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 . 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 , 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  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 . 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  and negative selection from exon 10 through the first few codons of exon 13 . 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 . 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 . 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 , or citalopram  subcutaneously with daily injections. All drugs were dissolved in saline . 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 . 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  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  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  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 . 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 . 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 . 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  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  and their fluorescence quantified by ScanArray Express software or by GenePix Pro 3.0 . 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 . 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 ; and 10 μl 33P dATP at 10 mCi/ . 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  for 15 min each. The filters were then washed for about 2 hrs at 55°C in high stringency buffer  with gentle shaking.Filters were analyzed with a Typhoon 8600 variable mode imager  and imaged with the software package Array Vision  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   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 .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


Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella.The complete nucleotide sequence of fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigth are functionally expressed and coassembled in the same flagellar filament in E. coli.The flagellar serotype H4 is encoded by closely related  Enterobacteriaceae species Escherichia coli are common as commensals in the intestinal flora of humans and warm-blooded animals [E. coli strains were developed in the early 1940ies when it became evident that certain E. coli strains were agents of infantile gastroenteritis [E. coli O- (lipopolysaccharide) and H-(flagellar) antigens which allowed the grouping of E. coli strains according to their O:H-types (serotypes) [E. coli (EPEC) strains from stools of diarrhoeic infants [E. coli strains from both humans and animals [Bacterial strains belonging to the  animals . Typing nteritis . In 1944rotypes) . Serotyp infants  and is s animals ,3.E. coli populations by multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) allowed the detection of clonal types of strains which carry specific virulence markers and are associated with disease in humans [E. coli [E. coli which can occur in different combinations in wildtype isolates of strains [E. coli serotyping and the laborious typing procedure restricts its usage to a few reference laboratories. Therefore, alternative typing methods were developed including molecular characterization of genes coding for the O- and H-antigens in E. coli [fliC genes by PCR was successfully employed for characterization of human pathogenic O157:H7 and O26:H11 strains [fliC genes coding for flagellar antigens H7 and H6 revealed large sequence similarities between strains sharing the same H-type but different O-types [fliC gene allows H-typing of non-flagellated (non-motile) E. coli isolates which cannot be analyzed for their flagella with H-specific antisera [The genetic analysis of n humans ,7. It wa[E. coli ,7. At pr strains ,3,5,8. H E. coli -12. Typi strains ,14. Anal O-types . Moreoveantisera .E. coli belonging to many different O-groups including strains of shiga toxin-producing E. coli (STEC) and extraintestinal pathogenic serotypes [). Moreover, a cryptic fliC-H4 gene was described to be present and to be spontaneously expressed in E. coli strain P12b (O15:H17) which carries another type of flagella called H17 which is not encoded by the fliC gene [E. coli strains belonging to different O-serogroups and pathotypes. We have used the published nucleotide sequence of the fliC gene present in the E. coli H4 reference strain U9-41 (accession AB028472) to develop a PCR which allows discrimination between the fliC-H4 gene variants. Nucleotide sequence analysis of the fliC gene was performed on other E. coli H4 strains that either showed deviations in the PCR analysis or were reported to harbour allelic types of the H4-fliC gene [fliC genes and have introduced them into the laboratory E. coli K-12 strain JM109 [The flagellar type H4 is frequently occurring in erotypes -20,  and P12b (O15:H17) [E. coli K-12 strain JM109 (O-rough:H48) (this study), which was used for expression studies were investigated for inhibition of motility in swarm-agar containing 1:600 dilutions of either H4, or H48-antiserum (see Methods). U9-41 and P12b were fully inhibited for their motility in the presence of H4 antiserum derived from strains U9-41 or from P12b but not in the presence of H48 specific antiserum. The E. coli K-12 strain JM109 was not inhibited for motility by H4 antisera but by H48 antiserum. These findings indicate that H4 antisera specifically inhibited the motility of E. coli H4 strains and that the antigenically different flagellar type H17 was not expressed or lost in our P12b isolate, similar as previously described with spontaneously arising variants of P12b [E. coli H17 strains would also carry the genes for expression of H4 type flagella as it was described for P12b [E. coli H17 strains  for their serological reaction with different H4 specific antisera and all strains showed specific positive reactions . Differences in agglutinating titers between H4U9-41 and H4P12b antisera were not more than twofold with either strain indicating that both sera were similar for their specificity (Table E. coli H-types for polymorphisms in the fliC gene by HhaI digestion of fliC-PCR products as described in the Method section.O15:H17)  and the  of P12b . We becafor P12b ,22,24. FE. coli H-serotypes [E. coli fliC genes coding for flagellar types H1 to H56 revealed individual patterns corresponding to the different H-serotypes  in the corresponding E. coli strains and the fliC HhaI patterns obtained from these strains do therefore not correspond to their H-serotypes [fliC gene in strain P12b codes for flagella of serotype H4. The relationship between fliC genes present in the different E. coli strains was further investigated by nucleotide sequencing.PCR products obtained with primers fliC-1 and fliC-2 were digested with HhaI to obtain H-serotype specific RFLP patterns from reference strains for the 53 different erotypes . HhaI-RFerotypes ,25-27. Herotypes  , P12b (O15:H17), U1-41 (O5:K4:H4), P7d (O68:H4), C107-74 (O15:H17) and E1541-68 (O154:H4) as listed in Table fliC genes present in these strains revealed in all cases a length of 1050 bp. As a control, the fliC gene of the strain U9-41 was sequenced and found to be identical to the previously deposited fliC sequence (accession AB028472). The identity of the fliC-H4 sequences ranges from 97,6 % to 100 %. The greatest sequence difference to the fliC gene coding sequence of strain U9-41 was found in the fliC gene of strain P7d (exchange of 20 nucleotides), whereas the fliC sequences of strains U1-41 and U9-41 were identical to each other. All deduced flagellins have a length of 349 amino acids. The greatest deviation in the primary structure to the FliC protein of the reference strain U9-41 was again observed for the FliC protein of strain P7d (exchange of 9 aa in a stretch of 349 aa). Fig. To obtain the entire coding region of the s U9-41 O:K1:H4, Ps U9-41 O:K1:H4, PfliC genes present in E. coli strains U9-41 and P12b with primers fliC-1 and fliC-2 resulted in the generation of a 953 bp internal PCR product with both strains  from 88 E. coli strains which showed agglutination reactions with H4 antisera (Table MboI restriction profiles which corresponded to the patterns obtained with strain U9-41 (O2:K1:H4) (Table Amplification of the fliC-H4 genes in a different genetic background we cloned the corresponding PCR products of strains U9-41 and P12b into the vector pLITMUS38 as described in the Methods section. The fliC coding regions were inserted downstream of the lacZ promoter of pLITMUS38 and the fliC recombinant plasmids were transformed into the laboratory E. coli K-12 strain JM109 [fliC-pattern as the E. coli reference strain P4 (O16:H48) [fliC-H4 genes in the JM109 derivative strains TPE1976 (fliC-H4U9-41 clone) and TPE1978 (fliC-H4P12b clone) was analyzed by tube agglutination with H4 and H48 antisera, respectively. The strains TPE1976 and TPE1978 showed agglutination with H48 and H4 sera whereas the parental JM109 strain reacted only with H48 serum  and H17 (P12b) flagellar antigen [fliC-H4 genes from different E. coli strains with primers which were generated on the basis of the previously published fliC sequence (accession AB028472) of the E. coli H4 reference strain U9-41. The comparison of the fliC gene sequence encoding H4 flagella in strain P12b with the fliC sequences of five other E. coli H4 or H17 strains revealed a high similarity on the DNA and on the amino acid level  is a 51.3 kDa protein consisting of 498 amino acids and thus much larger than the 36.3 kDa H4 flagellin which is composed of 349 amino acids. However, both flagellins share conserved N- and C-terminal sequences which are known to be involved in the structural assembly of flagella [The functional expression of the cloned ace Fig. . Single ace Fig.  and doubace Fig.  experimeflagella . H48 andflagella .fliC gene in E. coli can change the antigenic properties of the flagella made by this strain. Horizontal transfer of fliC genes may contribute to the diversity of flagellar serotypes by recombination within E. coli recipient strains. The mammalian host immune system is the driving force for continuous selection of new flagellar antigens in E. coli. Published data indicate that both mutation and recombination events in the fliC gene have taken place in the evolution of E. coli flagellar antigens [We were able to show that the introduction of an isolated antigens ,16.fliC sequence data have shown that the flagellar type H4 which is present in E. coli strains of clinical importance covers several genetic variants which are closely related to each other. We have shown for the first time that flagellins of different molecular size are expressed and coassembled into functional flagella in a laboratory E. coli K-12 strain.Our E. coli were obtained from the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark and are described elsewhere [E. coli isolates originating from humans and animals was investigated by PCR/RFLP typing for fliC-H4 genes. These strains were previously investigated for the O-types and for production of Shiga-toxins (Stx) and were isolated in different countries between 1941 to 2002 [E. coli K-12 strain JM109 is described elsewhere [The reference strains used for production of antisera for O- and H-typing of lsewhere . Origin  to 2002 ,19,30 (Tlsewhere .E. coli were prepared according to ∅rskov and ∅rskov [fliC encoded H4 antigen and produced flagellar type H4 (this work).Rabbit antisera against the different O- and H-antigens of d ∅rskov . Antiserd ∅rskov . Our strE. coli strains was tested by inoculating bacteria in tubes containing 10 ml portions of swarm-agar (L-broth + 0.3% agar) as described [E. coli strains in the presence of flagellar-specific antiserum (H4 and H48) was tested in swarm-agar containing a 1:600 dilution of the respective antiserum. Cultures which were inhibited for motility were observed over two weeks for possible switch to motility by phase variation.Expression of flagella and swarming of H-serotyping was performed as described . In briefliC genes present in the E. coli reference strains as described [E. coli reference strains for 53 different H-types [The oligonucleotide primers fliC-1 (5' CAA GTC ATT AAT AC(A/C) AAC AGC C 3') and fliC-2 (5' GAC AT(A/G) TT(A/G) GA(G/A/C) ACT TC(G/C) GT 3') were used for amplification of internal parts of escribed . The PCRescribed . PCR profliC-H4 sequence (AB028472) were used for the amplification of the entire fliC-H4 coding regions. The PCR was performed for 30 cycles: 30 sec at 94°C, 60 sec at 58.1°C and 90 sec at 72°C with primers fliC-5 (5'-TGA GTG ACC AGA CGA TAA CAG GG-3') and fliC-6 (5'-GGA CGA TTA GTG GGT GAA ATG AGG-3') and yielded a 1243 bp product. PCR products were purified with the QIAquick™ PCR Purification Kit  and used for sequencing. Sequencing reactions were carried out using the dye terminator chemistry  and separated on an automated DNA sequencer . The sequences were analysed using the Lasergene software  and the Mac Vector software  to assemblings and alignings.Two primers deduced from the published E. coli strain P12b (O15:H17) with a size of 1234 bp containing the fliC gene for flagellin has been submitted to EMBL data library under accession number AJ515904. The coding sequences of the different fliC genes from the following strains have been assigned the following accession numbers: AJ605764 for strain U1-41 (O5:K4:H4), AJ605765 for strain P7d (O68:H4), AJ605766 for strain C107-74 (O15:H17) and AJ536600 for strain E1541-68 (O154:H4). The origin of the strains is listed in Table The nucleotide sequence of the genomic region of fliC-H4 genes were obtained from genomic DNA prepared as described [E. coli strains U9-41 and P12b using primers fliC-5 and fliC-6. The amplification products were inserted into the vector pLITMUS38  digested with EcoRV. The orientation of the the insert PCR products was determined by using commercially available sequencing primers LITMUS forward 28/38 and LITMUS Reverse 28/38 (New England Biolabs).PCR products encompassing the complete coding sequences of the escribed  of E. coE. coli strains were produced by repeated passage on semi-solid agar followed by growth in L-Broth as described above. Cultures carrying recombinant pLITMUS38 plasmids were grown in the presence of 100 μg/ml ampicillin. IEM was performed using fresh, non-formalized cultures of motile bacteria. For IEM, aliquots of respective bacterial cultures were diluted 1:2 in PBS pH 7.2 and adsorbed onto glow-discharge treated 400 mesh grids coated with Pioloform and carbon  [E. coli strains were incubated with rabbit anti-H4 (1:1000) followed by anti-rabbit-IgG-5 nm gold and two washing steps on droplets of blocking buffer for 10 min. The samples were subsequently incubated with anti H48 antibody (1:1000) followed by incubation with anti-rabbit-IgG- 10 nm gold. Removal of surplus conjugate and negative staining were performed as detailed above.Motile cultures of Germany) . Grids wfliC recombinant plasmid carrying strains. AM and HG developed the IEM methodology and HG contributed to the data analysis. All authors participated in review and preparation of the final manuscript.LB conceived of the study and carried out PCR genotyping and coexpression studies. ES carried out sequence determination and alignments and construction of recombinant plasmids. SZ and SK performed serological assays and PCR genotyping. CS performed analysis of


The process of oxidative folding combines the formation of native disulfide bond with conformational folding resulting in the native three-dimensional fold. Oxidative folding pathways can be described in terms of disulfide intermediate species (DIS) which can also be isolated and characterized. Each DIS corresponds to a family of folding states (conformations) that the given DIS can adopt in three dimensions.The oxidative folding space can be represented as a network of DIS states interconnected by disulfide interchange reactions that can either create/abolish or rearrange disulfide bridges. We propose a simple 3D representation wherein the states having the same number of disulfide bridges are placed on separate planes. In this representation, the shuffling transitions are within the planes, and the redox edges connect adjacent planes. In a number of experimentally studied cases , the observed intermediates appear as part of contiguous oxidative folding pathways. can be obtained from V.A.Such networks can be used to visualize folding pathways in terms of the experimentally observed intermediates. A simple visualization template written for the Tulip package  Even though most of the conformational states are not accessible to experiment, graphic representations of the potential energy surface have played pivotal roles in explaining how the conformational space is gradually restricted during the process folding [The process of protein folding whereby a linear polypeptide chain reaches its native structure has been one of the most intensely studied biomolecular problems over the past 50 years (for current reviews see -3). Fold folding . Key conin vitro reaction can be promoted by an external redox system such as a mixture of oxidized and reduced glutathione, or cysteine and cystine, respectively. In vivo, the oxidative power comes from specific agents such as the molecular chaperones protein disulfide isomerases[The particular kind of folding that this article is concerned with is oxidative folding, which is the fusion of native disulfide bond formation with conformational folding -8. This somerases.redox reaction a protein disulfide bond is created (or abolished), i.e. the oxidative state of the polypeptide is changed. This is the case when one of the participants of the reaction (say RSH) is not part of the protein. ii) In a shuffling reaction both participants of the reaction are protein-bound, so the oxidative state of the polypeptide does not change. In view of these possibilities it becomes obvious that there are a great many ways in which disulfide bridges can form and rearrange during the folding process. Today it is generally accepted that non-covalent interactions guide the process of folding and formation of disulfide bridges will lock the protein into the right conformation. The advantage of oxidative folding as opposed to general protein folding is that disulfide intermediates can be chemically isolated and studied using such techniques as acid trapping of the intermediates and analysis of the disulfide bridges using a combination of enzymatic cleavage and mass spectrometry. There is a body of literature in describing the pathways of oxidative folding in terms of disulfide intermediates [The underlying chemical mechanism is disulfide interchange Figure . In thismediates -8, and oGraph theory has been applied to many aspects of protein research (for a recent review see ). The api) First, protein structure itself can be considered as a graph consisting of various interactions  as edges, the nodes being atoms or residues of the protein. For instance, one of the classical definitions of protein secondary structures is based on main/chain H-bond contacts between residues . Structuii) In the network descriptions of the folding space, on the other hand, the folding states are the nodes, and transitions are the edges between them. This approach was fostered by the finding that the robustness and stability of networks may be the result of simple topological properties that are invariant throughout various technical as well as biological systems . In the Our purpose is to describe the folding space of the oxidative folding process using graph representations. This is an intriguing task since, in contrast to "ordinary" protein folding, the number of states defined in terms of disulfide links is not exceedingly high, moreover the actual disulfide intermediates can be isolated and studied. We will approach the problem in two steps as: i) We will use graph theory to describe the disulfide intermediates, and to enumerate the states of the folding space. ii) Then we will represent the folding space as a network (graph) of all possible intermediates. We show with few examples that experimentally observed intermediates mapped onto this network appear as contiguous folding pathways.The disulfide topology of a protein is unequivocally defined by describing which cysteines are connected to each other. For example, a topology "1–3, 2–4" means that a protein with four cysteines has two disulfide bridges that connect cysteines  and cysteines  respectively. Cysteines can be labelled by their sequence position, or – as in the previous example – in a serial order from the N-terminus  can be deduced from simple combinatorial considerations as (2n)!/(n!*2n). According to this formula proteins with two disulfide bridges have 3 fully oxidized isomers, 3-disulfide proteins have 15 and 4-disulfide proteins have 105. In other words, the number of intermediates increases very fast as a function of the number of constituent cysteines.The number of fully oxidised (disulfide bonded) isomers in a protein chain with n cysteines, the n × n adjacency matrix of the graph is symmetrical; it will contain a value of 1 if two cysteines form a disulfide bond and zero otherwise. As one cysteine can form only one disufide bridge, each column and each row will have at most one value of 1. Examples are shown in Figure For a complete description of the folding process we have to consider both fully oxidized intermediates as well as the ones with free cysteine residues. For this purpose we will use a formal description of the intermediates as undirected graphs, with cysteines as nodes and disulfide bridges as edges (the main chain will not be represented). For the majority of naturally occurring protein structures the resulting graphs will be extremely simple. If the protein has NB is the number of disulfide bonds, calculated as the sum of the elements of the adjacency matrix.The transitions between folding intermediates can be conveniently described by comparing the adjacency matrices of the two states. For the enumeration of the transition reactions we introduce a few simple variables. The sum of elements in the i-th column plus the i-th row,Si measures of two adjacency matrices,is 1 if the i-th cysteine is part of a disulfide bridge and zero otherwise. The sum of the differences calculated between the shuffling reactions, the number of disulfide bridges NB remains the same by definition, and it is easy to show that SD will differ exactly by 2. In redox steps in which one disulfide bridge is established or lost, NB and SD will increase or decrease by one and two, respectively.shows how many cysteines gain or loose a bond as the molecule passes from one state to the other. Here we are interested only in the two kinds of elementary reactions depicted in Figure n cysteines, we first generate the graphs (adjacency matrices) of all possible intermediates, i.e. those with 0,1...(i ≤ n/2) disulfide bridges. Then we compare all pairs of intermediates in terms of the above parameters. A shuffling edge will be drawn between two intermediate states if |SD| = 2 and ΔNB = 0; redox edges will be drawn if |SD| = 2 and ΔNB = 1. If the values of |SD| and ΔNB are different from these two combinations, no edge will be drawn.On the above basis one can easily draw a network of all possible oxidative folding pathways. For a protein of The graph characteristics of a few systems are summarized in Table The pathways can also be graphically represented, and in order to simplify the resulting picture, we chose a 3D representation wherein the states (species) are grouped according to the number of disulfide bridges Figure . Speciesin vitro folding of the molecule [In vivo, the propeptide is subsequently cleaved, and in this way the structure is locked into the native disulfide configuration.Panel B shows a peptide with an odd number cysteines, such as granulocyte-colony stimulating-factor ,24 in whmolecule . In vivoIn principle, the oxidative folding pathways can be pictured as routes within the full network, starting at the fully reduced species and ending at the native state. In the literature there are a few well-studied examples in which folding intermediates have been determined. The experimentally observed disulfide intermediates of three examples, bovine pancreatic trypsin inhibitor, insulin-like growth factor and epidermal growth factor, are shown in Table The oxidative folding space of polypeptides can be represented as networks in which the nodes are the disulfide intermediates while the edges are transitions between them. A simple visualization tool written was developed to draw 3D pictures of such networks in which the states having the same number of disulfide bridges are placed on separate planes. These pictures provide a simple method for the visualization of oxidative folding pathways as studied by experimental methods. In the case of bovine pancreatic trypsin inhibitor, insulin-like growth factor and epidermal growth factor, the folding pathways appear as a small network of contiguous routes that connect the fully reduced state to the native state. A further plausible extension of this method would include colouring of the folding states by quantitative properties and look for correlations between the coloured areas of the network and the experimentally determined folding pathways.Even though the topology of the theoretically complete folding space appears to be highly regular, data currently available are insufficient to draw general conclusions on the topology of the experimentally observed folding pathways. Experimentalists find folding intermediates as a series of chromatographic peaks, and usually the disulfide bridges of more abundant species are analysed first. The question whether or not all the relevant intermediates have been analyzed is difficult to answer, and mapping the intermediates onto the graphs presented here may help one to decide whether or not the pathways found are contiguous.V.A. designed and implemented the algorithms and carried out the calculations. M.C. helped to compile the experimental data and to draft the manuscript. L.K. designed the representation of folding intermediates. The project was coordinated by S.P.


The long acting β2-agonists, salmeterol and formoterol, have been recommended, by some, as first line treatment of stable chronic obstructive pulmonary disease (COPD). We reviewed evidence of efficacy and safety when compared with placebo or anticholinergic agents in patients with poorly reversible COPD.After searching MEDLINE, EMBASE, HealthSTAR, BIOSIS Previews, PASCAL, ToxFile, SciSearch, the Cochrane Library, and PubMed, as well as Web sites, selected journals, reference lists, and contacting drug manufacturers, two reviewers independently screened reports of randomised controlled trials of parallel or crossover design lasting four weeks or longer and including patients with a forced expiratory volume in one second (FEV1) ≤ 75% of predicted, a ratio of FEV1 to forced vital capacity (FVC) ≤ 88% of predicted, and < 15% improvement from baseline FEV1 after a dose of a β2 agonist. We included trials comparing salmeterol or formoterol with placebo or with ipratropium bromide and reporting one of these outcomes: lung function; exercise capacity; quality of life scores; dyspnea; exacerbations; rescue inhaler use; incidence of tachycardia, hypokalemia, or dry mouth. Two reviewers assessed the quality of included reports using the Jadad scale and allocation concealment, and abstracted data.Twelve trials satisfied our inclusion criteria; eight were high quality (Jadad score >2) and four were low quality (≤ 2). The adequacy of allocation concealment was unclear in all of them. We did not perform a meta-analysis due to differences in trial design and how outcomes were reported.Two trials comparing salmeterol with ipratropium did not detect differences; one trial comparing formoterol and ipratropium described greater improvement with formoterol in morning PEFR .Of twelve trials comparing long acting β2 agonists with placebo, six reported no improvement in exercise capacity, eleven reported improvements in FEV1 lung function (one reported no improvement), six reported less rescue inhaler usage (one reported no difference) and five reported improved dyspnea scores (two reported no improvement). Differences in quality of life were detected in one salmeterol trial ; however, two salmeterol, and one formoterol trial reported no differences. Adverse effects of interest were not reported.In terms of clinical outcomes and safety, we could not find convincing evidence that salmeterol and formoterol have demonstrated advantages to ipratropium, a less expensive drug, for patients with stable COPD and poor reversibility. Compared to placebo, we found evidence of reduced rescue inhaler usage and improved spirometric outcomes without a significant impact on quality of life or exercise capacity. Bronchodilators are the primary agents used to manage chronic obstructive pulmonary disease (COPD). They modestly improve forced expiratory volume in one second FEV1) and reduce dynamic hyperinflation; breathlessness may lessen and exercise tolerance increase despite little improvement in spirometric measurements .-11.9-11]Canadian provincial drug plan managers have noted a substantial increase in the use of salmeterol and formoterol in recent years, an observation supported by data from International Medical Services Canada, which collects information on Canadian patterns of drug prescribing and estimates use: between 1997 and 2001, the use of salmeterol and formoterol for COPD increased 1,150% and 1,975%, respectively, whereas the use of ipratropium for COPD decreased by 37% .In light of the new trials and the recent changes in prescribing practices, we undertook a systematic review to evaluate the efficacy and safety of long acting β2 agonists when compared with placebo or anticholinergic agents in patients with stable, poorly reversible, COPD.2-agonists for maintenance treatment of stable chronic obstructive pulmonary disease in mixed population. The first search was performed on MEDLINE®, EMBASE®, HealthSTAR, BIOSIS Previews® in June 2001 using a sensitive search strategy. The second search performed in December 2002 had a more focused search strategy and included PASCAL, SciSearch and ToxFile databases in addition to MEDLINE®, EMBASE® and BIOSIS Previews® databases. As designed in the search strategy, this search captured all the studies included in CCOHTA's published review as well as some additional trials published since previous search date. Search details for both searches can be found in Appendix 2 [see Additional file We obtained published literature and conference abstracts for this document from two separate sources: (1) search results from the CCOHTA's published health technology review "Long-acting β2-agonists for maintenance therapy of stable chronic obstructive pulmonary disease: a systematic review"; and, (2) search results from CCOHTA's ongoing clinical review on long-acting βTwo reviewers (D R H and V K S) worked independently on these phases of the study. Disagreements were resolved by discussion and consensus; a neutral third party (M B) was consulted when necessary.The reviewers evaluated the 504 unique citations by reviewing titles and abstracts, discarding those deemed irrelevant . They then selected all reports of randomised controlled trials (RCTs) comparing salmeterol or formoterol with placebo or an anticholinergic agent, with or without the additional use of short acting β2 agonists. No restrictions were placed on dosage, but the trials had to be of either parallel or crossover design, have lasted four weeks or longer, and have included patients that met each of the following criteria.• Non-asthmatic.• Stable COPD: no infections, exacerbations, or hospitalizations in the past month.• FEV1 ≤ 75% of predicted.• Ratio of FEV1 to forced vital capacity (FVC) ≤ 88% of predicted.• After a dose of a short or long acting β2 agonist < 15% improvement in FEV1.Since bronchodilators are much more efficacious in asthma than in COPD, including patients with asthma would have influenced the findings. It may be difficult to determine whether chronic airflow obstruction with relatively large responses to short acting β2 agonists represents COPD with reversibility or asthma with incomplete reversibility. A suggestive feature in the differential diagnosis of COPD is irreversible airflow limitation. To betteIn addition, the trials had to have investigated one of the following outcomes.• Lung function, including FEV1 and peak expiratory flow rate (PEFR).• Exercise capacity: six minute or shuttle walking test.• Health related quality of life (QoL).• Dyspnea, including symptom diary scores.• Exacerbations of COPD.• Rescue use of salbutamol, a short acting β2 agonist.• Adverse effects, including tachycardia, hypokalemia, and dry mouth.The reviewers independently scored the quality of the included trial reports using a five-point scale described by Jadad , which aThe reviewers independently recorded characteristics of the trials and patients, as well as details of the interventions and outcomes. When outcome data were available only graphically, each reviewer estimated values, and the means of the two estimates were reported.When possible, we calculated mean differences with 95% confidence intervals (CIs) for continuous outcomes and odds ratios (ORs) with 95% CIs for binary outcomes for individual trial data using Statistics with Confidence software , We usedWe found that even commonly measured outcomes, such as FEV1, could not be combined by meta-analysis because of differences in how they were reported. For example, in the six trials comparing salmeterol with placebo, FEV1 was reported as a mean change in percent predicted, a mean Both reviewers agreed to tentatively accept 35 of the 58 potentially relevant reports. After further evaluation one reviewer disagreed with including 14 of the 35, which resulted in a moderate level of agreement . Discussion revealed that this difference related primarily to confusion surrounding interpretation of one of the criteria for eligibility, and ultimately the other reviewer agreed to reject the disputed reports. The reviewers then independently selected the same nine reports ,23,26,27The 13 reports were of 12 trials, all funded by manufacturers of the drugs. One report  describeThree trials ,23,26 thIn a 12 week trial  formoterTen trials -16,22,23As table Five trials,26,28,31Three trials,26,28.).Results  for six minute walk tests were reported from six trials ,22,26,29One study comparing formoterol, ipratropium, and placebo  reportedIn several trials ,26,28-31In five of six trials salmeterol treatment was associated with less salbutamol use than was placebo treatment ,22,28,29In one 12 week trial comparing salmeterol, ipratropium, and placebo  no signiHRQoL was evaluated in four trials,26,28,29In the 52 week study).), health In the trial comparing formoterol, ipratropium, and placebo  HRQoL waThree trials,22,28 reData on adverse effects of interest were not available from the reports.We identified thirteen reports of twelve randomized controlled trials describing the effect of administering the long acting β2-agonists, salmeterol and formoterol, to patients with poorly reversible COPD.It is not clear from the reports whether the twelve selected trials had sufficient power to detect significant differences between treatment and control groups in the various subjective and objective outcome measures. Since data were not pooled for meta-analysis, we were not able to conduct a sensitivity analysis based on the quality of trial reporting. Accordingly, we cannot comment on the possible influence of quality on the effect size of the outcome measures. Clinical heterogeneity among the trials limited assessment of the overall effect of the interventions. Since we did not perform a meta-analysis, statistical heterogeneity was not an issue.We selected reports that met our inclusion criteria, regardless of publication status, language and trial quality using a systematic research methodology; this approach has been shown to minimize potential selection and publication bias and lead to more reliable conclusions We made A potential limitation of our research is that we did not seek trials comparing long acting β2-agonists marketed outside of Canada  or those trials comparing long acting β2-agonists to agents other than ipratropium and placebo . Similarly, we excluded those trials in which the FEV1 response to a bronchodilator was not reported or greater than 15%. Thus, our results may not be generalisable to the greater population of patients who can be currently defined as having COPD. We planOur results are similar to those of an earlier review  that ideAnother review has recently been published. However,We believe our findings are in accord with current guidelines, such as the GOLD guidelines, that suggest bronchodilators should be prescribed according to individual patient responses. However, policymakers with limited health service resources need to be aware of an identifiable sub-population of patients with poorly reversible COPD for which long acting β2-agonists may result in reduced efficiency (cost-effectiveness).Our research also suggests clinical investigators of COPD trials should stratify trial participants into groups for which outcomes may consistently differ. Of the trials identified, four,30,31,40In terms of clinical outcomes and safety, we could not find convincing evidence that salmeterol and formoterol have demonstrated advantages to ipratropium, a less expensive drug, for patients with stable COPD and poor reversibility. Compared to placebo, we found evidence of reduced rescue inhaler usage and improved spirometric outcomes without a significant impact on quality of life or exercise capacity.Donald Husereau, Vijay Shukla, Michel Boucher, and Shaila Mensinkai have no competing interests to declare. CCOHTA is an independent, nonprofit health research agency funded by the federal, provincial, and territorial governments of Canada. Robert Dales sits on the advisory committees for GlaxoSmithKline  and Boeheringer Ingelheim .DH edited and prepared the final manuscript for publication. VS led development of the research protocol, supervised the literature review, and summarized results. DH and VS were responsible for reviewing articles, judging their relevance, assessing their quality, and extracting data. MB assisted in developing the research protocol and in conflict resolution during study selection. RD assisted in developing the research protocol and provided clinical expertise. SM designed and conducted the electronic searches and provided expertise in the area of information science. All authors either wrote sections or critically reviewed drafts of this article.The pre-publication history for this paper can be accessed here:Appendix 1. A list of reports considered in this review but excluded.Click here for fileAppendix 2. Search strategies including databases, time horizons and subject headings/keywords used to locate trials.Click here for file