Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif).Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection.Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi. Centromeres are the chromosomal loci where kinetochores assemble to serve as attachment sites for the spindle microtubules that direct chromosome segregation during mitosis and meiosis. Despite this essential conserved function in all eukaryotes, centromere structure is highly variable, ranging from the simple short centromeres of budding yeast, which have a consensus sequence of approximately 125 base pairs (bp) on each chromosome, to holokinetic centromeres that span the entire length of a chromosome -5. The fDrosophila [Arabidopsis [Although there is no discernable conservation of centromeric DNA sequences in disparate eukaryotes, considerable progress has been made in identifying common proteins that form the kinetochore . A univeosophila  and Arabbidopsis , this rabidopsis ,11,12.The finding of positive selection in a protein that is required at every cell division is remarkable. Ancient proteins with conserved function are expected to be under negative selection because they typically have achieved an optimal sequence, so new mutations tend to produce deleterious variants that are quickly eliminated from populations. The canonical histones are extreme examples of this type of protein. In contrast, recurrent positive selection generally occurs as a consequence of genetic conflict, for example in the 'arms race' between pathogen surface antigens and the immune-cell proteins that recognize them. In this case, a mutation in a surface antigen that allows the pathogen to escape detection and proliferate will trigger selection for a new immune receptor to fight the mutated pathogen, which can then mutate again, and so on. The evidence for positive selection of CenH3 proteins specifically in the regions that contact DNA thus suggests a conflict between centromeric DNA and a histone component of the nucleosome that packages it. Is it commonplace for eukaryotes to have such a conflict at their centromeres? Is the conflict unique to centromere-specific histones, or are other proteins that bind centromeres also involved in this conflict? Is conflict responsible for centromere complexity? To answer these questions, we investigated the evolution of a second common DNA-binding kinetochore protein.in vitro [in vivo [Of the handful of essential kinetochore proteins that are widely distributed among eukaryotes, only one class other than CenH3 has been shown to bind centromeric DNA: centromere protein C (CENP-C), a conserved component of the inner kinetochore in vertebrates -16. Humain vitro -19 and b[in vivo ,21. Vert[in vivo ,23 share[in vivo  and plan[in vivo . As expe[in vivo ,26-28.Other than the defining CENPC motif, these proteins are dissimilar in sequence across disparate phyla. Such a small stretch of sequence conservation, accounting for less than 5% of the length of these 549-943 amino-acid proteins, is unexpected considering that CENP-Cs are encoded by essential single-copy genes that are expected to be subject to strong negative selection. We therefore wondered whether the same evolutionary forces responsible for the rapid evolution of CenH3s cause divergence of CENP-Cs outside of the CENPC motif.Cenpc genes and test whether Cenpc genes are in general, like CenH3 genes, subject to positive selection. We find evidence for adaptive evolution of CENP-C in plants and animals, but we find negative selection in yeasts. Our results provide support for a meiotic drive model of centromere evolution.Here, we describe coding sequences from several unreported Drosophila and Arabidopsis [a) and synonymous substitution (Ks) between coding sequences from closely related species. These rates are expected to be equal if the coding sequences are evolving neutrally (Ka/Ks = 1). Negative selection is indicated by Ka/Ks < 1, and positive selection is indicated by Ka/Ks > 1.Previous work has shown that CenH3s are evolving adaptively in bidopsis ,10, but Mus musculus) CenH3, CENP-A [Rattus norvegicus) genomic clone (AC110465) that contains the predicted rat CENP-A coding sequence. The predicted CENP-A protein is encoded in four exons and is 87% identical in amino-acid sequence to mouse CENP-A, excluding a 25 amino-acid insertion that appears to derive from a duplication of the amino terminus  in both the tail and the histone-fold domains. Similar results were obtained when comparing either sequence with the Cenpa gene from Chinese hamster (Cricetulus griseus) [s = 0.45 rat, 0.67 mouse) makes the statistical conclusion near the limit of reliability (Ks ≤ ~0.5) because of the increased likelihood of multiple substitutions. Thus, CENP-A appears to have been under negative selection throughout its length in multiple rodent lineages.To determine whether stimator . Positivgriseus) , althougCenpa gene [Cenpa gene from chimpanzee (Pan troglodytes). A blastn search of the Genome Sequencing Center's assembly of the chimpanzee genome [Cenpa identified the chimp Cenpa gene encoded in four exons in Contig 286.218. We searched the NCBI trace archives [Cenpa gene differs from the human gene by six synonymous nucleotide substitutions and an indel (insertion or deletion) of two codons. This excess of synonymous substitutions indicates negative selection of CENP-A (p < 0.01). Overall negative selection of CENP-A appears also to extend to the bovine (CB455530) protein, given the relatively high degree of conservation seen for all regions, including the tail and Loop 1 regions that evolve adaptively in Drosophila  of maize (Zea mays) [Saccharum officinarum), and identified three that encode full-length CENH3 genes (CA119873, CA127217, and CA142604). The CenH3 proteins encoded by these ESTs differ from each other by 2-4 amino acids. Because sugarcane is thought to be octaploid, these variants may represent co-expressed homeologs. The coding regions of ESTs CA119873 and CA127217 differ by four synonymous and four nonsynonymous substitutions (Ks = 0.03, Ka = 0.01), suggesting negative selection. Comparison of either of these sequences with maize CENH3 by sliding-window analysis found that all windows had Ks > Ka, with overall negative selection . Thus, in contrast to CenH3s in Arabidopsis and Drosophila, CenH3s of rodents, primates, and grasses appear not to be evolving adaptively.We also found overall negative selection in CenH3s of grasses. We used the ea mays)  to searcea mays)  from sugThe evident lack of positive selection on CenH3 in mammals and grasses raises the possibility that another kinetochore protein is evolving in conflict with centromeric DNA in these organisms, in which centromeric satellite sequences are known to be evolving rapidly ,38. We fCenpc ESTs from rat. From these ESTs , we obtained and sequenced a full-length cDNA , and compared its coding sequence with that of the mouse Cenpc gene (68% predicted amino-acid identity). We found positive selection over most of the amino-terminal two-thirds of the coding sequence, interrupted by one region of significant negative selection (mouse codons 208-273), one region of nearly significant negative selection (mouse 410-464), and three short regions without significant selection  to search the assembled chimpanzee genome and the NCBI trace archives. We found that the chimpanzee genome contains a single copy of the Cenpc structural gene (contigs 375.88-375.100), as well as a processed Cenpc pseudogene (contigs 76.642-76.643), as has been found in humans [Cenpc coding sequence differs by 17 nucleotide substitutions from the human cDNA sequence, with Ks = 0.0054 and Ka = 0.0063. The > 99% identity of the human and chimp coding sequences provides little opportunity to detect selection, but using sliding-window analysis we found a single region of significant positive selection (human codons 278-585) that overlaps the central regions of positive selection found in the more divergent rat-mouse comparison, indicating that the central portion of CENP-C is under positive selection in both rodents and primates.To determine whether any of these regions is also under positive selection in primates, we identified the n humans ,18,39. Ta/Ks ratios. We compared the null model with two fixed site classes (Ka/Ks = 0 or 1) to a 'data-driven' model in which two classes of sites were estimated from the data. The data-driven model was found to be significantly more probable than the null model  with Ka/Ks = 0.20 for 57% of the 685 sites in the multiple alignment and Ka/Ks = 1.64 for 43% of the sites (data not shown). Similar results were obtained using either a DNA- or a protein-based tree, or testing more complex models. When the same tests were applied to the core region of 11 aligned Brassicaceae (mustard family) CenH3s, only 17% of residues were estimated to be in the positive selection class (Ka/Ks = 2.54)  in a region of positive selection identified by K-estimator analysis. Therefore, pairwise K-estimator and multiple PAML analyses yield similar results and reveal that large regions of mammalian CENP-Cs have been adaptively evolving.in vitro has been mapped by two groups of investigators. Sugimoto and colleagues [in vivo [Arabidopsis CenH3, adaptively evolving codons are found in close proximity to codons under strong negative selection [The regions of positive selection in rodent and primate CENP-Cs overlap some protein landmarks identified in functional analyses of human CENP-C. The binding activity of human CENP-C to DNA lleagues ,18 foundlleagues  identifi[in vivo . Compari[in vivo ,11. No pXenopus [Drosophila CenH3, where the adaptively evolving Loop 1 region has been shown to be necessary and sufficient for targeting when swapped between native and heterologous orthologs [In human CENP-C, three regions have been reported to confer centromere targeting. One targeting signal was recently reported in region 283-429 . A seconXenopus ,41-43. Arthologs . TherefoTargeting of native CENP-C proteins depends on other centromere proteins that vary according to species , but thein vitro or that are sufficient for targeting centromeres. For example, the region comprising the most amino-terminal 200 or so amino acids of rodent CENP-C has been evolving adaptively, but the orthologous region in human CENP-C fails to bind DNA in a southwestern assay [Drosophila CenH3, which was found to be neither necessary nor sufficient for targeting in vivo to homologous centromeres. In this case, Loop 1 was identified as the targeting domain, and the amino-terminal tail was hypothesized to help stabilize higher-order chromatin structure by binding to linker DNA, similar to the known binding activity of canonical histone tails [Drosophila, then CENP-C might be playing a comparable role in the stabilization of higher-order centromeric chromatin.Not all regions of CENP-C that display positive selection correspond to regions that bind DNA rn assay ,19 or torn assay . This sune tails . If CENPPositive selection in the central DNA-binding and centromere-targeting region of CENP-C offers an explanation for the lack of conservation of this region between chicken and mammals : as posiCenpc genes in plants had been characterized only in maize (Z. mays), so we needed first to identify Cenpc homologs from other plants to ascertain whether or not the gene is evolving adaptively.Our finding that adaptive evolution is occurring in animal CENP-Cs encouraged a similar survey of plant CENP-Cs, because centromeres from both animals and seed plants comprise rapidly evolving satellite sequences. At the time we began this study, Cenpc homologs have been described in maize: CenpcA, CenpcB, and CenpcC [Cenpc homolog (AC013453, At1g15660) in the genome of Arabidopsis thaliana by sequence similarity at both protein termini  revealed that the 705 amino-acid CENP-C protein of Arabidopsis is encoded in 11 exons, with the CENPC motif encoded in exon 10 . We obtained the cDNA clones corresponding to 16 of these ESTs and sequenced them completely . An alignment of the carboxyl termini encoded by cDNAs representing six angiosperm families revealed that the final 80 or so amino acids of CENP-C, including the CENPC motif, are highly conserved in plants . A full-length cDNA from barrel medic (Medicago truncatula) encodes a protein of 697 amino acids, which corresponds to a gene model of eleven exons when aligned to a genomic pseudogene , maize, and sorghum (Sorghum bicolor)  [Cenpcs in encoding an unrelated carboxyl terminus. CenpcC is 99.9% identical to maize CenpcA until it diverges downstream of the CENPC motif at the point corresponding to the end of exon 13 in our gene model. On the basis of an overlap with maize and Sorghum genomic sequence that spans the intron between exons 13 and 14, we conclude that the divergent 3' end of CenpcC derives from the unspliced intron 13 of CenpcA, and that all angiosperm CENP-Cs share a highly conserved carboxyl terminus.As an aid in identifying other conserved regions of angiosperm CENP-Cs, we developed gene models for full-length e Figure . We also) Figure . The maiF129859) , which dArabidopsis, barrel medic, maize, Sorghum, and rice, the limited conservation of the encoded amino-acid sequences and approximate correspondence of exon sizes suggest that the exons in the amino-terminal half and the final two exons of plant CENP-C are conserved  cDNA that appears to contain an unspliced intron. Our alignment reveals short regions of conservation throughout the amino terminus, as well as a high relative incidence of the dipeptide SQ in the poorly conserved exon 5  and barley (Hordeum vulgare) indicate that there are two tandem copies of exons 9 and 10 in these species . The S. bicolor pseudogene has a deletion that joins sequences just upstream of the initiation codon in exon 1 to sequences upstream of exon 2. Despite the presence of tandemly duplicated exons, the S. bicolor truncated pseudogene is more closely related to the full-length S. bicolor gene than it is to the S. propinquum gene. Exons 11 and 12 in the S. bicolor full-length gene are identical to 11b-12b in the pseudogene, but have 7 differences from 11a-12a. This suggests that the duplication of exons 11 and 12 preceded the divergence of S. propinquum and S. bicolor, and that the full-length S. bicolor gene may have been derived by loss of exons 11a-12a from a full-length ancestral gene similar to the truncated pseudogene.Exon duplications were also found for E < 0.0001 for both comparisons). We conclude that exon pairs 9-10 and 11-12 derive from a more ancient duplication event.We wondered why two different pairs of exons, 9-10 and 11-12, were each independently subject to duplication in the grasses. When we examined multiple alignments of the peptide sequences encoded by both exon pairs in Logos format, it became apparent that they resembled each other in length and composition Figure . Exons 9Sorghum lineage; fourth, two duplications generating rice exons 9a-10a, 9b-10b, and 9c-10c all within the rice 9q-10q lineage; and fifth, a partial duplication in rice exon 9c.To trace the likely ancestry of these duplication events, we used an alignment of the exons from multiple species to construct phylogenetic trees of duplicates of exons 9-10 and 11-12 Figure . This phS. bicolor gene, one of exons 11b-12b in the sugarcane genes, and one of the hypothetical rice 9p-10p. Alternatively, it is possible that the latter loss and one of the rice-specific duplications resulted from gene conversion of rice 9p-10p by a derivative of rice 9q-10q. Regardless of the exact number of duplication and deletion events, it is clear that the exon pair ancestral to grass exons 9-10 and 11-12 has been subjected to repeated episodes of duplication and deletion.There also appear to have been at least three losses of duplications: one of exons 11a-12a in the lineage leading to the full-length Cenpcs allowed us to analyze them for evidence of adaptive evolution. First, we compared Cenpcs from Arabidopsis species in which we had previously found adaptively evolving CenH3s. Using the A. thaliana genomic sequence to design primers, we amplified, cloned, and sequenced a Cenpc cDNA from A. arenosa . Comparing this sequence with that of A. thaliana, the predicted proteins differ by 87 amino-acid subtitutions out of 703 alignable residues, plus five indels of 1-3 amino acids.The delineation of gene models for plant A. thaliana and A. arenosa Cenpc. At three regions, Ka exceeded its 99% confidence interval for the null hypothesis, indicating that these regions are under positive selection , the 3' half of exon 6 (codons 376-441), and exons 8 and 9 (codons 486-618). In addition, a region encompassing most of exons 1 and 2 (codons 24-89) was found to be under positive selection with p < 0.03. We also determined that the 5' half of exon 6 (codons 255-386) and the conserved exons 10 and 11 (codons 595-703) are under negative selection with p < 0.01.We applied the sliding window option of K-estimator to the aligned coding sequences of  Figures ,3. TheseA. arenosa cDNA has a CAG (glutamine) codon that is absent in the A. thaliana cDNA, appears to be caused by the species-specific use of alternative acceptor splice sites, because the genomic sequence (data not shown) at this intron-exon boundary is identical in both species (...cag cag ^GAG GGT... or ...cag ^CAG GAG GGT...). The presence of species-specific alternative splicing of the same codon in an adaptively evolving region suggests that splicing variation can contribute to adaptive variability.Curiously, an indel at the beginning of exon 9, where the Cenpc is unique to Arabidopsis or occurs more generally in plants, we compared Cenpc genes from the two Sorghum species. We removed the duplicate exons 11a and 12a from the S. propinquum coding sequence in order to compare the sequence with the full-length gene from S. bicolor . For this comparison, Ka = 0.014 and Ks = 0.003. Ka exceeds the 99% confidence interval of the null hypothesis, and neutral evolution can be rejected in favor of positive selection. The limited divergence between these two sequences did not allow statistically significant conclusions to be drawn about positive selection in particular regions of the gene.To examine whether positive selection in S. bicolor sequence with the maize CenpcA coding sequence (75% amino-acid identity). Between maize CenpcA and S. bicolor, Ka = 0.12, Ks = 0.14, and there are seven indels of 1-11 codons. We identified positive selection for a single window in exon 1, for a region including all of exon 5, for a region in the second half of exon 6, and for a region from the end of exon 12 through most of exon 14 , two had identical coding sequences (Cenpc1), and the third (Cenpc2) differed by 13 nucleotide substitutions, suggesting that Cenpc1 and Cenpc2 may be homeologous genes in the polyploid sugarcane genome. We compared Cenpc1 to maize CenpcA and S. bicolor Cenpc. Regions of positive or negative selection identified in the maize/Sorghum comparison were generally found to coincide with regions under selection in the corresponding direction in maize/sugarcane and Sorghum/sugarcane comparisons, although in a few cases the selection was not found at the p < 0.05 level of significance in all comparisons  that was not under significant selection in the maize/ge Table . Therefo2 = 12.0; p = 0.003) with Ka/Ks = 0.00 for 51% of the 686 sites in the multiple alignment and Ka/Ks = 2.00 for 49% of the sites (data not shown). Using the PAML 'free-ratio' option that measures Ka/Ks differences between branches in a tree [2= 10.0; p = 0.007 for the data-driven over the null model) along both Sorghum lineages (Ka/Ks = 2.6) and along the sugarcane Cenpc2 lineage (Ka/Ks = 1.3) but not detectably along the sugarcane Cenpc1 lineage (Ka/Ks = 0.23). PAML analysis also confirmed that the carboxy-terminal region of maize CenpcA is adaptively evolving  with Ka/Ks = 1.4. Thus, different methods of analysis demonstrate that CENP-Cs are adaptively evolving in an episodic fashion in grasses that have multiple CENP-C copies.Phylogeny-based PAML analysis confirms that plant CENP-Cs are adaptively evolving and in an episodic fashion, consistent with inferences based on pairwise K-estimator analysis. As was found for mammalian CENP-C, the data-driven model for grass CENP-C was found to be significantly more probable than the null model , and positive selection from the end of exon 6 through exon 7 (codons 403-492, p < 0.01). Elsewhere the null hypothesis could not be rejected. Comparing CenpcB with S. bicolor, Ka = 0.13 and Ks = 0.11, and there are six in-frame indels between the sequences. We found positive selection in the first half of exon 6 (Sorghum codons 273-327, p < 0.03) and negative selection from exon 10 through the first few codons of exon 13 (codons 537-619, p < 0.02). Elsewhere the null hypothesis could not be rejected. In summary, regions under negative selection in other grass Cenpcs can be under positive selection in CenpcB, and regions under positive selection in other grass Cenpcs are under negative selection or evolving neutrally in CenpcB . Additional windows in exons 9p-10p and 12 had Ka >Ks (p > 0.05), suggesting that most of the duplicated region has been evolving adaptively , we detected only negative selection (p = 0.01), as though exon duplication allowed for adaptation.Just as gene duplication can result in different selective pressures on the two genes, duplications within a gene can lead to specialization and thus can change selective pressures on the region. Such specialization appears to have occurred between the anciently duplicated region encoded by exons 9-10 and 11-12 Figure . In maizy Figure . In contDrosophila CenH3s, which contain repeats of a minor groove-binding motif that are thought to provide DNA compositional preference [We find an approximate correspondence between adaptively evolving regions in angiosperms and those in mammals that overlap DNA-binding and centromere-targeting regions. Although no DNA-binding regions have been experimentally determined for plant CENP-Cs, the correspondence with animal CENP-Cs suggests that the different regions play comparable roles. One of the corresponding adaptive regions is repeatedly duplicated in grasses, and the distribution of basic residues in exons 10 and 12 suggests that the repeat unit binds DNA. A parallel situation again appears to be found for the amino-terminal tails of some MIF2 genes of Saccharomyces cerevisiae [S. paradoxus [a = 0.036 and Ks = 0.38. In sharp contrast to all pairwise comparisons of plant and animal Cenpc genes, Ka was much less than Ks for all of the 99 bp windows of yeast MIF2, indicating that it is under negative selection throughout its length (p < 0.001). In all pairwise comparisons among these two species and the additional species S. mikatae and S. bayanus [a << Ks . We also found strong negative selection for all 99 bp windows in pairwise comparisons of yeast CenH3 . Thus, adaptive evolution of both CenH3s and CENP-Cs appears to be limited to organisms with complex centromeres.If positive selection for CENP-Cs in plants and animals is related to centromere complexity, then we would expect conventional negative selection to operate in organisms such as budding yeast, which have simple centromeres. The revisiae  and S. paradoxus  are 93%  bayanus , we consDrosophila and Arabidopsis CenH3 [We have demonstrated that CENP-C has been adaptively evolving in multiple lineages of both plants and animals, a feature that had been previously shown for some CenH3s. Thus, the occurrence of adaptive evolution appears to be a general feature of proteins that bind to complex centromeres. Recurrent adaptive evolution implies an arms race, and an arms race involving centromeric DNA-binding proteins is remarkable given that centromeres have a conserved function. But centromeric DNA is rapidly evolving in plants and animals, so adaptation of the major centromere DNA-binding proteins would maintain an interface with the conserved kinetochore machinery. Indeed, regions of CENP-C that show evidence of positive selection include DNA-binding and specificity regions, in parallel with previous findings for is CenH3 ,11,44.A 'meiotic drive' model has been proposed to explain the rapid evolution of centromeric DNAs and CenH3s . AccordiThe lack of evidence for adaptive evolution in CenH3s from mammals and grasses does not seem to fit this scenario. But the extensive positive selection on the corresponding CENP-Cs provides a ready explanation for the absence of an adaptive signal for CenH3. The meiotic drive model predicts that over evolutionary time any mutation that restores centromere parity will be selected, suggesting that proteins besides CenH3 - and in particular other kinetochore proteins that contact centromeric DNA - may be positively selected to suppress centromere drive. Our demonstration of the adaptive evolution of CENP-C, especially in DNA-binding regions, fulfills this prediction of the centromere drive model. Apparently, in mammals and grasses CENP-C performs the function of a suppressor of meiotic drive.Drosophila species have the longest CenH3 tails known [Drosophila with centromeric satellites compensates for the absence of CENP-C and permitted its loss. This might explain why Drosophila CenH3s localize in a species-specific manner [The large size and lack of sequence conservation of CENP-Cs make them much larger mutational targets for suppression than CenH3s. Moreover, PAML analysis suggests that a larger proportion of CENP-C than CenH3 residues are evolving adaptively. Mammalian CENP-A consists of a well-conserved histone-fold domain with only a short unconstrained tail region. Conversely, ls known  but lackc manner , whereasc manner .Drosophila CenH3 gene [Centromere drive may have important consequences for karyotypic evolution. Centromeres of two acrocentric chromosomes frequently fuse (Robertsonian translocations), and metacentrics often misdivide to yield two acrocentrics. In humans, there is a bias in favor of Robertsonian translocations over their homologous acrocentric pair when transmitted by females, and male carriers have reduced fertility . This genH3 gene ,62. The Candida glabrata and Kluyveromyces lactis, which have greater average divergence from budding yeast in protein sequences than mammals have from fish [In contrast to CENP-Cs of plants and animals, yeast Mif2p appears to have evolved entirely under negative selection. This is consistent with Mif2p interacting with a stable centromere, rather than one that is rapidly evolving. In accordance with this observation, budding yeast centromeres are determined by the presence of a consensus DNA sequence that includes binding sites for the Cbf1 and CBF3 proteins . The conrom fish . We attrA. thaliana cDNA was amplified from a cDNA pool from whole plants of the ecotype Columbia, and the A. arenosa cDNA was amplified from a cDNA pool from leaves of Care-1 [A. thaliana genomic Cenpc sequence. Genomic DNAs from wheat and barley were generously provided by Andreas Houben. Exons 9p-10p and the intervening intron 9p were amplified from both wheat and barley using the primers 5'-AGATGAACCAATCCATCCAC-3' and 5'-AAATTCGTTTTCCTCTCTTTGCT-3'. Likewise, 9q-10q and intron 9q were amplified with the primers 5'-AGATAAGCCAATCCATACATCA-3' and 5'-CCCCTCTTTTCATTCTCTTCAA-3'. The first and last of these four primers were also used to amplify both exon pairs as a unit to confirm their contiguity and to determine the genomic sequence around the junctions of intron 10p with exons 10p and 9q. Amplifications used High Fidelity Platinum Taq polymerase (Invitrogen, Carlsbad, USA). The amplified fragments were cloned using the pCR2.1-TOPO-TA cloning kit (Invitrogen) according to the manufacturer's instructions. Sequencing was carried out using ABI Big Dye sequencing on both strands of all reported sequences. Sequencing primers were standard vector primers or were designed using Primer 3 [Genomic DNAs and cDNAs were obtained from several sources . The f Care-1 . Both ofPrimer 3 . SequencPrimer 3 . AccessiSequence similarities of genes and their encoded proteins were identified using the NCBI BLAST server ,67, as wa and Ks in comparisons of Cenpa/CENH3 or Cenpc genes from pairs of closely related species. Prior to analysis, gaps were removed from the coding sequences as indicated by the amino-acid alignments.K-estimator  was useda and Ks for windows of 99 nucleotides, positioned every 33 nucleotides. For candidate regions of positive selection, we determined the confidence intervals of Ka and Ks under the null hypothesis that Ka is equal to Ks using the default parameters (1,000 replicates). For individual windows of 99 nucleotides, or for regions defined by contiguous groups of overlapping windows, limited trial and error suggested that statistically significant positive selection was not supported if Ka/Ks < 1.5. Therefore, to find evidence of positive selection, we determined the confidence intervals for regions defined by sets of overlapping or immediately adjacent 99 nucleotide windows with Ka/Ks ≥ 1.5. For regions with Ks = 0, one or more flanking windows with Ks > 0 were included in the region analyzed, regardless of the value of Ka, so that a value for Ka/Ks could be defined. Similarly, we looked for statistically significant negative selection for regions defined by overlapping or adjacent 99 nucleotide windows with Ka/Ks ≤ 0.67. The codeml program of PAML version 3.13d [a/Ks ratios as previously described [We estimated Kon 3.13d  was alsoescribed .Cenpc ESTs and genomic sequences from GenBank. Additional data file Cenpc cDNAs and amplified genomic sequences. Additional data file The following are provided as additional data files. Additional data file Cenpc ESTs and genomic sequences from GenBankTable S1 reports accession numbers for selected Click here for additional data fileCenpc cDNAs and amplified genomic sequencesTable S2 reports accession numbers for Click here for additional data fileFigure S1 displays the conservation of the exon containing the CENPC motifClick here for additional data file


Before therapeutic effect is obtained after treatment with antidepressant drugs, like serotonin selective reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAO-Is) there is an initial lag-period of a few weeks. Neuronal adaptations on a molecular level are supposed to be involved in the initiation of the antidepressant effect. Transcription factor AP-2 is essential for neuronal development and many genes involved in the brainstem monoaminergic systems have binding sites for AP-2 in their regulatory regions. The genotype of the AP-2β isoform has been associated with e.g. anxiety-related personality traits and with platelet MAO activity. In addition, previous studies have shown that the levels of AP-2α and AP-2β in rat whole brain were decreased after 10 days of treatment with citalopram (SSRI) and imipramine (TCA), and were increased with phenelzine (MAO-I).In the present study, we report that treatment with citalopram for 1, 7 or 21 days did not have effect on the AP-2 levels in rat brainstem. However, after treatment with phenelzine for 1, 7 or 21 days the levels of AP-2α and AP-2β had increased after 7 days, but had returned to control levels at day 21.The decrease in AP-2 levels in rat whole brain previously seen after treatment with citalopram does not seem to be localised to the brainstem, it may rather occur in the monoaminergic terminal projection areas. The present data suggest that the increase in AP-2 levels previously seen in rat whole brain after subchronic treatment with phenelzine is located in the brainstem. It cannot, however, be excluded that other brain regions are involved. Well-known antidepressant drug effects are blockade of the serotonin 5-HT) and/or norepinephrine (NE) reuptake pumps, direct effects on the 5-HT- and/or NE receptors and inhibition of the monoamine oxidase A (MAO-A) enzyme. Irrespective of which drug or combination of drugs that is used, there is a delay of a few weeks before any therapeutic effect is noticed. This initial lag-period usually is associated with several side-effects, many of which fade away with the appearance of the therapeutic effect. Considering the lag-period needed for the antidepressant effect to emerge, it has been suggested that the antidepressant mechanisms involve secondary molecular neuronal adaptations, rather than being a result of the primary actions of the drugs ,2. Some eceptors ,4. Furth-HT and/oeceptors . Gainingeceptors -11. Traneceptors -14. Fiveeceptors -19. The eceptors . Severaleceptors -26 indiceceptors , indicateceptors ,29. The eceptors  and to peceptors , which teceptors .In a previous study, we reported that the levels of AP-2α and AP-2β were decreased in rat whole brain after treatment for 10 days with citalopram, imipramine and lithium, respectively . We haveThe mean relative amounts of AP-2α and AP-2β protein ± standard deviation (SD) for each of the citalopram-, phenelzine- and saline treated animal groups are shown in tables When comparing the untreated naive animals with the treated animal groups no differences with regard to the levels of AP-2α  or AP-2β  were found. Moreover, no differences in the levels of AP-2α and AP-2β were observed between the groups of animals treated with saline for the different time periods.In two independent studies, we have shown that the levels of AP-2α and AP-2β in rat whole brain were decreased after subchronic (7 or 10 days) treatment with citalopram ,34. We h1A receptors in hippocampus get an enhanced response to 5-HT after treatment with TCA (that blocks both 5-HT and NE reuptake) for a time-period that corresponds to the time required for initiation of the antidepressant therapeutic effect [1A and in other projection areas than hippocampus [The different responses on AP-2 of citalopram and phenelzine are likely to be explained by the different molecular mechanisms of the two drugs. Considerering the specific drug targets for citalopram and phenelzine, respectively, the target for citalopram, 5-HTT, is specific for membranes of the serotonergic system, while the target for phenelzine, MAO-A, is not located in the serononergic neurons . It has c effect . An incrpocampus . Thus, tpocampus , an effepocampus ), coinciIn a previous study, we reported higher levels of AP-2α and AP-2β in rat whole brain in untreated naive animals compared to animals treated with citalopram or saline for different time periods . In the As mentioned earlier, we have previously shown that the AP-2β genotype is associated with platelet MAO activity . Thus, iUnraveling of the molecular mechanisms involved in the initial phase of antidepressant treatment is essential for the development of new efficient antidepressant drugs with less side-effects. We find transcription factors, such as AP-2, with ability to regulate expression of specific genes involved in the monoaminergic mechanisms, to be interesting candidates as novel antidepressant drug targets.2 inhalation 24 hours after their last injection. A group of untreated naive animals (n = 6) was sacrificed after 21 days. After sacrifice the brainstem was dissected and nuclear extracts were prepared for measurement of AP-2 levels by Enzyme-Linked Immunosorbent Assay(ELISA). This study was carried out with permission from the local animal ethics committee in Uppsala, Sweden.Adult male Sprague-Dawley rats  were housed in groups of three and maintained on a 12 hour light /dark cycle with food and water freely available. Animals were administered phenelzine (n = 18, 10 mg/kg, Sigma, Sweden), or citalopram (n = 18, 10 mg/kg, Lundbeck AB, Helsingborg, Sweden) subcutaneously with daily injections. All drugs were dissolved in saline (NaCl, 9 mg/kg). Saline treated animals (n = 18) received saline injections in the same volume as that given the citalopram and phenelzine treated animals. Each group of animals was treated for 1, 7 or 21 days, respectively. All animals were sacrificed by CORat brainstem was homogenized in 3 ml buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, pH 7.9). The homogenate was incubated on ice for 15 minutes. To this 125 μl Nonidet P40 was added, and the homogenate was centrifuged for 30 seconds at 14000 rpm in 4°C. The pellet was resuspended in 500 μl buffer C 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, pH 7.9). Thereafter the tubes were put on a shaker for 15 minutes and centrifuged at 14000 rpm for 5 minutes (4°C). The supernatant i.e. the nuclear protein were aliquoted and stored at -80°C. The protein concentration for all nuclear extracts were determined by the method by Lowry at al. (1951) . The con2, pH 9.5). Thereafter, 50 μl substrate (Phosphatase substrate, 5 mg tablets, Sigma, Sweden, diluted in 5 ml Wash buffer II) was added to each well. The reaction continued for 30 minutes and was terminated by adding 50 μl of 0.1 M EDTA, pH 7.5. The plates were analysed in an ELISA reader (Molecular Devices, Thermo Max) at optical density (OD) 405/490. The OD of the AP-2 isoforms for each rat was correlated to a value in a standard curve, where known concentrations of antibody were plotted against optical density. The value form the standard curve was then divided with the concentration of total protein in the nuclear extracts. The quota was used as a value of the relative amount of AP-2α and AP-2β protein. Each rat were analysed twice for accuracy.96-well microtiter plates were coated (50 μl/ well) with nuclear extracts (10 μg/ml) diluted in 50 mM Carbonate-Bicarbonate buffer pH 9.0. The plates were covered with parafilm and incubated overnight at 4°C. Antigen solution was then removed and 200 μl blocking buffer (PBS, 1 % BSA) was added to each well and the plates were incubated for two hours in room temperature. Following this the blocking buffer was removed and the plates were washed with PBS. Primary antibody  diluted in blocking buffer was then added and the plates incubated overnight at 4°C. After incubation the antibody was removed and the plates were washed three times with Wash buffer I (PBS, 0.05 % Tween-20). Secondary antibody  diluted 1:350 in blocking buffer, was then added (50 μl) to each well and the plates were incubated for two hours in room temperature. After removal of the secondary antibody the plates were washed three times with Wash buffer I, and once with Wash buffer II  and Fisher's Protected Least Significant Difference (PLSD). To test if any of the groups of saline treated animals differed in the amounts of AP-2α and AP-2β protein ANOVA and PLSD test were used. All calculations were performed using Stat View 5.0 software (SAS Institute Inc., Cary, NC, USA). Results have been considered statistically significant when p < 0.05.CB planned the experiments, carried out the molecular and statistical analyses and drafted the manuscript, MD participated in planning the experiments and writing of the manuscript, LO conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Caenorhabditis elegans, a tiny nematode and the first animal to have its genome sequenced, to gather functional information on nearly 400 genes.The past ten years saw great progress in the field of molecular genetics, as new tools gave scientists the ability to investigate entire genomes instead of just one or two genes at a time. In this paper, Ronald Plasterk and colleagues developed a systemic approach using Many of the systemic approaches to discovering gene function involve either measuring or deleting messenger RNA (mRNA), the molecule that helps translate genes into proteins. The method used here, called RNA interference, or RNAi, follows the deletion approach by taking advantage of a cellular process bearing the same name. In nature, RNAi is thought to be an important part of the innate defense machinery in plants and animals, protecting them from invaders like viruses by interrupting the manufacture of viral proteins. To do this, short double-stranded RNA molecules with complementary sequences to the target gene inhibit the gene's function by disabling mRNA, which effectively shuts down the gene. By mimicking this natural process to turn off selected genes, scientists can find clues to how those genes might normally function by watching what happens when they are taken out of the picture.With the fully sequenced worm genome, it is possible to create interfering RNAs for all of its 20,000 or so genes. And because worms eat bacteria—which can themselves be used to deliver interfering RNAs—worms are the perfect RNAi model organism. The researchers fed the worms RNAi-producing bacteria, then observed the effects on the worm or its offspring to infer the function of the targeted gene. As previously reported, repeating this experiment for every gene in the worm genome, yields about 10% of the worms displaying abnormalities ranging from embryonic death to uncoordinated movement, suggesting defects in genes controlling development or muscle control, respectively.Having previously identified an RNAi-hypersensitive mutant worm strain, Plasterk and his colleagues repeated the experiment in the mutants and report proposed functions for 393 previously unknown genes. The types of abnormalities observed in the short-lived mutations induced by RNAi, they say, resemble the more stable mutations seen in the collection of worm mutations cataloged by worm researchers over the years. Though the DNA alterations for many of these mutations are not yet known, researchers know roughly where they occur in the genome. And the researchers show here that they can use their RNAi experimental results along with what is known about the mutants to identify several of the sequence alterations. They also performed what is believed to be the first analysis in which independently generated large-scale RNAi results were systematically compared to see how variable such RNAi results are, and the results have implications for similar approaches not just in worms but in plants and other animals.


Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays.Microarrays are an important tool with which to examine coordinated gene expression. Soybean (We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants.The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines.Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype. Genes of higher plants are expressed in a coordinated fashion during development of tissue and organ systems and in response to different environmental conditions. This regulation may be tightly linked for some sets of genes, for example, in a specific biochemical pathway. Expression of regulatory genes may modulate the expression of key genes or entire sets of genes in individual pathways. The investigation of single gene expression patterns as determined by RNA blotting or quantitative reverse transcriptase PCR have been used to understand how different temporal, developmental, and physiological processes affect gene expression. With recent advances in genomics, very large numbers of genes can now be simultaneously analyzed for their expression levels in a comparative fashion between two biological states using microarray or biochip technology. Several techniques for the 'global' analysis of gene expression have been described -4. TheseGlycine max), more than 286,000 5' EST sequences have been generated and deposited in public databases [[Methods for global expression analysis require either knowledge of the entire genome of an organism or accumulation of a large EST (expressed sequence tag) database for the organism. In soybean  resulting from a computer assembly of ESTs is an estimate of the number of unique genes in the organism. As the number of ESTs grows, the number of unique genes in the organism will continue to be refined. Our current contig analysis of the entire public EST collection for soybean of 286,868 sequences yields 61,127 'unigenes' of which 36,357 are contigs and 24,770 are singletons.Arabidopsis thaliana (of 247,429 ESTs), 36,976 for Medicago truncatula (of 189,919 ESTs), 31,012 for tomato (of 156,645 ESTs), 109,509 for wheat (of 494,195 ESTs) and 56,364 for maize (of 377,188 ESTs). The complete genome sequence for Arabidopsis has revealed an estimated 26,000 genes [9 bp which is about 7.5 times the size of the Arabidopsis genome and twice that of tomato, but less than half the size of the maize genome. Thus, it is not unexpected that soybean may have a larger number of unigene clusters than Arabidopsis or tomato, for example. Although soybean is not hexaploid in origin as is modern wheat, it is thought to be an ancient autotetraploid and many examples of duplicate loci exist in soybean.The finding of 61,127 soybean unigenes by EST cluster analysis agrees well with an independent contig and unigene assembly in the databases of The Institute for Genomic Research (TIGR) which shows 30,084 contigs and 37,601 singletons for a total of 67,826 tentatively unique sequences from among 334,730 sequences representing all publicly available sequences clustered in Release 11 [of 247,42 ESTs, 3633P-cDNA produced by reverse transcription of total mRNA isolated from immature soybean cotyledons. Figure Certain tissues contain large amounts of specialized transcripts. Developing soybean cotyledons, for example, contain large amounts of RNA transcripts representing highly expressed storage protein genes. In order to select for some of the weakly expressed cDNA clones from a mid-maturation stage cotyledon library, we used a virtual subtraction approach using high densisty filters. A total of 18,000 bacterial clones containing cDNAs from library Gm-c1007 (immature cotyledons of 100–300 mg fresh weigh range from Williams variety) was printed in high density on nylon filters and probed with High density cDNA arrays of bacterial cultures spotted on nylon membranes and probed with radioactively labeled transcripts are useful for gene discovery as illustrated in Figure To enable global expression studies, the ideal would be to have each gene represented at least once on an array. Toward this aim, we selected 27,513 of the cDNA clones by four successive clustering assemblies of the soybean cDNAs performed as the 5' EST data accumulated. Table EST clustering will overestimate the number of unique genes as some of the shorter ESTs will not overlap and thus are falsely counted as independent, unique sequences. However, the clustering analysis can also falsely lump non-identical members of gene families into the same contig based on conservation of sequence similarity in the coding region. The 3' sequencing is especially useful for resolving both of these issues as there is generally more variation in the 3' UTR in plant genes than in the coding region. For those reasons and as a quality control of the reracking process, we sequenced the 3' end of the reracked cDNAs. From the 27,513 total 3' sequencing attempts on the tentatively unique cDNAs represented in Table Since the clustering analyses were performed at successive intervals as the EST collection grew in size, we repeated the Phrap contig analyses separately using only the input sequences of each cDNA rerack for which both a 5' and 3' EST were known. We also performed a CAP3 analysis . Table 1The 3' ESTs were also separately subjected to CAP3 or Phrap analysis. The CAP3 values showed a slightly higher level of uniqueness (or lower level of redundancy) with 79.3 to 91.5% for CAP3 in the successive clustering analyses versus 75.5 to 78.2% unique sequences as determined by Phrap. An overall figure of 73.0% for the CAP3 analysis on the 21,048 total 3' sequences clustered was found versus 63.3% for Phrap.The differences between the 5' and 3' levels of uniqueness (i.e., 88.2% versus 73.0% for the entire sets as determined by CAP3) can be explained by the nature of reverse transcriptase action. The reverse transcriptase was primed using an oligo dT primer and so the cDNAs will begin at the 3' end and will terminate randomly at variable sites as the enzyme progresses to the 5' end of the mRNA template. Thus, 5' ESTs often begin at variable sites. Therefore, even though two 5' ESTs may have originated from the same mRNA transcript, they will not cluster if they are non-overlapping and will be counted as two separate ESTs. The 3' soybean EST reads begin just after the poly A tail and produce longer average read lengths than the 5' soybean ESTs; thus, the 3' ESTs are more likely to form an overlapping contig if there is any redundancy among them.-6. Table The 5' and 3' sequence (where known) of each soybean unigene were queried against the non-redundant (nr) database with BLASTX . AnnotatBradyrhozobium japonicum, 2-month old roots, and whole seedlings); and Gm-r1088 . As an example, the Gm-r1070 set contains 3,938 tentatively unique cDNAs that are directly derived from two flower cDNA libraries (Gm-c1015 and Gm-c1016) that were sequenced deeply with over 14,000 5' ESTs obtained from these two libraries. In addition, a total of 2,639 cDNAs on the array are directly derived from source libraries made from the immature stages of cotyledon development and representing over 11,000 input sequences from the cotyledon stages of seed development.The current 'unigene' collection (or tentatively unique sequences) represents low redundancy sets of cDNA clones. We have processed all of these cDNAs for microarrays, as outlined in the Methods section, into three sets of 9,216 cDNAs per array. As shown in Table E. coli cultures containing the plasmid DNA). All 27,513 PCR reactions were performed with generic M13 forward and reverse primers using a robotic pipettor. Approximately 25% of the purified PCR cDNA inserts were subjected to agarose gel electrophoresis for quality control. Of these, the average insert size was estimated to be 1,340 bp for library Gm-r1021, 1,110 bp for library Gm-r1070, 1,259 for library Gm-r1083, and 1,269 bp for library Gm-r1088.The cDNAs from the sequence-driven, reracked clone sets were amplified by PCR using the Qiagen-purified cDNA templates that were prepared for 3' sequencing (as opposed to amplification of the inserts directly from The 9,216 amplified inserts of each set were singly spotted onto glass slides as outlined in the Methods section. A set of 64 control or 'choice' clones was assembled by hand into one 96-well plate (designated Gm-b10BB) and printed eight times repetitively throughout each array. Thus, the total number of spots on the array is 9,728 consisting of 9,216 cDNAs from the unigene set plus 512 (64 cDNAs × 8 repeats) from the choice clones. The choice clones were selected for various reasons. Some represent constitutively expressed genes (such as ubiquitin and EF1). Some are cDNAs whose expression is restricted to a subset of specific plant tissues (such as Rubisco or seed storage proteins). Some are clones of enzymes representing commonly used antibiotic resistance markers in transgenic plants (as hygromycin or kanamycin resistance), and 32 are cDNAs that represent at least 13 different enzymes of the flavonoid pathway. The flavonoid pathway was chosen because the corresponding genes often respond to many biotic and abiotic stress conditions and it has been widely studied in plant systems.T locus. The T locus controls the color of the pigment in the trichome hairs on the stems, leaves, and pods of the plant and also modifies the composition of the flavonoids and the color of the seed coats. Total RNA extracted from developing seed coats of line XB22A (T/T genotype) was labeled with Cy3 and compared to RNA extracted from the same stage of developing seed coats from an isoline containing the spontaneous mutation 37609 (t*/t* genotype) that was labeled with Cy5. A replicate experiment with a dye swap was also performed. Hybridizations were performed to microarrays constructed with the low redundancy set Gm-r1070 representing cDNAs from seeds, seed coats, and flowers. Figure T/T line relative to the t*/t* line by approximately three-fold. These cDNAs correspond to the flavonoid 3' hydroxylase cDNAs that were repetitively spotted on the array as members of the 'choice' clone set. Table Figure t test showed that the mean ratio of the repeated cDNAs on replicate 1 (2.424) were statistically significant at a P value of 0.0001 when compared to an expected mean of a 2.0, or a two-fold expression difference. The low P values were also found for replicate 2 and for the mean value (3.245) of both replicates. Thus, the flavonoid 3' hydroxylase cDNAs are statistically significant outliers in the microarray analysis.An examination of the ratios of the 16 repetitively spotted flavonoid 3' hydroxylase cDNAs using a T/T line agree very well with RNA blot data which showed that the flavonoid 3' hydroxylase gene has reduced expression in the seed coats of the t*/t* isoline compared to the T/T lines [T locus by sequence data of other alleles of the locus and by genetic cosegregation data [T locus does determine the flavonoid and pigment compounds synthesized in various tissues including seed coats and trichomes, it is possible that the flavonoid compounds themselves modulate an additional effect on seed protein synthesis in the seed coats. Alternatively, the observed differences in the levels of these cDNAs could be due to an artifact during the dissection procedure. We know from Northern blots, that flavonoid 3' hydroxylase is highly expressed in the seed coats, but is not expressed in the cotyledons so any small amount of contaminating cotyledon cells due to imprecise dissection of the seed coats of one line versus the other could lead to observed differences in seed protein RNAs.The microarray data presented here and showing that the cytoplasmic levels of the flavonoid 3' hydroxylase are higher in the /T lines . In addiion data . We do nt allele at the T locus is the result of a premature stop codon and does not affect abundance of the flavonoid 3' hydroxylase mRNA to the same extent as does the t* mutation at that locus [As this example in Figure at locus .T locus which showed that relatively few cDNAs showed differential expression between the two very closely related lines, the soybean microarrays reveal larger numbers of cDNAs showing differential mRNA abundance levels in different tissue types of the same plants. For illustration, Figure In contrast to the results with near isogenic lines of the Rhizobia and therefore only a few nodulin-related genes were observed to be markedly upregulated: nodulin-26, nod factor binding lectin-nucleotide phosphohydrolase (GS50, Accession # AF207687) and a MtN19 homolog.Table The soybean proline-rich protein (SbPRP1 Accession # J05208) (represented on the array by AW309104, Gm-c1019-3688) is also among the root expressed clones. SbPRP1 has been shown to be expressed preferentially in the roots . A gene In leaves, genes typical for green tissues are upregulated as expected. These are the photosynthesis genes , and the vegetative storage proteins. Ribosomal proteins, cytochrome P450, catalase, and chitinase were also noted as overexpressed in the leaves.Our publicly available Gm-r1021 soybean unigene subset containing 4,098 cDNAs has also been used to examine differential gene expression in roots and shoots of older soybean plants . We haveThus, the soybean cDNA arrays that we have developed from the unigene cDNA set can be used to reveal the underlying physiological and biochemical pathways potentially operative in specific tissues, developmental stages, or environmental treatments. Obviously, cDNA arrays from soybean or any other organism that are constructed with PCR inserts representing an average size of 1.1 kb will generally hybridize with any RNAs from gene family members that share greater than 85% homology. Thus, cDNA arrays will generally not distinguish expression from closely related duplicated sequences. Oligo arrays spotted with synthetic 70-mers or Affymetrix short oligo arrays have greater potential to separate the expression from close related duplicated sequences if the oligos are chosen from the 3' or 5' non-coding regions that carry more sequence variability than the protein coding regions.in situ. The cDNA or oligo-based microarrays add to the developing suite of genome analysis approaches in soybean [T locus as an example, we demonstrated the potential of soybean cDNA arrays to be used for discovery of genes responsible for uncharacterized mutations. Future expression profiling of mutant phenotypes or of genotypes that differ in protein or oil content and other quantitative traits will yield significant clues to the genes involved in those pathways and traits.Although microarray data is limited from soybean and most plants other than Arabidopsis, the construction of the 27,513 member low redundancy 'unigene' cDNAs for soybean reported in this paper will greatly stimulate this area. The number of slides containing all 27,513 of the cDNAs is being reduced to one, or at most two slides, and the slides are publicly available. Spotted PCR products with average size of over 1 kb are useful not only for soybean, but for other legume species as cross hybridization to the long probes will be substantial. The 3' sequencing reported here is particularly useful for differentiating gene family members and for future design of gene specific oligo arrays of either 70-mers spotted on glass slides or by Affymetrix technology using short oligos synthesized  soybean . A few oE. coli sequences were removed. Quality checks included determining the number of ambiguous 'N' base calls in a sequence and trimming the leading and trailing poor quality (high-N) sections to obtain the best subsequence where the number of Ns was 4% or less of the total bases. The EST sequences were clustered into contig sets based on sequence overlap using the program Phrap [Raw sequence files of the 5' soybean EST data from Washington University or 3' data from the University of Illinois Keck Center were produced from sequence traces using the Phred base calling program ,19. The am Phrap . The proam Phrap . The disam Phrap .E.coli stocks representing those clones were reracked into new 384 well plates to form the sequence driven reracked libraries Gm-r1021 (4,089 cDNA clones), Gm-r1070 (9,216 cDNA clones) and, Gm-r1083 (4,992 cDNA clones). Initially, these were reracked from source 384-well plates to destination 384-well plates by Genome Systems (St. Louis, MO) using a Qbot and shipped on ice to the University of Illinois for extraction and 3' sequencing. Reracked library Gm-r1088 (9,216 cDNA clones) was reracked at the University of Ilinois Keck Center using a QPix robot, (Genetix, New Milton, Hampshire UK). Growth rates for the E.coli stocks were over 99.5%.Soybean cDNA clones corresponding to the 5' most representative member of a contig or to a singleton were selected using Oracle database tables and SQL queries. The The cDNA libraries were all constructed in either pSPORT 1 (Invitrogen, Carlsbad, CA) or pBluesciptII SK (+) (Stratagene, La Jolla, CA) plasmid vectors in DH10B host cells. Each 384-well plate of a bacterial library was split into four 96-well, 2 ml block plates, each corresponding to a different quadrant (A1, A2, B1, B2) and grown overnight in 1 ml LB media with 100 μg/ml ampicillin. High quality DNA templates were purified using a QIAGEN BioRobot 9600 or BioRobot 8000 with QIAprep 96 Turbo miniprep kits (QIAGEN, Germantown MD). Dideoxy terminator sequencing reactions for the 3' ends of the soybean cDNA clones were conducted by the University of Illinois Keck Center for Comparative and Functional Genomics  using stAll cDNA clones of the low redundancy reracked 'unigene' sets are available to the public through Biogenetic Services, Inc., Brookings, SD, or the American Type Culture Collection, Manasas, VA.-6. The top blast hit was used as the annotation for each of the 5' and 3' ESTs represented in the unigene sets printed on the microarrays.The 5' and 3' sequences of the 27,513 unigene cDNAs clones were annotated using BLASTX against the nonredundant (nr) protein database with cutoff E value of 10EIn some cases, the protein family assignments were also made using the Metafam program based on a BLASTX analysis against a protein sequence database consisting of a non-redundant set of sequences from SwissProt & TrEMBL , PIR & NAll pipetting steps involved in amplifying the cDNAs by PCR, purification of the cDNAs, and assembling them into 384-well spotting plates were conducted with a Multimek TM 96 Automated pippetor (Beckman Instruments, CA) to reduce errors associated with manual pipetting.2, 0.25 mM each of dGTP, dATP, dTTP, dCTP, 1 μM of M13 universal primer, 1μM of M13 reverse primer, and 0.05 U/μl of Taq polymerase (Invitrogen, Carlsbad, CA, cat no. 18038-042) was prepared and 48 μl were aliquoted into each well of a 96 well PCR reaction plate (MJ Research MSP-9621). A 0.5 μl aliquot of an undiluted plasmid template DNA was aliquoted into the 48 μl of master mix. The plates were briefly centrifuged for 1 min at 1500 rpm and placed into an MJ PTC-200 DNA Engine for 1 min of denaturation at 94°C, and 28 cycles of 92°C for 30 sec, 56°C for 45 sec, and 72°C for 30 sec and a final extension of 72°C for 5 minutes. A typical yield from the PCR was about 30–100 ng/μl.The same Qiagen plasmid DNA templates that were prepared for the 3' sequencing by a Qiagen robot at about 100+ ng/μl were also used for PCR amplification using Taq polymerase (Invitrogen, Carlsbad, CA), universal forward and reverse primers in 96 well plates using the MJ DNA Engine Tetrad . Four PCR reaction plates are prepared at a time, one from each quadrant of a 384-well library plate. A master mix consisting of final concentrations of 1X PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 2 mM MgClThe PCR products were loaded into Millipore multiscreen plates (Millipore #MANU 03050) and were subjected to a vacuum applied at 15 psi for about 10 min until the wells were completely empty. Then 60 μl of sterile water were added to each well using the Multimek automated pipettor and the PCR products were washed. The purified products were eluted in sterile water, retrieved and then stored in 96 well plates at -20°C. A 1 μl aliquot of each well from 3 rows from each 96 well plate is run on a gel to check the quality of the PCR and purification of the cDNA. The yield after purification was between 30 and 40 μls with concentrations around 15–50 ng/μl.The four quadrants were then reassembled into a 384-well spotting plate containing 6 μl per well: 4.5 μl of PCR product from the 96 well plates mixed with 1.5 μl of 4X Micro Spotting Solution Plus . Alternatively, in earlier prints, the spotting plates were assembled at a final concentration of 3X SSC, 0.01% N-lauroylsarcosine by mixing 3.5 μl of purified PCR product with 1.5 μl of 10X SSC, 0.033% Sarkosyl, pH 7.0 .A set of 9,216 prepared cDNA inserts from 24, 384-well spotting plates were single spotted onto amine coated glass slides  using a Cartesian PyxSys 5500 robot (Genomic Solutions, Ann Arbor, MI) equipped with 16 quill pins  and an environmental chamber. The cDNAs were printed at 55% ± 5% relative humidity setting within the chamber and in a room that was controlled for humidity to be between 45 and 60% using room dehumidifiers as needed. Control of humidity was critical for printing.All arrays contained 32 grids of spots arranged in an 8 × 4 matrix. Each grid had 19 rows and 16 columns of spots for a total of 9,728 spots per array. A total of 9,216 spots were the cDNAs prepared from the 'unigene' set to form 18 of the 19 rows with 288 spots per grid. After all of the 9,216 cDNAs were printed, an additional row of 16 spots was printed as the first row of each grid for a total of 32 grids × 16 spots = 512 additional spots. These cDNAs were printed from the choice clone spotting plate designated Gm-b10BB which contained 64 hand-picked clones. Thus, the 64 hand-picked, choice clones were printed 8 times each, i.e., each clone was printed in twice in four separate grids. In addition, since the Gm-r1021 library contained only 4089 cDNAs, an additional 135 were repeated in order to obtain an even 9216 cDNAs for printing when combined with the Gm-r1083 unigene set.The three microarray platforms were entered in the Gene Expression Omnibus database  with plaTo construct the choice plate Gm-b10BB, 64 clones were chosen to be used as negative and positive controls for expression analysis in all microarray slides. These 64 clones were chosen to represent certain constitutively expressed genes, or other markers for particular tissues, and is also highly representative of key genes of the soybean flavonoid pathway.The 64 clones were hand picked and grown over night in microfuge tubes containing 100 μl of YT media at 37°C, 250 rpm. The following day, microfuge tubes containing 200 μl of YT supplemented with 100 μg/ml ampicillin and 8% glycerol were inoculated with 5 μl from the previous culture and grown over night at 37°C and 250 rpm.E.coli stocks, 100μl of the previously grown culture were transferred to wells in columns A1 thru H8 and stored at -80°C. Wells in columns A9 thru H12 were left empty. A small database for the Gm-b10BB plate was prepared containing the name of each gene, its sequence, accession number, and the corresponding well in the Gm-b10BB plate. To make a replicate copy for sequencing, 100 μl of YT supplemented with 100 μg/ml ampicillin and 8% glycerol were inoculated with 5 μl of the -80°C E. coli stock and incubated overnight at 37°C and 250 rpm. Miniprep DNAs were isolated and sequenced at the University of Illinois Keck Center using a 5' M13 primer. The identity of each clone was confirmed by comparison of the sequences obtained from the Keck center with the sequences contained in our previously prepared database by using the Pairwise Blast tool available at the NCBI web page. All sequences showed >97% identity with the corresponding sequence in the database.To create a 96 well plate of these PCR amplification using the DNA miniprep plate as a source for templates was performed with the Mutimeck 96 automated pipetor (Beckman) as described above. All PCR products were purified and separated in 1% agarose gel to evaluate the purity of the amplified DNAs and determine their size. The purified and analyzed PCR products from the 96 well plate, Gm-b10BB, were used to assemble a 384 well spotting plate. The 384 well spotting plate contained 6 μl per well: 4.5 μl of PCR product from the 96 well plate aliquoted on each of the 4 quadrants and mixed with 1.5 μl of 4X Micro Spotting Solution Plus  after assembly.After all slides were printed, the cDNAs were UV-cross linked to the slide coating with 650 m Joule ultra violet light using a StrataLinker (Stratagene, La Jolla, CA). [Note: prior to cross-linking the spots were rehydrated if necessary. Rehydration was required for the slides printed with the SSC-Sarkosyl spotting solution but was not required for those printed with Telechem spotting solution. DNA spots were rehydrated by passing the slide over a gentle vapor of steam for a few seconds until spots glistened but did not coalesce and then were quick dried on a 70°C heating block]. To remove excess spotted DNA as well as to denature attached DNA to single strands, slides were treated with the following series of washes with agitation: 2 min with 200 mls of 0.2% SDS, two 1 min water rinses, 95°C water for 2 min, 0.2% SDS for 1 min, and finally two water rinses of 1 min each. Slides were subjected to low speed centrifugation for 2 min at 500 rpm to dry and were stored in a slide rack in a dust free container.Seed coats and cotyledons were dissected from plants grown to maturity in soil in the greenhouse. Roots and leaves were collected from soybean plants grown for 11 days after germination in an aerated hydroponic solution with normal nutrient conditions. Total RNA was extracted using phenol-chloroform and lithium chloride precipitation methods ,32. RNA For each RNA probe, 50 to 60 μg of purified total RNA was labeled by reverse transcription in the presence of Cy3- or Cy5-dUTP . BrieflyThe microarray slides were prehybridized by incubation in 5X SSC, 0.1% SDS, 1% BSA at 42°C for 45 to 60 min. For each slide, the labeled cDNA probe was brought to 30.5 μl with the addition of sterile water. A 1.5 μl aliquot of 10 μg/μl polyA was added and the probe was denatured at 95°C for 3 min. An equal amount (32 μl) of pre-warmed 2X hybridization buffer (50% formamide, 10X SSC, 0.2% SDS,  was adder) was computed and used to adjust the Cy3 values to Cy3 X sqrt(r) and the Cy5 values to Cy5/sqrt(r). A between-replicate correction was made using an ANOVA model, which equalized average grid or slide intensities between replicates, for Cy3 and Cy5 separately. The ratio of the resulting adjusted intensities of Cy5 to Cy3 was computed for each spot. The coefficient of variation (standard deviation/mean) across replicates was calculated for each spot to evaluate repeatability of the hybridizations.The hybridized slides were scanned with a ScanArray Express fluorescent microarray scanner (Perkin Elmer Life Sciences, Boston, MA) and their fluorescence quantified by ScanArray Express software or by GenePix Pro 3.0 (Axon Instruments, Union City, CA). A perl program was written for post analysis processing of the quantitated image files from the Scan Array Express or GenePix Pro3.0. Local background was subtracted from each spot intensity. Spots showing signal intensities below the 95th percentile of the background distribution in the Cy3 or Cy5 channel were filtered out. The ratio of Cy5 mean to Cy3 mean  was combined with 4 μl Oligo dT (0.5 μg/ul, 70 μM, Sigma Lot 29H9065). The mixture was heat treated for 10 min at 70°C and chilled on ice before adding the following: 6 μl of 5X first strand buffer (BRL/Life Tech Cat. #18064-014); 1 μl DTT; 1.5 μl each of 10 mM dGTP, dCTP, and dTTP; 1.5 μl reverse transcriptase (200 units/μl, SuperScript II RT from BRL/Life Tech Cat. #18064-014); and 10 μl 33P dATP at 10 mCi/ (NEN, 33P Cat#612H04029). After incubation at 37°C for 90 min, the probe was purified by a passage through a Bio-Spin 30 Chromatography Column (Bio-Rad Cat. #732-6006), then stored at 4°C until ready to be denatured and added to the pre-hybridized filter).Total mRNA from developing cotyledons was labeled with The filter was rolled and placed in a hybridization bottle containing 25 ml of pre hybridization solution without formamide  and was Adding the probe to the filter: Once the filter was pre-hybridized, the probe was denatured for 10 min at 95°C and then the entire radiolabeled probe was added directly to the prehybridization mixture (in the bottle with the filer). The hybridization was allowed to proceed for 12–18 hrs.The filters were washed twice in the pre-warmed (50–55°C) low stringency wash solution (2XSSC, 0.5% SDS, 0.1% Na pyrophosphate) for 15 min each. The filters were then washed for about 2 hrs at 55°C in high stringency buffer (0.1XSSC, 0.5% SDS, O.1% sodium pyrophosphate) with gentle shaking.Filters were analyzed with a Typhoon 8600 variable mode imager (Amersham Pharmacia Biotech, Inc, Piscataway, NJ) and imaged with the software package Array Vision (Imaging Research Inc., St. Catharines, Ontario, Canada) to correlate spot intensity and filter position. Spots with very low intensity of 1 to 500 were selected at random in order to enrich for cDNAs representing mRNAs of low abundance. Theses clones were reracked into 384-well plates to form library Gm-r1030 and sent for 5' sequencing at the Washington University Genome Center.Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes. The cDNA clones are available from the Biogenetic Services, Brookings, SD or the American Type Culture Collection, Manasas, VA. Microarrays are available on a cost recovery basis by contacting Lila Vodkin, University of Illinois.PCR polymerase chain reactionSSC standard saline citrateSDS sodium dodecyl sulfateLOV led the unigene and microarray development, coordinated the project, and drafted the manuscript; AK constructed multiple cDNA libraries included in the unigene cDNAs, performed over 18,000 PCR reactions, performed the library normalization by filter screening shown in Figure


This 'chromosomal organization and functional correspondence' is conserved in all bilaterians investigated. Genomic sequences covering the HoxD complex from several vertebrate species are now available. This offers a comparative genomics approach to identify conserved regions linked to this complex. Although the molecular basis of 'colinearity' of Hox complexes is not yet understood, it is possible that there are control elements within or in the proximity of these complexes that establish and maintain the expression patterns of hox genes in a coordinated fashion.Homeotic gene complexes determine the anterior-posterior body axis in animals. The expression pattern and function of HoxD complex of several primate, rodent and fish species. This analysis revealed an unprecedented conservation of non-coding DNA sequences adjacent to the HoxD complex from fish to human. Stretches of hundreds of base pairs in a 7 kb region, upstream of HoxD complex, show 100% conservation across the vertebrate species. Using PCR primers from the human sequence, these conserved regions could be amplified from other vertebrate species, including other mammals, birds, reptiles, amphibians and fish. Our analysis of these sequences also indicates that starting from the conserved core regions, more sequences have been added on and maintained during evolution from fish to human.We have compared DNA sequence flanking the Such a high degree of conservation in the core regions of this 7 kb DNA, where no variation occurred during ~500 million years of evolution, suggests critical function for these sequences. We suggest that such sequences are likely to provide molecular handle to gain insight into the evolution and mechanism of regulation of associated gene complexes. Conservation of these sequences among species is a strong indication of their functional significance. With the availability of genome sequences it is possible to identify such sequences taking a comparative genomics approach -4. The cosophila  and lateosophila . Recent m region .hox complexes in order to identify conserved regions with potential regulatory function. Here we report that the upstream regions of HoxD complexes of human, mouse, rat, sacred baboon, horn shark, zebra fish and puffer fish contain long stretches of extremely conserved sequences. In the 25 kb region upstream of the HoxD complex from these organisms we found an extremely conserved region spread in three blocks located within 7 kb from the 3' end of the Evx-2 gene. These conserved regions, designated as Conserved Region 1, Conserved Region 2 and Conserved Region 3 (CR1, CR2 and CR3)  of vertebrates. In order to trace back the evolutionary origin of such sequences, it will be of interest to investigate occurrence of these sequences at the corresponding region in the hox complexes of species of urochordata, cephalochordata or even agnatha. In the tunicate Oikopleura dioca, where hox genes are dispersed but the spatial pattern seen in other animals is still present [Universal occurrence of these sequences in all vertebrate classes was confirmed by their amplification using primers from human x Figure  followed present , we did Several recent reports using comparative genomics approach have identified conserved non-coding regions among different vertebrates -16 but nHoxD complex, no known regulatory element requires such extreme conservation extending up to hundreds of base pairs. It is, therefore, likely that these elements could be components of a novel mechanism common to all vertebrates that regulates this gene complex. We are tempted to suggest that such a strongly conserved region from fish to human linked to a gene complex that is known to determine body axis formation may be the key determinant of molecular basis of early ontogeny. Early embryos of all vertebrates show striking similarity and we suggest that these elements may control the early expression pattern of HoxD which leads to similar pattern of the embryo shape. The gradient of conservation seen in this region from fish to human may further signify the evolutionary history of this locus and diversification of the morphological features along the anterior-posterior body axis of the vertebrate classes.While such an extreme conservation of several hundred nucleotides over half a billion years in a region that does not code for any known proteins certainly implicates essential role for such sequences, probably in the regulation of Evx-2 and any of the Hoxd genes were downloaded and annotated using gene/ORF prediction tools. Similar approach was used for other hox complexes. Homology searches of the upstream sequences of HoxD region from human  was carried out using the BLAST program of NCBI. The sequences that showed significant homology were further used to analyze the extent of homology by BLAST 2 program. The conserved regions from each sequence was obtained and subjected to multiple sequence analysis using Clustal X. In order to identify the expressed sequences corresponding to the conserved sequence, the conserved sequences along with the unique sequences were BLASTed against EST databases (human, mouse and dbEST).The genomic sequences that contained HoxD were annotated using the tBLASTx program and searching the translated amino acid sequence in the Swissprot database. Repeat masker program was used to look for repeat content. Genebank sequences used in this study are as follows: AC116665 Papio hamadryas, AF224263 Heterodontus francisci, AC015584 Mus musculus, AC009336 Homo sapiens, CAAB01000449 Fugu rubripes and NW_042732 Rattus norvegicus.The contigs that showed significant homology to the upstream sequences of human Naja naja) were used while liver tissue of mouse and muscle tissue of frog (Bufo melanostictus) and zebra fish were used. Standard protocol of DNA isolation was followed which included lysis, RNase A and proteinase K digestions followed by phenol/chloroform extraction and precipitation. Concentration and quality of the genomic DNA was checked on 0.7% agarose gel and UV absorption spectrophotometry. Based on the sequence of conserved regions primers were designed to amplify the three regions CR1, CR2 and CR3.For the isolation of genomic DNA blood samples of human, chick and cobra (Primers used in this study to amplify conserved regions from different vertebrate species were:CR1 forward- GAGGCTGTTCACACTGGTGG,CR1 reverse- ATCATGCTCTCTGATGGACC,CR2 forward- GCATCGTAATCAGTTCGGTC,CR2 reverse- TGATACAAGCTGATACCGTC,CR3 forward- GCTATTCAAAATGTTATTTGAG and CR3 reverse- CTGTAATGAAGAAAAGATTTATG.The 25 μl reaction was performed using 100 ng template DNA and 5 pmol each of forward and reverse primers. PCR protocol was as follows: initial denaturation step of 94°C for 3 min was followed by 35 cycles of 94°C for 1 min, 57°C for 1 min and 72°C for 1.30 min and final extension step at 72°C for 7 min. Authenticity of the PCR products was confirmed by direct sequencing and Southern hybridization, using the corresponding human DNA as probe., [HOXD and, in agreement to our observation, noted only a "core" conserved region in fish, suggesting that additional parts of the ultraconserved region were innovations after the common ancestor with fish.An earlier version of this article was deposited in the 'Deposited Research' section of Genome Biology, , . While t, . InteresCS carried out the sequence analysis, PCR amplification and Southern analysis. SS participated in sequence analysis and DNA isolation from several organisms. AT carried out the sequencing of PCR products and participated in the sequence alignments. RKM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Size and degree of conservation of CR1, CR2 and CR3 in different vertebrates. Core of conserved regions and extended conserved regions between indicated species is shown as length of sequence and degree of conservation. Non-overlapping blocks of vertebrate conservation is indicated based on human, baboon, rat, mouse and shark comparison.Click here for file