Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module  or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included.Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes.Two new endoglucanases from the migratory nematodes We conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of the whole gene cassette, i.e. the GHF5 catalytic domain and the CBM2, rather than that it evolved by domain shuffling. Our evolutionary model for the gene structure in PPN GHF5 endoglucanases implies the occurrence of an early duplication event, and more recent gene duplications at genus or species level. Endo-1,4-beta-glucanases EC 3.2.1.4), or endoglucanases, can degrade the beta-1,4-linkages of cellulose, the most abundant component of plant cell walls. The endoglucanases are classified in different glycosyl hydrolase families (GHF) on the basis of sequence similarity and hydrophobic cluster analysis .1.4, or  from theet al. [Horizontal gene transfer (HGT) from bacteria has been proposed to explain the origin of GHF5 endoglucanases in nematodes . Howeveret al.  hypotheset al. .Domain shuffling  is a speThe original introns-early concept states that (nearly) all introns have been inherited by eukaryotic genes from prokaryotic ancestors -19. The Cavalier-Smith strongly disagrees with the introns-early view . On the A synthetic theory of intron evolution has been put forward by de Souza . This inIn order to determine the domain structure of the ancestral PPN GHF5 endoglucanase gene present in the PPN of the phylum Tylenchomorpha, the evolution of the GHF5 catalytic domain and the CBM2 cassette was investigated through phylogenetic analyses including sequences from fungi, plants, protista and bacteria.To study the subsequent evolution of the GHF5 endoglucanase gene family within nematodes, intron positions were used as phylogenetic characters and a model was developed for their evolution.Radopholus similis genes that were recently described [Pratylenchus coffeae and Ditylenchus africanus. The latter gene is the first GHF5 endoglucanase isolated from the superfamily Sphaerularioidea [Until now, evolutionary models for PPN GHF5 endoglucanases focused on genes from cyst and root knot nematodes and one migratory nematode, all belonging to the Tylenchoidea superfamily. In order to have a broader overview of the endoglucanase evolution in the infra-order Tylenchomorpha, the gene structure of six additional genes was incorporated in our study. These include the four escribed  and two arioidea .et al. has proven the validity of this hypothesis for an endo-1,4-beta-glucanase of the bacterium Myxococcus xanthus [Glycosyl hydrolase family 5 (GHF5) endo-1,4-beta-glucanase genes have been identified in various plant-parasitic nematodes (PPN). These genes have different structures: all have a signal peptide and a catalytic domain, some have an additional linker and carbohydrate-binding module  and others only have a linker but no CBM2. This modular domain structure could have arisen by exon or domain shuffling, a process by which a chimeric protein is created by domains or segments of two different genes through recombination in intron regions. In bacteria, all endo-1,4-beta-glucanases are supposed to originate from a few progenitor sequences by mutation and domain shuffling  and Quil xanthus . The autWhile the ancient origin of the PPN GHF5 endoglucanases  still iD. africanus and P. coffeae. This resulted in a fragment of 605 bp for D. africanus and a fragment of 443 bp for P. coffeae. Sequence analysis confirmed them to be endoglucanase fragments and the genes were named Da-eng1 and Pc-eng1 respectively. Full length sequences of the genes were obtained by genome walking. The length of these sequences from start till stop codon is 2007 bp for Da-eng1 and 1527 bp for Pc-eng1. The corresponding coding sequences were amplified from a cDNA pool and have respective lengths of 1395 bp and 1377 bp. Da-eng1 contains 5 introns whereas Pc-eng1 only has 3.Endo-1,4-beta-glucanase fragments were amplified by a degenerate PCR on genomic DNA of The putative endoglucanase proteins Da-ENG1 and Pc-ENG1 have an estimated molecular weight of 49 and 47 kDa respectively. Both have a predicted N-terminal signal peptide for secretion. A conserved domain search confirmed that the proteins are endoglucanases belonging to the glycosyl hydrolase family 5 . In addition to the catalytic domain, both proteins have a linker and a carbohydrate-binding module (CBM) of family 2 (pfam00553).In addition to the already known endoglucanase genes from PPN and the newly identified sequences in this study, we searched for the most homologous endoglucanase genes from other major lineages. Based on their sequence similarity to the PPN endoglucanases, one or more representative sequences were selected per lineage for further analyses. These included GHF5 endoglucanase sequences from the kingdom of Plantae, Animalia, Fungi, Protista and Bacteria  of the genes. Since not all GHF5 endoglucanase genes include a CBM, the alignment and hence also the phylogenetic tree for the CBM contained less taxa. For both domains, alignment of the sequences was performed at both DNA and protein sequence level.Alignments showed that plant sequences are not sufficiently similar to PPN GHF5 endoglucanases to be useful for phylogenetic analyses. Preliminary unrooted phylogenetic tree construction with the other endoglucanases revealed fungal sequences to be most divergent from PPN GHF5 endoglucanases. Therefore they were chosen as outgroup to construct rooted trees. All alignments were analyzed using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian methods.Mi-eng1, see further) because linkers show very little large-scale sequence homology. At DNA level, only linkers from very closely related genes show some degree of homology. This is not surprising as the same observation has been made for linkers from bacterial endoglucanases [No data from linker sequences have been included in this study . The divergence of Da-ENG1 at sequence and structural level is not surprising since Pratylenchus coffeae and P. penetrans with those of the root knot nematodes (genus Meloidogyne). Cyst nematode (Heterodera and Globodera) endoglucanases are grouped together with some endoglucanases from Radopholus similis (classified in the Pratylenchidae family), Rs-ENG1B and Rs-ENG3. Although the positions of Rs-ENG1A and Rs-ENG2 are unresolved, these observations partially confirm recent phylogenetic trees derived from 28SrRNA data [Radopholus similis to Heteroderidae than to Pratylenchidae. This distinction was also observed on a morphological level by Luc [Radopholus can be distinguished from other genera in Pratylenchidae by its strong secondary sexual dimorphism and the distinctive lip pattern.A well-confirmed cluster groups the GHF5 endoglucanases of RNA data  revealinl by Luc , who fouGlobodera rostochiensis (Gr-ENG3 and Gr-ENG4). Also, Rs-ENG1B is paralogous to some intra-species or intra-genus duplicates from Heterodera and Globodera spp.Paralogy between groups of proteins is well-confirmed by high posterior probabilities. For instance, Rs-ENG3 and Hg-ENG5 are paralogous to each other and to two orthologues from The Bayesian tree based on the CBM Figure  reveals Congruency between the obtained phylogenetic trees from the catalytic domain and the CBM was evaluated under the parsimony and likelihood criterion using the partition homogeneity and Shimodaira-Hasewaga (SH) tests. These analyses were run for all genes with a CBM as well as for a limited set excluding non-nematode taxa (PPN dataset). Interestingly, no statistical incongruence between all trees based on the GHF5 catalytic domain and on the CBM2 was found for the whole and the PPN dataset Table . This obFigure The exon/intron gene structure of the PPN GHF5 endoglucanases was compared with the multiple protein sequence alignment. All introns and their properties are represented in Figure Hg-eng1)  of PPN GHF5 endoglucanase genes is interrupted by maximum 8 introns  Figure  with an Mi-eng5) to 58% (intron 2 of Gr-eng1), with an average for all introns (n = 110) of 31%. Apparently there are differences in GC percentage according to the species. For instance, the average GC% of all introns of M. incognita endoglucanase genes (n = 23) is 22%, whereas for R. similis introns (n = 13) it is 42%. This is probably related to the overall GC% of the coding sequences of a certain species. M. incognita coding sequences generally have a relatively low GC content (37%) [R. similis coding sequences have a high GC content (54%) [The GC percentage per intron varies from 5% (intron 10 of nt (37%) , whereasnt (54%) .et al. [C. elegans . 49%  GC-AG splice site in a minority of the homologues. This non-classical splice site was already reported by Yan et al. [Exon-intron boundaries were compared for all introns observed in the available PPN GHF5 endoglucanase gene sequences. The great majority of the detected introns are of the GU-AG type. Only three intron positions  show the alternative, but rare  of the introns show a consensus splice site that is completely consistent with the protosplice site (MAG/R), pointing to a probable recent origin of these introns.Even though many clusters of closely spaced introns were detected in the aligned sequence, no evidence was found in our study, or in other studies, that proves their possible homology. Although intron sliding cannot be completely ruled out, the influence of this process is considered to be negligible (except for sliding by one base), and intron position diversity is indicated to be primarily arising by the gain or loss of introns during eukaryotic evolution . TherefoNoteworthy, there is a strong conservation of the location and phase of introns within the PPN GHF5 gene family . This coGr-eng1 and Gr-eng2 as well as Gr-eng3 and Gr-eng4 reveal remarkable similarity. This is also the case for introns in Hg-eng2, Hg-eng3 and Hg-eng4. The conserved introns of the Meloidogyne endoglucanase genes are also very similar, confirming the homology of these conserved introns and the paralogy of the genes.Since spliceosomal introns diverge so rapidly that sequence similarity indicative of homology quickly vanishes, significant intron sequence conservation can only be expected in genes resulting from very recent duplications. Sequence comparisons between introns that are conserved in the majority of the dataset indeed showed similarities between paralogous genes. For instance, introns of Mi-eng2 and Hg-eng6 form a well-confirmed cluster. This is also the case for some groups of paralogues and orthologues from H. glycines, G. rostochiensis and R. similis. Generally, resolved branches show similar clustering as catalytic domain and CBM-based trees implying that intron presence data reflect the evolutionary tract of PPN GHF5 genes.As could be expected based on the low number of informative sites (14), Wagner Parsimony of the 1/0-data for intron presence/absence shows very low resolution  and are therefore less likely to be involved in recombination.A model was constructed by combining the sequence-based evolutionary tracts with the intron data. Figure The evolutionary tree based on the catalytic domains shows the genes with the same structure grouped in the same terminal branches, although the length and sequence of introns have diverged.et al. [et al. noticed that nematodes have a particularly high rate of intron turnover compared to other animals [Our working hypothesis proposes that the divergence of the gene structure of the PPN GHF5 gene family is associated with the expansion of the number of members from an ancient or early eukaryotic ancestral gene. The possible congruent evolutionary tract of modules, as observed in this study Table , suggestet al.  observed animals . These t animals  our modeHg-eng6 and Mi-eng2 and their sequence divergence from the other genes indicate that they have probably originated from a different type of GHF5 gene (type A). This implies that the ancestral PPN GHF5 endoglucanase must have been duplicated already early in the Tylenchomorpha lineage. This early duplication event was also suggested by Ledger et al. [The exon/intron structure of r et al. . During Radopholus similis, a second gene duplication took place. These nematodes all contain two divergent types of GHF5 endoglucanases within their genomes.In the ancestral nematode of the lineage leading to the cyst nematodes and At intra-species level, the conserved gene structure of certain paralogues and the observation of some sequence conservation in their introns indicate a history of recent duplication events in the present-day genomes of plant-parasitic nematodes.et al. [et al. [Radopholus similis and the cyst nematodes) into consideration and is therefore confusing in regard of gene and species evolution.Ledger et al.  also pro [et al.  does notIn some cases, intra-species duplicates lost their CBM and occasionally also their linker region. Generally, the observed variation in exon/intron structures between homologous and paralogous genes implies that introns have been gained and lost along all major lineages of the tree and at different time points during evolution. Concordant with the fact that the conservation of protosplice sites strongly supports the recent origin of an intron , recentlOur model implies some special evolutionary gene structure processes for which little information is available and that are therefore difficult to prove.et al. [The phylogenetic hallmark of intron sliding is a distribution in which one intron is nested within the distribution of another . We propet al.  pertainset al. -50. AlteHg-eng5 is an example of a gene where this process might have happened. We propose a similar mechanism for Rs-eng1A and Rs-eng2, involving a reverse-transcribed mRNA containing only a catalytic domain, but in this case the recombination must have been followed by a duplication event. The loss of introns through reverse transcription of mRNAs followed by the recombination of the synthesized cDNA in the genome [Hg-eng5 and any other gene from the same species suggests that reverse transcription followed by a homologous reinsertion is most probable. For Rs-eng1A and Rs-eng2, the reverse transcription event is supposed to have taken place earlier in evolution, involving only the 5' end, and should have been followed by a duplication event, the loss of linker and CBM and a gain of intron 21 in one of the paralogues.The genes with no or only one intron probably have lost their introns by reverse transcription and subsequent homologous reinsertion of the synthesized cDNA, affecting either the whole gene or only a part of it. e genome -53 is hae genome . The impRadopholus similis are more related to genes from the Heteroderidae, while Pratylenchus spp. show a close relationship to the root knot nematodes.This study integrates previously known sequence information from mainly sedentary plant-parasitic nematodes and new data from different migratory nematode species. With the available data from PPN GHF5 endoglucanases and other major prokaryotic and eukaryotic lineages no evidence for an early domain shuffling event between the two domains  present in these genes was found, suggesting parallel evolutionary tracts. Moreover, the phylogenetic data confirmed that some endoglucanases of the migratory species Our evolutionary model for the PPN GHF5 gene family proposes an intronless ancestral GHF5 endoglucanase gene in plant-parasitic nematodes, consisting of a catalytic domain and a CBM2. Early in the evolution of plant-parasitic nematodes, the ancestral gene must have been duplicated, and numerous introns were gained. Later in evolution, some introns were lost, gained or shifted in certain lineages, and additional duplication events frequently took place. Our data reveal that loss of the CBM2 and in some cases also the linker region almost certainly occurred independently in different lineages.While our data show that exon shuffling most probably did not lie at the origin of the emergence of the PPN GHF5 endoglucanase domain structure, more recent exon/domain shuffling events at lower taxonomic levels cannot be ruled out.In this study the HGT hypothesis can neither be accepted nor rejected. Additional sequence data from other major eukaryotic lineages are awaited before a thorough and sound investigation of this problem can be realized. However, evidence was provided that it is very unlikely that an ancestral eukaryotic GHF5 catalytic domain was joined with a horizontally-acquired CBM2 in the early evolution of the PPN GHF5 domain structure. Therefore, HGT of the whole structural cassette (GHF5-linker-CBM2) remains one of the most likely explanations for its presence in both bacteria and PPN.Pratylenchus coffeae and Ditylenchus africanus were maintained on carrot disks in small petri dishes at a constant temperature of 25°C [D. africanus) or 8 weeks (for P. coffeae) after inoculation of the carrot disks. Collected nematodes of mixed stages were stored as a pellet at -20°C for DNA or RNA extraction. of 25°C . Nematodet al. [25VN), 10 mM DTT, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and 200 U Superscript II Reverse Transcriptase (Invitrogen). This reaction mixture was incubated for 2 h at 42°C.Genomic DNA was isolated from the nematodes according to Bolla et al. . RNA extP. coffeae and D. africanus. The reaction mixture contained 150 ng DNA, 0.5 μM of each primer, 4 mM each of dNTPs, 1.5 mM MgCl2, 20 mM Tris-HCl (pH8.3), 50 mM KCl and 1 U of Taq DNA polymerase (Invitrogen). The PCR conditions were as follows: 2' at 94°C followed by 35 cycles of 1' at 94°C, 1' at 52°C and 1' at 72°C. Resulting fragments were separated on a 0.5× TAE 1.5% agarose gel, excised and purified by the QIAquick Gel Extraction kit (Qiagen). Purified fragments were ligated into pGEM-T (Promega) and transformed in E. coli DH5α cells (Invitrogen). A colony PCR under standard conditions revealed the presence or absence of inserts. Plasmids of positive colonies were isolated using the Nucleobond AX kit (Machery-Nagel) and the insert was sequenced at the VIB Genetic Service Facility .A polymerase chain reaction (PCR) using degenerate primers ENG1 (TAYGTIATHGTIGAYTGGCA) and ENG2 (GTICCRTAYTCIGTIACRAA)  was perfP. coffeae fragment were Pc-eng1-up1 (TTGGAGTCAATGGCCCGGATGG), Pc-eng1-up2 (GATGTTCGGATTGGAGCCATATTGC) to walk upstream en Pc-eng1-down1 (TCAAATCTCTGCTACACTCTCCAC) and Pc-eng1-down2 (CAAACAATCCCTCAGGGATAAGGC) to walk downstream. For the D. africanus fragment, Da-eng1-up1 (CCAGCGTATTTTTGTGCCATTTGG), Da-eng1-up2 (CTCTCAACCAATATCTTTGACTTAACAGC), Da-eng1-down1 (AATAGTCGCGTTTAGGACGTGGACGTC) and Da-eng1-down2 (TCCACTTCTACGCCGGCACTCAC) were used to walk up- and downstream respectively. The longest obtained fragments were cloned and sequenced as described above. Corresponding cDNA sequences of the endoglucanase genes were amplified by PCR using gene-specific primers Da-eng1-start (ATGAAATTCTTCGCCAGCCTCG) and Da-eng1-stop (TCAGCAATATCCATAAGACACAACGC) on the cDNA pool of D. africanus and Pc-eng1-start (ATGGCATTCACTTTGCTTTCC) and Pc-eng1-stop (TGTGAGCGCCACTGCCTGCTAA) on P. coffeae cDNA. These cDNA sequences were cloned and sequenced as described above. Obtained sequences were submitted to the GenBank database .To obtain the 5' and 3' genomic sequences of the endoglucanase fragments, the Genome Walker Universal kit (Clontech) was used according to the manufacturer's instructions. Primers used for the http://us.expasy.org. Signal peptides were predicted using SignalP 3.0 [Putative protein sequences were obtained by translating the cDNA sequences using the EMBOSS program "Transeq" . The molnalP 3.0 .Using BlastX, a GenBank search for all available endoglucanase gene sequences from nematode species was performed on May 21, 2007. For comparison, the most homologous endoglucanases from other eukaryotic and prokaryotic lineages were included in the dataset , which has a known 3D structure. For the carbohydrate-binding module, the location of the beta strands was inferred by aligning the sequences to a CBM from an endoglucanase from Cellulomonas fimi (1EXG). GC percentages of the introns were calculated with the internet tool GC calculator http://www.genomicsplace.com/gc_calc.html.The presence, location and phase of introns were evaluated for each open reading frame (ORF) by comparing DNA and mRNA sequences. Intron positions were mapped onto the sequence alignments. Intron phase was named 0 when the intron was located in-between two consecutive codons; 1 if an intron was located between the first and second codon nucleotides and 2 if an intron was found between the second and third codon nucleotides. The intron positions were mapped onto the secondary structure  analyses were executed using PAUP* v4.0b10 , on the Intron presence was coded by binary scoring (1/0) for presence/absence. Introns were only considered to be homologous when they appeared at exactly the same position and the same phase in the orthologous/paralogous genes. Although Dollo Parsimony is frequently used in intron evolution , recent F) as designated by Johnson & Soltis [et al. [Congruence between the obtained Maximum Parsimony trees based on catalytic domain and CBM was tested using the partition homogeneity test   as impleBayesian analysis was run using MrBayes version 3.1.2 .For the DNA datasets the best-fitting evolutionary model, as identified using Modeltest 3.5, was applied. For the protein datasets a mixed model of amino acid evolution was applied to allow model-jumping for fixed-rate models. Bayesian inference was run for 1,000,000 generations, and the first 25,000 generations were discarded as burn-in.TK did the phylogenetic analyses and drafted the manuscript. AH did the experimental work, i.e. cloning the genes, and was also involved in the phylogenetic analyses and the drafting of the manuscript. GG participated in the coordination of this study and critically revised the manuscript.List of available data. Overview of all available endoglucanase gene sequences from nematode species and the most homologous endoglucanases from other eukaryotic and prokaryotic lineages that were included in our dataset. GenBank Accession Numbers and the type of available data are specified.Click here for fileMaximum Parsimony tree based on the intron data of GHF5 endoglucanases. Consensus tree of 34 Maximum parsimonious trees based on intron presence/absence data of GHF5 endoglucanases from different prokaryotic and eukaryotic lineages. Wagner Parsimony was applied. The bootstrap value is given on each node. The tree is rooted with the CBM of two genes from the fungus Trichoderma reesei.Click here for fileIntron positions of a typical PPN GHF5 endoglucanase in relation to the secondary gene structure. The position of alpha helices and beta strands was derived from comparison to known crystal structures of a GHF5 catalytic domain (1EGZC) and a CBM2 (1EXG). The signal peptide, catalytic domain, linker and CBM, are indicated in red, blue, yellow and green respectively. Transition zones are white. Arrows represent beta strands; boxes alpha helices. Introns are represented by a vertical black line and numbered in order of their occurrence, as specified in Figure Click here for file


The so-called group II introns perform some neat tricks. They are primarily found within genes and are transcribed into precursor RNA when the host gene is activated. That's when the fun starts. First, a portion of the intron RNA binds a ribosome and dictates production of an “intron-encoded protein,” or IEP. Next the IEP binds to the intron RNA, coaxing it to fold up into a catalytically active structure, a “ribozyme,” which then cuts the intron—that is, itself—out of the precursor RNA. Still bound to the IEP, the intron then seeks out a new home elsewhere in the DNA, base-pairing its sequence to an appropriate target site and splicing itself into one strand of the double-helix. Next, the IEP reverse-transcribes the intron to make the complementary DNA sequence for the other strand. Host enzymes complete the process and tidy up the loose ends, and presto—the intron has copied itself into the genome.PLoS Biology, Georg Mohr, Eman Ghanem, and Alan Lambowitz suggest an answer, one that relies in part on higher temperatures thought to exist at the time that eukaryotes first emerged.Group II introns are found in bacteria and eukaryotic organelles. They also resemble eukaryotic spliceosomes, molecular machines that remove introns from pre-messenger RNA within the eukaryotic nucleus, and biologists have hypothesized that spliceosomes evolved from group II introns that came aboard with the bacterial ancestors of our mitochondria. But most bacteria carry very few group II introns, suggesting their proliferation is tightly controlled, and it has been unclear how they might have multiplied sufficiently to provide the numbers found in eukaryotes. In this issue of Thermosynechococcus elongatus, contains plenty of group II introns—28 in all—amounting to more than one percent of its genome. The 28 are all closely related and constitutes 6 families. Each family has a slightly different target specificity, which is likely one mechanism explaining their ability to proliferate in the bacterial genome.The authors began with the observation that at least one bacterial species, the heat-loving Three of the 28 introns are only fragments, having lost large chunks of their sequence, but 25 are intact. Of these, eight appear to have complete IEP sequences, while the IEP sequences in 17 have become corrupted and can no longer code for a functional protein. But that doesn't appear to have prevented their proliferation—all 17 of them seem to have descended from a single intron after a deletion corrupted its IEP sequence. That deletion may have only improved their reproductive ability—because they are smaller, they may evade the host cell's defenses more readily. They still need an IEP though, and IEPs from other introns appear to pitch in, having evolved to become less stringent in their specificity for their intron or origin.The high temperatures preferred by the bacterium don't impair the intron's ability to move within the genome—on the contrary, the mobility efficiency of one intron increased dramatically when the temperature rose from 37°C to 48°C. One likely factor increasing that efficiency is that high temperature increases DNA “melting,” or dissociation of the two DNA strands. This both improves the intron's access to the DNA for base pairing and reduces the role of the IEP in selecting the site, hence increasing the number of potential insertion sites.T. elongatus.The combination of variation in target specificity, reduced size, less stringent IEP specificity, and high temperature appears to account for the greater proliferation of introns within The authors speculate that this facilitation of intron mobility at high temperatures may echo the situation in which eukaryotes first evolved, believed to be at temperatures between 50°C and 65°C, and may have contributed to the large number of group II descendants found throughout the eukaryotic genome. Over time, early eukaryotic group II introns may have evolved to lose most of their IEPs, leading to the adoption of a common splicing mechanism, which ultimately evolved into the eukaryotic spliceosome.Mohr G, Ghanem E, Lambowitz AM (2010) Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns. doi:10.1371/journal.pbio.1000391


The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions.We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs.We obtained robust estimates of the contribution of parallel gain to the observed sharing of intron positions between eukaryotic species separated by different evolutionary distances. The results indicate that, although the contribution of parallel gains varies across the phylogenetic tree, the high level of intron position sharing is due, primarily, to evolutionary conservation. Accordingly, numerous introns appear to persist in the same position over hundreds of millions of years of evolution. This is compatible with recent observations of a negative correlation between the rate of intron gain and coding sequence evolution rate of a gene, suggesting that at least some of the introns are functionally relevant. The presence of spliceosomal introns and the concurrent splicing machinery are one of the principal distinctive features of eukaryotic genomes -3. IndeeThanks to their ubiquity and potential major role in the evolution of eukaryotes, intron evolution has drawn considerable attention -3,10. ItRecently, we have obtained more conclusive results by developing a comprehensive probabilistic model of intron evolution, and by compiling a data set that is considerably larger than any one previously used ,20. The Here, we describe, in detail, the developed model of intron evolution and the derivation of an improved version of the EM algorithm used to estimate its parameters. Previously, we have applied this comprehensive model to a set of 391 conserved genes from 19 eukaryotic species, to investigate evolutionary trends in gene structure both at the lineage level  and at tIn the present work, we focus on the process of intron gain, and address the causes of the high level of intron position conservation between eukaryotic taxa. It had been already noticed that many intron positions are shared between distant eukaryotic taxa ,13. For G sets of aligned protein-coding sequences of orthologous genes from S species. Each nucleotide in these alignments is substituted by 0 or 1 depending on whether or not an intron is present following the respective position. We allow for missing data by using a third symbol (*) to indicate lack of knowledge about the presence or absence of an intron. Consequently, every site in the alignments, called pattern, is a vector of length S over the alphabet  and is denoted by ω. Let Ω be the total number of unique patterns in the entire set of G alignments, and let ngp be the count of the number of times pattern ωp  is found in the multiple alignment of gene g. Assuming that the sites evolve independently, the set Mg =  fully characterizes the multiple alignment of the g th gene.The primary input component in the study of intron evolution consists of T be a rooted, bifurcating phylogenetic tree with S leaves , describing the evolutionary relationships between the S species above. The total number of nodes in T is N = 2S - 1, and we index them by t = 0,1, ..., N - 1, with the convention that zero is the root node. The state of node t is described by the variable qt, which can take the values 0 and 1 (and * in leaves). We use Vt for the set of all leaves such that node t is among their ancestors. The entire collection of leaves is, obviously, V0. The parent node of t is denoted P(t). We use the notations qt) P(and Vt)P and R(t), and we use the notations qt)LRLR of the t th branch. Hereinafter we assume that the tree topology, as well as all the branch lengths Δ1, ..., ΔN-1, are known.Let A bifurcating phylogenetic tree can be viewed as a graphical model depicting the probabilistic modelπi = Pr(q0 = i) to describe the prior probability of the root, and Tij = Pr to describe the transition probability for gene g along branch t. In our model, we assume that this transition probability depends on both the gene and the branch, and that it takes the explicit formWe use the notation ηg and θg are non-negative parameters which determine, respectively, the intron gain and loss rates per site for gene g. That is, along branch t the gene's contribution to intron gain and retention probabilities per site is branch-specific intron gain coefficient, ξt, as well as an intrinsic branch-specific intron loss coefficient, φt, both of which are bounded, 0 ≤ ξt, φt ≤ 1.Here, rate variable, r, which scales, for each site, the time units of the phylogenetic tree, Δt ← r·Δt. This rate variable is a random variable that is distributed according to a distribution function with non-negative domain and unit mean, typically, the unit-mean gamma distribution. The rate variability captures rate variations among sites of the same gene. Specifically, there are fast-evolving sites (r >> 1), as well as slow-evolving ones (r << 1). In our model of intron evolution, we extend this idea by assuming that the gain and loss processes are subject to rate variability, independently of each other. Hence, a site can have any combination of gain and loss rates, for example, it can be fast to gain introns but slow to lose them. To implement this approach, we use two independent rate variables, rη and rθ, that are used to scale, for each site, the gene-specific gain rate, ηg ← rη·ηg, and the gene-specific loss rate, θg ← rθ·θg, respectively. We further assume that the distributions of these rate variables are independent of the genes, and are explicitly given byIn other fields of molecular evolution, it had been long realized that the precision of the analysis significantly improves if one allows for rate variability across sites -26. Typix; λ) is the unit-mean gamma distribution of the variable x with the shape parameter λ, δ(x) is the Dirac delta-function, and ν is the fraction of sites that are assumed to have zero gain rate. The existence of these zero sites reflects the notion that introns cannot be gained at any location within genes, but rather are preferentially inserted at specific locations, contingent on particular sequence motifs known as proto-splice sites [ν measures the density of potential intron insertion sites. Importantly, using the same value of ν for the entire tree does not mean that the proto-splice sites are constant throughout the evolution, or are identical for different lineages. It only means that, on the average, the fraction of potential insertion sites, whatever is their concrete nature, is similar across the lineages throughout the course of eukaryotic evolution. The incorporation of such invariant sites in a rate variability model appears natural for intron evolution and has proved beneficial also in other fields of molecular evolution [Here, Γ are rendered discrete . We assut = {ξt, φt} be the set of parameters that are specific to branch t, and let Ξ =  be the set of all branch-specific parameters. Similarly, let Ψg =  be the set of parameters that are specific for gene g, and let Ψ =  be the set of all gene-specific parameters. Additionally, we denote by Λ =  the "global" parameters that determine the rate variability and the prior probability of an intron absent in the root. When the distinction between the different sets of parameters is irrelevant, we shall use Θ =  as the set of all the model's parameters. We achieve further succinctness in notations by denoting the actual gene-specific rate values for particular values kk'g = .For notational clarity, we aggregate the model's parameters into a small number of sets. To this end, let ΞWe estimate the parameters of the model using maximum likelihood. As the probability model includes observed random variables (state of the tree leaves) as well as hidden random variables , the expectation-maximization algorithm is a natural tool to use . This isG + 4S, where G is the number of genes and S is the number of species. For data sets in the hundreds of genes, this sums up to >1000 parameters. For infinite data, maximum likelihood estimators are known to be nonbiased and efficient. However, as each gene typically goes through a small number of intron-related events during its lifetime, the information content of our data is limited, and cannot straightforwardly support the estimation of such a large number of parameter. To overcome this, we adopt a two-phase approach in the data analysis. In the first phase, that we denoted homogeneous evolution, it is assumed that all the genes have identical intron gain and loss rates, formally, that θg = θ0 and ηg = η0 for any gene g. In the second phase, denoted heterogeneous evolution, all the global and branch-specific parameters are fixed, which allows for estimation of the gene-specific parameters θg and ηg that can now take different values for different genes. Except for the rate variability within genes, the model of evolution under the homogeneous phase resembles the branch-specific models, and consequently the EM algorithm used in this part has similar structure to the EM algorithm developed by Nguyen et al. [The total number of parameters in the model is 2n et al. ,36.v, the rate variability within genes can be ignored without having any significant effect on the results  of the impact of multiple factors such as differential tendencies to be inserted into different proto-splice sites, local densities of pre-existing introns, and degree of chromatin exposure. Furthermore, this density estimation strongly depends on the specific data set as the parameter N0 = 289,902 our calculations yield 39,962 sites that can gain introns . Accordingly, the 5,755 sites that are actually occupied by introns in the genes analyzed here comprise 14.4% of all sites that can potentially gain introns . Thus, even when data from 19 species are combined, the density of the sites actually occupied by introns is still far below the density of sites capable of hosting introns, which emphasizes the inadequacy of any analysis that considers only those sites in which introns are, actually, observed.With the total number of sites Ni and Nj be the number of intron positions in species i and j, respectively; let Sij be the number of intron positions shared by the two species; and let N be the total number of sites capable of gaining introns. Then, approximately, we would expect that NiNj/N positions will be shared between the two species by chance alone. We define the conservation levelNumerous intron positions are shared even between taxa that had diverged during the early stages of the eukaryotic evolution ,13. To qSij exceeds random expectation, whereas negative values indicate that Sij is below expectation. Even if we take for N its lower 95% confidence interval bound of 20,891 (see the preceding section), almost all pairs show a positive value, namely, a greater than random number of shared positions  that do not have even a single shared position.as the log-ratio between the observed number and the number expected by chance. Positive values designate that p = NiNj/N2, and N experiments. Therefore, it is easy to associate a p-value with any observed number of shared positions, Sij measuring how improbable it is to obtain by chance this value or a greater one. An overall significance level of 0.05 is equivalent to a Bonfferoni-corrected significance level of 0.0003 . The calculations indicate that only 20 species pairs out of 171, all involving the intron-poor S. cerevisiae or one of the apicomplexans, had a number of shared positions that was indistinguishable from the random expectation; all other pairs had a significant excess of shared intron positions . Accordingly, regions of high sequence conservation might gain introns at exactly the same position.The two explanations have opposing impacts on our understanding of the evolution of eukaryotic genes. If introns are persistent, many must have been gained early in the eukaryotic evolution, and the later evolution involved multiple losses ,38. By cIn order to estimate the extent of parallel gain, we examined all the sites that host introns in at least two species. Assuming that these sites are not invariant, the probability was estimated that the corresponding pattern had arisen from parallel gain, by computing the probability of the last common ancestor of the intron-bearing species to lack an intron. This approach assumes that, if the last common ancestor had an intron in a particular position, no parallel gain occurred, i.e., highly unlikely scenarios involving at least two gains and one loss at the same site are neglected. The probabilities of parallel gain for all the patterns observed in our data set are summarized in Additional file N. crassa, the probability is >0.99 that it results from parallel gain. Considering somewhat more frequent patterns, 11 sites harbor introns in C. intestinalis, A. thaliana and O. saliva, with the probability of parallel gain ~0.875, and another 12 sites harbor introns only in C. elegans and C. intestinalis, with the probability of parallel gain ~0.8.Importantly, however, the contributions of parallel gains to the emergence of different patterns of intron sharing are widely different; in particular, some rare patterns are explained  entirely by parallel gain and do not reflect evolutionary conservation , all patterns that have 1s in the two sub-clades stemming from t, t is in state zero was computed. By this analysis, in which 728 unique patterns were included, we found that nearly 20% of the shared intron positions between plants and unikonts, thought to have diverged more than a billion and a half years ago [Aspergillus versus Neurospora, and flies versus mosquitoes). Table Generally, the distribution of parallel gains in comparisons of specific clades is, obviously, more informative than overall counting Table . To obtaears ago , are dueet al. [Given that most of the other studies report overall estimates on a smaller (8 species) data set , a direcet al.  used simet al. . An evenet al. .B denote the set of branches that comprise a path in the tree. Then, intron retention probability along this path isWe conclude, therefore, that a substantial majority of the shared intron positions are due to evolutionary conservation, hence intron positions tend to be retained for long times. To further validate this conclusion, we explicitly computed the probability of an intron to survive along given paths in the phylogenetic tree. To this end, let t, and k'.where In accord with the above conclusions on the high level of evolutionary conservation, this probability is, typically, high, even for very long paths in the phylogenetic tree Table . For exaC. elegans, H. sapiens and D. melanogaster has a probability of 0.44, 0.69, and 0.68, respectively, to have been present in the last common ancestor of metazoa.A complementary approach involves the computation of the probability of extant introns to be of ancient origin. To calculate this probability, we assumed that a site is known to host an intron in one species, and that no information is available on this site in other species . Then, we used our model to compute the probability that this intron was present in any of the ancestors of that species Table . ClearlyDespite the extensive attention given to the evolution of eukaryotic gene structure over the last three decades, the fundamental characteristics of this process remain controversial. In particular, depending on the methods and data sets used, different researchers have reached opposite conclusions on the causes of the remarkably high fraction of shared introns in orthologous genes from distant eukaryotic species. Some attribute it  entirely to a remarkable evolutionary conservation of intron positions and others, largely, to parallel gain of introns. To resolve these contradictions, it is important to analyze the evolution of introns by using a probabilistic model that minimally relies on arbitrary assumptions. To this end, we developed a model that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we found that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform such that there are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains might have contributed up to 20% of the shared intron positions. Given the distinctly non-uniform distribution of the inferred gain events over the phylogenetic tree of eukaryotes [We estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, many of the extant introns are ancient. The reasons for this remarkable endurance of a substantial fraction of the introns are not clear. One possibility is mechanistic, i.e., removing existing introns might be imprecise and hence potentially deleterious. Another possibility is functional, i.e., introns might have acquired many functional roles since entering eukaryotic genomes. The latter possibility is compatible with the recent observation of a negative correlation between the rate of intron gain and coding sequence evolution rate of a gene  which suCaenorhabditis elegans, Strongylocentrotus purpuratus, Ciona intestinalis, Danio rerio, Gallus gallus, Homo sapiens, rodents (Mus musculus and Rattus norvegicus combined), Drosophila melanogaster, Anopheles gambiae); 5 fungi ; two plants ; two apicomplexans ; and the amoebozoan Dictyostelium discoideum.The methods and criteria used in compiling the data set have been described previously ,20. BrieThroughout this paper we assumed the traditional "crown-group" tree topology  is guaranteed to increase as long as we maximize the auxiliary functionAccording to the well-known EM paradigm , log L asand the second term asSubstituting (8) and (9) back into (7) giveswgpkk' and Qgpkk' the first and second square brackets, respectively, this expression becomesDenoting by And, consequently.Q or, equivalently, the set of coefficients wgpkk' and Qgpkk' is computed by using inward-outward recursion on the tree.In this step, the function t (except for the root) a vector Vt (which are a subset of the pattern ωp) for a gene g, when the gain and loss rate variables are t is known to be in state i. By definition, this function is initialized at all leaves (t ∈ V0) byHere we suggest a variation on the well-known Felsenstein's pruning algorithm . Let us γ. For all internal nodes (except for the root), γ is computed using the recursionHere and in the derivations to follow, we omit the superscript from γj(L(t))γ j(R(t)). This is easy to see, aswhere Tij . Given qt, qt = j) = γj(tL) . By similar argument, the third term is simply Pr(qt = j) = γj(tR). Substituting these results in (14), we recover the recursion formula (13).The first term is, simply, the definition of γ-recursion allows for computing the likelihood of any observed pattern ωp, given the values of the rate variables:The q0, Given andOne of the useful features of this recursion is that is allows to treat missing data fairly easily. Only a single option has to be added to the initialization phase (12),γ-recursion is computed, we can use it to compute a second, complementary, recursion. To this end, let us associate with each node t (except for the root node) a matrix t (except for the root node) a vector α, β is readily computed, too. Again, omitting the superscripts, α can be initialized from its definition on the two direct descendants of the root,Once the α is computed using the outward-recursionHere, D(0) stands for any one of the direct descendants of the root, and α,To prove this recursion, let us start with the definition of V0 = Vt + t is not among their ancestors. But, given t is independent on and make the decomposition From Bayes formula,qt, Vt is independent of P(t) and thereforeBut, given Combining (22) and (21) in (20), we getwhich is just another form of (18). Finally, for each leaf that is not a descendant of the rootWhen missing data are present, two simple modifications are required. First, we have to add to the initialization phase (17) an optionSecond, we have to add to the finalization phase (23) an optionα computes marginal probabilities of pairs ), but such pairs are nothing but maximal cliques on rooted bifurcating trees.These inward-outward recursions are the phylogenetic equivalent of the backward-forward recursions known from hidden Markov models, and other versions of this method have been developed previously ,48. The γ-recursion is sufficient to compute the coefficients wgpkk'. From the definition, x, y | z) = Pr/Σx, y Pr, we can rewrite it asHere we show that the wgpkk' reduces toBut ωp for gain and loss rate variables γ-recursion, using (15).The function α, β-recursion. By definition,Here we show that those coefficients require the ωp, σ | Ξ, Ψgkk') is the likelihood of a particular realization of the tree, thus from (1)The probability Pr is the Kronecker delta function, which is 1 for a = b and 0 otherwise. Denote the expectation over Eσ. Applying it to (25), we getHere, But Qgpkk' is given byHence, Substituting (26) in (11), we obtain an explicit form of the function whose maximization guarantees stepping up-hill in the likelihood landscape,Q is sufficient to guarantee an increase in the likelihood, suggesting that the precise maximization of Q is not particularly important. Therefore, we speed up the computations by performing low-tolerance maximization with respect to each of the parameters individually. Except for the parameters λη and λθ, it is easy to differentiate Q twice with respect to any parameter. This lends itself to using simple zero-finding algorithms of which we chose the Newton-Raphson algorithm [Actually, any increase in lgorithm .Q with respect to the shape parameters λη and λθ is more involved, as Q depends on these parameters only through the discrete approximation of the rate variability distributions (3). In our implementation, we used Yang's quantile method [k = 2, ..., Kη and k = 1, ..., Kθ. To perform the maximization in this case, we used Brent's maximization algorithm that does not require derivatives [Maximizing e method  to compuivatives .α, β, γ-recursions on the tree with the final model parameters, it is straightforward to reconstruct the history of intron evolution, and to assign gains and losses to specific branches. The number of introns in an internal node t for a gene g, assuming gain rate variable ωp, is t is Given the In order to obtain confidence intervals on the model parameters, we used the profile likelihood technique. In brief, if ϑ is one parameter in the model, and L(Θ) = maxΘ Pr(Θ), then the likelihood ratioThat is, we compute the maximum likelihood under the constraint that the value of ϑ is given. If we denote the overall maximum likelihood by 0 is distributed according to the χ2 distribution with one degree of freedom. In order to find the 95% confidence interval of the parameter ϑ around its optimal value ϑ0, we find the value of ϑ for which the likelihood ratio (28) exceeds the value 3.84 (95%-percentile of the χ2(1) distribution). This value is found numerically using Ridder's method [under the hypothesis that ϑ = ϑs method .LC contributed to the development of the probabilistic model, developed the EM algorithm, and drafted the manuscript; IBR collected the data and contributed to the development of the probabilistic model; YIW contributed to the development of the probabilistic model; EVK conceived of the study, provided the biological interpretation of the results, and finalized the manuscript. All authors read and approved the final manuscript.Maximum likelihood estimation of the model parameters. Each parameter is associated with three values – the lower 95% confidence bound, the optimal value, and the upper 95% confidence bound. The confidence intervals were computed using the profile likelihood technique.Click here for fileComparison of the number of shared intron positions with the number expected by chance. All pairs of species (20), where the number of shared intron positions is not significantly greater than the number expected by chance alone.Click here for filePattern-by-pattern analysis of parallel gains. The order of species in each pattern is Dicdi, Caeel, Strpu, Cioin, Danre, Galga, Homsa, Roden, Drome, Anoga, Cryne, Schpo, Sacce, Aspfu, Neucr, Arath, Orysa, Thepa, and Plafa. The frequency of a pattern is the number of times it was observed in our data.Click here for fileCaenorhabditis elegans), Strpu (Strongylocentrotus purpuratus), Cioin , Danre (Danio rerio), Galga , Homsa (Homo sapiens), roden (Mus musculus and Rattus norvegicus combined), Drome (Drosophila melanogaster), Anoga (Anopheles gambiae), cryne (Cryptococcus neoformans), Schpo (Schizosaccharomyces pombe), Sacce (Saccharomyces cerevisiae), Aspfu (Aspergillus fumigatus), Neucr (Neurospora crassa), Arath , Orysa (Oryza sativa), Thepa (Theileria parva), Plafa , Dicdi (Dictyostelium discoideum), AME (Ancestor of Multicellular Eukaryotes).The phylogenetic tree of eukaryotes used in the present study. Species and lineage abbreviations: Caeel (Click here for file


The nature of soluble factors that regulate fibroblast proliferation have not been finally characterized. Our aim was to study the role of tumour necrosis factor α (TNF-α) and interleukin-1 (IL-1) in the suppressive activity of alveolar macrophages on autologous lung fibroblasts proliferation in sarcoidosis. We found that supernatants recovered from alveolar macrophages suppressed the proliferation of alveolar fibroblast in sarcoidosis by 35.5 ± 1.13% compared to 3 ± 16% in controls (p < 0.001 between the two groups). This suppression correlated with high content of TNF-α and IL-1 in sarcoidosis patients stage II-III . Both cytokines in sarcoidosis stage I were within the normal ranges. Exogenous TNF-α (1000-0.5 ng/ml) and IL-1 (500-0.24 ng/ml) had an additive suppressive activity on fibroblast proliferation which was partially reversed by indomethacin.


A reliable and valid clinical tool to capture symptoms and signs of diabetic sensorimotor polyneuropathy (DSP) for use in clinical research trials is urgently needed. The validated Toronto Clinical Neuropathy Score (TCNS) was modified to improve sensitivity to early DSP changes. We aimed to assess the reproducibility of this modified tool, the mTCNS and to determine its validity relative to the precursor TCNS.Sixty-five patients  with diabetes duration 13 ± 8 years were accrued from four study sites and examined on 2 days for internal consistency and inter- and intra-rater reliability of the mTCNS. In the absence of a single quantitative gold-standard measure for DSP, results of the mTCNS were compared with the precursor TCNS for the purpose of estimating validity.Internal consistency of the two domains within the mTCNS was good . Very good inter-rater reliability for the mTCNS was demonstrated by an intra-class correlation coefficient for the mTCNS of 0.87 , which was similar in magnitude to that of the TCNS . Intra-rater reliability testing of the mTCNS showed moderate to good correlation for individual symptoms and sensory tests . The mTCNS shared moderate correlation with the precursor TCNS .The mTCNS, a clinical score with higher face validity for tracking mild to moderate DSP, has sufficient reliability and validity relative to its precursor TCNS for use in clinical research. Although therapeutic agents such as aldose reductase inhibitors have the potential to restrict the progress of diabetic sensorimotor polyneuropathy (DSP), none have proven efficacious . A possiThe Toronto Clinical Neuropathy Score TCNS;  can be uIn a nerve biopsy study of the aldose reductase inhibitor ranirestat, with a 15-month extension study, the TCNS showed a statistically significant improvement in the drug-treated patients compared with baseline assessment ,12. In tIn view of these potential advantages of the mTCNS over the TCNS for clinical research involving drugs that could modify the early pathophysiology of DSP, we aimed to assess the reproducibility of the mTCNS (measured by inter- and intra-rater reliability). Secondly, in the absence of a single quantitative gold-standard measure for DSP, we aimed to simply validate the mTCNS relative to its precursor, the validated TCNS.We performed a multi-centre study at four centres, three in the USA and one in Canada (see below for list of study centres). The local Institutional Review Board at each of the four sites approved the study protocol prior to enrolment of patients.Sixty-five patients were enrolled (10–22 patients per site). Inclusion criteria included age 18–70 years, presence of diabetes mellitus, a sural nerve amplitude response of 1.0 µV or more and the presence of symmetrical distal DSP as defined by the modified San Antonio Criteria, in which two of symptoms, signs, abnormal nerve conduction parameters or abnormal vibration perception thresholds (VPT) were required . PatientEach patient was examined on the same day by 1–3 raters for inter-rater reliability testing and then tested again within 48 h by one rater for the intra-observer reliability testing. The raters were all trained at a single training session before starting the study in order to standardize the clinical assessments. Ten raters participated in the study in four study sites.At the first visit, demographic information was collected and the TCNS and mTCNS were performed. Details of the TCNS have been presented previously . The mTCOther procedures done at the first visit included: MNSI, MDNS, the NDS, nerve conduction studies (NCS) and quantitative sensory testing (QST). NCS included motor nerve conduction studies of the dominant peroneal and tibial and non-dominant median nerves with corresponding F wave latencies and sensory nerve conduction studies of bilateral sural, non-dominant median, radial and ulnar nerves. QST included VPT and cooling detection thresholds measured using the method of limits and the Medoc device .On day 2, each patient had the TCNS and mTCNS performed by 2–4 raters, including the rater from day 1. The assessments were performed at least 15 min apart to reduce memory effects. The time interval of less than 48 h between assessments was selected to minimize the potential for change in symptoms. All examinations were masked to the results of all other testing, including the results of previous TCNS and mTCNS tests. The TCNS was selected as the criterion standard for DSP in this study in the absence of a single, universally accepted gold standard for DSP and the prior demonstration of validity of the TCNS against the morphological criterion of sural nerve fibre density .The NCS, QST, TCNS and mTCNS were reviewed by a central Neurological Core Laboratory for quality control according to established control procedures .To determine the acceptability of the mTCNS to patients, a cognitive debriefing substudy was conducted in 12 randomly selected patients (three from each study site) after the completion of their two study visits. The cognitive debriefing constituted a qualitative semi-structured one-to-one interview that included questions pertaining to the importance of each component of the mTCNS in the opinion of the patients.n = 2–4) were considered random effects. Intra-rater reliability reflects the agreement between responses when the instrument is administered by the same rater on more than one occasion. It was determined by the Cohen's kappa statistic [Internal consistency was assessed for the mTCNS and TCNS using Cronbach's alpha . Inter-rtatistic ,18 for ttatistic . ValiditConstruct validity was assessed by correlations of the mTCNS with the TCNS, NCS, QST, MDNS and NDS.All 65 patients completed the two visits and all study procedures. Details of their clinical characteristics are shown in Ten raters, eight of whom were physicians, participated from the four centres, with a minimum of two raters per site. Three of the raters were neurologists or neurophysiologists, one was an endocrinologist and six had other specialties. The 10 raters had a mean of approximately 3.5–4 years’ experience with the TCNS and other similar clinical scoring systems. They had less experience with the mTCNS (mean 2.6 years). Four (40.0%) raters indicated that the TCNS was the scale with which they were most familiar; other scales were most familiar to 1–3 raters each. The raters had a mean of 11.5 years of experience with neurological examinations and approximately 8 years of experience with monofilament sensory tests and vibration perception threshold testing.The components of both the mTCNS and the TCNS showed good to very good internal consistency. The total scores showed good internal consistency . Within the symptom score and sensory test domains, internal consistency was very good for the mTCNS . Rather than very good, the internal consistencies for the equivalent TCNS domains were good .Inter-rater reliability was very good for the total scores of the mTCNS and TCNS and comparable in magnitude . The corIntra-rater reliability was assessed only for individual symptoms and signs and not the total or domain scores because of the nature of Cohen's kappa that is used to analyse discrete parameters. It uses the counts of cases where there is agreement between two assessment times. Ninety-nine per cent of paired values were concordant within a maximum absolute difference of three points for the mTCNS. For precise concordance of the exact integer scores, generally Cohen's kappa values indicated moderate to very good agreement for the symptoms  and sensory tests . Measures of intra-rater reliability were non-significantly higher for the TCNS as compared with the mTCNS and are summarized in the second section of The mTCNS demonstrated moderate correlation with the TCNS (Pearson correlation coefficient of 0.58). Comparison of the symptom score and sensory test domains showed very good and good correlation . Other than the moderate correlation seen between the mTCNS and the MNSI , in most cases correlation was poor to fair with other clinical scales. For example, the Pearson correlation coefficient was 0.40 with the MDNS and 0.04 with the NDS. Correlation with individual quantitative objective tests was also poor. For example, correlation was 0.13 with VPT and –0.13 with the summed sensory amplitudes from NCS. However, the Pearson correlation coefficients with the sensory score domain of the mTCNS were higher for these parameters—for example, correlation of this domain with summed sensory amplitude was –0.45. As such, lower (impaired) sensory amplitudes correlated with higher sensory test scores.The results of the cognitive debriefing indicated that most patients understood the meaning of the questions relating to DSP symptoms that are part of the mTCNS. In the opinion of the 12 respondents, they generally reported that the symptoms in the mTCNS are ‘important’ to ‘very important’ in terms of their relevance to DSP. Details for each symptom in the mTCNS scoring system are shown in This study demonstrates that the modification of a well-validated score, designed to capture the manifestations of the early processes of DSP better than the precursor score, produces a measure with similar internal consistency, inter-rater reliability and intra-rater reliability. The resultant measure, the mTCNS, maintains acceptable correlation with the precursor score, the TCNS.Although this speaks generally to the validity of the mTCNS simply because the TCNS represents well the morphological and functional change in peripheral nerves seen in those with diabetes ,10, fullAlthough the mTCNS correlates with the TCNS and other clinical scales measuring DSP, in many cases the degree of correlation is lower than would be expected from scales that make use of similar symptoms and signs. For example, MNSI shares a Pearson correlation coefficient of 0.63 with the mTCNS, even although they have in common similar elements on the semi-structured questionnaire and examination. However, perfect correlation between the mTCNS and the other clinical scales  would indicate that the new modified scale would offer no potential advantage over the existing metrics for neuropathy. As such, the degree of correlations observed might indicate advantages of the mTCNS over the other scales that generally include variables associated with advanced neuropathy and may therefore be less sensitive to milder degrees of change in nerve function. Specifically, the MNSI includes examination of the feet for deformities and reflexes (not components of the mTCNS), while the NDS excludes assessment of symptoms in place of reflexes.Similarly, correlation with specific quantitative sensory tests (cold detection thresholds) may also relate to the inability of this test to represent the full DSP phenotype as well as a scale that includes more sensitive components such as a quantitative representation of symptoms. Furthermore, cold detection thresholds are characterized by significant error variability that may also explain poor correlation with the mTCNS.The reproducibility of a quantitative measure for DSP is of fundamental importance as a tool for identifying changes in DSP severity that are of small magnitude. The very good inter-rater reliability of the mTCNS in this study (Kraemer's kappa 0.87) is comparable with that reported previously from a study of the TCNS . That thAlthough serving to quantify the reliability of the mTCNS and to establish its validity in a patient group with mild to moderate DSP severity, this analysis has some limitations. First, the assessment of any proxy score for DSP is hindered by the lack of a single quantitative gold standard measurement for neuropathy. As such, the validity of the mTCNS can only be inferred by its association with the TCNS and components of objective tests. Second, although performance of the mTCNS was generalizable among study centres by different study staff, these centres represent a certain expertise that may limit our knowledge of the applicability of this test in broader clinical settings, such as in primary care clinics.Taken together, the results of this multi-centre study have implications for future treatment trials in DSP. Patients in this study were selected for mild to moderate levels of DSP as evidenced by the presence of bilateral sural nerve responses. This feature of the study group makes it similar to the patient populations in prior therapeutic studies of agents such as aldose reductase inhibitors ,21. As cVB and RAB have been reimbursed by Dainippon Sumitomo Pharma for consultation fees. ST is an employee of Dainippon Sumitomo Pharma USA. BAP has nothing to declare.


Eupatoriopicrin (EUP), a sesquiterpene lactone from Eupatorium cannabinum L., possesses cytostatic activity. This was demonstrated for FIO 26 cells in vitro with the aid of a clonogenic assay and in vivo by tumour growth delay in FIO 26 and Lewis lung tumour-bearing mice. In vitro the IC50 for 1 h exposure to EUP was 1.5 microgram ml-1 (4.1 nmol ml-1). This concentration depleted about 25% of its cellular GSH concentration. Pretreatment of FIO 26 cells with BSO, resulting in greater than 99%. GSH depletion, enhanced the cytotoxic effect of EUP. The dose-enhancement factor at the level of 10% cell survival was 2.3. Growth inhibition of the Lewis lung carcinoma and the FIO 26 fibrosarcoma, solidly growing in C57Bl mice, was found after i.v. injection of 20 or 40 mg kg-1 EUP, at a tumour volume of about 500 microliters. Pretreatment with BSO at a dose of 4 mmol kg-1 i.p., 6 h before EUP administration, resulted in a significantly stronger growth delay of both tumours compared with EUP only. At the time of EUP treatment, cellular GSH in the tumours was reduced by BSO treatment to about 60%. It is concluded that EUP possesses antitumour activity in vivo and that chemosensitisation of EUP may be accomplished by pretreatment with BSO, indicating that endogenous GSH protects against the cytostatic action of EUP.


Furthermore, some introns also contain promoter sequences for alternative transcripts. Nevertheless, such regulatory roles do not require long conserved sequences, so that introns are relatively free to evolve faster than exons: this feature makes them important tools for evolutionary studies and provides the basis for the development of DNA molecular markers for polymorphisms detection. A survey of introns functions in the plant kingdom is presented.Spliceosomal introns in higher eukaryotes are present in a high percentage of protein coding genes and represent a high proportion of transcribed nuclear DNA. In the last fifteen years, a growing mass of data concerning functional roles carried out by such intervening sequences elevated them from a selfish burden carried over by the nucleus to important active regulatory elements. Introns mediate complex gene regulation  Thirty years have elapsed since the discovery of interrupted genes in Adenovirus2 mRNA, then defined as “amazing rearrangements” . Now, thS. cerevisiae to hundreds of thousands in higher plants and animals. Nevertheless, their number is not proportional to organism complexity: i.e. Drosophila melanogaster and Caenhorabditis elegans have about the same percentage of intronic DNA (29.1% and 30.4%) and number of introns per gene (4.22 and 5.46 respectively) [Introns represent a large proportion of the vast genomic noncoding DNA from which only a small percentage (1-2%) of coding DNA sequences differentiate. Introns length and density across the genomes of different eukaryotes vary enormously, from very few introns in some fungi such as ctively) . Intron Introns can be no more regarded as junk DNA, in the light of the increasing amount of data provided by genome sequencing projects and because of the discovery of regulatory functions that can be attributed to them. If their presence has certainly contributed, early in eukaryotic evolution, to increase the number of protein structures through genome rearrangements and exon shuffling , intronsDrosophila species [Introns neutrality in evolution, apparently implicit in the overall high variability of their sequences, has to be clearly reconsidered. In fact, evidence for the existence of some selective constraints in noncoding DNA regions including introns, has emerged by cross-comparison of genomic sequences of closely related  species  or analy species . Selecti species  and in d species . It has 1).The reason why, despite massive losses in some branches of the tree, introns have been maintained in the course of evolution, to the extent that a significant proportion of intron positions is conserved across the millions of years separating plants from animals , could hC. elegans have roughly the same number of protein coding genes [Assigning regulatory functions to the ncRNAs, that represent something like 98% of the transcriptional output of a human cell, also helps to explain the G-value paradox, emerged from genomic and transcriptomic projects. It has been shown that the estimated number of protein coding genes does not correlate with the organism complexity as it was previously presumed: i.e. humans and ng genes . This inng genes .Nowadays we are witnessing an epochal shift with regard to the genome fundamental unit: the previous proteo-centric view is being replaced by a more widely distributed and multifunctional model that is centred on transcripts .Introns represent a fraction of the transcriptome to which different regulatory functions have been already assigned and new ones are likely to be added in a short time. In plants, introns have mainly been investigated for their role in gene expression, since they are capable of increasing and addressing it. Hereafter, we will briefly review the more recent information about splicing in plants, to then discuss the different roles played by introns in the light of most recent information. These latter can be summarized as follows:Introns, especially first introns, may contain binding sites for transcription factors or may act as classical transcriptional enhancers.In plants, several introns may influence the expression of their own genes by increasing transcript levels. This is frequently observed for the first or second intron of a given transcription unit. Some introns are also responsible for tissue or development specific gene expression. Some introns, mainly first introns, may act as internal promoters to produce alternative mRNAs. When such intronic promoters lay within leader introns, the alternative transcripts differ only for their first, noncoding exon. Introns may release trans-acting factors such as microRNA (miRNA) and small nucleolar RNA (snoRNA).Even if the debate on introns origin is still open, the contraposition between the “introns early” model, sustaining the existence of introns in the ancestors of prokaryotes and eukaryotes , 17 and If the presence of introns in LUCA, the putative Last Universal Common Ancestor of all leaving organisms is still debated , new datExtensive studies on eukaryotes genomes aimed to asses the rate of intron gain or loss that has occurred during evolution are often biased by the different assumptions that each author makes followed up with the choice of different datasets and statistical methods. This may sometimes lead to opposite conclusions. Furthermore, some apparently conserved intron positions may actually occur due to parallel insertions at proto-splice sites  as earlyet al. [et al. [Marchantia polymorpha (a liverwort) may correlate with the transition to terrestrial habitats. Data from orthologous genes in chlorophycean green algae displayed largely different intron patterns.To this regard, very few analyses have been performed on plant genomes and the results are also discordant. A study from Lin et al.  reportedet al.  from theet al. . By anal [et al.  concludeThe basic spliceosomal machinery seems to be roughly conserved in all eukaryotes even if many eukaryotic lineages have acquired their own specificities , 31.Arabidopsis thaliana and rice (Oryza sativa) indicate that in both species, about 80% of coding regions contain introns, with a similar intron density of about 4 introns per gene.Data from the two completely sequenced plant genomes of Data from the Arabidopsis genome sequencing led to the filing of ASRG (Arabidopsis Splicing-Related Genes database) in which 74 snRNA genes and 395 genes coding for splicing-related proteins have been inserted, based on sequence comparison and motif searches . SplicinA. thaliana [Adh1 intron1 (57% AU-rich) are not efficiently spliced in dicot cells [Bronze2 gene, enhances splicing efficiency while a reduction in the GC content diminished splicing [One of the main differences between splicing in plants and in vertebrates concerns splice site recognition: while vertebrates bear introns that may span hundreds of thousands of base pairs, plants have no more than 1-1,5 Kbp long introns, rarely 2 or 3 Kbp. The exon definition model proposed for splicing in mammals is sustained by the finding of splicing enhancer and silencer sequences within exons, and applies well to those organisms. By converse, plants bear shorter introns whose base composition is strongly different from that of exons (10-15% more AT rich) as it is also observed in invertebrates such as insects or nematodes , 35. AU-thaliana . Nucleotthaliana , 39. Conthaliana . This isot cells , 41. Monot cells . Similarot cells . All theot cells . Even thot cells , in whicsplicing , 44. Fursplicing .Aequorea victoria was identified due to the poor expression of the reporter gene in transgenic Arabidopsis plants. The same sequence in tobacco is spliced with 40% efficiency, implying differences in splice site recognition between these two plant species. These observations demonstrate that the plant kingdom is much more differentiated at the molecular level than previously thought and inference from one or two model species to larger phylogenetic groups is not straightforward. In other words, working on plant species from both distantly related and lower phylogenetic groups is also important to gain a more detailed view of plant complexity.All these studies have been made on the assumption that rice or maize are good model plants for all monocot species, but recent evidences pose the question on how representative the Poales (Graminae) are for the monocots as a whole. Recent data from onion EST  highlighAlternative splicing (AS) accounts for the discrepancy between the number of putative coding genes in sequenced genomes and the larger number of EST found in eukaryotes. Together with alternative transcription start sites, this mechanism is responsible for generating even tens of different transcripts from one gene, leading to the production of different or slightly different proteins. Dscam gene that codes for the axon guidance receptor .FCA gene, have been recently documented in plants [In plants, the phenomenon was considered scarcely relevant, but is now emerging as important as it is in animals . Severaln plants , but thiThe most recent estimation of the number of alternatively by spliced genes is about 20% for both Arabidopsis and rice but this number may even increase since estimation in humans has recently raised to more than 60% . From thIn the case of intron retention, the functional significance of AS is not so evident and leaves the doubt that incorrectly spliced transcripts constitute a background of “escaped” mRNA that are not eliminated by the cell. However this is unlikely, since most retained introns within coding sequences contain stop codons, supposed to drive mRNAs to Nonsense mediated decay (NMD). Furthermore, in some cases it has been demonstrated that intron-containing transcripts are not retained in the nucleus (as they should be in case of incomplete or inefficient splicing) but are found associated to polyribosomes .Alternatively, introns may be retained to play a functional regulatory role. In vertebrates, it has been suggested that the coupling of AS and NMD could be an important post-translational mechanism to adjust the level of transcript isoforms . IndirecIntrons in 5’-and 3’UTRs are more commonly retained than those constitutively spliced. This is likely to have a regulatory meaning since UTRs are important regulators of gene expression by influencing mRNA stability and translational efficiency. This can occur through the presence of upstream ORFs, the presence of secondary structures or binding sites for tissue-specific proteins or short RNAs like miRNA and short interfering RNAs , 58. MulIn plants, AS is regulated in a tissue-specific manner and is strongly influenced by stresses and hormones. The regulation of alternative splicing is quite complex and involves splicing regulators, such as SR proteins, protein kinases, hnRNPs, probably driving it in a concentration-dependent mode. The synthesis of splicing regulators is in turn controlled by stress-dependent alternative splicing so that environmental stimuli may control the AS pattern of co-ordinated sets of related genes . In animFurthermore, not only protein coding RNA, but even noncoding RNAs, may be alternatively spliced, as it is the case for the recently discovered NRON RNA  a represIn a proteo-centric world, AS is a mechanism that increases the number of protein isoforms encoded by genes supporting the synthesis of different or slightly different products. Although undoubtedly true, AS is not limited to this output. In fact it is frequently found that splice variants also produce different transcripts with the same coding sequences. This is the case for retained introns within 3’ or 5’ UTR or for transcripts with alternative first noncoding exons. Furthermore, 30-40% of AS in Arabidopsis leads to premature stop codon formation, targeting transcripts for NMD.In the human genome, it has been estimated that 10-18% of genes express alternative 5’UTR by the use of multiple promoters . Recent An analysis of 91,425 rice 5’ end ESTs detected 46 (4%) potential Alternative First Exon (AFE) clusters. RT-PCR analysis of 12 of these clusters, in six different tissues and stages, revealed tissue-specific expression of the alternative first exon for 5 of them.2): in the first (type I), the two first exons are mutually exclusive; in the second (type II), the first exon of an alternative mRNA, exon 1b in Fig. (2), falls internal to the other.A more recent work identified about 5-6% of AFE clusters in both rice and Arabidopsis . Many ofWhen alternative exons are distant (more then 500 base pairs) they are usually associated with alternative promoters, the most downstream of which lays within an intron. Estimations from available datasets indicated that about 58% of rice and 23% of Arabidopsis AFE containing genes may be derived from alternative promoters. The functional significance of splice variants encoding the same protein is likely to be merely regulative and may occur for the following reasons: alternative promoters may vary for strength, tissue or condition-specificity thus binding different transcriptional factors or repressors.mRNAs with different 5’UTRs may differ in stability or translational efficiency and this may cause a finer tuning of expression levels, i.e. they may contain uORFs that reduce correct translation. 5’UTR may also be the target for regulatory miRNAs (see below).the structure of loci containing alternative promoters is such that the downstream promoter may lay within an intron sequence that is spliced from the upstream transcript. Hence, alternative first exons mean alternative introns. Since plant introns can specifically influence expression at post-transcriptional level, this latter may obviously be a mechanism by which gene regulation is further influenced. Promoter function is thus an additional function for introns located in the 5’ region of a gene. To this regard, the following few examples have so far been reported in plants.Genes encoding pyruvate orthophosphate dikinase (PPDK) from maize  and ArabPvSR2 [Bean PvSR2  is a heaOstub4 and CDPK2, the first encoding for a beta tubulin isotype, the second for a Ca++-dependent protein kinase [Ostub4 e CDPK2 genes the alternative first exons were noncoding ones, so that the protein products from the alternative transcripts remained the same. In the case of Ostub4, the intronic promoter showed a very low activity in transient transformation assays, while the upstream promoter sustained high level of GUS activity [et al. 2008, submitted)1. In the case of CDPK2, the intronic promoter has a strong activity, but only in the presence of an intron located further downstream [Furthermore, similar data were reported for two unrelated rice genes, n kinase -70. Whilactivity . This laIn all these cited examples, intron sequences may probably exert their function at different levels, since they can act as transcriptional promoters and as intronic enhancers, probably by a post-transcriptional mechanism (see next paragraph).Recently, after years of “promoter leadership” that followed the discovery of transcription factors, the involvement of other regulatory sequences in gene expression has rapidly emerged, revealing that all the downstream elements present within transcribed sequences  may also play an important role in gene expression contributing to a variety of post-transcriptional regulatory mechanisms .In the case of introns, the general observation that intron-containing DNA sequences are better expressed than their cDNA counterparts was originally made in mammalian cells  and lateAdh1, Bz1, Act1, Shrunken1) have on gene expression, date back to the late eighties. The inclusion of such intron sequences in reporter-containing expression plasmids greatly enhanced downstream reporter gene expression [Adh1 intron 9 and Hsp81 intron 1 [Physcomitrella patens [In plants, early observations on the effect that some introns of maize genes  may selectively act at either DNA or RNA level. As recently discussed by Mattick and Makunin , RNA molACT1 [AGAMOUS gene of Arabidopsis  have been shown to contain enhancer-like transcriptional elements, but in most cases, the presence of introns within the transcription unit , and in introns  and petu [PhADF1 ), is chi [PhADF1 . These rAdh1, Hsp82 and Sh1 genes [Further effects on translation have been suggested in one case where no increase in the mRNA level was observed  and in oh1 genes , 81, 92 h1 genes . Pat1 at different locations within the transcribed sequence greatly influences IME: longer is the distance from the 5’ –end of the gene, lesser is the effect [Sh1 leader intron [Tpi first intron [RpoT gene, while all efficiently spliced, have different enhancing effect when placed in different positions within the luciferase coding sequence with some of these locations that could even cause negative effects [The position of the intron also appears to be an important factor. Insertion of intron 1 of thee effect . Similarr intron  and for t intron . Three d effects .PhADF1 intron1 is required for detectable gene expression in transgenic tobacco. If replaced by intron 2 of the same gene, the level of expression falls below that observed with a construct that lacked introns [In any case, splicing per se does not seem to be sufficient for IME to occur since the degree of expression enhancement varies greatly in relation to the intron used. In fact, as already mentioned, it has also been reported that some correctly spliced introns fail to exert any effect.  introns .Shrunken1 leader intron extended up to 85% of the sequence, but preserving the splicing sites, can still support the same enhancement of expression as observed with the intron in the native configuration, but a stretch of Ts, not required for splicing, is necessary for enhancement [AtEF1-A3 seem to reveal a correlation between intron length and intron-mediated enhancement [PhADF1 first intron suggested that the 5’ distal region is more important for enhancement than the intron 3’ region [In most cases, large internal intron deletions are tolerated without significant effects on IME , 88, sugancement . Conversancement . Deletio’ region .So, despite the many informations that have been obtained up to now, more experiments with additional and specific promoter-intron combinations are required to reach a better understanding about the mechanisms underlying intron-mediated enhancement of gene expression, in an effort to identifying the true determinants.cat-1) driven by the 35S CaMV promoter, can enhance GUS expression in rice protoplasts and leaves, while it has no effect in transgenic tobacco plants. This finds explanation with the fact that the intron was efficiently spliced in rice but not in tobacco [Ultimately, it should be noted that the requirement of introns for gene expression may be different in monocots compared to dicots and differences in these latters can also be scored. The first intron of the castor bean catalase gene  gene of Arabidopsis [Early evidence for the need of intragenic sequences for tissue-specific expression in plants came from works on sucrose synthase n potato ,100 and bidopsis .AG promoter indicated that the pattern of GUS expression driven by a 6 kb upstream sequence in transgenic Arabidopsis plants did not match that obtained from in situ hybridization experiments. The addition of a 3.8 kb intragenic sequence inclusive of the first two introns restored a correct expression pattern with overlapping profiles between the two experimental approaches.Studies performed on the A subsequent work clarified the original finding since it demonstrated that enhancer-like elements that specifically regulate expression in carpels and stamens were found in the large second intron of the AG gene. A correct pattern of expression was also maintained by inserting this intron in the unstranscribed region of the gene, independently of its orientation .Sus3 and Sus4, two sucrose synthase genes from potato, bear long introns within their leader sequences. It has been demonstrated, both in transgenic potato and tobacco plants, that such introns are necessary for the correct level and site of expression of a downstream reporter gene. Sus4 leader intron is required for high-level tuber expression and for sucrose induction. Introns contain both positive and negative regulatory elements, whose deletion caused either ectopic expression or absence of expression. Whether their regulatory role is exerted at the transcriptional or post-transcriptional level was not investigated.Ostuba1, the effect was exerted at level different than DNA. The first intron of the coding region of rice OstubA1 was found to be necessary for the correct expression pattern of GUS in transgenic rice plants. In its absence, GUS expression shifted in roots from the apex to the elongation zones, while in leaves became diffusely distributed rather than being confined to the innermost of leaf sheaths. In this case the enhancing effect was lost when the intron was inserted in the 5’ untranscribed region or after the GUS sequence. Similar data have been confirmed for the cognate Ostua2 gene and for rice beta tubulin gene Ostub4 1.In another case, that of rice ACT1 gene is mainly expressed in pollen and ovules, although expression in meristematic regions of roots and shoots is also observed. In transgenic Arabidopsis plants, the precise deletion of the whole leader intron from a chimeric ACT1::GUS gene, abolished expression in all tissues while reducing pollen-specific GUS activity to about 10% [ACT1 leader intron with that of ACT2, an actin gene mainly expressed in vegetative tissues and not in reproductive organs, can rescue activity in meristematic tissues but not in pollen. This indicates that pollen-specific elements are contained within the ACT1 leader intron sequence.Arabidopsis bout 10% . The samPRF5 gene with that of the vegetative PRF2 isotype, completely re-directed expression driven by the PRF5 promoter in transgenic Arabidopsis from an anther-specific to a constitutive, generic pattern [PRF5 gene with that of the petunia actin-depolymerizing factor1 gene (PhADF1): the intron re-addressed GUS expression to vegetative tissues .A recently investigated case is that of the Arabidopsis profilin gene family members: replacing the first intron of the reproductive  pattern . SimilarLast but not the least, spliceosomal introns can influence expression by encoding for trans-acting regulatory RNAs, such as intronic miRNA, snoRNA and others RNA molecules that are processed from intron precursors by specific RNAse/protein complexes. The issue of short regulatory RNAs such as short interference RNAs (siRNA) and micro RNAs (miRNA) represents a very recent yet greatly expanding field of investigation. Many review works have thoroughly analyzed the state of the art including plants -106**. HSmall nucleolar RNAs (snoRNAs) are the oldest family of short RNAs. They are short RNAs assembled in ribonucleoprotein complexes whose function is the nucleotide modification of ribosomal and spliceosomal RNAs . Almost C. elegans. They work by base-pairing to mRNAs, targeting them to degradation or inhibiting translation. They derive from longer precursors, cleaved by two type III RNAse/protein complexes, Drosha and Dicer. Again, a great number of miRNAs is processed from intron precursors, and their synthesis is regulated contextually to the gene in which they are hosted [miRNAs are short single-stranded 20-24 nucleotide long RNAs first identified in e hosted . This sue hosted .CARPEL FACTORY and DICER-LIKE (DCL) genes. Mutations in CARPEL FACTORY indicate that miRNAs may play pivotal roles in plant development [A growing number of miRNA species have been also found in plant cells, many of which are conserved between distantly related species , while oelopment -106. miRDrosophila and Caenorhabditis [Of recent, miRNAs that directly originate from debranched introns (mirtrons), bypassing Drosha processing whose function is replaced by the spliceosome, have been identified in habditis . MirtronThe existence in the human transcriptome of long, unspliced, antisense intronic transcripts can reveal a new class of regulatory RNAs. Antisense intronic transcripts whose level is regulated by androgens in a prostate cancer cell line have been recently reported . The genAt the beginning of this review we have reported on how introns are being used as key elements for molecular evolution studies, the aim of which is to identify LUCA, the Last Universal Common Ancestor of the living organisms. These studies are mainly based on the analysis of eukaryotic genomes with respect to intron conserved position rather than sequence composition. This latter is in fact quite variable and makes it difficult to identify conserved nucleotide motifs in paralogous or orthologous intron pairs. Whatever is the identity of LUCA, it is a fact that introns are largely abundant in Eukaryotes where they contribute to increase the variability of protein isoforms, mainly through the mechanism of AS. We have also learnt that AS can lead to the production of different transcripts coding for the same product and that introns may be part of non-coding exons. Moreover, introns can host promoters and transcriptional start sites. Introns influence gene expression through IME and IDSE and are the elements from which miRNAs and snoRNAs are produced. Due to all this bundle of regulatory functions, it should not come to surprise that introns length and sequence composition can be highly variable and that no appreciable significant nucleotide homology can be found when comparing introns of orthologous genes and gene-families. At the same time, the position of introns is not randomly scattered within genes and across the genomes. On the contrary, intron position is amazingly retained across unrelated eukaryotic species , 23. ThiILP markers have several advantages, the most important of which is transferability across the plant species. It has been shown that out of 51 pairs of rice-based ILP primers, only 6 of them (11.8%) failed to generate PCR products from genomic DNA extracted from 8 different plant species while 24 pairs (47.1%) yielded similar amplification products as observed in rice . As justIt is clear that introns have regained general interest by virtue of their recently uncovered multi-functionality. This also explains why they have been conserved along great evolutionary distances.While positioning within genomes has been largely maintained, nucleotide sequence has substantially diverged with the exception of short conserved elements responsible for intron specific functions (i.e. miRNAs formation). Introns can regulate the level and the pattern of gene expression during plant growth and morphogenesis and under different environmental conditions. Introns are key elements of the new molecular genetics, dealing with unannotated TUFs and the role they have in the general control of eukaryotic gene expression.