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Since the publication of draft sequences for the human and mouse genomes, several groups have run large-scale comparisons of the sequences to detect regions of conserved sequence. An initial survey of these was published along with the draft mouse genome , with additional comparisons appearing since then . Briefly, protein coding genes are – as we might expect – among the most strongly conserved regions, but homologous sequences can be found throughout the genome. In total, it is possible to align up to 40% of the mouse genome to human sequence , but it seems likely that at least some of this is just random "comparative noise" – regions of sequence which serve no particular purpose but which, purely by chance, have not yet accumulated enough mutations to make their evolutionary relationship unrecognisable. However, it is widely accepted that some of the noncoding-but-similar regions, especially those with the highest levels of sequence identity between the two species, are preferentially conserved because they perform some important function. It has been estimated that around 5% of the genome is under purifying selection , indicating that mutations in these regions have deleterious effects: a strong suggestion of some important function. | 15369604_p0 | 15369604 | Background | 4.404693 | biomedical | Study | [
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Here, we apply the Eponine Windowed Sequence (EWS) sequence analysis method method which uses a Relevance Vector Machine (RVM) to extract a minimal set of short motifs which are able to discriminate between two sets of sequences: in this case, a positive set of conserved non-coding sequences and a negative set of randomly picked non-coding sequences. The EWS model is an adaption of the Eponine Anchored Sequence (EAS) model, first applied for transcription start site prediction in and subsequently used to predict a range of additional biological features including translation start sites and transcription termination sites [A. Ramadass, unpublished] While EAS is designed to classify individual points in a sequence – a feature which allows the model to predict precise locations for features such as transcription start sites – EWS classifies complete blocks (windows) of sequence. The basis functions (inputs) of the RVM are sums of position-weight matrix scores across the whole window. | 15369604_p1 | 15369604 | Background | 4.207819 | biomedical | Study | [
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We considered a set of alignments made by the blastz program between release NCBI33 of the human genome and release NCBIM30 of the mouse genome. Since unprocessed blastz aligns around 40% of human sequence to the mouse genome, we chose to focus on the 'tight' alignments. These are a subset of alignments which are rescored and thresholded using a set of parameters given in , and cover only around 5.6% of the human genome – a proportion much closer to the fraction of bases thought to be under purifying selection . | 15369604_p2 | 15369604 | Results | 4.083272 | biomedical | Study | [
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In total, the tight blastz set contained 787173 blocks of sequence with high-scoring alignments between the two genomes. We considered only those blocks assigned to human chromosome 6, a 170 Mb chromosome which has recently undergone manual annotation of gene structures and other features . This chromosome included 44105 (5.6%) of the total alignments. These varied in length from 34 to 9382 bases, with a length distribution skewed towards relatively short alignments, as shown in figure 1 . | 15369604_p3 | 15369604 | Results | 4.109158 | biomedical | Study | [
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Since we were interested in non-coding features of the genome, we ignored all regions where an alignment overlaps an annotated gene structure. This removed 20.8% of aligned bases. It is possible that some genes, and especially pseudogenes, have been missed by the annotation process, so we also removed portions covered by ab initio gene predictions from the Genscan program . This eliminated an additional 4.3% of aligned bases. Finally, repetitive sequence elements annotated by the programs RepeatMasker and trf (5.9%) were removed from the working set. The remainder of the aligned regions were split into non-overlapping 200 base windows, ignoring any portions less than 200 bases. This gave a set of 13925 sequences which are well-conserved between human and mouse – and therefore likely to be functional – but which are very unlikely to be part of the protein-coding repertoire. These formed the positive training set for our machine learning strategy. | 15369604_p4 | 15369604 | Results | 4.135834 | biomedical | Study | [
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A negative training set of equal size was prepared by picking 200-base windows at random from the non-coding, non-repetitive portions of chromosome 6, using the same criteria to define repeats and coding sequence. While it is probable that this set also included some functional sequences, we would expect them to be represented at a substantially lower level than in the conserved set. | 15369604_p5 | 15369604 | Results | 3.802903 | biomedical | Study | [
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These two sets of sequence were presented to the Eponine Windowed Sequence machine learning system, as described in the methods section. Randomly chosen 5-base words were used as seed motifs, and three independent training runs were performed, each for 2000 cycles. The set of motifs used in model 1 is shown in table 1 . | 15369604_p6 | 15369604 | Results | 3.006421 | biomedical | Study | [
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While the exact set of motifs used in the model varied somewhat from run to run, testing pairs of models on non-overlapping windows from a 1 Mb region of human chromosome 22 and plotting the scores showed that the model outputs were highly correlated . We calculated the Pearson correlation coefficient for all pairs, and in all cases this was greater than 0.96. From this strong correlation, we concluded that any variations in the model were simply the result of the trainer picking one representative from a group of motifs which provide similar information. | 15369604_p7 | 15369604 | Results | 3.993884 | biomedical | Study | [
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We scanned genomic sequences using these models at a range of thresholds, and examined the results on the Ensembl genome browser using a Distributed Annotation System server. Visual inspection showed that many of the highest-scoring regions were localised near the start of genes. This prompted us to look at the distribution of high-scoring sequences with respect to the starts of a set of well-annotated genes. We considered the GD_mRNA genes from version 2.3 of the human chromosome 22 annotation. These are confidently annotated genes with experimental evidence as described in , which confirms at least the approximate location of the ends of the transcripts, and are independent from the chromosome 6 training data. Figure 3 shows the density of predictions with EWS scores ≥ 0.90 relative to the annotated 5' ends of these genes. This shows a strong peak of predictions close to the annotated starts, demonstrating that the model is predicting some sequences commonly located around the transcription start site of genes. Combining this observation with the fact that the model was trained from conserved (and therefore presumed functional) sequences, we believe that it is detecting signals found in the promoter regions of genes. | 15369604_p8 | 15369604 | Results | 4.193588 | biomedical | Study | [
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Evaluation of promoter-prediction methods on a large scale is a difficult exercise, since there are no large pieces of genomic sequence for which we can be certain we know the complete set of transcribed regions, and even in the case of well-known genes we often do not know the precise location at which transcription begins. In , we developed a pseudochromosome, derived from release 2.3 of the chromosome 22 annotation. As described above, this includes a subset of 284 experimentally verified gene structures. The pseudochromosome was constructed to include these genes while omitting all other annotated genes (which could be substantially truncated). We considered predictions (groups of one or more overlapping windows which all have scores greater than some chosen threshold) to be correct if they lie withing 2 kb of an annotated gene start, and false otherwise. Plotting accuracy (proportions of predictions which are correct) against coverage (proportion of transcript starts which are detected by one of the correct predictions) gives a Receiver Operating Characteristic (ROC) curve. Using this criterion, a totally random set of predictions would be given an accuracy of around 0.07. ROC curves are plotted for the three independently trained models in figure 4 . Firstly, this shows that predictive performance for all three models is rather similar. It also shows that they can function as accurate promoter predictors, with accuracy rising to a plateau of around 0.7, much higher than expected for random predictions. | 15369604_p9 | 15369604 | Results | 4.15755 | biomedical | Study | [
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We picked model 1 for further study. Using a score threshold of 0.91, this gives an accuracy of 0.68 and a coverage of 0.31. We compared the set of genes correctly detected by this model to two other methods: firstly, the EponineTSS predictor described in , and secondly, the published results from the PromoterInspector program . PromoterInspector results were mapped to pseudochromosome coordinates using the procedure described in . Figure 5 shows how the set of promoters detected by these three distinct methods overlaps. There are clearly strong correlations between all three methods. In particular, at this threshold the EWS homology model detects 98 promoters which were found by at least one of the other methods, but only 4 novel promoters. | 15369604_p10 | 15369604 | Results | 4.101628 | biomedical | Study | [
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We investigated the robustness of the signal learned by this process by retraining models with a variety of seed word sizes, from 2 to 6 bases. During training, motifs can be trimmed to lengths shorter than that of the seed words (down to a minimum of 2 bases) but can never grow longer than the seed word size. When evaluated on the pseudochromosome, the resulting models always showed a preference for regions around gene starts, regardless of word length, as shown in figure 6 . However, the accuracy was reduced when using short seed words – particularly words of length of 2. The best accuracy was seen for a seed word length of 5, and decreased somewhat for words of length 6. | 15369604_p11 | 15369604 | Results | 4.075315 | biomedical | Study | [
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This suggests that a large fraction (but not all) of the information learned by these models can be encoded in dinucleotide frequencies. It is well known that many transcription start sites are close to regions of relatively high CpG dinucleotide composition (CpG islands) . To investigate the contribution that CpG dinucleotides make to our models, we deleted all CpG dinucleotides from the training data, then re-evaluated the resulting models on the pseudochromsome (also with CpG dinucleotides removed), as shown in figure 7 . Perhaps not surprisingly, dinucleotide models now show very little tendency to detect gene starts. However, as the word size increases, the preference for gene starts gradually increases, until a seed size of 6 gives an accuracy comparable to that see when CpG dinucleotides are included, although the maximum coverage before accuracy begins to drop rapidly is somewhat lower. Broadly similar results are seen if CpG dinucleotides are randomly replaced with other dinucleotides. | 15369604_p12 | 15369604 | Results | 4.104287 | biomedical | Study | [
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We have shown here that, when presented with a set of non-coding sequences which are strongly conserved between human and mouse, a simple motif-oriented machine learning system consistently builds models which are able to detect a substantial fraction of human promoter regions with good accuracy. This strongly suggests that this promoter signal represents the most widely used motif-based signal in functional non-coding sequence. While the model learned here can clearly be applied for the purpose of genome-wide promoter annotation, in practise existing methods offer better coverage and (in the case of the EponineTSS predictor) predictions for the precise location of the transcription start site. | 15369604_p13 | 15369604 | Conclusions | 4.131089 | biomedical | Study | [
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It is interesting that the promoter model learned by this technique detected substantially the same set of promoters as found by the EponineTSS and PromoterInspector methods. It has previously been remarked that these two methods detect similar sets , but this could perhaps be explained by the fact that both methods were initially derived from similar sets of known promoter sequences (in both cases, training data was extracted from the EPD database . In the case of the homology models described here, there is no connection with EPD, or any similar set of known promoters: the training data was picked purely on the basis of its high similarity to corresponding portions of the mouse genome. These results therefore support the alternate view that there is a particular 'easily detected' subclass of promoter sequences. | 15369604_p14 | 15369604 | Conclusions | 4.135934 | biomedical | Study | [
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One distinct group of promoters, which previous results show may correspond to this easily detected family, is the set of promoters associated with CpG islands . However, while a number of the motifs listed in table 1 are G/C rich and/or contain the CpG dinucleotide, by no means all of the motifs match this description, and indeed one motif containing CpG has a negative weight in the linear model – its presence in a sequence will reduce the model's output score – while some A/T rich motifs have positive weights. We therefore believe that the signals detected here are significantly more complex than a simple over-representation of CpG dinucleotides. Experiments with smaller seed-word sizes support this assumption: while dinucleotide-based models were also able to predict promoter regions, the accuracy was lower than for models including longer motifs. Finally, we show that while the predictive capacity of dinucleotide models is largely eliminated once CpG dinucleotides are removed from the sequence, models including longer words are still able to make correct promoter predictions in many cases. So while CpG dinucleotides are an important contribution to the promoter signal, they are clearly not the only component. | 15369604_p15 | 15369604 | Conclusions | 4.253947 | biomedical | Study | [
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Human genome sequence release NCBI33 and mouse genome release NCBIM30 were extracted from Ensembl databases , which also contained gene predictions from Genscan and repeat data from RepeatMasker and trf . Curated annotation of gene structures on human chromosome 6 was obtained from the Vega database . Vega and Ensembl data was extracted directly from the SQL databases using the BioJava toolkit with biojava-ensembl extensions . | 15369604_p16 | 15369604 | Genomic sequence and annotation | 4.105514 | biomedical | Study | [
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Human-mouse genome alignments were generated by the blastz alignment program. These were subsequently re-scored and filtered to give a 'tight' set of high-confidence alignments, as described in . We downloaded the tight alignment set from the UCSC genome website . | 15369604_p17 | 15369604 | Genome alignments | 3.715925 | biomedical | Study | [
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A 16.3 Mb pseudochromosome sequence was produced based on version 2.3 of the curated annotation for human chromosome 22. This includes all the experimentally-validated gene structures and their upstream regions, while omitting regions containing genes that are predicted but not fully verified. In the case of a pair of divergent genes where one has been verified and the second has not, their shared upstream region was cut at the midpoint. More information about pseudochromosome construction is given in . | 15369604_p18 | 15369604 | Pseudochromosome for testing promoter-finding methods | 4.167931 | biomedical | Study | [
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The Eponine Windowed Sequence (EWS) model is designed by analogy to the Eponine Anchored Sequence model first described in , but rather than targeting individual points in the sequence, it is designed to classify small regions or windows of a sequence, based purely on their own sequence content. | 15369604_p19 | 15369604 | Eponine Windowed Sequence learning | 3.175628 | biomedical | Other | [
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The EWS model uses the Relevance Vector Machine algorithm to drive the training process. Relevance Vector Machines solve classification and regression problems by building Generalised Linear Models (GLMs) as weighted sums of a "working set" of basis functions. During the training process, those basis functions which are not informative are given weights close to zero and eventually discarded from the working set. To explore very large sets of possible basis functions, it is possible to add extra basis functions during the course of the training process . | 15369604_p20 | 15369604 | Eponine Windowed Sequence learning | 3.0407 | biomedical | Other | [
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The "sensors" of the EWS model are DNA position-weight matrices , which make convenient models of short sequence motifs. When using weight matrices to analyse sequence windows, we sum the weight matrix probability scores for all possible positions within the sequence. Normalising for the length of the sequence being inspected and the size of the PWM, the basis functions of the model take the form: | 15369604_p21 | 15369604 | Eponine Windowed Sequence learning | 3.87899 | biomedical | Other | [
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where W ( s ) is the probability that sequence s was emitted by weight matrix W , | S | is the sequence length, | W | is the weight matrix length, and denotes a subsequence from i to j . | 15369604_p22 | 15369604 | Eponine Windowed Sequence learning | 3.226629 | biomedical | Other | [
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An initial set of basis functions is proposed by taking all possible DNA motifs of a specified length (typically 5) and generating weight matrices which preferentially recognise these motifs. As the relevance vector machine trainer removes non-informative basis functions from the working set, they are replaced by applying one of the following sampling strategies to a basis function picked randomly from the working set: | 15369604_p23 | 15369604 | Eponine Windowed Sequence learning | 3.890726 | biomedical | Study | [
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• Generate a new weight matrix in which each column is a sample from a Dirichlet distribution with its mode equal to the weights in the corresponding column of the parent weight matrix. | 15369604_p24 | 15369604 | Eponine Windowed Sequence learning | 2.41156 | biomedical | Other | [
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• Generate a new weight matrix one column shorter than the parent by removing either the first of the last column. | 15369604_p25 | 15369604 | Eponine Windowed Sequence learning | 1.393535 | other | Other | [
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By using these sampling rules, the trainer is able to explore motif space. The process of generating candidate motifs using these rules then selecting the most informative using the RVM can be seen as a form of genetic algorithm. | 15369604_p26 | 15369604 | Eponine Windowed Sequence learning | 2.300513 | biomedical | Other | [
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TD and TH conceived and designed this study, and analysed results. TD implemented the Eponine machine learning system and drafted the manuscript. All authors read and approved the final manuscript. | 15369604_p27 | 15369604 | Authors' contributions | 0.954128 | other | Other | [
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Development of eukaryotic cells towards particular cell fates is regulated by complex and dynamic changes in gene expression. These changes, when monitored on a genome-wide scale, provide a detailed framework for the analysis and modeling of cellular development. To monitor patterns of gene expression it is important to be able to isolate cells at precise stages along a developmental pathway. Well-developed procedures for cell culture and single-cell PCR techniques have allowed genome-wide changes in gene expression to be monitored during animal cell differentiation . | 15535861_p0 | 15535861 | Background | 4.029372 | biomedical | Study | [
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Transcriptomic studies of single cell types in plants have focused on diploid sporophytic cell types, including undifferentiated cell suspensions , leaf epidermal and mesophyll cells , stomatal guard cells and cultured mesophyll cells . These studies have provided valuable information about gene expression in single cell types; however, their coverage of the transcriptome has been limited and/or hampered by low RNA yields from individual cells, requiring mRNA preamplification steps that can bias the complementary RNA (cRNA) . Moreover, such studies have not involved the use of the most comprehensive tools for monitoring gene expression that are now available for Arabidopsis - which include the Affymetrix ATH1 gene arrays. Recently, a significant advance in transcriptome analysis of plant cell types has been achieved through fluorescence-activated cell sorting of cell-type marked and protoplasted root cells using Affymetrix ATH1 micorarrays . This has provided a near-comprehensive transcriptomic view of cell-fate determination at three developmental stages in five different domains of the root apex. | 15535861_p1 | 15535861 | Background | 4.364785 | biomedical | Study | [
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In contrast to such enabling technologies and procedures developed for sporophytic cell types there have been no studies that provide a genome-wide perspective of cell fate determination and differentiation during haploid gametophyte development. The haploid male gametophyte generation of flowering plants has a simple and well-defined pathway of development and consists of two- or three-celled pollen grains that deliver two sperm cells via the pollen tube to the embryo sac at fertilization. The highly reduced cell lineage and functional specialization of the male gametophyte are thought to be key factors in the reproductive fitness and evolutionary success of flowering plants. Moreover, pollen ontogeny provides an attractive model of cellular development in which to dissect the regulation of cell growth and division, cellular differentiation and intercellular communication (for reviews see ). | 15535861_p2 | 15535861 | Background | 4.207327 | biomedical | Review | [
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Recent progress in understanding of molecular and cellular aspects of pollen development has emerged from genetic studies that have identified mutants in Arabidopsis that affect all phases of pollen development . In parallel, cDNA libraries and databanks have been obtained for sperm cells in maize, lily, tobacco and Plumbago zelanica . Despite such advances there is limited information about developmental changes in gene expression associated with particular phases of male gametophyte development. Our objective was to develop procedures to enable the isolation of populations of microspores and developing pollen grains at precise developmental stages in Arabidopsis and to analyze changes in gene expression from unicellular microspores to mature differentiated pollen grains. A particular advantage of the male gametophyte generation is that developing microspores and pollen grains are symplastically isolated. This facilitates access to viable cell populations at different stages of haploid development without contaminating sporophytic cells. | 15535861_p3 | 15535861 | Background | 4.197473 | biomedical | Study | [
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Some initial progress has been made towards the definition of the male gametophytic transcriptome of Arabidopsis using serial analysis of gene expression (SAGE) and Affymetrix AG microarrays that harbor probes for approximately 8,000 different genes . These studies have provided valuable insight into the complexity of gene expression in mature pollen and the extent of overlap between male gametophytic and sporophytic gene expression (reviewed in ). However, these studies monitored the expression of only 30% of the annotated genes in Arabidopsis and analyzed mRNA populations only in mature differentiated pollen grains. These studies do not, therefore, provide a developmental perspective of gene expression during development and differentiation of the male gametophyte. | 15535861_p4 | 15535861 | Background | 4.144932 | biomedical | Review | [
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Here we describe spore isolation procedures for Arabidopsis and the use of Affymetrix ATH1 Genome Arrays to analyze transcript expression profiles throughout four successive stages of male gametophyte development in Arabidopsis . Isolated spore populations were large enough to enable RNA extraction for direct microarray hybridization without any preceding amplification step that could lead to bias in expression signals between stages or between genes within individual stages. Progression from proliferating microspores to terminally differentiated pollen was characterized by large-scale repression and the activation of a unique collection of late-program genes during pollen maturation. Putative male gametophyte-specific genes and distinct clusters of coexpressed genes are identified, including key groups of regulatory factors including cell cycle, transcription and translation factors. Bioinformatic and experimental data are used to address the importance of transcription and translation during pollen germination and tube growth | 15535861_p5 | 15535861 | Background | 4.233756 | biomedical | Study | [
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Transcriptome profiling throughout microgametogenesis in Arabidopsis required the introduction of a procedure for the isolation of homogeneous populations of viable spores at precisely defined stages of development. The method was based on centrifugation of isolated mixed spores in a Percoll step-gradient . Large homogeneous spore populations at three developmental stages were collected: uninucleate microspores (UNM), bicellular pollen (BCP) and immature tricellular pollen (TCP). In addition, a homogeneous mature pollen grain (MPG) population was isolated from open flowers according to Honys and Twell . | 15535861_p6 | 15535861 | Isolation and characterization of developing spores | 4.129242 | biomedical | Study | [
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Microscopic examination of isolated spore populations revealed no contaminating sporophytic cells and little or no other cellular debris . Vital staining revealed more than 90% viable spores at each stage (data not shown). The purity of spore populations was evaluated by DAPI staining . The UNM population was the most homogeneous, containing 95% uninucleate microspores, 2.5% tetrads and 2.5% bicellular pollen. The BCP population was 77% pure, but also contained some tetrads (3.5%), microspores (12%) and tricellular grains (7.5%). The TCP population comprised 88% tricellular pollen and 12% bicellular pollen. The MPG population was 100% pure with approximately 2% aborted pollen. | 15535861_p7 | 15535861 | Isolation and characterization of developing spores | 4.187575 | biomedical | Study | [
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Arabidopsis ATH1 Genome Arrays were used to explore the dynamics of gene expression throughout male gametophyte development in comparison with sporophytic tissues. Microarrays were hybridized with cRNA probes made from total RNA purified from isolated spores. Hybridization data from two biological replicates derived from independently grown populations of plants were compared. Only genes with a positive hybridization signal and a detection call value of 1 in both experiments were scored as expressed. Microarray data from each pair of replicates were highly correlated, with correlation coefficients of 0.986 (UNM), 0.972 (BCP), 0.991 (TCP) and 0.971 (MPG). Complete microarray data are publicly available at the European Arabidopsis Stock Centre (NASC) microarray database . Sporophytic ATH1 Genome Array datasets were downloaded from the NASC website . This provided transcriptome data for seedlings at open cotyledon stage (COT, stage 0.7 ), leaves (LEF, stage 6.0), petiole (PET, stage 3.9), stems (STM, stage 6.1), roots (ROT), root hair zone (RHR, stage 1.02), and suspension cell cultures (SUS). Genes that were consistently expressed in replicate sporophytic datasets were identified using the same algorithm used for gametophytic data. | 15535861_p8 | 15535861 | Developmental changes in the male gametophytic transcriptome | 4.1635 | biomedical | Study | [
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We have previously confirmed and validated the expression pattern of 15 putative pollen-specific genes identified using Affymetrix AG arrays by reverse transcription-PCR analysis . Similarly we validated the current ATH1 datasets by RT-PCR analysis in two separate experiments that included analysis of 41 genes encoding predicted glycosylphosphotidylinositol-anchored proteins (GAPs) and 16 cation/proton exchanger proteins . In both experiments the expression patterns of all genes tested that were identified as pollen-expressed, or pollen-specific by ATH1 analysis were confirmed by RT-PCR. | 15535861_p9 | 15535861 | Developmental changes in the male gametophytic transcriptome | 4.093526 | biomedical | Study | [
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The ATH1 Genome Array harbors oligonucleotide probes representing 22,591 genes based on the Arabidopsis Genome Initiative annotation. This represents 80.7% of the most recent estimate of 28,000 protein-coding genes in Arabidopsis . Of these, 13,977 genes gave a consistently positive expression signal in at least one stage of male gametophyte development, representing 61.9% of the unigene targets on the microarray. The majority of these were expressed in the two earliest developmental stages; 11,565 in microspores and 11,909 in bicellular pollen . After pollen mitosis II, there was a sharp decline in the number of diverse transcripts to 8,788 in tricellular pollen and 7,235 in mature pollen. | 15535861_p10 | 15535861 | Developmental changes in the male gametophytic transcriptome | 4.215282 | biomedical | Study | [
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To identify genes expressed preferentially or specifically in developing male gametophytes, hybridization data was compared with sporophytic ATH1 datasets (COT, LEF, PET, STM, ROT and RHR; see Additional data file 1). Transcripts with a consistent positive expression signal in at least one stage of male gametophyte development and a zero signal in any sporophytic dataset were considered male gametophyte-specific. In total, 1,355 specific transcripts were identified, representing 9.7% of the male gametophytic transcriptome. The number of male gametophyte-specific transcripts ranged from 857 (BCP) to 625 (MPG). Thus, in contrast to the decline in the total number of diverse transcripts expressed, the representation of male gametophyte-specific transcripts increased, from 6.9% and 7.2% at UNM and BCP-stages to 8.0% and 8.6% at TCP and MPG-stages respectively. | 15535861_p11 | 15535861 | Developmental changes in the male gametophytic transcriptome | 4.125002 | biomedical | Study | [
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Analysis of the distribution of transcripts among three abundance classes: high (up to 10-fold less than the maximum signal), medium (10- to 100- fold less) and low (more than 100-fold less) , revealed a decrease in the proportion of transcripts forming the high-abundance class during development from 20% to 12%. On the contrary, there was sharp increase in the proportion of mRNAs forming the low-abundance class after pollen mitosis II from 4% (UNM) to 14% (MPG). Moreover, 55% of low-abundance transcripts at MPG stage represented repressed mRNAs expressed more abundantly at earlier stages. Thus, the dramatic decrease in the number of transcripts expressed between bicellular and tricellular stages is paralleled by redistribution of mRNA from the high to the low abundance classes. These changes may be associated with reduced cellular activities and cell differentiation processes together with preferential expression of certain classes of genes during pollen maturation. This finding is in accord with the over-representation of cytoskeleton, cell-wall and signaling-related genes that comprise 26% of the high-abundance transcripts at MPG stage. In particular, the average expression signals of cytoskeleton, cell-wall and signaling-related transcripts were increased by 3.1, 3.7 and 2.3-fold, respectively, compared with the UNM stage. | 15535861_p12 | 15535861 | Developmental changes in the male gametophytic transcriptome | 4.331929 | biomedical | Study | [
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Scatter-plot analysis was used to examine in more detail the complexity of the mRNA decline after PMII. The expression levels of individual genes were normalized using a scale of 0 to 100. Genes coexpressed in pairs of datasets were plotted using a logarithmic scale and a correlation coefficient ( R value) calculated . There was an extremely high correlation ( R = 0.96) between the transcriptomes of UNM and BCP, the two earliest developmental stages . These stages are closely related, with a moderate increase in the expression of a number of genes at BCP stage. The profiles of the two latest developmental stages, TCP and MPG, were also very similar , but with greater deviation than the early stages. The scatter plot of TCP and MPG revealed the shift between extreme mRNA abundance classes as described above. This was more evident when bicellular and tricellular stages were compared . The scatter of gene expression values and the low correlation ( R = 0.541), provide evidence that the major quantitative shift in transcriptome size between BCP and TCP stages is not simply the result of repression, but also involves the activation of new groups of genes associated with pollen maturation. The lack of correlation ( R = 0.194) between gene expression profiles in uninucleate microspores and mature pollen , also reflects the pronounced change in cell status from proliferating microspore to terminally differentiated pollen. | 15535861_p13 | 15535861 | Developmental changes in the male gametophytic transcriptome | 4.268426 | biomedical | Study | [
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The relationship between cell proliferation activities and transcriptome profiles was examined by comparison of early UNM and late MPG stages with a publicly available suspension cell culture dataset. These comparisons demonstrated that the microspore transcriptome was significantly more similar to that of cell suspensions ( R = 0.474) than to mature pollen ( R = 0.194). This is also in accord with the lack of correlation between transcriptome profiles of mature pollen and cell suspensions ( R = 0.13). | 15535861_p14 | 15535861 | Developmental changes in the male gametophytic transcriptome | 4.124085 | biomedical | Study | [
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To identify gametophytic genes that may form co-regulated clusters, all 13,977 male gametophyte-expressed genes were hierarchically clustered using EPCLUST clustering and analysis software. Application of a threshold value of 0.05 resulted in the definition of 39 gene clusters covering all phases of male gametophyte development . Cluster 37 contained 735 early genes (5.3% of all gametophytic genes) with positive expression signals only at UNM stage. Transcriptome data reflect steady-state mRNA profiles that result from the combination of transcription and mRNA turnover rates. In this regard, some transcripts grouped in early cluster 37 may be inherited through meiosis and/or from the tetrad stage. | 15535861_p15 | 15535861 | Co-regulated clusters of gametophytic genes | 4.15751 | biomedical | Study | [
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The majority of male gametophyte-expressed genes (52%) were grouped into four clusters (25, 27, 29 and 35) comprising early expressed genes repressed after PMII. Several large gene clusters collectively containing 1,899 genes (13.6%) were associated with pollen maturation. These were activated or upregulated between BCP and TCP stages, forming clusters 5, 7, 11, 13, 18-24, 26, 28, 38 and 39. In contrast, a discrete set of 298 genes forming cluster 17 was upregulated only after TCP stage. In total, 3,342 late genes (24%), forming clusters 1-3, 6, 8 and in particular, cluster 17, encode proteins that are likely to function during post-pollination development. | 15535861_p16 | 15535861 | Co-regulated clusters of gametophytic genes | 4.220909 | biomedical | Study | [
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We focused our further analysis on three key categories of genes with likely regulatory significance in male gametophyte development; core cell-cycle genes, transcription factors and core translation factors . Core cell-cycle genes were defined according to TAIR . Genes comprising Arabidopsis transcription factor families were derived by compilation of data available at The Ohio State University Arabidopsis Gene Regulatory Information Server , data published in and databases homology searches. Recent annotations of the MADS-box and bHLH transcription factor gene families were defined according to , respectively. Core translation factors were defined according to TAIR . | 15535861_p17 | 15535861 | Expression of regulatory genes throughout male gametophyte development | 4.048983 | biomedical | Study | [
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Among 61 core cell-cycle genes, 55 genes were present on the ATH1 GeneChip and 45 (82%) of these were expressed in the male gametophyte (see Additional data file 1). Representative(s) of all families and subfamilies were expressed. The majority of gametophytic core cell-cycle genes showed similar expression profiles , with a decline in mRNA abundance after UNM stage to zero (or low levels) at TCP and MPG stages. This pattern is consistent with the termination of proliferation of the microspore and generative cell before pollen maturation. | 15535861_p18 | 15535861 | Core cell-cycle genes | 4.129916 | biomedical | Study | [
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We identified 1,594 genes encoding putative transcription factors that were divided into 34 gene families (see Additional data file 1). Their representation on the ATH1 GeneChip was 1,350 (85%). Of these, 608 (45%) were expressed in the male gametophyte, including 54 (15.7%) that were male gametophyte-specific. There were distinct differences in the representation of large transcription factor families (with over 25 members) in the gametophyte. Among those over-represented were the p-coumarate 3-hydroxylase (C3H) family (67% of family members present on the ATH1 GeneChip), the CCAAT family (64%), C2H2 zinc finger proteins (57%), the WRKY family (53%), the bZIP family (51%), the TCP family (50%) and the GRAS family (50%). In contrast, the AUX/IAA (20%), HSF (33%), bHLH (34%), NAC (34%), AP2-EREBP (35%), HB (36%), R2R3-MYB (37%), MADS (37%) and C2C2 zinc finger (37%) gene families were all under-represented. | 15535861_p19 | 15535861 | Putative transcription factors | 4.212017 | biomedical | Study | [
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The dominant expression pattern of transcription factor genes reflected the general repression of mRNA diversity between BCP and TCP stages . Besides a limited number of constitutively expressed genes, two major transcription factor gene groups could be distinguished. One contained a major group of early-expressed genes and the second a smaller group of genes that were more abundantly expressed late during pollen maturation. The same general tendency was apparent when the profiles of individual transcription factor families were analyzed . Several gene families comprised predominantly early-expressed genes. These were the NAC, WRKY, TCP, ARF, Aux/IAA, HMG-box and Alfin-like gene families . Complete lists of transcription factor gene families and their expression profiles are presented in Additional data files 1 and 3. | 15535861_p20 | 15535861 | Putative transcription factors | 4.140104 | biomedical | Study | [
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Among 100 annotated core translation factor genes, 82 were present on the ATH1 GeneChip and 75 (91%) of these were expressed in the male gametophyte (see Additional data file 1). The vast majority of translation factor genes belonged to the early group and these were strongly expressed . Reflecting the constitutive requirement for protein synthesis, only six genes showed male gametophyte-specific expression. These were: AtPAB3 , AtPAB6 , AtPAB7 , AteIF2 - B3 , AteIF4G -like and AteIF6 - 2 . There was a striking over-representation of poly(A)-binding (PAB) proteins among the male gametophyte-specific genes; seven out of eight PAB genes were male gametophyte-expressed, three of which were specific. Moreover, two of these gametophyte-specific PAB genes were among the few late pollen genes encoding translation initiation factors . | 15535861_p21 | 15535861 | Core translation factors | 4.187117 | biomedical | Study | [
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The rapid decline of mRNAs encoding translation initiation factors after bicellular stage and the parallel de novo synthesis of a new set of late pollen transcription factors, suggested storage of translation factors and ongoing transcription after pollen germination. Therefore we investigated the dependence of Arabidopsis pollen germination and tube growth on transcription and translation. Pollen was cultured with increasing concentrations of actinomycin D and cyclohexmide to examine the importance of transcription and translation, respectively. Actinomycin D had only moderate effects on both pollen germination and tube growth even at high concentrations . Similar results were observed when another transcription inhibitor, cordycepin, was used (data not shown). In contrast, cycloheximide had a dramatic effect on pollen tube growth . The presence of 0.1 μg/ml cycloheximide only inhibited pollen germination by 40%, but pollen tube growth was inhibited by 90%. At higher concentrations, 40% of pollen was still able to germinate, but further pollen tube growth was blocked. We conclude that active pollen tube growth is strictly dependent upon protein synthesis, and that pollen germination and tube growth are relatively independent of transcription. | 15535861_p22 | 15535861 | Integrating transcriptomic and experimental data | 4.413005 | biomedical | Study | [
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To identify patterns of gene expression involved in Arabidopsis male gametophyte development, we compared the transcriptomes of isolated spores at four discrete developmental stages using ATH1 microarrays. ATH1 microarrays harbor probe sets for 22,591 annotated genes . Of these, 61.9% (13,977 genes) gave positive hybridization signals in at least one stage of male gametophyte development. A comparable proportion of active genes was reported for isolated root cells which expressed 10,492 genes (46%) on ATH1 microarrays . Moreover, in similar studies of animal cell development, 53% of 13,179 arrayed genes were found to be expressed during early murine adipocyte differentiation . | 15535861_p23 | 15535861 | Discussion | 4.17686 | biomedical | Study | [
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As the proportion of known genes embedded on the ATH1 array is 80.7%, we estimate the total number of genes expressed throughout Arabidopsis male gametophyte development to be more than 17,000. Similarly, the total number of genes expressed at individual developmental stages is estimated to be 14,300 at UNM stage, 14,800 at BCP stage, 10,900 at TCP stage and 9,000 at MPG stage. Previous gene-by-gene approaches identified only 21 different genes expressed during Arabidopsis male gametophyte development (for a review see ). Moreover, only three of these genes were shown to be expressed at microspore stage . The data sets reported here include more than 11,000 microspore-expressed genes, representing a 3,600-fold increase in knowledge of gene expression in haploid microspores. | 15535861_p24 | 15535861 | Discussion | 4.177797 | biomedical | Study | [
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Two recent studies of the Arabidopsis mature pollen transcriptome using Affymetrix 8K AG arrays led to the identification of 992 and 1,584 pollen-expressed mRNAs, respectively . Results obtained with ATH1 and AG arrays are considered comparable and largely independent of the different probe sets used . However, there was a significant discrepancy in the number of incorrectly annotated genes between both arrays, with 6.3% of probe sets on the AG array being incorrectly annotated in comparison with only 0.4% on the ATH1 array . Therefore, results from ATH1 arrays are more accurate as well as more comprehensive. Accordingly, the use of the more complete ATH1 array and more accurate microarray normalization protocols led to an increase in the estimated total number of genes expressed in mature pollen from around 3,500 to around 9,000 (this study). The proportion of these genes that are considered male-gametophyte specific is strongly dependent on the choice of the set of reference sporophytic datasets. In the work reported here, the availability of more comprehensive sporophytic datasets and the application of more stringent criteria therefore led to a decrease in the estimated number of putative pollen-specific genes from around 1,400 to around 800 (this study). This number could be reduced further if cell-type-specific expression within an organ limits detection of overlap with pollen expression. Our data highlight the extensive overlap between sporophytic and gametophyte gene expression and reveal the subset of the transcriptome that is strongly enhanced or specifically expressed during male gametophyte development. Considering all stages of microsporogenesis the total number of putative male-gametophyte-specific genes was 1,355 with the proportion of specific genes increasing from 6.9% at UNM-stage to 8.6% at MPG-stage. Among the male-gametophyte-specific genes identified there was an increase in the collective proportion of cell-wall, cytoskeleton, signaling and transport-related genes from 22% at UNM stage to 34% in MPG stage. This reflects the increasing functional specialization of mature pollen in preparation for a dramatic change in the pattern of cell growth during pollen germination and pollen tube growth. | 15535861_p25 | 15535861 | Discussion | 4.471972 | biomedical | Study | [
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Developmental analysis of transcriptome data revealed two striking features, a sharp reduction in transcript diversity after BCP stage and a major shift in mRNA populations between BCP and TCP stages. The decline in mRNA diversity after BCP stage is associated with terminal differentiation as well as the documented phenomenon of protein storage in pollen (see , and this study). Moreover, this large-scale repression associated with termination of cell proliferation after PMII is accompanied by the selective activation of new groups of genes that are likely to function during pollen maturation and post-pollination development. | 15535861_p26 | 15535861 | Discussion | 4.268936 | biomedical | Study | [
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It is interesting that the expression profiles of UNM stage and BCP stages are similar despite the presence of two different cell types in pollen grains at BCP stage - the larger vegetative cell and the smaller generative cell. Given the limited volume of cytoplasm associated with the generative cell, developmental changes in gene expression in the gametic or male germline cells are likely to be masked by the predominant contribution of the vegetative cell cytoplasm. Therefore, our male gametophytic gene expression profiles largely reflect the passage of the microspore through cell division and changes in gene expression associated with the differentiation of the vegetative cell. Large-scale changes in gene expression occur between BCP and TCP stages, and therefore do not coincide with asymmetric division of the microspore. UNM expression patterns persist into the bicellular stage, which is consistent with experiments that demonstrate that vegetative cell fate is specified independently of cell division at pollen mitosis I . | 15535861_p27 | 15535861 | Discussion | 4.252969 | biomedical | Study | [
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In contrast, generative cell fate appears to be dependent on asymmetric division at pollen mitosis I . Sperm-cell cDNAs and databanks recently established in maize, lily, tobacco and Plumbago zelanica provide valuable gametic gene-expression data in other species. Although our data do not provide direct information about gametic gene expression in Arabidopsis , further development of cell gamete isolation sorting would allow genome-wide identification of generative- and sperm-cell-specific genes in comparison with the datasets generated here. | 15535861_p28 | 15535861 | Discussion | 4.106032 | biomedical | Study | [
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Hierarchical cluster analysis provided detailed evidence for the dramatic switch between early and late developmental programs. We identified 39 gene clusters that could correspond to co-regulated genes. These included early clusters, several clusters of late genes, those with constitutive expression profiles and clusters showing transient expression with peaks at BCP or TCP stages. The large size of cluster 29 (4,464 genes) documents the homogeneity in expression profiles of most early genes. In contrast, late gene clusters included a significant number of genes with similar profiles between BCP and TCP stages, followed by expression profiles that deviated between TCP and MPG stages. Cluster 1, and in particular cluster 17, contained genes strongly upregulated in TCP and MPG, with likely functions in post-pollination events. The differential fate of certain late gene clusters is likely to be a feature of their requirement during pollen maturation or post-pollination events. | 15535861_p29 | 15535861 | Discussion | 4.261786 | biomedical | Study | [
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Our analysis revealed completely different expression profiles of transcription factors when compared to core translation factors. The majority of core translation factors belonged to the early-group genes with few that were male gametophyte-specific. This may be expected, given that many genes are involved in general cellular activities. However, genes encoding PAB proteins did not follow the general trend. Seven out of eight Arabidopsis PAB mRNAs were gametophytically expressed. Three PAB genes ( PAB3 , PAB6 and PAB7 ) appeared to be male gametophyte-specific and PAB5 was preferentially expressed in pollen. Moreover, PAB3 and PAB5 are the most abundant early and constitutive PAB mRNAs and PAB6 and PAB7 belong among the few late core translation-factor genes. Although these data suggest-specific expression, our data do not rule out expression in other sporophytic tissues, particularly in flowers. Indeed, previously published expression data confirmed the expression of these PABs in other reproductive tissues together with pollen . | 15535861_p30 | 15535861 | Discussion | 4.216772 | biomedical | Study | [
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Conversely, transcription factors showed more diverse spectra of expression profiles including early, constitutive and late. There was a considerable variation in the expression profiles of individual transcription factor families. The most over-represented was the C3H family, members of which are known to have roles in lignin and other phenylpropanoid pathways in plants . Although sporopollenin synthesis is believed to be under strict sporophytic control (see ), the diversity of gametophytic C3H transcription factors might suggest a function for these genes in regulating chemical interactions between phenylpropanoid precursors secreted by the tapetum. One candidate is the At1g74990 gene encoding a putative RING finger protein, which is abundantly and preferentially expressed at UNM and BCP stages. | 15535861_p31 | 15535861 | Discussion | 4.194817 | biomedical | Study | [
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The majority of core translation factors belonged to the early gene clusters. In contrast, a significant number of transcription-factor genes were strongly expressed during pollen maturation. These data alone did not obviously support the fact that pollen germination and early tube growth in many species are largely independent of transcription, but vitally dependent on translation . Similarly, we found that Arabidopsis pollen germination and tube growth were relatively independent of transcription, and that active pollen-tube growth, and to a lesser extent pollen germination, were dependent upon protein synthesis. It is known for some plant species that mRNAs and rRNAs accumulate during pollen maturation and are stored for use during pollen germination . Our results show that Arabidopsis pollen is charged with a diverse complement of stored mRNAs that could be used to support pollen germination and pollen tube growth. Moreover, the early synthesis of mRNAs encoding translation factors strongly suggests that these are preformed and stored in mature pollen grains to support rapid activation upon hydration and germination. We also suggest that some abundant late transcription factors could regulate maturation-associated genes or act as repressors of inappropriate transcription in growing pollen tubes. | 15535861_p32 | 15535861 | Discussion | 4.396548 | biomedical | Study | [
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The key impact of this work is that it provides a genome-wide view of the complexity of gene expression during single cell development in plants. Analysis of the male gametophytic transcriptome provides comprehensive and unequivocal evidence for the unique state of differentiation that distinguishes the developing male gametophyte from the sporophyte. Male gametogenesis is accompanied by large-scale repression of gene expression that is associated with the termination of cell proliferation and the selective activation of new groups of genes involved in maturation and post-pollination events. Development is associated with major early and late transcriptional programs and the expression of about 600 putative transcription factors that are potential regulators of these developmental programs. This wealth of information lays the foundation for new genomic-led studies of cellular functions and the identification of regulatory networks that operate to specify male gametophyte development and functions. | 15535861_p33 | 15535861 | Conclusions | 4.252004 | biomedical | Study | [
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Arabidopsis thaliana ecotype Landsberg erecta plants were grown in controlled-environment cabinets at 21°C under illumination of 150 μmol/m 2 /sec with a 16-h photoperiod. Isolated spores from three stages of immature male gametophyte were obtained by modification of the protocol of Kyo and Harada . After removal of open flowers, inflorescences from 400 plants were collected and gently ground using a mortar and pestle in 0.3 M mannitol. The slurry was filtered through 100 μM and 53 μM nylon mesh. Mixed spores were concentrated by centrifugation (50 ml Falcon tubes, 450 g , 3 min, 4°C). Concentrated spores were loaded onto the top of 25%/45%/80% Percoll step gradient in a 10-ml centrifuge tube and centrifuged (450 g , 5 min, 4°C). Three fractions were obtained containing: (1) microspores mixed with tetrads; (2) microspores mixed with bicellular pollen; and (3) tricellular pollen . Fraction 2 was diluted with one volume of 0.3 M mannitol loaded onto the top of a 25%/30%/45% Percoll step gradient and centrifuged again under the same conditions. Three subfractions of immature pollen were obtained: (2.1) microspores; (2.2) microspores and bicellular pollen mixture; and (2.3) bicellular pollen. Spores in each fraction were concentrated by centrifugation (eppendorf tubes, 2,000 g , 1 min, 4°C) and stored at -80°C. The purity of isolated fractions was determined by light microscopy and 4',6-diaminophenylindole (DAPI) staining according to . Viability was assessed by fluorescein 3',6'-diacetate (FDA) treatment . Mature pollen was isolated as described previously . Pollen tubes were cultivated in vitro for 4 h according to . Pollen was scored as germinated when pollen tubes were longer than half a pollen grain diameter. Pollen-tube growth was scored by counting those with tubes longer than two pollen grain diameters. | 15535861_p34 | 15535861 | Plant material and spore isolation | 4.256904 | biomedical | Study | [
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Total RNA was extracted from 50 mg of isolated spores at each developmental stage using the RNeasy Plant Kit (Qiagen) according to the manufacturer's instructions. The yield and RNA purity was determined spectrophotometrically and using an Agilent 2100 Bioanalyzer at the NASC. | 15535861_p35 | 15535861 | RNA extraction, probe preparation and DNA chip hybridization | 3.909609 | biomedical | Study | [
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Biotinylated target RNA was prepared from 20 μg of total RNA as described in the Affymetrix GeneChip expression analysis technical manual. Double-stranded cDNA was synthesized using SuperScript Choice System (Life Technologies) with oligo(dT) 24 primer fused to T7 RNA polymerase promoter. Biotin-labeled target cRNA was prepared by cDNA in vitro transcription using the BioArray High-Yield RNA Transcript Labeling Kit (Enzo Biochem) in the presence of biotinylated UTP and CTP. | 15535861_p36 | 15535861 | RNA extraction, probe preparation and DNA chip hybridization | 4.132455 | biomedical | Study | [
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Arabidopsis ATH1 Genome Arrays were hybridized with 15 μg labeled target cRNA for 16 h at 45°C. Microarrays were stained with streptavidin-phycoerythrin solution and scanned with an Agilent 2500A GeneArray Scanner. | 15535861_p37 | 15535861 | RNA extraction, probe preparation and DNA chip hybridization | 3.874706 | biomedical | Study | [
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Sporophytic data from public baseline GeneChip experiments used for comparison with the pollen transcriptome were downloaded from the NASC website . The list of dataset codes was as follows: COT (three replicates), Cornah_A4-cornah-wsx_SLD_REP1-3; LEF (three replicates), A4-LLOYD-CON_REP1-3; PET (three replicates), Millenaar_A1-MILL-AIR-REP1-3; STM (two replicates), Turner_A-7-Turne-WT-Base1-2_SLD; ROT (two replicates), Sophie_A1-Fille-WT-nodex_SLD, Sophie_A5-Fille-WT-nodex_SLD; RHR (two replicates), Jones_A1-jones-WT1, SLD, Jones_A1-jones-WT2_SLD; SUS (three replicates), A1-WILLA-CON-REP1-3. | 15535861_p38 | 15535861 | Data analysis | 3.498385 | biomedical | Study | [
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All gametophytic and sporophytic datasets were normalized using freely available dChip 1.3 software . The reliability and reproducibility of analyses was ensured by the use of duplicates or triplicates in each experiment, the normalization of all 26 arrays to the median probe intensity level and the use of normalized CEL intensities of all arrays for the calculation of model-based gene-expression values based on the Perfect Match-only model . A given gene was scored as 'expressed' when it gave a reliable expression signal in all replicates. Expression signal value '0' means that the detection call value was not 'present' in all replicates provided. All raw and dChip-normalized gametophytic datasets are available at the Institute of Experimental Botany AS CR website . Although a RT-PCR validation of microarray data was not performed specifically for the purpose of this publication, our confidence in the quality of the data presented is based on our previously published RT-PCR validation of the expression of 70 genes . | 15535861_p39 | 15535861 | Data analysis | 4.124419 | biomedical | Study | [
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Microsoft Excel was used to manage and filter the microarray data. For annotation of genes present on the ATH1 Array, the Arabidopsis Genome Annotation Release 3.0 published by The Institute for Genomic Research was used. Genes were sorted into functional categories created according to data mined from the Munich Information Center for Protein Sequences Arabidopsis thaliana Database , Kyoto Encyclopedia of Genes and Genomes and TAIR . Hierarchical clustering of expressed genes was performed using expression-profile data clustering and analysis software EPCLUST , with correlation measure based distance and average linkage clustering methods. | 15535861_p40 | 15535861 | Data analysis | 3.984634 | biomedical | Study | [
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The following additional data is available with the online version of this article: Additional data file 1 is an Excel file containing the following items. The table Data contains the complete transcriptomic datasets used. Data were normalized using dChip 1.3 as described in Materials and methods. Expression signal value '0' means that the detection call value for particular gene was not 'present' in all replicates provided. In the column 'Cluster', the appropriate cluster for each male gametophyte-expressed gene is shown. The table Clusters gives the number of genes comprising all 37 clusters of genes coexpressed during male gametophyte development. The table Cell-cycle data lists core cell-cycle genes showing their expression values in male gametophytic datasets. Genes were defined according to . The chart shows expression profiles of male gametophyte-expressed core cell-cycle genes. The table Transcription data lists transcription-factor genes, showing their expression values in male gametophytic datasets. Genes comprising Arabidopsis transcription factor families were derived by compilation of data available at the Ohio State University Arabidopsis Gene Regulatory Information Server , data published in and database homology searches. MADS-box and bHLH gene families were defined according to and , respectively. The table Translation data lists core translation-factor genes showing their expression values in male gametophytic datasets. Genes were defined according to the FIAT database . The chart shows expression profiles of male gametophyte-expressed core translation-factor genes. The Transcription table summarizes transcription factor gene families showing the number of genes expressed during male gametophyte development. Additional data file 2 lists a complete set of 39 clusters of genes coexpressed during male gametophyte development. Clusters were determined using EPCLUST software with a threshold value of 0.05. The list of genes comprising each cluster is given in Additional data file 1 . Additional data file 3 gives the expression profiles of male gametophyte-expressed transcription factors sorted into individual gene families. Expression data are given in Additional data file 1 . | 15535861_p41 | 15535861 | Additional data files | 4.147919 | biomedical | Study | [
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The catecholamines (dopamine, norepinephrine and epinephrine) are a group of biogenic amines possessing a substituted 3, 4-dihydroxy phenyl ring that are widespread in the animal kingdom; but they have also been detected in plants . The role of catecholamines in plants is poorly documented, but it is clear that they are involved in many aspects of growth and development. They were proposed as precursors for various alkaloids and to be associated with processes such as ethylene production, nitrogen fixation, defence against herbivores, flowering, prevention of 3-indole acetic acid (IAA) oxidation and gibberellin signalling . Analogous with animal cells in which catecholamines stimulate glycogen mobilization a similar role for catecholamines in the regulation of plant carbohydrate metabolism was suggested. Transgenic plants over-expressing tyrosine decarboxylase, which controls an important step of catecholamine synthesis, were characterized by highly increased concentrations of norepinephrine and soluble sugars, whereas starch level was dramatically decreased. Observed changes indicated a positive correlation of norepinephrine with soluble sugars and a negative correlation with starch . The physiological action of catecholamines in animal cells is mediated by their interaction with G-protein coupled receptors that stimulate or inhibit the enzyme adenylyl cyclase (AC). In most animal cells cyclic AMP (cAMP) exerts its effect by activating cAMP dependent, serine-threonine protein kinase (PKA). Recently strong evidence for the occurrence and function of cAMP in higher plants has emerged . It was demonstrated that cAMP levels in tobacco bright yellow 2 (TBY-2) cells are tightly connected to cell cycle progression . The involvement of cAMP in gibberellin and ABA action has also been suggested . Molecular evidence has shown the existence of plant protein kinases containing a high degree of sequence homology with PKA . Moreover, molecular techniques led to the identification of cAMP response element-binding proteins (CREBs) , cyclic nucleotide-gated cation channels and cAMP binding enzymes . These data strongly indicate the involvement of catecholamines in regulating plant carbohydrate metabolism, possibly by a mechanism similar to that in animal cells. However, this suggestion is limited by the fact that to date no catecholamine receptor has been identified in plants. In the present study we characterize potato plants transformed with a cDNA encoding human dopamine receptor (HD1). The receptor is a rhodopsin-like integral membrane protein of 446 amino acids, seven transmembrane domains and molecular mass of 49 kDa. Our analysis revealed a regulatory effect of HD1 on carbohydrate metabolism including changes in key enzyme activities. | 15701180_p0 | 15701180 | Background | 4.558156 | biomedical | Study | [
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Solanum tuberosum plants transformed with pHD1-BinAR, a plasmid carrying a cDNA for the human dopamine receptor under the control of the CaMV 35S promoter , were pre-selected by means of PCR with the primers for neomycin phosphotransferase (kanamycin) gene and then selected by northern blot analysis with a HD1 specific cDNA as a probe . Four transgenic lines showing the highest mRNA expression of the expected length were chosen for further analysis by western blot (HD1.10, HD1.27, HD1.35 and HD1.36). Using commercially available monoclonal rabbit IgG anti-HD1 antibody a ~37 kDa protein band was detected in transgenic plants. It was absent from control plants . Surprisingly the protein was ~10 kDa smaller than expected which may suggest posttranslational modification. Careful inspection of the cDNA sequence revealed the presence of two translational signals (232 bp and 313 bp) that would result in 40 kDa and 37 kDa proteins, respectively. However, as the translational machinery is very similar in plants and animals we suggest that the short form of receptor resulted from proteolytic action rather than de novo synthesis. It should be pointed out that a stronger signal for HD1 expression was accompanied by a stronger protein signal (lines HD1.10; HD1.35; HD1.36). Conversely in plants with weak HD1 expression, the protein signal was comparatively weak (line HD1.27). HD1 extraction with 1% Triton was much more efficient than extraction with 0.1%Triton in agreement with the expected membrane localization of the HD1 protein. | 15701180_p1 | 15701180 | Transgenic plant selection | 4.287649 | biomedical | Study | [
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Tubers of HD1 plants grown in a field were harvested after four months and analyzed. All examined transgenic lines produced more tubers per plant. The yield was not significantly changed since increase in tuber number was accompanied by decrease in tuber weight (Table 1 ). There were no obvious morphological differences between aerial parts of wild type Desiree and HD1 plants. | 15701180_p2 | 15701180 | Phenotype analysis | 2.946006 | biomedical | Study | [
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In order to develop an easy and reliable assay for the quantitative and qualitative determination of catecholamines in plants, the suitability of gas chromatography coupled to a quadruple mass spectrometer (GCMS) was recently investigated. A sensitive GCMS method based on the analysis of the trimethylsilylated catecholamine derivatives was developed to monitor the presence and concentration of these compounds and related metabolites. Based on the retention times and the mass spectra of standards the presence of dopamine, norepinephrine and a new compound normetanephrine in potato leaves and tubers was clearly detected . | 15701180_p3 | 15701180 | Catecholamine level | 4.123064 | biomedical | Study | [
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In contrast to the previous studies performed on plants over-expressing tyrosine decarboxylase , which controls an important step of catecholamine synthesis, the goal of our work was to stimulate an alternative signalling pathway by introducing human dopamine receptor. Surprisingly the expression of dopamine receptor resulted in a more than two-fold increase of dopamine, norepinephrine and normetanephrine in all transgenic lines examined . This increase of catecholamines content was accompanied by significant increase of tyramine and L-DOPA, which are direct precursors of dopamine . Despite changes in concentration of catecholamines and their precursors, the level of tyrosine, which serves as a precursor for tyramine and L-DOPA, was not altered (data not shown). | 15701180_p4 | 15701180 | Catecholamine level | 4.096666 | biomedical | Study | [
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Following our recent finding that the action of dopamine and norepinephrine in potato is on starch mobilization, we decided to analyze transgenic tubers expressing dopamine receptor for soluble sugars and starch content. All transgenic plants showed decreased starch content, with levels that ranged from 20 to 60% percent of the wild type. This was accompanied by a significant increase in soluble sugar concentration ranging from 2.7 to 1.15 fold in comparison to Desiree . Concentrations of starch and soluble sugars were highly correlated. The calculated correlation coefficients between catecholamines content and the levels of glucose, sucrose, fructose and starch were 0.38, 0.69, 0.38 and -0.95, respectively. | 15701180_p5 | 15701180 | Determination of carbohydrate content in tubers of transgenic plants | 4.097103 | biomedical | Study | [
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Changes in carbohydrate are most likely responsible for the altered phenotype of transgenic HD1 tubers. Reduced tuber mass can be explained by decreased starch content whereas increased tuber number by the increase of soluble sugar concentration. | 15701180_p6 | 15701180 | Determination of carbohydrate content in tubers of transgenic plants | 3.249133 | biomedical | Study | [
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Under normal growth conditions the major flux in potato tuber carbon metabolism is the conversion of sucrose through hexose phosphates to starch . | 15701180_p7 | 15701180 | Sucrose – starch metabolism | 3.084377 | biomedical | Other | [
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Since HD1 plants were characterized by changed concentrations of both soluble sugars and starch we measured the activities of enzymes involved in this pathway. Sucrose transported from leaves is symplastically unloaded from the phloem and degraded by sucrose synthase (SuSy). ADP-glucose phosphorylase (AGPase) converts glucose-1-phosphate (Glu-1-P) into ADP-glucose, an immediate precursor of starch. Both SuSy and AGPase are considered as key enzymes for starch synthesis . | 15701180_p8 | 15701180 | Sucrose – starch metabolism | 4.15633 | biomedical | Study | [
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0.00006085172572056763
] | en | 0.999997 | {
"en": 0.9999965239061759
} |
Activities of AGPase and SuSy were significantly decreased in HD1 plants to 56% and 68% of the wild type level, respectively . In agreement with their roles in starch synthesis, and their proposed coordinated regulation, activities of both enzymes and starch content were all significantly correlated (cor >0.9). | 15701180_p9 | 15701180 | Sucrose – starch metabolism | 4.05737 | biomedical | Study | [
0.9991021156311035,
0.0002436786744510755,
0.0006542539340443909
] | [
0.9995410442352295,
0.00019745194003917277,
0.00022095987515058368,
0.000040578328480478376
] | en | 0.999997 | {
"en": 0.9999967857621159
} |
Phosphoglucomutase (PGM) catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate. Tubers are characterized by the presence of cytosolic and plastidial isoforms of phosphoglucomutase. Repression of either of them results in plants with decreased starch levels pointing out the importance of the enzyme for starch accumulation . The activity of PGM was significantly decreased in all transgenic lines, most likely contributing to the reduction in starch synthesis . Activities of other enzymes involved in sucrose-starch conversions (hexokinase, UGPase and starch synthase) were not changed significantly . In most of the transgenic lines inhibition of starch synthesis was accompanied by increased hexose-6-phosphates (Table 2 ). | 15701180_p10 | 15701180 | Sucrose – starch metabolism | 4.192892 | biomedical | Study | [
0.9992818236351013,
0.00025739011471159756,
0.0004608033050317317
] | [
0.9995003938674927,
0.00020650270744226873,
0.00024652708088979125,
0.00004663483923650347
] | en | 0.999996 | {
"en": 0.9999959062081216
} |
To establish whether enhanced starch mobilization also contributed to the observed decreases in starch content we measured the activity of starch phosphorylase. In two out of the four examined transgenic lines the activity of starch phosphorylase was significantly increased, further contributing to decreased starch content of HD1 plants | 15701180_p11 | 15701180 | Sucrose – starch metabolism | 3.891469 | biomedical | Study | [
0.9985058307647705,
0.000293849065201357,
0.0012003141455352306
] | [
0.9993792772293091,
0.0004041420470457524,
0.0001669589546509087,
0.00004964924664818682
] | en | 0.999997 | {
"en": 0.9999973134909386
} |
Moreover HD1 expression led to activation of sucrose phosphate synthase (SPS), responsible for sucrose production. Maximum SPS activity (measured wih saturating substrates, Vmax) only changed in two lines, whilst activity of the enzyme measured in the assay that contained limiting substrate concentration (Vmax/Km) and as a consequence 1/Km increased in all the lines. | 15701180_p12 | 15701180 | Sucrose – starch metabolism | 4.120253 | biomedical | Study | [
0.9993693232536316,
0.00021347728034015745,
0.00041719546425156295
] | [
0.999314546585083,
0.0004269224009476602,
0.00020172253425698727,
0.00005681549373548478
] | en | 0.999998 | {
"en": 0.9999976558813314
} |
1/Km, correlated well with the sucrose content of transgenic tubers (cor -0.81) . | 15701180_p13 | 15701180 | Sucrose – starch metabolism | 3.275309 | biomedical | Study | [
0.9937518239021301,
0.0005207686335779727,
0.005727387499064207
] | [
0.9939550757408142,
0.005525962915271521,
0.0003743274137377739,
0.00014463586558122188
] | en | 0.999998 | {
"en": 0.9999979137129739
} |
The high concentrations of glucose and glc-6-P measured in the HD1 plants indicated changes in the glycolytic pathway. However, activities of glycolytic enzymes (hexokinase, phosphofructokinase and enolase) were not changed. The only exception was pyruvate kinase, which showed a significant decrease of activity in all transgenic lines . To investigate if this reduction of activity led to changes in carbon metabolism via the TCA cycle we measured the content of TCA intermediates. In all transgenic lines citric acid, isocitric acid and malate were significantly reduced, while fumarate showed a significant increase (Table 2 ). | 15701180_p14 | 15701180 | Glycolysis/TCA cycle | 4.136599 | biomedical | Study | [
0.9993144273757935,
0.0002880146203096956,
0.00039753978489898145
] | [
0.9995667338371277,
0.00015869809431023896,
0.0002208345104008913,
0.00005379934736993164
] | en | 0.999997 | {
"en": 0.9999965930692547
} |
In contrast to the vast knowledge concerning the role and action of catecholamines in mammals, very little is known about the physiological significance of catecholamines in plants. Since most of the components of animal catecholamine signaling pathway have been also identified in plants (G-proteins, cAMP, PKA homologs) the involvement of catecholamines in plant signalling pathways is possible. Recently, the analysis of transgenic plants over-expressing tyrosine decarboxylase, which accumulate a high quantity of catecholamines, suggested a possible signalling effect on plant primary metabolism. The increase of catecholamines resulted in decreased starch concentration but increased soluble sugars . | 15701180_p15 | 15701180 | Discussion | 4.136292 | biomedical | Study | [
0.9994632601737976,
0.0001691511133685708,
0.0003675886837299913
] | [
0.9961705803871155,
0.0005123745067976415,
0.003236171556636691,
0.0000808901313575916
] | en | 0.999997 | {
"en": 0.9999970408373068
} |
The only component of mammalian catecholamine signaling pathway that to date has not been identified in plants is the catecholamine receptor. We transformed potato plants with a cDNA encoding human dopamine receptor (HD1) in order to analyze whether the presence of a receptor affects the endogenous catecholamine action. Western blot analysis showed that the protein was produced in transgenic plants and biochemical analysis of transgenic tubers revealed vast changes in carbohydrate metabolism and carbohydrate content. Surprisingly the catecholamine level was changed as well. It has to be pointed out that in contrast to plants over-expressing tyrosine decarboxylase, those expressing human dopamine receptor are characterized by increases of all known tuber catecholamines (dopamine, norepinephrine and normetanephrine). Whereas norepinephrine content was positively correlated with soluble sugars and negatively with starch, normetanephrine was considered as the product of norepinephrine turnover. Increased catecholamine content was accompanied by an increase of their precursors, tyramine and L-DOPA, suggesting upregulation of the biosynthetic pathway, mediated by tyrosine decarboxylase and tyrosine hydroxylase, respectively. It is hard to explain how expression of a human receptor triggers a positive loop leading to enhanced catecholamine synthesis and turnover. It is interesting to compare data on tuber carbohydrate levels from plants over-expressing tyrosine decarboxylase (TD) with those expressing human dopamine receptor. In both cases starch content is strongly decreased, this decrease was larger for HD1 plants (from 40% to 80%) than for TD tubers (from 12% to 60%) although the norepinephrine content was higher in TD plants . The norepinephrine content in TD plants was about four folds higher than in HD1 plants. | 15701180_p16 | 15701180 | Discussion | 4.354183 | biomedical | Study | [
0.9993658661842346,
0.00037302137934602797,
0.00026114666252397
] | [
0.9992020726203918,
0.00024734920589253306,
0.00045824196422472596,
0.00009231259173247963
] | en | 0.999996 | {
"en": 0.9999961243017546
} |
Therefore we suggest that the exogenous receptor activates catecholamine action in potato plants. A difference in enzyme activities involved in starch biosynthesis was noted. The sucrose level was comparable in HD1 and TD plants and consistent with enhanced activity of SPS. Activity of starch phosphorylase was significantly increased in both TD and HD1 plants but the decreases in activity for AGPase, SuSy and PGM was seen only for HD1 plants. | 15701180_p17 | 15701180 | Discussion | 4.109247 | biomedical | Study | [
0.9992179870605469,
0.00019103946397081017,
0.0005909492028877139
] | [
0.9995285272598267,
0.00024924983154051006,
0.00018088905198965222,
0.00004136958159506321
] | en | 0.999996 | {
"en": 0.9999962960479848
} |
The previously reported positive correlation between catecholamine level and soluble sugars content and negative correlation with starch level for tubers of potatoes over -expressing tyrosine decarboxylase and in tubers stored at 4°C , was also found in our study. Expression of a dopamine receptor resulted in increased catecholamine content and was accompanied by decreased starch level and increases of glucose, fructose and sucrose content. It seems likely that the introduced dopamine receptor activates catecholamine action in carbohydrate metabolism. The question now arises whether catecholamine activates starch breakdown or inhibits its synthesis or whether both processes are affected. | 15701180_p18 | 15701180 | Expression of HD1 in potato ( Solanum tuberosum ) results in altered carbon metabolism | 4.093778 | biomedical | Study | [
0.9994567036628723,
0.0001940033835126087,
0.0003492863615974784
] | [
0.9994227886199951,
0.000229922792641446,
0.00029618493863381445,
0.00005116233660373837
] | en | 0.999999 | {
"en": 0.9999985998639311
} |
In mammalian systems epinephrine and norepinephrine regulate glycogen turnover by stimulating glycogen mobilization and inhibiting glycogen synthesis. This appears similar in potato, with decreased starch content in HD1 tubers being a consequence of both inhibition of starch synthesis and enhanced starch mobilization. AGPase and SuSy, two key enzymes involved in starch biosynthesis, showed 44% and 32% decreases in their activities respectively. Also the activity of PGM was significantly decreased; we have not determined the contribution of the different isoforms (cytosolic and plastidial) to the observed changes. Increased content of hexose-6-phosphates demonstrates that a direct inhibition of AGPase, rather than a substrate shortage may cause inhibition of starch synthesis. Alternatively the increased hexose phosphate levels may be due to increased starch degradation in response to elevated catecholamine levels. This is supported by the increased activity of starch phosphorylase in two of four transgenic lines. The inhibition of starch synthesis and accumulation of hexose phosphates was accompanied by an increase of sucrose synthesis. Two factors should be taken into consideration. First, that SPS is subject to allosteric activation by Glc-6-P and inhibition by Pi. Second, elevated catecholamine content led to a decrease of SPS Km suggesting increase of the enzyme catalytic activty. Sucrose phosphate synthase has many potential sites of phosphorylation and three of them were shown to influence its catalytic activity. In spinach leaf, phosphorylation of Ser 158 is responsible for enzyme downregulation in darkness, phosphorylation of Ser 229 enables binding of 14-3-3 proteins and down- regulates the enzyme whereas phosphorylation of Ser 424 under stress conditions stimulates SPS activity. There is a growing body of correlative evidence that the potato tuber SPS is regulated in an analogous manner to the leaf enzyme . | 15701180_p19 | 15701180 | Expression of HD1 in potato ( Solanum tuberosum ) results in altered carbon metabolism | 4.718836 | biomedical | Study | [
0.9990531802177429,
0.0005847412976436317,
0.00036207708762958646
] | [
0.9976575374603271,
0.0006296201609075069,
0.0014207307249307632,
0.0002920459082815796
] | en | 0.999997 | {
"en": 0.9999970379994723
} |
Since the level of 14-3-3 proteins was not changed in any of the transgenic lines (data not shown) it is thus suggested that enzyme phosphorylation targeted to the stress site is responsible for its activity enhancement in HD1 plants. | 15701180_p20 | 15701180 | Expression of HD1 in potato ( Solanum tuberosum ) results in altered carbon metabolism | 3.046356 | biomedical | Study | [
0.99591463804245,
0.00039142800960689783,
0.003693877486512065
] | [
0.9917601943016052,
0.007661499548703432,
0.00038709279033355415,
0.00019134202739223838
] | en | 0.999998 | {
"en": 0.999997677306778
} |
In mammals the action of epinephrine and norepinephrine is mediated by phosphorylation of enzymes involved in glycogen mobilization and synthesis. Very recent studies reported direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation in the control of starch anabolism and catabolism . Therefore, it is possible that catecholamine action in plants could also involve phosphorylation of enzymes involved in starch metabolism. | 15701180_p21 | 15701180 | Expression of HD1 in potato ( Solanum tuberosum ) results in altered carbon metabolism | 4.070583 | biomedical | Study | [
0.9994494318962097,
0.00013148618745617568,
0.0004189899191260338
] | [
0.9867450594902039,
0.004476399626582861,
0.008616817183792591,
0.00016172548930626363
] | en | 0.999995 | {
"en": 0.9999953794789794
} |
In mammalian systems, catecholamines serve as stress hormones increasing as a result of stress. In order to see whether or not a similar response occurs in plants, leaves of potato plants were wounded and catecholamines levels prior to and 5, 10 and 13 min after wounding were determined. Although the data varied, there was a consistent increasing trend in concentration of dopamine, norepinephrine and normetanephrine . Very recently a similar increase in norepinephrine was measured in potato leaves subjected to ABA and water stress treatment. Activities of both tyrosine hydroxylase (1.5 and 1.7 fold) and tyrosine decarboxylase (2.33 and 1.2fold) were increased . Under normal growth conditions the major flux in potato tuber carbon metabolism is the conversion of sucrose through hexose phosphates to starch . During environmental perturbations like wounding water stress , high temperature and hypoxia this balance is disturbed and, consequently, large changes in tuber metabolite levels occur. Elevated temperature or water stress leads to increased respiration, a decline in 3-phosphoglycerate (3PGA), inhibition of AGPase and consequently an inhibition of starch synthesis. Decreased starch was accompanied by a stimulation of sucrose synthesis caused by increased hexose posphate levels and activation of SPS via protein phosphorylation. The activity of SuSy was decreased whereas starch mobilization was suggested to increase. These changes in carbohydrate metabolism and carbohydrate content are very similar to those observed in HD1 plants, making it conceivable that catecholamines might play a role in plant stress responses by modulating tuber primary carbon metabolism. | 15701180_p22 | 15701180 | Catecholamines – the new stress hormones in plants? | 4.46999 | biomedical | Study | [
0.9993453621864319,
0.0003712550678756088,
0.00028345349710434675
] | [
0.9980677962303162,
0.0003286123101133853,
0.0014847195707261562,
0.00011880094825755805
] | en | 0.999998 | {
"en": 0.9999980692650419
} |
Introducing humane dopamine receptor into plant cells can be considered as controversial but the obtained data would argue for the value of our approach. | 15701180_p23 | 15701180 | Conclusions | 1.969746 | biomedical | Other | [
0.9753209948539734,
0.0010548575082793832,
0.02362413890659809
] | [
0.3600408434867859,
0.6330552101135254,
0.005269109271466732,
0.0016348488861694932
] | en | 0.999996 | {
"en": 0.9999961155449526
} |
Vast changes in the activities of key enzymes mediating carbon metabolism of potato tuber (in HD plants) led to a dramatic reduction of starch but increased sucrose content. The relation between catecholamine, primary carbon metabolism and stress seems possible. We speculate that similarly to situation in animal cells expression of HD1 in potato resulted in activation of the cAMP mediated signalling pathway. This can be supported by the result obtained for potato plants expressing another isoform of human dopamine receptor, HD2. In contrast to HD1, HD2 receptor does not affect activity of adenylate cyclase in animal cells. | 15701180_p24 | 15701180 | Conclusions | 4.158563 | biomedical | Study | [
0.9992349147796631,
0.0001824799837777391,
0.000582605367526412
] | [
0.9988897442817688,
0.0007487176335416734,
0.00029248945065774024,
0.00006910124648129568
] | en | 0.999998 | {
"en": 0.9999975099809085
} |
Similarly plants expressing HD2 showed no changes in carbohydrate metabolism (data not shown). The obvious next step would be further investigation of our plants with respect to their kinase activity as well as cAMP levels. In parallel we have made efforts to identify a plant dopamine receptor. | 15701180_p25 | 15701180 | Conclusions | 2.451626 | biomedical | Study | [
0.9911333918571472,
0.0005146369221620262,
0.008351976051926613
] | [
0.9602277278900146,
0.038580697029829025,
0.0008018825319595635,
0.00038971920730546117
] | en | 0.999995 | {
"en": 0.9999953578834833
} |
Potato plants ( Solanum tuberosum L. cv. Desiree) obtained from "Saatzucht Fritz Lange KG" (Bad Schwartau, Germany) were cultivated in a greenhouse in soil under 16 h light (22°C) and 8 h dark (15°C) regime. Plants were grown in individual pots and watered daily. For analysis, the leaves were harvested at noon from 30-day-old greenhouse grown plants and the tubers were harvested in September, 3 months after the transfer of the tissue culture plants to the greenhouse. | 15701180_p26 | 15701180 | Plant material | 3.4222 | biomedical | Study | [
0.9921680092811584,
0.0003758993116207421,
0.007456135004758835
] | [
0.9904265999794006,
0.008960584178566933,
0.000482355710119009,
0.00013054355804342777
] | en | 0.999997 | {
"en": 0.9999969751363339
} |
The 1.3 kb SmaI, XbaI cDNA encoding HD1 from Homo sapiens ((kindly provided by Marc G.Caron (Duke University, Medical Center); [EMB: XX55760])), was ligated in the sense orientation into the same restriction site of the plant binary vector under the control of the 35S CaMV promoter and Nos terminator. The vector was introduced into the Agrobacterium tumefaciens strain C58C1:pGV2260 and the integrity of the plasmid was verified by restriction enzyme analysis. Young leaves of wild-type potato S. tuberosum L.(cv. Desiree) were transformed with A. tumefaciens by immersing leaf explants in bacterial suspension. A. tumefaciens inoculated leaf explants were subsequently transferred to callus induction medium and shoot regeneration medium. Transgenic plants were pre-selected by using PCR with the primers for the respective phosphotransferase (kanamycin resistance) gene and then selected by northern blot analysis with a HD1 specific cDNA fragment as probe. | 15701180_p27 | 15701180 | Construction of a transgenic plant | 4.22032 | biomedical | Study | [
0.9993709921836853,
0.00031851971289142966,
0.0003104748611804098
] | [
0.9988012313842773,
0.0007934047025628388,
0.00032120285322889686,
0.00008408342546317726
] | en | 0.999995 | {
"en": 0.9999952052045702
} |
Total RNA was prepared from frozen plant material using the guanidinium hydrochloride method. Following electrophoresis (1.5% (w/v) agarose, 15% formaldehyde (w/v)), RNA was transferred to nylon membranes (Hybond N, Amersham, UK). Membranes were hybridised overnight at 42°C in 250 mmol sodium phosphate buffer (pH 7.2) containing 7% (w/v) SDS, 1% (w/v) bovine serum albumin (BSA) and 1 mM EDTA. Radioactively labelled full-length cDNA was used as a hybridisation probe. Filters were washed three times in 1 × SSC containing 0.5% (w/v) SDS at 65°C (highly stringent condition) or in the same buffer but at 42°C (medium stringent condition) for 30 min. | 15701180_p28 | 15701180 | Northern blot analysis | 4.103244 | biomedical | Study | [
0.9993300437927246,
0.0002511450438760221,
0.0004188290040474385
] | [
0.9975970387458801,
0.0019109962740913033,
0.0004139058873988688,
0.0000781402486609295
] | en | 0.999997 | {
"en": 0.9999973166567537
} |
Proteins were extracted from frozen plant material using extraction buffer E (100 mM Hepes-NaOH, pH 7.4, 10 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 20%glycerol (v/v), 0.5 mM PMSF, 70 mM beta-mercaptoethanol) supplemented either with 0.1% or 1% TritonX- 100 (v/v). The assessment of the expression of HD1 gene by means of western blot analysis using rabbit IgG anti HD1 protein was conducted as described previously. Briefly, solubilised protein was run on 12% SDS polyacrylamide gels (w/v) and blotted electrophoretically onto nitrocellulose membranes (Schleicher and Schuell). Following transfer, the membrane was sequentially incubated with blocking buffer (5% (w/v) dry milk), and then with antibody directed against the HD1 protein . Formation and detection of immune complexes were performed as previously described . Alkaline phosphatase-conjugated goat ant rabbit IgG at a dilution of 1:1500 was used as a secondary antibody. | 15701180_p29 | 15701180 | Western blot analysis | 4.105913 | biomedical | Study | [
0.9992635846138,
0.00023853140010032803,
0.0004979193327017128
] | [
0.9993545413017273,
0.00036079439450986683,
0.0002421697718091309,
0.00004242960494593717
] | en | 0.999998 | {
"en": 0.9999983347251001
} |
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