ExInt: an Exon/Intron database

The Exon/Intron (ExInt) database incorporates information on the exon/intron structure of eukaryotic genes. Features in the database include: intron nucleotide sequence, amino acid sequence of the corresponding protein, position of the introns at the amino acid level and intron phase. From ExInt, we have also generated four additional databases each with ExInt entries containing predicted introns, introns experimentally defined, organelle introns or nuclear introns. ExInt is accessible through a retrieval system with pointers to GenBank. The database can be searched by keywords, locus name, NID, accession number or length of the protein. ExInt is freely accessible at http://intron.bic.nus.edu.sg/exint/exint.html     

Conserved intron positions in ancient protein modules

Background  

The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.     

RNA sequences upstream of the 3' splice site repress splicing of mutantyeast ACT1 introns.

A yeast ACT1 intron in which both the first and last intron nucleotides are mutated, the /a-c/ intron, splices 10% as well as wild type. We selected for additional cis-acting mutations that improve the splicing of /a-c/ introns and recovered small deletions upstream of the 3' splice site. For example, deletion of nucleotides -9 and -10 upstream of the 3' splice site increased the splicing activity of the /a-c/ intron to 30% that of the wild-type ACT1 intron. To determine if the increased /a-c/ splicing was due to changes in intron spacing or sequence, we made mutations that mimicked the local sequence of the delta-9, -10 deletion without deleting any nucleotides. These mutants also increased /a-c/ splicing, indicating that the increased splicing activity was due to changes in intron sequence. The delta-9, -10 deletion was not allele specific to the /a-c/ intron, and improved the splicing efficiency of many mutant introns with step II splicing defects. To further define the sequences required for improved splicing of mutant introns, we randomized the region upstream of the ACT1 3' splice site. We found that almost all sequence alterations improved the splicing of the /a-c/ intron. We postulate that this sequence near the 3' end of the intron represses the splicing of mutant introns, perhaps by serving as the binding site for a negative splicing factor.     

Malin: maximum likelihood analysis of intron evolution in eukaryotes

Malin is a software package for the analysis of eukaryotic gene structure evolution. It provides a graphical user interface for various tasks commonly used to infer the evolution of exon-intron structure in protein-coding orthologs. Implemented tasks include the identification of conserved homologous intron sites in protein alignments, as well as the estimation of ancestral intron content, lineage-specific intron losses and gains. Estimates are computed either with parsimony, or with a probabilistic model that incorporates rate variation across lineages and intron sites. Availability: Malin is available as a stand-alone Java application, as well as an application bundle for MacOS X, at the website http://www.iro.umontreal.ca/~csuros/introns/malin/. The software is distributed under a BSD-style license.     

Thermal drive contributes to hyperventilation during exercise in sheep

The etiology of exercise hypocapnia is unknown.The contributions of exercise intensity (ExInt), lactic acid,environmental temperature, rectal temperature(T_re), and physicalconditioning to the variance in arterialCO₂ tension(Pa_CO2) in the exercising sheep werequantified. We hypothesized that thermal drive contributes tohyperventilation. Four unshorn sheep were exercised at ~30, 50, and70% of maximal O₂ consumption for30 min, or until exhaustion, both before and after 5 wk of physicalconditioning. In addition, two of the sheep were shorn and exercised ateach intensity in a cold (<15°C) environment.T_re andO₂ consumption were measured continuously. Lactic acid and Pa_CO2 weremeasured at 5- to 10-min intervals. Data wereanalyzed by multiple regression onPa_CO2. During exercise,T_re rose andPa_CO2 fell, except at the lowest ExIntin the cold environment. T_reexplained 77% of the variance in Pa_CO2,and ExInt explained 5%. All other variables were insignificant. Weconclude that, in sheep, thermal drive contributes to hyperventilation during exercise.

     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Investigating the intron recognition mechanism in eukaryotes

Recent studies indicate that many introns, as well as the complex spliceosomal mechanism to remove them, were present early in eukaryotic evolution. This study examines intron and exon characteristics from annotations of whole genomes to investigate the intron recognition mechanism. Exon definition uses the exon as the unit of recognition, placing length constraints on the exon but not on the intron (allowing it a greater range of lengths). In contrast, intron definition uses the intron itself as the unit of recognition and thus removes constraints on internal exon length forced by the use of an exon definition mechanism. Thus, intron and exon lengths within a genome can reflect the constraints imposed by its splicing. This study shows that it is possible firstly to recover valid intron and exon information from genome annotation. We then compare internal intron and exon information from a range of eukaryotic genomes and investigate possible evolutionary length constraints on introns and exons and how they can impact on the intron recognition mechanism. Results indicate that exon definition-based mechanisms may predominate in vertebrates although the exact system in fish is expected to show some differences with the better characterized system from mammals. We also raise the possibility that the last common ancestor of plants and animals contained some type of exon definition and that this mechanism was replaced in some genes and lineages by intron definition, possibly as a result of intron loss and/or intron shortening.     

Widespread intron loss suggests retrotransposon activity in ancient apicomplexans

Several facets of spliceosomal intron in apicomplexans remain mysterious. First, intron numbers vary across species by 2 orders of magnitude, indicating massive intron loss and/or gain. Second, previous studies have shown very different evolutionary patterns over different timescales, with 100-fold higher rates of intron loss/gain between genera than within genera. Third, the timing and dynamics of nearly complete intron loss in Cryptosporidium species, as well as reasons for retention of the few remaining introns, remain unknown. We compared intron positions in 785 orthologous genes between 3 moderate to intron-rich apicomplexan species. We estimate that the Theileria-Plasmodium ancestor had 4.5 times as many introns as modern Plasmodium species and 38% more than modern Theileria species, and that subsequent intron losses have outnumbered intron gains by 5.8 to 1 in Theileria and by some 56 to 1 in Plasmodium. Several patterns suggest that these intron losses occurred by recombination with reverse-transcribed mRNAs. Intriguingly, this finding suggests significant retrotransposon activity in the lineages leading to both Theileria and Plasmodium, in contrast to the dearth of known retrotransposons and intron loss within modern species from both genera. We also compared genomes from Cryptosporidium parvum and C. hominis and found no evidence of ongoing intron loss, nor of intron gain. By contrast, Cryptosporidium introns are less evolutionary conserved with Toxoplasma than are introns from other apicomplexans; thus the few remaining introns are not simply indispensable ancestral introns.     

Statistical analysis and prediction of the exonic structure of human genes

Summary Nonhomologous fully sequenced human protein-coding genes were studied. Three sets of exon-exon junctions were formed defined by the intron (shadow) position relative to the reading frame. For the analysis of intron shadow signals in exons, information content and discrimination energy approaches were used with the correction allowing one to ignore the influence of a protein-coding message. The corrected formulas allow one to define the consensuses for the three types of intron shadow signals as aAG/guwn, cAG/GUnn, and cAG/gunU, and provide better recognition than the original formulas. The analysis of the codon usage in the signal positions leads to the conclusion that the prevalence of some amino acids in corresponding protein sites is caused by the signal requirements and not vice versa. The distribution of potential intron shadow signals in exons contradicts the hypothesis of intron insertion into suitable preexisting sites. There exists a correlation between the intron types and/or the exon length modulo 3.     

Generation of a database containing discordant intron positions in eukaryotic genes (MIDB)

MOTIVATION: Intron sliding is the relocation of intron-exon boundaries over short distances and is often also referred to as intron slippage or intron migration or intron drift. We have generated a database containing discordant intron positions in homologous genes (MIDB--Mismatched Intron DataBase). Discordant intron positions are those that are either closely located in homologous genes (within a window of 10 nucleotides) or an intron position that is present in one gene but not in any of its homologs. The MIDB database aims at systematically collecting information about mismatched introns in the genes from GenBank and organizing it into a form useful for understanding the genomics and dynamics of introns thereby helping understand the evolution of genes. RESULTS: Intron displacement or sliding is critically important for explaining the present distribution of introns among orthologous and paralogous genes. MIDB allows examining of intron movements and allows mapping of intron positions from homologous proteins onto a single sequence. The database is of potential use for molecular biologists in general and for researchers who are interested in gene evolution and eukaryotic gene structure. Partial analysis of this database allowed us to identify a few putative cases of intron sliding. AVAILABILITY: http://intron.bic.nus.edu.sg/midb/midb.html     

Complex selection on intron size in Cryptococcus neoformans

We conducted a genome-wide analysis of the roles of mutation and selection in sculpting intron size in the fungal pathogen Cryptococcus neoformans. We find that deletion rate is positively associated with intron length and that insertion rate exhibits a weak negative association with intron length. These patterns suggest that long introns as well as extremely short introns in this unusually intron-rich fungal genome are in mutation-selection disequilibrium and that the proportion of constrained functional sequence in introns does not scale linearly with size. We find that untranslated region introns are longer than coding-region introns and that first introns are substantially longer than subsequent introns, suggesting heterogeneous distribution of constrained functional sequence and/or selective pressures on intron size within genes. In contrast to Drosophila, we find a positive correlation between d(N) and first intron or last intron length and a negative correlation between d(N) and internal intron length. This contrasting pattern may indicate that terminal introns and internal introns are differentially subject to hypothesized selection pressures modulating intron size and provides further evidence of widespread selective constraints on noncoding sequences.     

The interaction between the first and last intron nucleotides in the second step of pre-mRNA splicing is independent of other conserved intron nucleotides.

Virtually all pre-mRNA introns begin with the sequence /GU and end with AG/ (where / indicates a border between an exon and an intron). We have previously shown that the G residues at the first and last positions of the yeast actin intron interact during the second step of splicing. In this work, we ask if other highly conserved intron nucleotides also take part in this /G-G/ interaction. Of special interest is the penultimate intron nucleotide (AG/), which is important for the second step of splicing and is in proximity to other conserved intron nucleotides. Therefore, we tested interactions of the penultimate intron nucleotide with the second intron nucleotide (/GU) and with the branch site nucleotide. We also tested two models that predict interactions between sets of three conserved intron nucleotides. In addition, we used random mutagenesis and genetic selection to search for interactions between nucleotides in the pre-mRNA. We find no evidence for other interactions between intron nucleotides besides the interaction between the first and last intron nucleotides.     

A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.     

Excision of the Sinorhizobium meliloti group II intron RmInt1 as circles in vivo

Excision of group II introns as circles has been described only for a few eukaryotic introns and little is known about the mechanisms involved, the relevance or consequences of the process. We report that splicing of the bacterial group II intron RmInt1 in vivo leads to the formation of both intron lariat and intron RNA circles. We determined that besides being required for the intron splicing reaction, the maturase domain of the intron-encoded protein also controls the balance between lariat and RNA intron circle production. Furthermore, comparison with in vitro self-splicing products indicates that in vivo, the intron-encoded protein appears to promote the use of a correct EBS1/IBS1 intron-exon interaction as well as cleavage at, or next to, the expected 3' splice site. These findings provide new insights on the mechanism of excision of group II introns as circles.     

A chemical phylogeny of group I introns based upon interference mapping of a bacterial ribozyme

Despite its small size, the 205 nt group I intron from Azoarcus tRNA(Ile) is an exceptionally stable self-splicing RNA. This IC3 class intron retains the conserved secondary structural elements common to group I ribozymes, but lacks several peripheral helices. These features make it an ideal system to establish the conserved chemical basis of group I intron activity. We collected nucleotide analog interference mapping (NAIM) data of the Azoarcus intron using 14 analogs that modified the phosphate backbone, the ribose sugar, or the purine base functional groups. In conjunction with a complete interference set collected on the Tetrahymena group I intron (IC1 class), these data define a "chemical phylogeny" of functional groups that are important for the activity of both introns and that may be common chemical features of group I intron catalysts. The data identify the functional moieties most likely to play a conserved role as ligands for catalytic metal ions, the substrate helix, and the guanosine cofactor. These include backbone functional groups whose nucleotide identity is not conserved, and hence are difficult to identify by standard phylogenetic sequence comparisons. The data suggest that both introns utilize an equivalent set of long range tertiary interactions for 5'-splice site selection between the P1 substrate helix and its receptor in the J4/5 asymmetric bulge, as well as an equivalent set of 2'-OH groups for P1 helix docking into most of the single stranded segment J8/7. However, the Azoarcus intron appears to make an alternative set of interactions at the base of the P1 helix and at the 5'-end of the J8/7. Extensive differences were observed within the intron peripheral domains, particularly in P2 and P8 where the Azoarcus data strongly support the proposed formation of a tetraloop-tetraloop receptor interaction. This chemical phylogeny for group I intron catalysis helps to refine structural models of the RNA active site and identifies functional groups that should be carefully investigated for their role in transition state stabilization.     

甘丙肽重构基因GAL（intronⅡ）的克隆及其过表达转基因载体的构建

目的克隆甘丙肽重构基因GAL（intronⅡ）,构建小鼠神经系统特异性表达甘丙肽（galanin,GAL）的转基因载体pUC18/PDGF-β-promoter/GAL（intronⅡ）,为制备GAL转基因小鼠做准备。方法通过常规PCR、RT-PCR及重叠延伸PCR（overlap extension PCR,OE-PCR）扩增出插入GAL第二内含子的GAL全长cDNA的重构基因GAL（intronⅡ）,将GAL（intronⅡ）克隆入T载体进行酶切、测序鉴定;同时从psisCAT6α中切下血小板源性生长因子β链（platelet-derived growth factor beta polypeptide,PDGF-β）启动子片段连接到空载体pUC18上构建出重组载体pUC18/PDGF-β-promoter;然后将GAL（intronⅡ）定向克隆于pUC18/PDGF-β-promoter下游,构建转基因载体pUC18/PDGF-β-promoter/GAL（intronⅡ）。结果 PCR和限制性内切酶分析鉴定,表明获得转基因载体pUC18/PDGF-β-promoter/GAL（intronⅡ）。通过对重构基因GAL（intronⅡ）的GALcDNA和第二内含子序列测序证实其与GenBank中的标准序列一致,GAL（intronⅡ）中第二内含子插入的位置准确,且无移码。结论使用重叠延伸PCR扩增重构基因GAL（intronⅡ）并成功构建转基因载体pUC18/PDGF-β-promoter/GAL（intronⅡ）,为转基因小鼠制备奠定一定的实验基础。     

Evolutionary dynamics of intron size, genome size, and physiological correlates in archosaurs

It has been proposed that intron and genome sizes in birds are reduced in comparison with mammals because of the metabolic demands of flight. To test this hypothesis, we examined the sizes of 14 introns in a nonflying relative of birds, the American alligator (Alligator mississippiensis), and in 19 flighted and flightless birds in 12 taxonomic orders. Our results indicate that a substantial fraction (66%) of the reduction in intron size as well as in genome size had already occurred in nonflying archosaurs. Using phylogenetically independent contrasts, we found that the proposed inverse correlation of genome size and basal metabolic rate (BMR) is significant among amniotes and archosaurs, whereas intron and genome size variation within birds showed no significant correlation with BMR. We show statistically that the distribution of genome sizes in birds and mammals is underdispersed compared with the Brownian motion model and consistent with strong stabilizing selection; that genome size differences between vertebrate clades are overdispersed and punctuational; and that evolution of BMR and avian intron size is consistent with Brownian motion. These results suggest that the contrast between genome size/BMR and intron size/BMR correlations may be a consequence of different intensities of selection for these traits and that we should not expect changes in intron size to be significantly associated with metabolically costly behaviors such as flight.     

Spatial patterns of photobiont diversity in some Nostoc-containing lichens

Patterns of photobiont diversity were examined in some Nostoc -containing lichens using the nucleotide sequence of the cyanobacterial tRNA^Leu (UAA) intron. Lichen specimens collected in northwestern USA were analysed and the sequence data were compared with tRNA^Leu(UAA) intron sequences previously obtained from lichens in northern Europe. Generally, it is the species identity of a lichen rather than the geographical origin of the specimen that determines the identity of the cyanobiont. Identical intron sequences were found in Peltigera membranacea specimens collected in Oregon (USA) and in Sweden, and very similar sequences were also found in Nephroma resupinatum thalli collected in Oregon and Finland. Furthermore, in mixed assemblages where two Peltigera species grew in physical contact with each other, the different lichen species housed different photobiont strains. There is however not a one-to-one relation between mycobiont and photobiont as some intron sequences were found in more than one lichen species, and different intron sequences were found in different samples of some lichen taxa. Peltigera venosa exhibited a higher level of photobiont diversity than any other lichen species studied, and several intron sequences could for the first time be obtained from a single thallus. It is not clear whether this is evidence of lower cyanobiont specificity, or reflects an ability to exhibit different degrees of lichenization with different Nostoc strains. In one specimen of P. venosa , which contained bipartite cyanosymbiodemes and tripartite, cephalodiate thalli, both thallus types contained the same intron sequence.     

Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion)

We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms. We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI). This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii. The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity). A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non- Chlamydomonad green algae surveyed, nor in land plants. A parallel survey of homologues of a previously described and similar intron/ORF pair (C. reinhardtii chloroplast CrLSU/A. castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage. The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site- 2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba- like organism, probably in the direction chloroplast to mitochondrion. The data also suggest that both introns could have been acquired in a single event.      

Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.