The cells were lysed in detergent and the lysates were centrifuged at 100,000 × g to isolate crosslinked complexes. Following cleavage of the crosslinks and analysis by two-dimensional gel electrophoresis, it was found that the crosslinker increased the level of various proteins, and particularly actin, in this pellet fraction. However, GAP-43 was not present, suggesting that GAP-43 was not extensively crosslinked to proteins of the cytoskeleton and membrane skeleton and did not sediment with them. GAP-43 also did not sediment with the membrane skeleton following nonionic detergent lysis. Calmodulin, but not actin or other proposed interaction partners, co-immunoprecipitated with GAP-43 from the 100,000 × g supernatant following crosslinker addition to cells or cell lysates. Faint spots at 34 kDa and 60 kDa were also present. Additional GAP-43 was recovered from GAP-43 immunoprecipitation supernatants with anti-calmodulin but not with anti-actin. The results suggest that GAP-43 is not present in complexes with actin or other membrane skeletal or cytoskeletal proteins in these cells, but it is nevertheless possible that a small fraction of the total GAP-43 may interact with other proteins.It has been suggested that GAP-43 (growth-associated protein) binds to various proteins in growing neurons as part of its mechanism of action. To test this hypothesis  The growth-associated protein GAP-43 participates in neuronal development and is also present in certain regions of the normal adult brain, retina, and cornea –4. DurinProteasome digestion studies suggested that GAP-43 is primarily unfolded at physiological ionic strength . It is oin vitro studies demonstrated the binding of GAP-43 to existing F-actin -labeled methionine and cysteine. The labeled cells received either DMSO alone as a control or DSP at a final concentration of 1 mM. This was followed by lysing the cells with SDS, addition of buffer containing NP-40 substitute, and shearing the lysates through a needle. The lysates were not centrifuged and received either monoclonal anti-GAP-43 or monoclonal anti β-galactosidase as a control, followed by incubation with protein G-agarose. The samples were divided and the material bound to the protein G-agarose was analyzed by SDS/PAGE either without DTT (lanes 1–4) or with DTT (lanes 5–8). The addition of DSP to cells caused the formation of large crosslinked complexes that associated with the agarose. In the experiment of DMSO- and DSP-treated cells were lysed and sheared as in 35S]-methionine and [35S]-cysteine is sufficient to label all of these proteins. It has been shown that spectrin [35S]-methionine for 3 hours or less. A six hour incubation was used to label the two Goα isotypes with [35S]-methionine in differentiated N1E-115 cells and each protein was found to have a half-life of approximately 49 hours [Immunoprecipitation of GAP-43 from membrane fractions of rat cerebellum growth cones yielded co-immunoprecipitated Goα, but only after GTPγS treatment of the membranes . Two isospectrin , Rab5 efspectrin , and SNAspectrin  all beco49 hours , which iIn The same proteins are present in In The GAP-43 and calmodulin spots were cut out of various gels, using the films as templates, followed by determination of the amount of radioactivity present. These results are shown in The experiment of If actin crosslinked to GAP-43 is blocking access of GAP-43 antibodies, then a polyclonal antibody to actin should recover this GAP-43. A supernatant received normal rabbit serum as a control  while anIn The lysate was centrifuged at low speed to remove nuclei and the cytoskeleton, and the supernatant was centrifuged at 100,000 × g for 1 h to sediment the membrane skeleton. In Sodium fluoride was included in the lysis buffer to inhibit the dephosphorylation of GAP-43. The nuclei and cytoskeleton were removed by low speed centrifugation, and supernatants received either DMSO as a control or DSP at a final concentration of 1 mM. After incubation for 15 min at room temperature, the unreacted DSP was quenched by the addition of glycine, and SDS was added at a final concentration of 0.1%. The lysates were then sheared through a needle and centrifuged at 100,000 × g for 1 h. Immunoprecipitation with monoclonal anti-GAP-43 was then carried out. Comparing in vivo if GAP-43 interacts directly with F-actin and other proteins that have been proposed, based on in vitro studies, to be interaction partners. Addition of DSP to living cells caused an increase in the amount of actin sedimenting at 100,000 × g following SDS lysis and the addition of nonionic detergent, but no GAP-43 was found in this pellet fraction, indicating that GAP-43 did not become crosslinked to F-actin and did not sediment with it. GAP-43 was found in the 100,000 × g supernatant, but no co-immunoprecipitated actin was seen. Following nonionic detergent lysis of cells, GAP-43 was bound to the membrane skeleton [Chemical crosslinking was used in this study to investigate skeleton  and co-pskeleton . In the in vitro [Although GAP-43 binds to F-actin in vitro –17, it min vitro . Filopodin vitro . GAP-43 in vitro . Evidencin vitro  and the in vitro . Evidencin vitro . An altein vitro  or when in vitro . GAP-43 in vitro  would bein vitro . When a in vitro . Finallyin vitro . When crin vitro , which iin vitro . FollowiCalmodulin co-immunoprecipitated with GAP-43 following DSP treatment of cells and additional GAP-43 was recovered from the immunoprecipitation supernatant with anti-calmodulin. Combining these results indicates that approximately 25% of the total GAP-43 was crosslinked to calmodulin. The fact that the remainder of the GAP-43 was not crosslinked to calmodulin is consistent with the cells being in a stimulated, differentiated state. The calmodulin crosslinking is also consistent with the fact that both unphosphorylated GAP-43 and GAP-43 phosphorylated by protein kinase C can exist in the same stimulated neuron, with phosphorylated GAP-43 predominating at the growth cone and unphosphorylated GAP-43 being present in the cell body and proximal axon [2, as proposed [2 in some regions and to calmodulin in other regions of the plasma membrane. In cultured hippocampal neurons that were not treated with any agent to activate protein kinase C, approximately 50% of the total GAP-43 could be crosslinked by DSP to calmodulin [What is the biological significance of GAP-43 binding to calmodulin in these cells? If the function of GAP-43 is to bind and sequester PIPproposed , it is nlmodulin . These clmodulin , in thatThe lack of quantitative recovery of GAP-43 by immunoprecipitation following DSP treatment of cells leaves open the possibility that GAP-43 becomes crosslinked to other proteins in the cell. The results tend to rule out GAP-43/actin interactions in these cells. It is clear from the present study that extensive crosslinking of GAP-43 to cytoskeletal and membrane skeletal proteins does not occur in living N1E-115 cells, suggesting that GAP-43 function in these cells may derive in part from its interaction with the lipid bilayer.N1E-115 cells (CRL-2263) were obtained from the American Type Culture Collection, Manassas, VA. Mouse monoclonal anti-β-galactosidase and rabbit antibody to GAP-43 (whole molecule as antigen) were from Affinity Bioreagents, Golden, CO. Mouse monoclonal anti-GAP-43 (clone 91E12) was from Millipore. Protein A-agarose and protein G plus agarose were from Santa Cruz Biotechnology, Santa Cruz, CA. Calmodulin Sepharose 4B was from Amersham Biosciences. Cell culture grade water, pH 3.5–10 ampholines, Hybri-max sterile DMSO, mouse monoclonal anti-calmodulin, rabbit anti-actin, Kodak Biomax Light film, 2-mercaptoethanol, rabbit skeletal muscle actin, and all cell culture grade chemicals used in preparing modified Puck’s D1 solution were from Sigma. Nonidet P 40 (NP-40) substitute was from Fluka. Dithiobis (succinimidyl propionate) (DSP) was from Pierce. HPLC grade DMSO was from Sigma-Aldrich. Fetal bovine serum, dialyzed fetal bovine serum, and penicillin/streptomycin solution were from Invitrogen. Sodium pyruvate (100 mM) was from Mediatech, Herndon, VA. Dulbecco’s modified Eagle’s medium (DMEM), Met, Cys-free DMEM, and TRAN35S-Label were from MP Biomedicals, Solon, OH. Complete Mini protease inhibitor tablets were from Roche. All materials used in gel electrophoresis, except those given above, were obtained from Bio-Rad, Hercules, CA. Soluene 350 tissue solubilizer and Enlightning autoradiography enhancer were from Perkin-Elmer.2 in air. The cells were plated on untreated 60-mm cell culture dishes, and 24 h later each dish received 5 mL of differentiation medium, which consisted of the above DMEM supplemented with 1% fetal bovine serum, 1% penicillin/streptomycin, 1 mM sodium pyruvate, and 2% Hybri-Max DMSO. Each dish received 5 mL of fresh differentiation medium every 24 h and the cells were used in crosslinking experiments after 4 days.N1E-115 cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose, 4 mM L-glutamine, 3.7 g/L sodium bicarbonate, and no pyruvate, and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidifed atmosphere of 10% COThis was performed as described n the literature . Differe2, 1.2 mM MgSO4, 25 mM HEPES (pH 7.4), and 5.6 mM glucose. Dishes 1 and 2 then received 4.8 mL of KRH containing 0.2 mL of DMSO, while dishes 3 and 4 received 4.8 mL of KRH containing 0.2 mL of freshly prepared 25 mM DSP in DMSO. This yielded a final DSP concentration of 1 mM and a final DMSO concentration of 4%. Incubation was carried out for 15 min at room temperature.The labeled cells were washed three times at room temperature with 5 mL of Krebs-Ringer’s-Hepes solution (KRH), which was prepared as described . KRH conFollowing removal of the DMSO and DSP solutions in KRH, each dish received 0.1 mL of 1% SDS in 103 mM glycine (pH 7.5). The glycine quenched unreacted DSP. Each dish then received 0.9 mL of NP-40 lysis buffer, which contained 20 mM sodium phosphate (pH 7.4), 1% NP-40 substitute, 150 mM NaCl, and 1 mM EDTA. Ten milliliters of this solution received one Complete Mini protease inhibitor tablet immediately before addition to the dishes. Each lysate was then sheared by passage six times through a 23 gauge needle. The lysates were centrifuged at 30,000 rpm  for 1 h at 4 °C in a Beckman SW50.1 rotor. The pellets were dissolved in sample buffer for two-dimensional electrophoresis. The supernatants were used for immunoprecipitation as described below.The membrane sleleton was isolated as described . Each diLabeled cells on 60-mm dishes were lysed by the addition of 0.9 mL of NP-40 lysis buffer. The nuclei and cytoskeleton were removed by centrifugation, and supernatants 1 and 2 each received 36 μL of DMSO while supernatants 3 and 4 each received 36 μL of 25 mM DSP in DMSO. After incubation at room temperature for 15 min, each tube received 100 μL of 2% SDS in 0.113 M glycine (pH 7.5). After 15 min, each lysate was passed six times through a 23 gauge needle and was centrifuged in the SW50.1 rotor at 30,000 rpm  for 1 h at 4 °C. The supernatants were used for immunoprecipitation as described below.The 100,000 x g supernatants from cells lysed in either SDS or NP-40 substitute received 1 μL of mouse monoclonal anti-β-galactosidase as a control or 2 μL of mouse monoclonal anti-GAP-43 (clone 91E12), or 7 μL of mouse monoclonal anti-calmodulin solution. Each tube then received 20 μL of protein G plus agarose suspension, followed by rotation overnight at 4 °C. The agarose pellets were washed three times with 1 mL of 20 mmol/L sodium phosphate (pH 7.4), 1% NP-40 substitute, 0.1% SDS, 150 mM NaCl, 1 mM EDTA. The pellets were suctioned dry and the bound proteins were eluted by the addition of 100 μL of sample buffer for two-dimensional electrophoresis. A rabbit polyclonal antibody to GAP-43, which was obtained using full-length GAP-43 as immunogen, was also used to immunoprecipitate GAP-43. The 100,000 × g supernatants received 2 μL normal rabbit serum as a control, 9 μL of rabbit polyclonal anti-GAP-43 solution or 2 μL of rabbit anti-actin serum. Twenty microliters of protein A-agarose suspension was added to each sample followed by rotation overnight a 4°C. The pellets were washed three times as above and were treated with 100 μL of sample buffer for two-dimensional electrophoresis.One-dimensional electrophoresis was performed as described . ElectroThe dried gels were overlaid with the x-ray film, and radioactive spots were cut out of the gel with a razor blade. The spots were rehydrated with 0.1 mL of water for 1 h at room temperature. Each vial then received 1 mL of a 9:1 (v/v) mixture of Soluene 350 tissue solubilizer and water, followed by incubation at 50 °C for 6 h. Each vial received 50 μL of glacial acetic acid and 10 mL scintillation fluid, followed by determination of radioactivity in an LKB Wallac model 1209 Rackbeta liquid scintillation counter.


Opposed to this gain mechanism, is another which implies that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss. The importance of both mechanisms (loss and gain) is matter of debate. Here we report the sequence of the cox1 gene of the button mushroom Agaricus bisporus, the most widely cultivated mushroom in the world. This gene is both the longest mitochondrial gene  and the largest group I intron reservoir reported to date with 18 group I and 1 group II. An exhaustive analysis of the group I introns available in cox1 genes shows that they are mobile genetic elements whose numerous events of loss and gain by lateral transfer combine to explain their wide and patchy distribution extending over several kingdoms. An overview of intron distribution, together with the high frequency of eroded heg, suggests that they are evolving towards loss. In this landscape of eroded and lost intron sequences, the A. bisporus cox1 gene exhibits a peculiar dynamics of intron keeping and catching, leading to the largest collection of mitochondrial group I introns reported to date in a Eukaryote.In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp) are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a “Homing Endonuclease Gene” ( The borders of these introns, their group and sub-group were deduced from the design of each secondary structure . The 19 introns are scattered over the whole gene sequence (iAbi13) to 2,736 nt (iAbi2). The intron sequences represent 28,318 nt, i.e. 94.7% of the gene. The Abi cox1 gene is the longest mitochondrial gene reported to date in all kingdoms. Indeed, the next longest mitochondrial genes are the cox1 gene of the Ascomycota Podospora anserina  which carries 14 group I and one group II introns Trametes cingulata  with 15 group I introns The complete sequence  of the sequence . The siziAbi2) of the 19 introns belongs to group II (subgroup IIA1). This large intron  contains an eroded ORF (402 aa) encoding a putative protein possessing significant amino acid (aa) sequence identity (up to 38%) with the central and C-terminal regions of several reverse transcriptases (RT) encoded by plant and fungal mitochondrial group II introns. However, this encoded protein lacks the first 200 aa of the RT N-terminal region. All these RT genes are carried by group II introns possessing the same insertion site in the orthologous cox1 genes. The highest amino acid (aa) identities (id.) and similarities (sim.) of the iAbi2 putatively encoded RT were obtained with the phylogenetically distant Ascomycota Cryphonectria parasitica  and the liverwort (Viridiplantae) Marchantia polymorpha . In Abi, this intron might be undergoing an elimination process, as suggested by the erosion of the encoded reverse transcriptase.Only one  contain eroded heg leading to non-functional HE  of a second complete heg (invader). Both heg are in an open reading frame.The ding heg . This iniAbi11 recipient heg possesses a percentage of aa identity ranging from 73% with heg encoded by the Ascomycota Podospora anserina i9 or Gibberella zeae i5 to 40% for an orthologous group I intron widely distributed in plants. All these orthologous heg are carried by introns located at the same position in fungal and plant cox1 genes.The iAbi11 invading heg is orthologous to a heg carried by an intron located in another mitochondrial gene: the cob gene of several Ascomycota species. The highest percentage of aa identity is obtained with G. zeae (50%) and P. anserina (47%).The heg are located in introns of the cob and cox1 genes, respectively. On the contrary, in A. bisporus, both heg are located in the same intron of the cox1 gene.In these two species, the invading and recipient heg do not possess sequence homology, the iAbi11 chimeric heg can not be generated by a recombinational event. Hence, the invasion of the iAbi11 intron heg by a heg arising from an other gene (cob) suggests a recent transfer activity of the enzyme encoded by the mobile invading element, as previously reported in the T4 phage and in the fungus Podospora curvicollaAs the recipient and invading Abi group I introns, all the complete cox1 genes in databases were compiled (Monosiga brevicollis) cox1 genes from the Eumetazoa lack group I intron, except two different intron types described in Cnidaria To understand the origin and dynamics of the compiled  as descrcox1 genes . They were sorted in 35 position classes (Pcls) defined first by the same location in the cox1 CDS sequence. This distribution, which is based on the precise insertion site of each intron, is correlated with the highest sequence identities at the nucleotide level as well as with the amino acid identities of the putative encoded HE. Hence, each Pcl is also a sequence identity class. Indeed, each Pcl was shown to be constituted by introns inserted at the same position in the cox1 CDS and, when present, carrying an orthologous heg. The orthologous character was deduced first from BLASTx analysis. When using a heg described in a cox1 intron as a query in a BLASTx search, the homologous sequences with the higher scores  and consequently, the lowest E-values (<e-20) were analyzed. In the large majority of cases, these sequences corresponded to heg located in introns with the same location in the cox1 CDS. If the sequence of the cox1 gene was complete and fully annotated, these heg were included in the same Pcl. Moreover, the analysis of all these complete cox1 genes allowed the highlight of some additional heg whose encoded protein was eroded or undescribed. The corresponding introns were added to the corresponding Pcl. In a few cases, a sequence with similar E-values but located in a mitochondrial gene different from the cox1 gene (nad5 or cob) was present in the BLAStx results. These sequences have been considered as an invader with a cox1 intron origin and not included in our analysis of the cox1 introns. In all cases, we did not find any non-homologous intron heg located at the same precise site of the cox1 CDS. This is in accordance with the fact that the insertion site of the intron appears to be determined by the DNA sequence precisely recognized by its encoded HE 240 group I introns were found in 53 fungal cox1 CDS. Since all the 35 Pcls are represented in at least one of the six longest fungal cox1 genes, an alignment of the corresponding COXI proteins is used in cox1 genes are found in the Basidiomycota Abi (18 group I introns), in the Ascomycota Podospora anserina (Pan: 14 introns) and Gibberella zeae (Gze: 12 introns), in the Chytridiomycota Rhyzophidium sp. (Rsp: 14 introns), in the Blastocladiomycota Allomyces macrogynus (Ama: 11 introns) and in the Glomeromycota Glomus intraradices (Gin: 11 introns).The 35 Pcls were named in alphabetic order starting from the 5′-end of the cox1 introns assigned to each Pcl varied greatly from 25 in the Pcl K to a single one in 8 different Pcls (Gin and in the distant Ascomycota Pan (Rsp) out of six Chytridiomycota, in one species out of two Blastocladiella, in the Glomeromycota species, in one out of three species of the basal fungal lineages, in seven out of ten Basidiomycota and in 14 out of 31 Ascomycota. Abi contains15 introns belonging to the 25 widely distributed Pcls, while Schizophyllum commune (Sco) which belongs to the same Agaricales order is intronless. Thus, the observed wide and patchy distribution of the group I fungal introns appears to result from numerous losses and gains of these mobile genetic elements.The number of fungal ent Pcls . Hence, cota Pan . The 25 cox1 CDS in the Viridiplantae, Amoebozoa or Metazoa kingdoms. As previously established with fungal introns , the heg of the introns from all these kingdoms belonging to the same Pcl are orthologous genes. These Pcls represent 49% (17/35) of the Pcls and up to 60% (15/25) of the widely distributed ones. Except for two group I introns specifically harbored by coral species (classes Co1 and Co2), all the cox1 group I introns belong to Pcls previously defined by representatives from the fungal kingdom.Among the 35 fungal intron Pcls, 17 contained orthologous introns defined by the same position in the In heg sequences of orthologous introns of the same Pcl. The HE encoded by the introns of Pcl K show percentages of aa identity (and similarity) ranging from 31% aa id. (54% aa sim.) between the fungal Ascomycota Gze and the Viridiplantae Mpo to 56% aa id (73% aa sim.) between the Basidiomycota Abi and Aae. These sequence divergences between orthologous introns of the same Pcl, which are distributed over up to four kingdoms but are also observed between closely related species, can be explained by (i) a high divergence of vertically inherited introns maintained in the compared species during the course of evolution or (ii) independent gains due to lateral transfer.A high sequence divergence can be seen between the heg and others with an eroded heg. The Pcl AG is an exception containing four representatives with only intact heg. Erosion of the heg is thought to be a preliminary step before the complete elimination of the intron heg, even in closely related species strengthens the hypothesis that numerous events of loss and gain occurred during evolution.Moreover, most of the Pcls of the widely distributed introns  contain cox1 genes of the Viridiplantae, Amoebozoa and Metazoa kingdoms lack intron and only a small number of species harbor 1 to 6 group I introns. On the contrary, the majority (45 genes) of the 53 cox1 sequences of the fungal kingdom possess one to 18 introns, while only eight genes do not contain any intron.Most cox1 gene was established. This curve does not fit with the Gaussian distribution that would be expected if the numbers of introns were to reflect random events of losses and gains. The plots can be represented by a theoretical curve showing a logarithmic decrease. Together with the high frequency of eroded heg carried by these introns , the curve can be interpreted as a trend of these group I introns towards loss from the cox1 genes. However, the six long genes distributed over five different fungal divisions behave as group I intron reservoirs, with 11 to 18 introns. They contain at least one representative of each Pcl, except for the introns reported in corals. As the eight rare introns detected once were reported in five of these six long genes, the ability to bear specific or rare introns appears to be a characteristic of these reservoir species.To analyse the dynamics (loss and gain) of group I introns in eukaryotes, a curve  represencox1 genes from the basal fungal lineages carry eroded heg: 10 introns out of the 11 of Ama, 9 out of the 11 of Gin and 7 out of the 14 of Rsp. This suggests that these introns are being eliminated from these species. In principle, the erosion of the heg should definitively prevent the harboring intron from spreading. On the contrary, most of the introns of the long cox1 genes of Dikarya  maintain a putative functional heg.Moreover, most of the introns described in the long Abi cox1 gene has to be considered as the largest group I intron reservoir described to date. It possesses representatives of more than half (18/35 = 51%) of the Pcls, and more than 64% (16/25) of the widely distributed ones and all but two of its 18 group I introns possess intact and potentially functional heg.In this context, the Abi cox1 gene is the most amazing representative to date, pose an appealing and still unresolved question about why and how some fungal species organize their mitochondrial genes in such an expensive manner.It appears that the evolution of the mitochondrial genome of eukaryotes is still moving in the direction of group I intron loss. However, in some species and particularly in fungi, complete elimination of the group I intron seems counterbalanced by the gain mechanism resulting from the lateral transfer of these mobile genetic elements. Furthermore, the long genes described in the Dikarya fungi, whose the Abi cox1 complete gene was sequenced by conventional procedures Abi cox1 gene) has been deposited in the GenBank (Accession number EU314927).The Agaricus bisporus strain Bs518 was cultured in Roux vials containing complete CYM medium supplemented with 10% (v/v) compost extract BamH1 and EcoRI mitochondrial DNA libraries were constructed in E. coli XL1-Blue Abi cox1 cDNA and by cloned PCR products .Taq polymerase from Promega Corp. and couples of primers .PCR amplifications were carried out using the Go 32P] dCTP (110 TBq/mmol) from Amersham Pharmacia Biotech., using the Promega Corp. “Random Primers DNA Labeling Kit”. The probes had a specific radioactivity higher than 108 cpm/µg DNA.The different probes were recovered after agarose gel electrophoresis by using the “Wizard SV gel Kit”, then labeled with 925 kBq of [αCloned PCR products and the isolated mitochondrial genomic fragments were sequenced by the primer walking methods using the Big Dye® Terminator Cycle Sequencing kit v 1.1 (Applied Biosystems). Sequencing was performed in the genotyping and sequencing facility at University Victor Segalen Bordeaux 2.cox1 sequences were compiled and analyzed with the DNA Strider 1.2 software . Comparisons with sequences of the GenBank and EMBL databases were performed using the BLAST search algorithm The overlapping cox1 gene sequences was performed by compiling the results (orthologous sequences) given by a BLASTx analysis using first the A. bisporus cox1 CDS as query. This analysis revealed most of the fungal cox1 sequences available in the Genbank and also numerous cox1 genes from plants. Second, all the intron heg carried by these sequences were used independently as a query for BLASTx analysis. This allowed the recovery of some additional split cox1 sequences from plants and also from other distant species such as Palythoa sp. JVK-2006 (Porifera), and species belonging to the Ichthyosporea, Choanoflagellida and Dictyosteliida phylla, which have been added to our analysis. Finally, the coral (Cnidaria) cox1 genes were added to the analysis because they contain the only introns reported to date in the Eumetazoa kingdom without homology with introns from other kingdoms. Alignments of nucleotide and protein sequences were carried out with Clustal W (1.8) software Compilation of the complete The intron secondary structures .Restriction map of the Abi mitochondrial genome region carrying the cox1 gene. The exon sequences are represented by coloured boxes. The cloned BamH1 and EcoRI restriction fragments used to determine the Abi cox1 gene sequence are indicated and named according to the restriction map. The sequenced PCR products and the location of the corresponding primers are indicated (CoxU: (0.18 MB PPT)Click here for additional data file.Figure S2Secondary structures of the A. bisporus introns iAbi1 to iAbi19. Exonic sequences and intronic sequences are in lower-case and upper-case letters, respectively. The base-paired regions P1 to P9 are shown on the secondary structures, according to the standard scheme for group I introns described by Michel and Westhof (0.74 MB PPT)Click here for additional data file.Figure S3Alignement (A) and representation of the Total Score (B) obtained when comparing the 21 intact HE of the most widely distributed Pcl K. The Total Scores higher than 200 are in red, those between 80 and 200 in pink. In C are reported the percentages of aa identity (a) and aa similarity (b) between I-AbiVII-P and the 20 others orthologous HE.(0.07 MB DOC)Click here for additional data file.


PLoS Biology by Howard Ochman and colleagues counters the prevailing view that diet shapes the makeup of gut microbes, revealing that the host animal is a stronger determinant of these bacterial worlds in our digestive tracts.The maxim “you are what you eat” goes only so far. The bacteria inhabiting our guts, which outnumber our own cells by perhaps 10 to 1, are commonly thought to reflect our diets. But other factors from geography to host physiology can also affect gut microbes, and sorting out their provenance is critical because they can affect our health for good or ill, from enhancing immune function to increasing the risk of stomach cancer. New research in this issue of Helicobacter pylori, which is found in about half of people and is linked to stomach ulcers and cancer. However, work on great apes had suggested that diet is the major determinant of gut microbe composition.On the face of it, gut microbe composition could be determined by either the host or its environment. Mammals are born with sterile digestive tracts and usually get their first gut microbes from their mothers, but thereafter acquire new ones from their environment. Moreover, there is evidence on both sides of the host versus environment debate. The evolutionary relationships of some gut bacteria are known to match those of their hosts, notably To sort out whether gut microbe composition depends primarily on the host or its environment, Ochman and colleagues compared fecal samples from five closely related hominid species: Eastern and Western lowland gorillas, bonobos, and three subspecies of chimpanzees from several African countries as well as people from Africa and North America. By using fecal samples from locations separated by such considerable distances, the researchers effectively knocked out any effects of geography and the local environmental on gut microbes.The researchers constructed two phylogenetic trees from the fecal samples: one of the great ape hosts, which was based on mitochondrial DNA, and the other of their gut microbe communities. Gut microbe diversity and abundance were based on small subunit ribosomal RNA genes, which are unique to bacteria and other prokaryotes. In turn, the “gut microbe” tree was based on the relative abundance of the various microbe species in each fecal sample, much as physical measures such as femur lengths from, say, mouse to elephant are used to infer evolutionary relationships.The resulting gut microbe tree mirrored that for hominids; the patterns of relationships among the gut microbe communities were identical to those among the five great ape species. This congruence neatly establishes hosts as the major force behind the makeup of gut microbe communities. Further, the concordance of the two phylogenetic trees suggests that the gut microbe communities were shaped by divergences in host physiology over the course of great ape evolution.The disparity with previous findings is partly because this new work entailed surveying many more fecal bacteria sequences per host, with a median of 28,000 sequences. In contrast, in previous studies only 100 to 200 bacterial sequences were surveyed per host, which is insufficient for gauging the diversity and abundance of species in the complex microbe communities living in our digestive tracts.This work brings us closer to understanding the acquisition and evolution of the gut flora that can affect our health for better or for worse. Even though new microbes are constantly arriving in digestive tracts, their makeup is largely determined by the distinctive physiologies of people and our closest relatives. Gut microbe communities are thus predictable rather than variable, as they would be if determined largely by the host's external environment. Perhaps this is not altogether surprising, given that we hosts essentially are the environment for the worlds of enteric microbes within us. In this case at least, what we are trumps what we eat.Ochman H, Worobey M, Kuo C-H, Ndjango J-BN, Peeters M, et al. (2010) Evolutionary Relationships of Wild Hominids Recapitulated by Gut Microbial Communities. doi:10.1371/journal.pbio.1000546


Gedunin, a family of natural products from the Indian neem tree, possess a variety of biological activities. Here we report the discovery of deoxygedunin, which activates the mouse TrkB receptor and its downstream signaling cascades. Deoxygedunin is orally available and activates TrkB in mouse brain in a BDNF-independent way. Strikingly, it prevents the degeneration of vestibular ganglion in BDNF −/− pups. Moreover, deoxygedunin robustly protects rat neurons from cell death in a TrkB-dependent manner. Further, administration of deoxygedunin into mice displays potent neuroprotective, anti-depressant and learning enhancement effects, all of which are mediated by the TrkB receptor. Hence, deoxygedunin imitates BDNF's biological activities through activating TrkB, providing a powerful therapeutic tool for treatment of various neurological diseases. Employing this assay, we have successfully identified TrkA agonist gambogic amide As described in this manuscript, this screen identified a number of gedunin derivatives. Gedunin, a tetranortriterpenoid isolated from the Indian neem tree (Azadirachta indica), has demonstrated the ability to exhibit antimalarial, insecticidal, and most recently anticancer activity Oxygen-Glucose Deprivation) is an in vitro model for ischemia stroke. BDNF reduces ischemic injury 2 and 5% CO2 at 37°C). Deoxygedunin, alpha-dihydrogedunol (epoxy ring down) and dihydrodeoxygedunin exhibited potent protective effects on hippocampal neurons under OGD. Titration assay reveals that deoxygedunin protected neurons in a dose-dependent manner . Hence, deoxygedunin directly binds TrkB ECD and triggers its association. Deoxygedunin also elicited tyrosine phosphorylation in TrkB but not in TrkA or TrkC receptor in transfected HEK293 cells. TrkB-KD displayed negligible phosphorylation compared to wild-type TrkB . Glutamate-provoked caspase-3 activation was substantially blocked by 7,8-DHF and deoxygedunin in wild-type but not TrkB −/− neurons. However, the control compound imipramine failed to blocked caspase-3 activation by glutamate. In contrast, amitriptyline weakly suppressed caspase-3 activation in both wild-type and TrkB −/− neurons , another positive compound from the screening, also selectively activated TrkB but not TrkA. The tricyclic antidepressant drugs amitriptyline but not imipramine activated both TrkA and TrkB . Since 1NMPP1 selectively inhibits TrkB F616A activation by deoxygedunin, we hypothesized that blockade of TrkB F616A signaling by 1NMPP1 in mice would make the neurons vulnerable to KA-provoked neuronal cell death. We pretreated the mice with 1NMPP1, followed by deoxygedunin and KA treatment. In 5 days, we monitored neuronal cell death in hippocampal region by immunoblottings. As predicted, 1NMPP1, deoxygedunin alone or 1NMPP1 + deoxygedunin combined treatment had no effect on apoptosis in TrkB F616A mice. KA caused marked caspase-3 activation, and pretreatment of 1NMPP1 mildly elevated KA-provoked apoptosis in TrkB F616A, supporting that TrkB signaling is critical for neuronal survival. Deoxygedunin evidently suppressed KA-provoked apoptosis, whereas 1NMPP1 pretreatment greatly diminished deoxygedunin's protective effect in F616A mice. TrkB activation status inversely correlated with TrkB activation by deoxygedunin  into BDNF cortex conditional knockout mice and sacrificed the mice at 4 h. Immunoblotting analysis with the cortical lysates demonstrated robust TrkB activation in both wild-type and BDNF −/− mice , underscBDNF plays an essential role in mediating antidepressants' therapeutic effects in vivo to enhance cue-dependent fear learning.We next wished to examine whether this TrkB agonist would enhance learning in a whole animal model of learning and memory, in which BDNF-dependent TrkB activation is required. Activation of TrkB by BDNF has been repeatedly shown to be associated with, and required for, the acquisition of classical conditioned fear in rodent models In the present study, we have demonstrated that deoxygedunin directly binds the ECD of TrkB and promotes its dimerization and activation. Deoxygedunin provokes TrkB activation in primary neurons and in mouse brain. It strongly protects neurons from apoptosis in a TrkB- dependent manner. Further, it activates TrkB in BDNF conditional knockout mice, indicating that BDNF is not implicated in the stimulatory effect of deoxygedunin. When it is injected in animals, deoxygedunin mimics BDNF and exerts neuroprotective and antidepressant actions and enhances learning processes. Moreover, administration of 7,8-dihydroxyflavone and deoxygedunin into pregnant BDNF +/− mothers substantially rescues vestibular ganglia, which are significantly degenerated in BDNF −/− pups.To determine which chemical group or moiety is critical for the neurotrophic activity of gedunin, we have conducted a structure-activity relationship study. The results suggest that the epoxy ring confirmation in the D ring is essential for gedunin's agonistic activity. It has to be in a down conformation for the agonist to be effective. Nevertheless, the alpha-beta unsaturated ketone in ring A is variable. Reduction of the unsaturated C = C bond did not impair its stimulatory activity, suggesting that this site can tolerate a potential chemical modification, which might be useful for improving its water solubility and biological activity. When the epoxy group is in down conformation, changing ketone into alcohol did not cripple its agonistic effect either regardless of alpha or beta conformation of the resulting hydroxy group. Nevertheless, deoxygedunin displays the strongest agonistic effect, suggesting that ketone might be more active than alcohol in ring A . We have3H-deoxygedunin administration. 3H-labeled compounds were concentrated in olfactory bulb and hippocampus in the brain. Clearly, the brain as a whole organ was the major tissue in the body where 3H-labeled compound was accumulated, followed by the lung  in order to improve their biological activity.Gedunin, a tetranortriterpenoid isolated from the Indian neem tree (Azadirachta indica), and was recently shown to manifest anticancer activity via inhibition of the 90 kDa heat shock protein (Hsp90) folding machinery and to induce the degradation of Hsp90-dependent client proteins similar to other Hsp90 inhibitors the lung . Obviousd ∼10−10 M). Although the detailed molecular mechanism of how a ligand provokes Trk receptor activation remains unclear, a conformational change hypothesis has been proposed. For instance, a mutation at P203A can cause spontaneous dimerization and activation of TrkA Deoxygedunin binds to the ECD domain of TrkB . InteresWe have shown that both 7,8-DHF and deoxygedunin bind to the ECD of TrkB and provoke the receptor dimerization autophosphorylation. They both display comparable agonistic activity on TrkB receptor. Moreover, they both minic BDNF by exhibiting robust neuroprotective effect in stroke, neuroexcitotoxicity, and vestibular ganglia survival assays. In addition, these two compounds also display strong activity in learning and memory and depression in animal models. Forced swim test revealed that deoxygedunin might possess more robust antidepressant effect than 7,8-DHF .Taken together, these two small molecules basically share the same biological and therapeutic actions. Nonetheless, 7,8-DHF is water soluble, but deoxygedunin is not. The latter has more complicated structure scaffold than the former. The flavonoids are easier for medicinal modification to improve their biological effect than gedunin family members.in vitro receptor/ligand binding assay, in vivo TrkB activation and neuronal survival experiments, we finally obtained a few potent and selective TrkB agonists that virtually mimic BDNF's biochemical and physiological actions, and 7,8-DHF and deoxygedunin are the most promising lead compounds. Employing a variety of animal models, we have established that deoxygedunin exhibits potent neuroprotective actions in kainic acid neuroexcitotoxicity and stroke animal models .TrkB2 atmosphere in a humidified incubator 2 atmosphere in DMEM medium containing 1 mM pyruvate and 10% FBS. T48 and T62 cells, stably transfected with rat TrkB in SN56 cells were cultured in the same medium containing 300 µg/ml G418 F616A mice have been described previously 3H]Acetic acid, sodium salt was purchased from Perkin Elmer . Deoxygedunin was purchased from Gaia Chemicals, Inc. All other chemicals were purchased from Sigma or Alfa Aesar.Human embryonic kidney HEK293 cell lines were grown in medium A  and 100 units penicillin-streptomycin) at 37°C with 5% COTrkB stable transfected SN56 cells (T48 cells) were seeded in a 96-well plate at 10,000 cells/well in 100 µl complete medium. Cells were incubated overnight, followed by 30 min pretreatment with 10 µM compounds in DMSO (10 mM stock concentration from The Spectrum Collection library). The cells were then treated with 1 µM staurosporine for 9 h. One h before the termination of the experiment, 10 µM MR(DEVD)2, a cell permeable caspase-3-activated fluorescent dye was introduced. Cells were fixed with 4% paraformaldehyde for 15 min. Cells were washed with PBS and incubated with 1 µg/ml of Hoechst 33342 for 10 min. Cover slides were washed with PBS, mounted, and examined using a fluorescence microscope.3H]Acetic acid, sodium salt  is syringed into a heavy-walled glass vial bearing a magnetic stirrer. The ethanol is removed under vacuum and replaced with 0.5 mL of THF at 0°C. Isobutylchloroformate  was then added and the reaction mixture was stirred for 1 h at 0°C. A solution of 7-deacetyldeoxygedunin , prepared by acetyl deprotection of deoxygedunin with K2CO3 in MeOH, in 0.5 mL THF was then added dropwise. The reaction was stirrer for another 1 h. Solvent was removed under vacuum and the product was purified by preparative thin layer chromatography  to give 3 mg (58%) of 32-[3H]3deoxygedunin. Note: Preparation of 32-[3H]3deoxygedunin occurred only after preparation of deoxygedunin under identical reaction conditions in order to confirm product formation. in 1 ml binding buffer (0.05 M Na/K phosphate buffer (pH 7.1), 200 mM NaCl) (1 nM [3H]deoxygedunin ∼82300 cpm) at 4°C for 10 min. After the incubation, the reaction mixture was loaded on filter paper. The mixture was washed with 3×5 ml Tris buffer . The dried filter paper was put into a small vial and subjected to liquid scintillation counter analysis. The value of the dissociate constant and the number of sites were obtained from Scatchard plots by using the equation r/[L]free  =  n/Kd - r/Kd, where r is the ratio of the concentration of bound ligand to the total protein concentration and n is the number of binding sites.Purified TrkB ECD or ICD proteins (10 µg/each) were incubated with different -deoxygedunin (2×106 cpm)/DMSO/PBS solution. In 4 h, various brain regions (A) and different organs (B) and were analyzed by liquid scintillation counter.(0.62 MB TIF)Click here for additional data file.


Saccharomyces cerevisiae and some have been shown to be functionally significant. Here we used transcriptome-wide approaches to evaluate the mechanisms underlying the generation of alternative transcripts in Yarrowia lipolytica, a yeast highly divergent from S. cerevisiae.Hemiascomycetous yeasts have intron-poor genomes with very few cases of alternative splicing. Most of the reported examples result from intron retention in Y. lipolytica gene models identified several cases of alternative splicing, mostly generated by intron retention, principally affecting the first intron of the gene. The retention of introns almost invariably creates a premature termination codon, as a direct consequence of the structure of intron boundaries. An analysis of Y. lipolytica introns revealed that introns of multiples of three nucleotides in length, particularly those without stop codons, were underrepresented. In other organisms, premature termination codon-containing transcripts are targeted for degradation by the nonsense-mediated mRNA decay (NMD) machinery. In Y. lipolytica, homologs of S. cerevisiae UPF1 and UPF2 genes were identified, but not UPF3. The inactivation of Y. lipolytica UPF1 and UPF2 resulted in the accumulation of unspliced transcripts of a test set of genes.Experimental investigation of Y. lipolytica is the hemiascomycete with the most intron-rich genome sequenced to date, and it has several unusual genes with large introns or alternative transcription start sites, or introns in the 5' UTR. Our results suggest Y. lipolytica intron structure is subject to significant constraints, leading to the under-representation of stop-free introns. Consequently, intron-containing transcripts are degraded by a functional NMD pathway. Yarrowia lipolytica is rather atypical among hemiascomycetous yeasts sequenced to date dCTP, with the Amersham Megaprime™ DNA labeling kit , and hybridizations were performed in Denhardt's solution-containing buffer at 65°C , [EMBL:FP680607-EMBL:FP690338] and [EMBL:FP690350-EMBL:FP700052] for the expo, stat and oleic libraries, respectively.Total RNA was extracted from cells grown in three different sets of culture conditions . We isolated mRNA from the total RNA preparation with the Oligotex mRNA kit (Qiagen) and the three libraries were constructed with the CloneMiner™ cDNA Library Construction Kit , based on GatewayY. lipolytica genes  was performed as previously described [4Cl (5 g/l), sodium potassium phosphate buffer, pH 6.8 (50 mM), agar (2%) and uracyl (100 mg/ml) or leucine (100 mg/ml). Gene deletion was checked by PCR, with primers external to the disruption cassette, upstream from P and downstream from T.The complete deletion of escribed . Primersescribed  were intescribed , with abURA3 or LEU2 cassettes, for comparison of their growth rates with that of the wild-type strain, W29.Auxotrophic mutants were complemented with the Y. lipolytica strain E150 included 6,703 CDSs (genes and pseudogenes [Y. lipolytica strain E150, including the version updated with our data, are available from the Génolevures database [At the beginning of this study, the genome annotation of database .Y. lipolytica CDS using BLAST [The sequences of the cDNA clones were compared with sequences in a nucleotide sequence database of ng BLAST . Only thDNA logos were created with WEBLOGO version 2.8.1 ,88.3'ss: 3' splice site; 5'ss: 5' splice site; AS: alternative splicing; bp: base pair; BP: branch point; CDS: coding sequence; CTD: carboxy-terminal domain; NMD: nonsense-mediated mRNA decay; PTC: premature termination codon; UTR: untranslated region.CN conceived and designed the experiments; MM, IBL, CO and CN performed the experiments. CC, CDS and PW performed the cDNA sequencing; CN and MM analyzed the data; CN and CG wrote the paper. All authors read and approved the final manuscript.Supplementary Table S1.Click here for fileSupplementary figures.Click here for fileSupplementary Table S2.Click here for fileSupplementary Table S3.Click here for fileSupplementary Table S4.Click here for fileSupplementary Table S5.Click here for fileSupplementary Table S6.Click here for fileSupplementary Table S7.Click here for fileSupplementary Table S8.Click here for file


Eukaryotic pre-mRNA gene transcripts are processed by the spliceosome to remove portions of the transcript, called spliceosomal introns. The spliceosome recognizes intron boundaries by the presence of sequence signals (motifs) contained in the actual transcript, thus sequence changes in the genome that affect existing splicing signals or create new signals may lead to changes in transcript splicing patterns. Such changes may lead to previously excluded (intronic) transcript regions being included (exonic) or vice versa. Such changes can affect the encoded protein sequence and/or post-transcriptional regulation, and are thus a potentially important source of genomic and phenotypic novelty. Two recent papers suggest that such changes may be a major force in remodeling of eukaryotic gene structures, however the rate of occurrence of such changes has not been assessed at the genomic level.Cryptoccocus fungi. Among 28,256 studied introns, canonical GT/C...AG boundaries are nearly universally conserved across all four species. Among only 40 observed cases of cDNA-confirmed non-conserved intron boundaries, most are likely to involve alternative splicing. I find only five cases of "intronization," intron creation from an internal exonic region by de novo emergence of new splicing boundaries, and no cases of the reverse process, "de-intronization." I find no more than ten clear cases of true movement of an intron boundary of a possibly constitutively spliced intron, and no clear cases of true "intron sliding," in which changes in the positions of both intron boundaries could lead to a movement of the intron position along the coding sequence.I studied four closely related species of These results suggest that intronization, de-intronization, and intron boundary movement are rare events in evolution. The spliceosome is a large RNA-protein complex that processes pre-mRNA transcripts to remove some regions of transcripts, called spliceosomal introns . Some genes exhibit alternative splicing, with some genic regions being included in some but not all mRNA transcripts . Intron-Intron-exon structures can change due to large-scale gain/loss of sequence in several ways. Precise loss of an intronic sequence can occur by genomic recombination with a reverse transcribed mRNA -5. IntroSmaller-scale sequence changes may also lead to changes in intron-exon structure, with formerly exonic sequence becoming intronic or vice-versa. Transcript splicing patterns are determined by generally short sequence motifs, including intron boundary motifs  and an internal 'branch point' motif which plays a key role in the splicing reaction , as well as various more diffuse intronic and exonic sequence signals . As suchSuch sequence changes can increase or decrease the total number of introns/exons in the gene, or simply change the position of an existing splicing boundary. Intron/exon number can increase by emergence of new splice boundaries, either (i) within an intron, leading to 'exonization' of an internal portion of an intron ; or (ii) within an exon, leading to 'intronization' of an internal portion of an exon ,23. IntrRecently, two sets of authors suggested that sequence changes leading to changes in splicing patterns could be important forces in the evolutionary remodeling of eukaryotic gene structures. First, Tarrio-Rodriguez et al.  presenteCryptococcus neoformans species .In order to understand tempo and mode of evolution of intron boundaries, I studied four-species genomic alignments of splicing ,26, and splicing ,28. Mysesplicing . Hughes m length . Other iI studied genomic regions corresponding to cDNA-confirmed introns in the reference species JEC21, previously studied by Hughes et al. . Intron Cryptococcus species  Could some of the above 'complex' cases represent de-intronization events? In de-intronization, newly exonic sequence would be expected to respect coding frame – thus in all species without splicing boundaries the corresponding regions would expected to be a multiple of three basepairs and to lack in frame stop codons. This pattern is not found for any of the complex cases Table , thus thP = 5.8 × 10-5 by a binomial test for equal probabilities). In 66% (24/35) of cases, there was a candidate alternative in frame boundary (GT or AG) within 9 nts of the JEC21 boundary position , and many of these alternative boundaries had extended consensus sequences  of changes at the 3' boundary (P = 0.12) for both 5' changes (4/5) and 3' changes (12/20). Use of the alternative boundary was observed  for 4 out of 15 cases for which multiple spliced cDNA transcripts were available, and an additional out-of-frame 3' boundary was found to have been used in a single cDNA for one additional case. In addition, cDNAs with the intron sequence retained were found in 2/25 cases , and showed weaker overall adherence to 5' boundary consensus . The difference was not significant when intronic sequence positions were considered separately, except for the sixth position, at which introns undergoing changes were less likely to contain a consensus T than expected, at a P = 0.017 level, correcting for multiple comparisons.I compared the 40 introns with observed boundary sequence changes with 10,000 randomly generated sets of 40 introns chosen from among all JEC21 introns. Introns undergoing changes were longer than expected  as an estimate of the amount of mutation within the history of these species, 0.46 × 4 changes interrupting the canonical GT/C..AG splicing boundaries would be expected per intron in the history of these species, or a total number of splicing interrupting mutations equal to 28,256 introns × 6.2% × 0.46 × 4 = 3235 de-intronization events. While this estimate is clearly quite rough, that no such events are observed indicates nearly universal efficient selection opposing such insertions of moderate length.The lack of de-intronization is striking. In the In most cases, non-conserved splicing boundaries lie near clear candidates for alternative splicing boundaries. In some cases cDNAs confirm alternative use of the two boundaries in JEC21. The non-conserved JEC21 boundary may dominate, but the alternative boundary may be used (at some level of efficiency) in the absence of the non-conserved boundary. Indeed, given the low overall level of transcript coverage, the 'alternative' boundary may be most frequently used even in JEC21, and by chance the few available transcript sequences reflect the less frequent boundary, thus loss of frequently-used splicing boundaries may be even less common than this study suggests.S. cerevisiae, which require extended splice boundaries  for splicing, and more frequent in species with more lax splice boundaries, which tend to rely on more distant splicing factors . Alternative boundaries are likely to be less frequent in intron-poor species such as ferences ,33).Cryptococcus, which is estimated to have a relatively large effective population size [The case for strength of selection is less straightforward. Recently, Lynch and colleagues have presented a series of arguments for a major role of slightly deleterious events in the evolution of genome structure e.g., ,34,35). ,35. 34,3In discerning the role of effective population size in determining genome structures, it is important to keep in mind that the selective effects on different mutations, even superficially similar ones such as analogous mutations at different intron boundaries, are likely to vary widely. Different intron boundaries do or do not have available alternative splice boundaries, alternative boundaries will have different splicing efficiencies across introns, the impacts of intron boundary shifts on mRNA transcripts and resulting proteins will vary across introns, strength of selection against inefficient splicing is likely to vary across introns, and so forth.Cryptococcus – relatively weakly and relatively strongly selected – are preserved by selection. As any of the above variables seems likely to vary by at least an order of magnitude, overall strength of selection on intron boundaries is likely to vary by multiple orders of magnitude. Thus, while a large decrease in effective population size relative to Cryptococcus could lead to some intron boundaries becoming effectively neutral, it would likely require a tremendous change in effective population size to lead to a significant rate of change for a substantial fraction of boundaries.What is notable, then, is that nearly all boundaries in -8 to 5 × 10-9, per year [Cryptococcus introns of around 4 × 10-11 or 4 × 10-12 per year. Thus, over roughly two billion years of eukaryotic evolution, the probability of a given intron in a given lineage having undergone a boundary change would be on the order of 0.008 to 0.08. On the simplest model, the probability of an intron undergoing 'sliding,' by change at both boundaries, would be the square of this probability, or 0.00006 to 0.006 . These very rough estimates suggest that intron sliding may not be a major contributor to intron position diversity among homologs.These results allow us to make rough estimates of the contribution of these processes to modern intron-exon structures. Among the species studied, divergence at synonymous sites is roughly 0.46, and degree of intron boundary change is roughly 10 changes for 28,256 introns, or 0.00077 times the rate of divergence at silent sites, per intron. Rates of divergence at synonymous sites have been estimated for a few divergent eukaryotic lineages, and are typically around 5 × 10per year -40. Thisc(1-L), where c is the probability that the intron becomes constitutive (rather than reverts) and L is the probability that it is subsequently lost. Thus the total contribution of intronization events to a modern species would be equal to I × c(1-L), where I is the number of initial intronization events occurring in the history of the species . Here, I found five intronization events occurring over an evolutionary time period corresponding to 0.10 of silent site divergence . Over tc-L, where introns ,42, in w species ,35, in wHow robust are the present results likely to be to the limitations of current genome assemblies and annotation? There are three major potential concerns: intron prediction, sequencing errors, and genome alignments. First, the presence of cDNA transcripts reflecting that splicing event confirms that annotated 'intron' is in fact spliced, whether constitutively or alternatively. Second, sequencing errors at intron boundaries are unlikely to be a major contributor to these results, since for the vast majority of observed apparent changes either (i) there is a nearby alternative boundary, providing a simple explanation for the absence of the boundary; or (ii) at least two species lack GT..AG boundaries, which cannot be explained by rare sequence errors. Third, the low degree of divergence across these species leads to clear sequence similarity across the entire studied region: as is clear in the figures, changes in boundaries often represent single nucleotide changes within a long region of conserved sequence, allowing confidence in the true homology and quality of alignment of the studied regions overall. Notably, each of these concerns involve false positives, potentially inflating numbers of boundary changes; thus the central finding of very little change of intron boundaries is robust to such concerns.It is also important to be clear about what sorts of changes are and are not detectable in this study. A change in intron-exon splicing boundary need not require change in GT...AG boundaries, but instead could simply be due to the emergence of a more robustly recognized boundary that overshadows the ancestral boundary. While the initial boundary movement in such a case would not be detectable in the current study, the now defunct boundary would be expected to eventually incur a mutation, which would be detected by this study. Given that for each such change both a true (unobserved) boundary change and a mutation knocking out the initial boundary are expected, the rate of mutations knocking out initial boundaries is expected to equal the true rate of boundary change, thus this scenario cannot explain the lack of observed change.Cryptococcus. Previously it has been shown that intron loss and gain are very infrequent among these species [These results attest to the slow general rate of change of intron-exon structures in  species , and tha species . The preTotal branch length in terms of rate of silent site substitutions (dS) has previously been estimated as 0.46 across the entire clade , thus moCryptococcus. These results suggest that genic sequence changes have not played as large a role in the evolution of intron-exon structures as previously suggested.These results show that intronization, de-intronization, and intron sliding have been rare events in the recent evolution of a genus of intron-rich fungi. Along with previous findings that intron loss and intron gain is rare among these species, this finding shows a picture of highly static intron-exon structures in I obtained genome-level alignments for regions corresponding to 28,256 coding sequence introns, previously studied by Hughes et al. , and ideSWR conceived the study, designed the experiments, performed the analyses, and wrote the manuscript. The author read and approved the final manuscript.


Alu and messenger RNA (mRNA) in trans, and probably also SVA and non-coding RNA. The structures of L1 copies and trans-mobilized retrocopies are variable and can be classified into three categories: full-length; 5'-truncated; and 5'-inverted insertions. These structures may be generated by different 5' integration mechanisms.Long interspersed nuclear element-1 (LINE-1 or L1) is a dominant repetitive sequence in the human genome. Besides mediating its own retrotransposition, L1 can mobilize trans, including Alu, SVA and mRNA retrocopies and also full-length and 5'-inverted L1, were not associated with short TSDs, indicating the difference of 5' attachment between retrotransposition in cis and retrotransposition in trans. Target sequence analysis suggested that short TSDs were generated in an L1 endonuclease-dependent manner. The MHs were longer for 5'-inverted L1 than for 5'-truncated L1, indicating less dependence on annealing in 5'-truncated L1 insertions.In this study, a method to correctly characterize insertions with short target site duplications (TSDs) is developed and extranucleotides, TSDs and microhomologies (MHs) at junctions were analysed for the three types of insertions. Only 5'-truncated L1 insertions were found to be associated with short TSDs. Both full-length and 5'-truncated retrotransposed sequences in in vivo. The method used in this study can be used to characterize elements without any apparent boundary structures.The results suggest that insertions flanked by short TSDs occur more often coupled with the insertion of 5'-truncated L1 than with those of other types of insertions  Long interspersed nuclear element-1 (LINE-1 or L1) is the only active non-long terminal repeat (non-LTR) retrotransposon family in humans and constitutes one sixth of the human genome . It encoThe insertion of L1 is initiated by single-strand DNA cleavage catalyzed by EN ,12 -K10 [Proteins encoded by L1, as well as other LINEs contribute to short interspersed nuclear element (SINE) mobilization ,28. Thern genome . Alu canL1 ORF1p ,30. ThisERV)-K10 ,31,32; iAlu are usually flanked by ~15-bp TSDs [Human L1 and -bp TSDs ,33,34. T-bp TSDs . However-bp TSDs . The advAlu, SVA and non-coding RNA (ncRNA) retrocopies were characterized by a newly developed method that can distinguish insertions from deletions and can characterize insertions with short TSDs. Only 5'-truncated L1 insertion is associated with short TSDs. Different target site structures were found on 5'-truncated retrotranspositions in cis and retrotranspositions in trans.In this study, human-specific insertions and chimpanzee-specific insertions of L1, In this study, 6197 human-specific insertions and 2910 chimpanzee-specific insertions which satisfied all the following criteria were found: (1) length between 50 bp and 10,000 bp; (2) ≥5-bp direct repeats at both junctions; (3) not a part of segmental duplications; (4) not inserted in repetitive sequences; and (5) orthologous locus can be determined in either orangutan or macaque. Criteria 1-3 were applied in order to enrich the insertions of LINEs and SINEs. Criterion 4 was applied because the orthologous locus in orangutans and macaques could not be characterized for insertions inside of repetitive sequences. Criterion 5 was used to distinguish species-specific insertions from species-specific deletions.Two primate genomes were used as outgroups to characterize the pre-inserted empty sites for each insertion; the orangutan and macaque genomes were complementary. Although empty sites for 3927 human-specific insertions and for 1281 chimpanzee-specific insertions were identified in the genomes of both species, empty sites for 2270 human-specific insertions and for 1629 chimpanzee-specific insertions were found only in the genome of either orangutan or macaque.Alu and SVA [Since there are many studies of insertions of L1,  and SVA ,27,37-39Alu and SVA retrotransposition , small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), 7SK RNA, and 7SL RNA  was very small.As the 5'-end of SVA is composed of variable numbers of degenerate CCCTCT repeats , full-leAlu, and full-length Alu  5'-inverted L1 insertions were free from MHs. Approximately 70% showed MHs of 2-5 bp. Nearly 80% of all the 5'-truncated L1 insertions were also associated with MHs, but the MH length distributions of these insertions were distinct from that of the 5'-inverted L1 insertions; 5'-truncated L1 appeared less dependent on MHs than 5'-inverted L1. The chi-square test supported the different dependency .As the length distributions of TSDs and MHs are distinguishable features between retrotransposition in et al. [in vivo. TSDs only between 5 bp and 30 bp in length were collected in this study, and further study to characterize shorter and longer TSDs is needed to clarify other radical alterations in vivo.The most significant finding in this study was that a bias towards shorter TSDs in truncated L1s, although insertions with longer TSDs still predominate even in the 5'-truncated elements. The TSD length distribution of 5'-truncated L1 was different from those of full-length and 5'-inverted L1. In the retrotransposition assay in HeLa cells performed by Gilbert et al. , all 6 ftrans is not associated with short TSDs. What causes the difference between retrotransposition in cis and retrotransposition in trans? There have been many papers describing the differences between L1 and Alu retrotransposition [Alu. Therefore, the binding ability to signal recognition particles [5'-truncated retrotransposition in position ,30,48. Harticles  and the et al. [trans-mobilized elements, top strand cleavage before the completion of first-strand cDNA synthesis could be nearly an exclusive feature of L1 retrotransposition [It is difficult to propose a model from the data described here, but one possible explanation is that the longer time for reverse transcription of L1 RNA, longer than other RNAs, would attract cellular DNA repair machineries. Gilbert et al.  proposedet al. . From thposition  and possIn this study, large-scale identification of retrotransposon insertions was achieved. Only closely related genomic sequences and at least one genomic sequence as an obvious outgroup were needed for analysis. One important improvement is that insertions could be distinguished from deletions, enabling the characterization of insertions with no signatures, and with drastic rearrangements.trans-mobilized elements by L1 are relatively long among the transposable elements. Many non-LTR retrotransposons show shorter TSDs [The TSD length analysed in this study was restricted between 5 and 30 bp because of the limit of the computational power and manual adjustment, but all insertions with or without TSDs could be characterized in the method developed in this study. TSDs of L1 and ter TSDs , DNA trater TSDs . The metin vivo.The newly developed method using outgroup genome sequences can distinguish insertions from deletions and can characterize insertions with short TSDs correctly. This method can be applied to characterize elements without any obvious boundary structures. This study confirmed the present knowledge of L1-dependent retrotransposition, and revealed the relatively frequent appearance of short TSDs coupled with 5'-truncated L1 Genome sequences of human, common chimpanzee, Sumatran orangutan, and rhesus macaque and the pairwise alignment between human and chimpanzee genomes were downloaded from the University of California, Santa Cruz Genome Browser website .Alu and SVA insertions were characterized by using RepeatMasker [L19088), AluY [Human- and chimpanzee-specific insertions were collected as described previously . L1, AluatMasker  with a l8), AluY  and SVA 8), AluY . RepeatM8), AluY  in orderSequence logos of the target sequences were generated by using WebLogo .et al. [et al. [To identify MHs at 5' junctions, RepeatMasker was used with two different queries; one included only sequences of inserts and the other included sequences of both inserts and TSDs. If repetitive elements were extended into TSDs, the extended sequences were considered to be MHs. Nucleotides corresponding to both inverted and noninverted segments of repetitive elements were counted as MHs at inversion junctions, and the MHs at 3' junctions were manually counted in the published data sets ,47. Sen  [et al.  reportedbona fide 5'-truncated SVA insertions were obtained by collecting SVA insertions without CCCTCT repeats. Consequently, 91 5'-truncated SVA insertions were found. In the case of mRNA retrocopies, because the transcription start site is considered to be heterogeneous, the retrocopies starting at > 50 bp downstream from the +1 position of annotated transcripts were considered as 5'-truncated mRNA retrocopies, and 53 5'-truncated retrocopies were identified.EN: endonuclease; HERV: human endogenous retrovirus; LINE: long interspersed nuclear element; L1: LINE-1; LTR: long terminal repeat; MH: microhomology; MMEJ: microhomology-mediated end joining; NHEJ: nonhomologous end joining; ORF1p: open reading frame 1 protein; ORF2p: open reading frame 2 protein; RT: reverse transcriptase; SINE: short interspersed nuclear element; TPRT: target-primed reverse transcription; TSD: target site duplication; TANT: target analysis of nested transposons.The author declares that he has no competing interests.Non-coding RNA retrocopies.Click here for fileAlu and SVA.Structures of 5'-inverted Click here for fileSequences around 5-bp assumed target site duplications.Click here for fileSequences around 6-bp assumed target site duplications.Click here for file


Idiopathic Pulmonary Fibrosis (IPF) is an unresolved clinical issue. Phosphodiesterases (PDEs) are known therapeutic targets for various proliferative lung diseases. Lung PDE6 expression and function has received little or no attention. The present study aimed to characterize (i) PDE6 subunits expression in human lung, (ii) PDE6 subunits expression and alteration in IPF and (iii) functionality of the specific PDE6D subunit in alveolar epithelial cells (AECs).In vitro, PDE6D protein depletion was provoked by transforming growth factor (TGF)-β1 in A549 AECs. PDE6D siRNA-mediated knockdown and an ectopic expression of PDE6D modified the proliferation rate of A549 AECs. These effects were mediated by increased intracellular cGMP levels and decreased ERK phosphorylation.PDE6 subunits expression in transplant donor (n = 6) and IPF (n = 6) lungs was demonstrated by real-time quantitative (q)RT-PCR and immunoblotting analysis. PDE6D mRNA and protein levels and PDE6G/H protein levels were significantly down-regulated in the IPF lungs. Immunohistochemical analysis showed alveolar epithelial localization of the PDE6 subunits. This was confirmed by qRT-PCR from human primary alveolar type (AT)II cells, demonstrating the down-regulation pattern of PDE6D in IPF-derived ATII cells. Collectively, we report previously unrecognized PDE6 expression in human lungs, significant alterations of the PDE6D and PDE6G/H subunits in IPF lungs and characterize the functional role of PDE6D in AEC proliferation. IPF is a progressive interstitial lung disease of unknown etiology associated with high morbidity and mortality , and furPDEs comprise a family of related proteins which can be subdivided into 11 families based on their amino acid sequences, sensitivity to different activators and inhibitors and their ability to preferentially hydrolyze either cAMP or cGMP, or both . Of theset al. demonstrates that PDE6D can modify cGMP hydrolytic activity in preparations of broken rod outer segments . Apart from IPF subjects, tissue was also obtained from 6 donor lungs, which could not be utilized due to size limitations between donor and putative recipient (mostly single lobes) or due to incompatibility between donor and recipient.Explanted lung tissues from IPF subjects (n = 6) or donor (n = 6) were obtained during lung transplantation at the Department of Cardiothoracic Surgery, University of Vienna, Austria. Diagnosis was established on the basis of a proof of a usual interstitial pneumonitis (UIP) pattern in the explanted lungs from lung transplant recipients -Thymidine uptake assay. For studies with inhibitors, A549 cells were rendered quiescence for 24 h in 0.1% FBS DMEM F12 medium and pretreated with U 0126 or SB 203580 for 30 min prior to serum stimulation for 12 h and 24 h. The effects on cell growth were measured by [3H]-Thymidine uptake assay.A549 cells were transfected under starvation conditions for 6 h, rendered quiescence for 24 h in 0.1% FBS DMEM F12 medium and then subjected to serum stimulation (10% FBS) for 24 h. The effects on cell growth were measured by 3--2,5-diphenyltetrazolium bromide (MTT) and [3H] Thymidine  was used at a concentration 0.1 μCi per well. The [3H]-Thymidine content of the cell lysates was determined by a scintillation counter  and the values were expressed as counts per minute (cpm)/number of cells -cGMP  at 37°C for 15 min. The samples were boiled for 3 min, subsequently cooled for 5 min and incubated with 25 μl Crotalus atrox snake venom  for 15 min at 37°C. After being chilled on ice, the samples were applied to QAE Sephadex A-25  mini-chromatography columns and eluted with 1 ml ammonium formate . The elutes were collected in 2 ml scintillation solution  and counted by a beta-counter with CPM (counts per minute) values. Each assay was performed in triplicate and repeated twice independently. Data are expressed as picomoles of cGMP per minute per milligram of protein. (pmol cGMP/minute/mg protein).The A549 cell protein was extracted with RIPA buffer  and equalized to the same concentration for use. The reactions were performed with 10 μg protein in 100 μl HEPES buffer (40 mM) at pH 7.6 consisting of MgClAt the end of culture, cells were washed with PBS twice and lysed in 0.1 M HCl at room temperature for 10 min. After centrifugation the supernatants were equalized to the same protein concentration for use. 50 μl protein samples which were pre-diluted to 0.3 μg/ml and standard solutions were incubated with 50 μl tracer and 50 μl antibody in darkness at 4°C overnight. After washing 5 times, plates were incubated with Ellman's solution for 90-120 min at room temperature with gentle shaking. The plates were read at a wavelength of 405 nm and the concentration was calculated by the ready-made Cayman EIA Double workbook. The standard curve was made as a plot of the %B/B0 value (%Bound/Maximum Bound) vs concentration of a series of known standards using a linear (y) and log (x) axis. Using the 4-parameter logistic equation obtained from the standard curve, the cGMP concentration of samples was determined and is given as nmol/mg protein. Each sample was determined in duplicate and repeated twice.t-test, or a 1-way ANOVA with the Bonferroni's post hoc test for studies with more than 2 groups. Statistical significance was assumed when P < 0.05.All data were expressed as the means ± S.E. Data were compared using a two-tailed Student's The mRNA expression of each PDE6 subunit in lung tissue homogenates of donors and IPF patients was analyzed by qRT-PCR technique. As illustrated in Figure The protein content of the PDE6 subunits in whole lung tissue homogenates of donors and IPF patients was quantified by immunoblotting. As illustrated in Figure The cellular localization of the PDE6 subunits was assessed by serial immunohistochemical stainings on tissue sections from donor and IPF lungs. As shown in Figure To confirm the AEC localization pattern, the PDE6 subunits were qRT-PCR amplified from primary human donor and IPF-derived ATII cells. All PDE6 subunits  were found to be expressed by these cells Figure . Notablyin vitro AEC model. Firstly, the cells were characterized for the expression of PDE6 subunits. mRNAs of all PDE6 subunits (except for PDE6C and PDE6H) and the complete set of PDE6 proteins were found to be expressed by these cells  cGMP hydrolyzing PDE activity, (ii) intracellular cGMP levels and (iii) serum induced phosphorylation of ERK protein in A549 cells. cGMP hydrolyzing PDE activity was decreased in PDE6D siRNA as compared to non-targeting siRNA and mock transfection 24 h post serum stimulation. In corroboration, intracellular cGMP determined by EIA assay was increased 1.6 fold by PDE6D down-regulation Figure  and 7B. 3H]-Thymidine uptake 12 h and 24 h post serum stimulation as compared to control (no DMSO) and DMSO treated A549 cells. The effects of U 0126 were dose dependent. Additionally, we used the p38α/β inhibitor (SB 203580) as a control. SB 203580 had no effect on [3H]-Thymidine uptake by A549 cells  and p38α/β (SB 203580) pharmacological inhibitors, we showed that ERK1/2 inhibitor (U 0126) significantly inhibits [s Figure  and 7G.in vitro by TGF-β1 in A549 cells, suggesting a link between the observed PDE6D down-regulation in IPF specimens and the pathogenesis of the disease [in vitro AECs model, we were able to show that PDE6D modulates the proliferation rate of these cells (siRNA and ectopic expression studies). More interestingly, we showed that mechanisms accounting for PDE6D effects on AEC proliferation is related to PDE6D increasing the intracellular cGMP levels and suppressing the phosphorylation of ERK.In the present study, we report previously unrecognized PDE6 expression in the human lung. The members of the PDE family, PDE1, PDE2, PDE3, PDE4 and PDE5 are highly expressed in the lung and have been shown to potentially contribute to the pathogenesis of various lung diseases ,28. Neve disease . Furtheret al. demonstrated that the catalytic core of the rod PDE6 enzyme can be synthesized in human kidney cells with consequent expression of enzymatic activity [This finding is further supported by the reports that have demonstrated solitary PDE6 subunit expression in a variety of non-retinal tissues. The rod catalytic PDE6A and PDE6B subunits were found to be weakly expressed in brain ,31. Piriactivity . The regactivity ,33. Idenactivity .et al. [Although at present the physiological roles of the PDE6 subunits in the lung are unknown and the functionality of the PDE6 enzyme in IPF needs to be explored, the study of Wang et al.  does proet al. .In fact, based on the data obtained from human donor and IPF lungs, it is not possible at present to determine whether PDE6 functions as a complex or each PDE6 subunit has a solitary function. However, considering the requirement for multiple subunits assembly to produce functional rod and cone PDE6 enzymes and the difficulties in expressing functionally active rod and cone PDE6 enzymes in various systems , we herehttp://www.unleashedinformatics.com). In line with these studies, PDE6D (-/-) mice are consistently smaller in size, indicating a plausible involvement of PDE6D in growth arrest [Impaired AECs proliferation is a significant finding in IPF . Multiplh arrest . Thus, iERK activation has been shown to be of critical importance for ATII cell proliferation . ERK sigin vivo level, transgenic mice with epithelial cell-specific PDE6D knockout would have to be generated. Hence, we can, right now, only postulate that decreased PDE6D expression in IPF might be involved in attenuation of type II cell hyperplasia. Further, it is tempting to speculate that therapeutic prevention of PDE6D down-regulation and/or PDE6D overexpression in animal models of pulmonary fibrosis may be beneficial to boost up alveolar re-epithelization and may represent a therapeutic option in IPF.In conclusion, these data demonstrate previously unrecognized PDE6 expression in human lung, significant alterations of the PDE6D and PDE6G/H subunits in IPF-derived lungs and characterize the functional role of PDE6D in AEC proliferation. For a further consolidation of the proposed pathomechanistic link between PDE6D content and type II cell proliferation on an The authors declare that they have no competing interests.Conceived and designed the experiments: SN, NW, HAG, RTS, SSP. Performed the experiments: SN, RS, SSP. Analyzed the experiments: AG, WS, FG. Contributed reagents/Materials: MK, OE, WK, RV. Wrote the paper: SN, RTS, SSP. All authors read and approved the manuscript.