich protein has been identified in an Arabidopsis thaliana genomic library. The gene codes for a 339 amino acid protein and is interrupted by a 686 bp intervening sequence. The gene is present in one copy only. Transcripts accumulate mainly in hypocotyls and stems and the highest level is observed in rosettes before the start of stem elongation. The protein contains 71% of glycine residues and is highly hydrophobic.

The nucleotide sequence and the 5′ flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria the P. littoralis rbcL gene is more closely related to that of a β-purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996–4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599–608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.