Patent Application
20110314573
The present invention relates to a novel method for screening proteins related to and/or involved in plant cell cycles. It further relates to proteins isolated with the method and the use of those proteins and/or the genes encoding those proteins for modulating plant yield and plant growth.
Knowledge of the basic cell cycle machinery is a prerequisite to grasp how signaling pathways impinge on and regulate cell proliferation during plant growth and development in a changing environment. The fundamental underlying mechanisms of cell division are conserved among all eukaryotes, however, due to their sessile lifestyle, plants have evolved unique features. Plant genome sequence analysis revealed the existence of an unexpected high number of genes involved in cell proliferation (Capron et al., 2003; Vandepoele et al., 2002; Menges et al., 2005; Schultz et al., 2007), compared to other organisms. Microarray analysis showed that a lot of these follow a cell cycle-dependent expression profile (Menges et al., 2005), sustaining their role in cell cycle regulation.
To elucidate which molecular machines are involved in plant cell division, we isolated cellular complexes by TAP from Arabidopsis thaliana cell suspension cultures (Van Leene et al., 2007; Van Leene et al., 2008) using “core” cell cycle proteins as baits. The dataset was corrected for non-specific interactions and divided in a “core” dataset of interactions that were biologically confirmed in at least two independent repeats, and a “non-core” dataset. Surprisingly, we found that the datasets, apart from known cell cycle proteins, were also comprising proteins of which the role in cell cycle never has been illustrated before.
The robustness of both the core and non-core datasets is demonstrated through different computational analysis and through the biological interpretation of the network. The interactome serves as an excellent hypothesis-generating tool, and the power of it is reached in particular when integrated with other data. Combining our interactome data with cell cycle-related expression profiles, for example, gives insight in which CDK/cyclin complexes are active during cell division and when. The data prove that the high numbers of cell cycle regulators in plants are not the sole consequence of redundancy, but that different cell cycle regulators are combined in complexes providing functional diversity leading to increased complexity and flexibility of the plant cell cycle.
Disclosed herein is a method to isolate novel cell cycle-related proteins, comprising (1) performing a tap analysis using a known cell cycle protein as bait, (2) correcting the results for non-specific interactions, and (3) reconfirming the corrected results. “Reconfirming the result,” as used here, can be done either by repeating the tap-tag experiment or by carrying out a reversed tap-tag, wherein the original prey is now used as bait.
Also disclosed herein is the use of a cell cycle-related protein isolated with the method of the invention for the modulation of plant growth and/or yield. In a particular embodiment, the plant is a crop plant, preferably a monocot or a cereal, even more preferably it is a cereal selected from the group consisting of rice, maize, wheat, barley, millet, rye, sorghum and oats.
The use, as indicated here, is the use of the protein and/or the use of a nucleic acid encoding this protein, or the complement thereof It includes, but is not limited to, genomic DNA, cDNA, messenger RNA (including the 5′ and 3′ untranslated regions) and RNAi; the use can result, as a non-limiting example, in overexpression or repression of the expression of the gene. Overexpression or repression of expression of a target gene can be obtained by transfer of a genetic construct, intended for the overexpression or the repression of expression into a plant. The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is a fairly routine technique known to the person skilled in the art. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include, but are not limited to, agrobacterium-mediated transformation, the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection.
Preferably, the cell cycle-related proteins are selected from the group consisting of At1g56110, At3g17020, At3g21140, At5g25460, At5g60790, At4g38900, At3g49240, At5g24690, At1g06070, At4g34150, At1g20480, At5g20920, At3g15970, At5g13030, At1g01880, At5g07310, At2g46610, At1g10690, At3g04710, At3g24690, At4g16130, At2g05830, At1g29220, At1g55890, At1g60650, At1g70830, At2g43140, At1g77180, At5g18620, At5g02530, At5g14170, At1g52730, At2g33340, At1g03060, At3g62240, At4g38740, At5g61220, At3g53880, At3g56860, At1g01970, At1g19520, At1g14620, At2g03820, At3g01280, At3g56690, At5g41190, At5g03740, At1g42440, At2g28450, At1g09760, At1g10840, At3g11830, At5g54900, At1g31760, At1g61870, At3g11760, At1g05805, At1g29200, At4g13850, At4g38780, At1g71380, At3g13640, At5g25060, At1g43700, At2g46020, At3g55760 and At5g21160 or a variant thereof. “Variants,” as used here, are including, but not limited to, homologues, orthologues and paralogues of the cell cycle-related proteins. “Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene. Preferably, the homologue, orthologue or paralogue has a sequence identity at a protein level of at least 50%, 51%, 52%, 53%, 54% or 55%, 56%, 57%, 58%, 59%, preferably 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, more preferably 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, even more preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more as measured in a BLASTp (Altschul et al., 1997; Altschul et al., 2005).
Preferably, the use is overexpression of the gene encoding the cell cycle-related protein, even more preferably it is overexpression of a cell cycle-related protein according to the invention, selected from the group consisting of At1g56110, At3g17020, At3g21140, At5g25460, At5g60790 (SEQ ID NOS:1-5), or a variant thereof. Preferably, overexpression results in an increase of plant growth and/or yield. Increase of plant growth and/or yield is measured by comparing the test plant, comprising a gene used according to the invention, with the parental, non-transformed plant, grown under the same conditions as control. Preferably, increase of growth is measured as an increase of biomass production. “Yield” refers to a situation where only a part of the plant, preferably an economically important part of the plant, such as the leaves, roots or seeds, is increased in biomass. The term “increase” as used here means at least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to control plants as defined herein. “Increase of plant growth,” as used here, is preferably measured as increase of any one or more of total plant biomass, leaf biomass, root biomass and seed biomass. In one preferred embodiment, the increase is an increase in total plant biomass. In a preferred embodiment, the plant is a crop plant, preferably a monocot or a cereal; even more preferably, it is a cereal selected from the group consisting of rice, maize, wheat, barley, millet, rye, sorghum and oats.
Still another aspect of the invention is a novel cell cycle-related protein, isolated with the method according to the invention.
Cloning of transgenes encoding tag fusions under control of the constitutive Cauliflower tobacco mosaic virus 35S promoter, transformation of Arabidopsis cell suspension cultures, protein extract preparation, TAP purification, protein precipitation and separation were done as previously described (Van Leene et al., 2007). The adapted protocol used for purification of protein complexes incorporating GS-tagged bait is described elsewhere (Van Leen et al., 2008). For identification by mass spectrometry, minor adjustments were implemented compared to previously described protocols (Van Leene et al., 2007), as described below.
After destaining, gel slabs were washed for 1 hour in H2O, polypeptide disulfide bridges were reduced for 40 minutes in 25 mL of 6.66 mM DTT in 50 mM NH4HCO3 and, sequentially, the thiol groups were alkylated for 30 minutes in 25 mL 55 mM IAM in 50 mM NH4HCO3. After washing the gel slabs three times with water, complete lanes from the protein gels were cut into slices, collected in microtiter plates and treated essentially as described before with minor modifications (Van Leene et al., 2007). Per microtiter plate well, dehydrated gel particles were rehydrated in 20 μL digest buffer containing 250 ng trypsin (MS Gold; Promega, Madison, Wis.), 50 mM NH4HCO3 and 10% CH3CN (v/v) for 30 minutes at 4° C. After adding 10 μL of a buffer containing 50 mM NH4HCO3 and 10% CH3CN (v/v), proteins were digested at 37° C. for 3 hours. The resulting peptides were concentrated and desalted with microcolumn solid phase tips (PerfectPure™ C18 tip, 200 nL bed volume; Eppendorf, Hamburg, Germany) and eluted directly onto a MALDI target plate (Opti-TOFTM384 Well Insert; Applied Biosystems, Foster City, Calif.) using 1.2 μL of 50% CH3CN:0.1% CF3COOH solution saturated with α-cyano-4-hydroxycinnamic acid and spiked with 20 fmole/μL Glu1 Fibrinopeptide B (Sigma Aldrich), 20 fmole/μL des-Pro2-Bradykinin (Sigma Aldrich), and 20 fmole/μL Adrenocorticotropic Hormone Fragment 18-39 human (Sigma Aldrich).
A MALDI tandem MS instrument (4700 and 4800 Proteomics Analyzer; Applied Biosystems) was used to acquire peptide mass fingerprints and subsequent 1 kV CID fragmentation spectra of selected peptides. Peptide mass spectra and peptide sequence spectra were obtained using the settings essentially as previously presented (Van Leene et al., 2007). Each MALDI plate was calibrated according to the manufacturers' specifications. All peptide mass fingerprinting (PMF) spectra were internally calibrated with three internal standards at m/z 963.516 (des-Pro2-Bradykinin), m/z 1570.677 (Glu1-Fibrinopeptide B), and m/z 2465,198 (Adrenocorticotropic Hormone Fragment 18-39) resulting in an average mass accuracy of 5 ppm±10 ppm for each analyzed peptide spot on the analyzed MALDI targets. Using the individual PMF spectra, up to sixteen peptides, exceeding a signal-to-noise ratio of 20 that passed through a mass exclusion filter, were submitted to fragmentation analysis.
PMF spectra and the peptide sequence spectra of each sample were processed using the accompanied software suite (GPS Explorer 3.6, Applied Biosystems) with parameter settings essentially as previously described (Van Leene et al, 2007). Data search files were generated and submitted for protein homology identification by using a local database search engine (Mascot 2.1, Matrix Science). An in-house non-redundant Arabidopsis protein database called SNAPS Arabidopsis thaliana version 0.4 (SNAPS=Simple Non-redundant Assembly of Protein Sequences, 77488 sequence entries, 30468560 residues; available at WorldWideWeb.ptools.ua.ac.be/snaps) was compiled from nine public databases. Protein homology identifications of the top hit (first rank) with a relative score exceeding 95% probability were retained. Additional positive identifications (second rank and more) were retained when the score exceeded the 98% probability threshold. Because identifications were done with different versions of the SNAPS database (Van Leen et al., 2007), and with the goal to obtain more uniformity between the identifications, all identifications from the core and the non-core dataset were resubmitted to Mascot and identified with the protein sequence repertoire from the latest TAIR database (TAIR8.0). Furthermore, an additional restriction was implemented to reduce the number of false positive identifications and, as so, identifications for which more than 50% of the corresponding peptides had a trypsin miss-cleavage, were discarded.
Analysis of over- and under-representation of GO terms was done with the BiNGO tool (Maere et al., 2005) in Cytoscape (Shannon et al., 2003). The hypergeometric test was chosen at a significance value of 0.05 with the Benjamini and Hochberg False Discovery Rate Correction for multiple testing. The Arabidopsis gene annotation file used in the analysis was downloaded from the gene ontology website on the 4th of October 2008.
For the periodic gene enrichment analysis, a list of 1258 genes showing cell cycle-regulated and cell cycle-associated expression was compiled from two datasets (Menges et al., 2003; Jensen et al., 2006). Genome wide corresponds to all 23834 genes present on the Affymtetrix ATH1 microarray. Genes containing E2F or M-specific activator (MSA) motifs in their promoter sequence were in silico determined by combining transcript expression data and comparative genomics (Vandepoele et al, 2006). Here, genome wide corresponds to 19173 genes for which a unique probe set is available on the ATH1 microarray. Proteins containing the CDK consensus phosphorylation site [ST]PX[KR], a known hallmark of CDK substrates (De Veylder et al., 1997), were considered as potential CDK substrates. The presence of the consensus motif was screened with the patmatch tool available at TAIR and, hence, genome wide corresponds here to all 27235 proteins present in the TAIR8.0 release. For all enrichment analysis, p-values were calculated with the hypergeometric cumulative distribution function of the Matlab 7.5 software. Proteins that could not be assigned to a specific gene locus were discarded from all enrichment analysis.
Overlap with Protein-Protein Databases
To assess the novelty of the cell cycle interactome, we screened for the overlap of our datasets with the following databases containing protein-protein interactions: TAIR (Huala et al., 2001), InTact (Kerrien et al., 2007), Arabidopsis Reactome (Tsesmetzis et al., 2008), AtPID (Cui et al., 2008), Reactome (Vastrik et al., 2007) and The Bio-Array Resource (BAR) for Arabidopsis Functional Genomics (Geisler-Lee et al., 2007).
Transcript Pearson Correlation Coefficients (PCC), representing the degree of co-expression of gene pairs, were calculated based on an Arabidopsis ATH1 micro-array compendium of 518 experiments focused towards cell cycle or plant growth and development (Table 7). We compared the PCC distribution of both datasets with the PCC distribution of 100 randomized datasets with an equal number of randomly chosen proteins and interactions.
The overexpressing constructs were produced by using gateway cloning technology. The cDNA of the genes of interest (table, sheet OE produced and LOF requested) were amplified by PCR from reverse transcribed RNA extracted from tissues of Arabidopsis thaliana ecotype Columbia. The PCR reactions were performed using the Phusion High fidelity DNA polymerase (Finnzymes) according to the manufacturer's instructions. The PCR fragments, corresponding to complete cDNA of the genes of interest were introduced into pDONr 201 using the Gateway system (Invitrogen) by attBXattP recombination sites and subsequently recombined into the pK7WG2 expression vector by attL XattR sites recombination. The sequence was confirmed by sequencing. The constructs containing the genes of interest under the control of the CaMV 35S promoter were used to transform Arabidopsis thaliana by the flowerdip method (Clough and Bent, 1998).
Transgenic lines were identified by selection on MS medium (half-strength Murashige and Skoog medium (Duchefa, Haarlem, The Netherlands), Sucrose 1%) supplemented with 50 mg/l kanamycin and later transferred to soil for seed production. A second selection on MS plus kanamycine allowed the selection of lines containing one site of insertion of the transgene. Plants were grown under a 16-hour day and 8-hour night regime at 21° C.
For the biomass measurement, the vegetative part of a 20-day-old plant grown on MS medium was harvested and the fresh weight was measured by weighing about 60 plants of each line.
As baits, we used 73 “core” cell cycle regulatory proteins (Vandepoele et al., 2002; Menges et al., 2006; Perez et al., 2007), four mitotic checkpoint proteins (Menges et al., 2005), eight anaphase promoting complex (APC) subunits and six APC activators (Capron et al., 2003), one 26S proteasome subunit (Brukhin et al., 2005), ten proteins involved in DNA replication or repair (Schultz et al., 2007), and as proof of concept, six proteins for reverse TAP experiments (Table 1). Of the 108 TAP fusions, 102 were expressed successfully. In total, 303 purifications were performed with at least two independent purifications per bait.
Purified proteins were identified via MALDI-TOFTOF. Non-specific proteins, determined by control purifications, were subtracted from the hit lists (Table 2), generating a non-redundant dataset of 857 interactions among 393 proteins. This dataset was divided in a “core” dataset of 371 interactions among 196 proteins, containing interactions that were biologically confirmed in at least two independent repeats or in the reciprocal experiment, and a “non-core” dataset with the remainder 486 interactions among 320 proteins.
To assess the quality of the interactome, we performed different enrichment analysis on the core and non-core preys. In both datasets, the GO term “cell cycle” was highly enriched (Table 3). Additional GO enrichments demonstrate that cell cycle is linked to a myriad of biological processes including growth and development, response to stress and hormone stimuli, energy production, chromatin remodeling and others. Next, we observed an enrichment in the core dataset for genes periodically expressed during cell cycle (
In a quest for new cell cycle-related proteins, we integrated different cell cycle-related features (Table 4). The distribution of the number of features per gene shows that a collection of known cell cycle genes (Table 5) is enriched for these features compared to the whole gene pool (
The robustness of the data is further exemplified by the observation that 46% of the core and 8% of the non-core dataset interactions are between baits, as our baits are supposed to act in common pathways. Screening our data for overlap with existing protein-protein interaction databases learned that 66% of the core and 95% of the non-core dataset interactions are new. On the other hand, this implicates that one-third of the core dataset is validated by other means. Finally, interactions from both datasets tend to be more co-expressed compared to interactions from randomized datasets as assessed by calculation of the transcript Pearson Correlation Coefficient (PCC) (
Key players in cell cycle progression are cyclin-dependent kinase (CDK) complexes. CDKA;1, the Arabidopsis ortholog of yeast cdc2/cdc28, co-purified with all tested D-type cyclins and with A3-type cyclins, but not with the mitotic A1-, A2- or B-type cyclins. Combining our interactome data with expression data (Menges et al. 2005), we speculate that at cell cycle reentry and early in G1-phase, CDKA;1 binds CYCD3;3 and CYCD5;1. Further on in G1-phase and at the G1/S checkpoint, CDKA;1 binds a variety of D-type cyclins, such as CYCD4;1, CYCD4;2, CYCD3;1, CYCD6;1 and CYCD7;1. In addition, CDKA;1 interacts with S-phase specific A3-type cyclins. The other A- and B-type cyclins, of which most possess a peak of expression at the G2/M-boundary, bind the plant-specific mitotic B-type CDKs. B1-type cyclins associate exclusively with B2-type CDKs, while the remainder A- and B-type cyclins preferentially bind B1-type CDKs. Although transient interactions are more difficult to screen with TAP, our interactome contains different potential CDK substrates. As predicted (Geisler-Lee et al., 2007), CDKA;1 is present as a highly connected hub in the core network. It co-purified the unknown protein AT4G14310, which was further present in complexes with CKS1, CKS2, CYCA3;1, CyCA3;4 and KRP2 and the reverse purification confirmed interaction with CDKA;1 and CKS2 and revealed interaction with the plant-specific kinesin motor protein KCA2 involved in division plane determination. Next, CDKA;1 was pulled down with the 26S proteasome complex, purified through RPN1a, possibly reflecting cell cycle regulation of the 26S proteasome. CDKA;1 further interacted with three proteins from the UDP-xylose biosynthesis pathway, coupling cell cycle regulation with cell wall synthesis (Siefert, 2004). With three A-type cyclins, we picked up a DNA repair protein and with CYCB1;3, we found γ-tubulin and a spindle pole body component, two proteins involved in microtubule nucleation during, e.g., assembly of the preprophase band, a plant-specific structure required for polarity determination during cell cycle (Erhardt et al., 2002). Furthermore, some interesting chromatin-remodeling proteins were identified with different cyclins: CHR17, an E2F-up-regulated ISWI protein (Huanca-Mamani et al., 2005) interacted with CYCD3;2 and CYCD5;1. CHC1 associated with CYCA1;1, CYCD7;1, CYCB2;3 and CKS2, and BRAHMA, a SWI/SNF chromatin-remodeling ATPase implicated in the formation and/or maintenance of cotyledon boundary cells during embryogenesis (Kwon et al., 2006), was identified with CYCB1;3 and CYCB2;3.
For full activity, CDKs require, next to cyclin binding, phosphorylation of a threonine residue within the T-loop by CDK-activating kinases (CAK). The Arabidopsis genome encodes four CAKs, namely, three D-type CDKs, homologous to human CDK7, and one cyclin-independent CAK-activating kinase (CAKAK) CDKF;1. Here, we show that both CDKD;2 and CDKD;3 form a trimeric complex with CYCH;1 and the CAK assembly factor MAT1. Like in rice (Rohila et al., 2006), CDKD;2 is also part of the basal TFIIH complex involved in transcription and DNA repair, as three members co-purified (UVH6/XPD, AT1G55750 and AT4G17020). In this complex, CDKD;2 activates transcription through phosphorylation of the C-Terminal Domain (CTD) of RNA polymerase II. With UVH6 and MAT1 as baits, we confirmed interaction with CDKD;2 and purified two more proteins of the TFIIH complex. CDKD;2 further co-purified proteins involved in nucleotide biosynthesis, namely, three ribose-phosphate pyrophosphokinases. More upstream, the monomeric CAKAK CDKF;1 activates CDKD;2 in a cyclin-independent manner. On the other hand, CDKF;1 also binds CDKG;2. The G-type CDK class has two members in Arabidopsis, and is homologous to the human cytokinesis-associated p58 galactosyltransferase protein. Here, we discovered CYCL1, a cyclin with a SR-like splicing domain (Forment et al., 2002), as the regulatory cyclin partner of both G-type CDKs, validating the clustering of CYCL1 with CDKG;2 in a tissue-specific gene expression analysis (Menges et al., 2005). Both core and non-core interacting proteins hint for a function of CDKG/CYCL complexes in regulation of transcription and splicing, so activation of CDKF;1 could lead to altered splicing events during cell proliferation.
Negative regulation of cell cycle progression is achieved by docking of small proteins to the CDK/cyclin complexes. Arabidopsis encodes seven proteins related to the mammalian Kip/Cip inhibitors, known as Kip-related proteins (KRPs). Here, we show that all KRPs, except KRP1, interact with both CDKA;1 and D-type cyclins. With three KRPs, CDKB1;2 and two APC activators, we found an ethylene responsive AP2 transcription factor (TF), and with KRP2 we picked up a bZIP TF also found with B-type cyclins and CDKB1;2. In plants, a second family of cell cycle inhibitor proteins exist that are up-regulated by abiotic and biotic stress, comprising SIAMESE (SIM) and SIAMESE-Related (SMR) proteins (Peres et al., 2007; Churchman et al., 2006). SIM is a nuclear protein promoting endoreduplication in trichomes by suppression of mitosis. It was proposed that it inhibits mitosis through inhibition of CDKA;1/CYCD complexes (Churchman et al., 2006). In our dataset, however, SIM co-purifies CDKB1;1 and not CDKA;1, so endoreduplication may be triggered directly by inhibiting mitotic CDKB/cyclin complexes. Next to SIM, also SMR1 and SMR2 associate with CDKB1;1, and the CDKB1;1 interactor CYCB2;4 binds AT2G28330, an additional member of the SMR family. In contrast, SMR3-5 bind CDKA;1 and D-type cyclins. Besides, with CDKA;1 and different D-type cyclins as bait, we picked up two new members of the SMR clan, AT5G40460 and AT1G10690, and reverse purifications confirmed these interactions. As AT5G40460 was almost 20-fold induced in plants overexpressing E2Fa and DPa (Vandepoele et al., 2005), it may inhibit CDKA;1/CYCD complexes during S-phase preventing re-initiation of DNA replication. SMR1 further co-purified bZIP69, a TF also found with KRP3 and KRP5. Importins often co-purified both with KRPs and SMRs, supporting the importance of the regulation of their subcellular localization for their activity.
At the G1/S boundary, CDKs activate the E2F/DP pathway by phosphorylation of the repressor RBR, inducing transcription of genes mainly involved in nucleotide synthesis, DNA replication and DNA repair. We demonstrate that E2Fa and E2Fb can associate both with DPa and DPb, and that all E2F and DP proteins co-purify RBR. Since CDKB1;1 interacted with DEL3, an atypical E2F protein lacking the trans-activation domain, we propose that DEL3 is regulated by CDKB1;1 activity, consistent with a second expression peak of DEL3 at G2/M (Menges et al., 2005). Interestingly, the mitotic CDKB1;1, and not CDKA;1, co-purified with RBR, providing further evidence that the E2F/DP/RBR network is not only active at G1/S but also at G2/M transitions, as was previously suggested in plants (Magyar et al., 2005) and mammalian cells (Ishida et al., 2001), or that mitotic CDK/cyclin complexes are active during S-phase as in yeast (Wuarin et al., 2002). We further identified some complexes involved in DNA replication, like the MCM complex, possessing helicase activity for unwinding of double-stranded DNA during DNA replication. This complex was isolated with MCM6 as bait, together with the recently published (Takahashi et al., 2008) and highly co-expressed E2F-target gene 1 (ETG1). The co-purified fraction of proliferating cell nuclear antigen 1 (PCNA1), a sliding clamp for DNA polymerase and thus a key actor in DNA replication, contained PCNA2, two DNA polymerase delta subunits (POLD1-2), of which one also interacted with CYCA2;3, an armadillo/beta-catenin repeat family of unknown function and a DNA binding protein. Furthermore, we prove the existence of the alternative Ctf18 replication factor C complex in plants, required for sister chromatid cohesion in yeast (Mayer et al., 2001) and a protein complex involved in stabilization of single-stranded DNA during replication, repair and transcription, including RPA2, two RPA3 proteins and a putative replication protein (Schultz et al., 2007).
Thirty-two genes were cloned into expression vectors using Gateway cloning to produce plants overexpressing the genes of interest under the control of the CaMV35S promoter (table, sheet OE produced and LOF requested). These constructs were used for Arabidopsis thaliana transformation using the flower dip method. Primo-transformants were selected for all the constructs and grown for seed production. The seeds from eleven constructs (At5g25460, At3g01280, At1g31760, At3g21140, At3g17020, At1g05805, At1g10690, At1g56110, At5g24690, At5g60790, At1g09760) were harvested and used to select lines having one site of insertion of the transgene. A biomass test has been carried out on these segregating populations containing one insertion site. In this population, it is expected to find ¼ of wt, 2/4 of hemizygous and ¼ of homozygous plants. When the overexpression of a transgene leads to the production of larger plants, a fraction (¼ or ¾) of the plants analyzed will show an increased biomass. This method allows a fast screening of genes of which the overexpression gives a positive effect on plant growth. Plants were then grown under in vitro condition and the rosette fresh weight of approximately 60 plants was measured 20 days after stratification (table, sheet OE data+average). The control plants used in this experiment correspond to segregating plants transformed with an empty vector (C1 and C2). As shown in
Arabidopsis thaliana.
thaliana}
laevis}; contains Pfam profiles PF02483: SMC family C-terminal
sapiens)
sativa (japonica cultivar-group)) GI: 13516746; contains InterPro entry
sapiens, EMBL: U02609
THALIANA] (GB: Q9FG23); SIMILAR TO OS06G0320100 [ORYZA
SATIVA (JAPONICA CULTIVAR-GROUP)] (GB: NP_001057510.1);
SATIVA (JAPONICA CULTIVAR-GROUP)] (GB: BAD13052.1);
THALIANA] (TAIR: AT5G48600.1
THALIANA)|CHR3: 18476664-18477361 FORWARD|ALIASES:
CRUZI) GI: 12005317; CONTAINS PFAM PROFILE PF00134:
CRUZI) GI: 12005317; CONTAINS PFAM PROFILE PF00134:
sativa) GI: 13536993; contains Pfam profile PF00098: Zinc knuckle
laevis, SWISSPROT: XCPE_XENLA
CRUZI) GI: 12005317; CONTAINS PFAM PROFILE PF00134:
CRUZI) GI: 12005317; CONTAINS PFAM PROFILE PF00134:
SAPIENS)|CHR5: 9311885-9315478 REVERSE|ALIASES: NONE
Agrobacterium induced tumor vs control; Tumour tissue
Agrobacterium tumefaciens
Agrobacterium induced tumor vs control; Inflorescence
| Number | Date | Country | Kind |
|---|---|---|---|
| 08171187.1 | Dec 2008 | EP | regional |
This is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2009/066856, filed Dec. 10, 2009, published in English as International Patent Publication WO 2010/066849 A1 on Jun. 17, 2010, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 08171187.1, filed Dec. 10, 2008.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP09/66856 | 12/10/2009 | WO | 00 | 8/31/2011 |
