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PubMed1998

A unique exon-intron organization of a porcine S100C gene: close evolutionary relationship to calmodulin genes.

We found a unique exon-intron structure of the porcine S100C gene, a member of the S100 family, in which all other genes characterized to date have common exon-intron organization. The genomic DNA encoding the porcine S100C was cloned and the entire nucleotide sequence of the gene was analyzed. The gene is present as a single copy and consists of three exons and two introns with a total size of 5.3 kb. The first intron is located after the ATG translation initiation codon. Such an intron has never been found in other S100 genes, but is found in almost all calmodulin genes. The gene structural similarity suggests a close evolutionary relationship between the S100C gene and calmodulin genes.

Animals↗
PubMed1998

Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV.

Starting with crude yeast mitochondria, the intron homing endonuclease, I-SecIV, was purified to near homogeneity. This highly purified enzyme differs from some other well-characterized yeast mitochondrial intron-encoded endonucleases in terms of its structure and DNA cleavage specificity. The enzyme is a heterodimer with a native molecular mass of 92 kDa. A small catalytic subunit (32 kDa) is probably encoded largely or entirely by intron 5 alpha of the cytochrome oxidase subunit I gene. A larger polypeptide subunit (60 kDa) may be a nuclear factor necessary for intron mobility. I-SceIV exhibits a low DNA sequence specificity as it cleaves a variety of DNA substrates. Analysis of kinetic parameters shows that the purified enzyme has a very high affinity for DNA and exhibits low turnover which may have implications for subsequent steps in the intron homing process.

Animals↗
PubMed2000

A common intron 3 mutation (IVS3 -48c-->g) leads to misdiagnosis of the c.845G-->A (C282Y) HFE gene mutation.

Amplification of the region of the HFE gene that contains the c.845G-->A (C282Y) mutation is usually performed using one amplimer that binds to a sequence in intron 3 and another that binds to a sequence in intron 4. Previously, a mutation that interferes with efficient binding to the intron 4 site has been described. We now find that another common mutation in intron 3, IVS3 -48c-->g, prevents binding of the amplimer to that site. This polymorphism occurs at a gene frequency of 0.128 in the African-American population and at a frequency of only 0.006 in the European population. DNA samples heterozygous for the IVS3 -48c-->g polymorphism and C282Y were undistinguishable from samples homozygous for the C282Y mutation when they were examined by allele-specific oligonucleotide hybridization (ASOH) and showed only a weak normal band when examined by electrophoresis following restriction endonuclease digestion. Although the polymorphism occurs in a DNA sequence almost identical to the intron 3 splice donor site, we found no evidence of alternative splice forms. Moreover, serum iron, transferrin saturation, and serum ferritin levels were normal in subjects heterozygous for the polymorphism. It appears, therefore, that the main importance of this polymorphism is that it may lead to misdiagnosis of heterozygotes for the C282Y mutation as homozygotes. We therefore recommend exonic amplimers that avoid sites that contain polymorphisms and that can be multiplexed for detection of the c.187C-->G (H63D) and c.845G-->A (C282Y) mutations.

Amino Acid Substitution↗
PubMed2001

Intron mobility results in rearrangement in mitochondrial DNAs of heterokaryon incompatible Aspergillus japonicus strains after protoplast fusion.

Mitochondrial transmission was carried out under selective conditions between incompatible Aspergillus japonicus strains always using an oligomycin-resistant mitochondrial donor and selecting for recipient nuclei and oligomycin-resistant mitochondria. All attempted intraspecific mitochondrial transmissions were successful, but the transmission between closely related A. japonicus and A. aculeatus failed. Under selection pressure, resistant progeny harbor the mitochondrial DNA (mtDNA) of the donor strain, which may remain unchanged or may be modified by the introns of the recipient mitochondrial genome. Detailed analysis of a certain strain harboring rearranged mtDNA suggests that the mtDNA profiles of recombinant-like progeny are strongly influenced by the characteristics and mobility of introns of both parental mtDNAs. Both intron loss and intron acquisition play a role in the rearrangement of mtDNA. In certain parental combinations, a particular intron was lost very frequently.

Aspergillus↗
PubMed1994

Identification of a novel tandemly repeated sequence present in an intron of the glucose phosphate isomerase (GPI) gene in mouse and man.

Glucose phosphate isomerase (GPI, glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) is a housekeeping gene expressed in all tissues and organisms that utilize glycolysis and gluconeogenesis. Deficiency in humans leads to a rare form of nonspherocytic hemolytic anemia. We have isolated a 3.2-kb mouse cDNA containing glucose phosphate isomerase coding sequence and a 2.1-kb intronic sequence and a large proportion of the human gene (approaching 55 kb) in four phage lambda recombinants. A 4-kb intronic fragment from the human gene showing homology to the mouse intronic sequence has been isolated and sequenced. The fragment contains approximately 1.5 kb of sequence that is composed of 30 repeat units of a novel 50-bp tandemly repeated units. The mouse intronic sequence contains 18 similar units. The human consensus sequence differs from the mouse consensus sequence at only 7 positions out of 50 (positions 16, 26, 27, 42, 43, 47, and 48). A probe containing the repeat element detects polymorphisms, specific to glucose phosphate isomerase, in human DNA. The repeat element does not appear to be present at any other loci in human DNA. The conservation of this intronic repeat element extends to pig and Chinese hamster.

Animals↗
PubMed1995

Sequence of human tryptophan 2,3-dioxygenase (TDO2): presence of a glucocorticoid response-like element composed of a GTT repeat and an intronic CCCCT repeat.

Abnormalities in serotonin levels have been implicated in a wide range of psychiatric disorders. Tryptophan 2,3-dioxygenase is the rate-limiting enzyme in the catabolism of tryptophan, the precursor of serotonin. As such it is a potential major candidate gene in psychiatric genetics. The regulatory, intron, and exon regions of the human TDO2 gene have been sequenced. Twelve exons were identified. The amino acid sequence of the enzyme was 88% homologous to that of the rat. Compared to the rat, the regulatory region of the human TDO2 gene had an insertion of approximately 1064 bp of random DNA beginning at -293 bp and extending to -1357 bp. This displaced the glucocorticoid response element (GRE) occurring at -1174 bp in the rat to -1500 in the human. The proximal GRE at -419 in the rat was missing in the human. However, within the DNA insert there was a GRE-like microsatellite region containing multiple GTT repeats plus additional GT(n) sequences. This could produce several staggered regions of the sequence TGTTGTnnnTGTTGT similar to a GRE consensus sequence of TGTTCAnnnTGTTCT. The intron regions 5' and 3' to each exon were sequenced This showed a HIS --> Val mutation polymorphism in exon 7. Three introns, 1,5, and 6, were completely sequenced and examined for polymorphism. This identified two polymorphisms consisting of G -- >T and G --> A mutations 2 bp apart in intron 6. The 3' end of intron 5 showed an extensive CCCT pentanucleotide repeat that was markedly polymorphic. These polymorphisms allow the TDO2 gene to be examined for a possible role in psychiatric disorders.

Amino Acid Sequence↗
PubMed1993

The conserved terminal guanosine of a group I intron can help prevent reopening of the ligated exons.

The highly conserved terminal guanosine of the Tetrahymena group I intron was mutated to an adenosine. The intron still excised itself but more slowly than the wild-type. At very low concentrations of GTP only ligated exons were produced, but at high concentrations of GTP unligated exons accumulated and the 3' exon acquired a GTP at its 5' end. A series of experiments suggested that GTP was primarily reopening the ligated exons, rather than directly cleaving the 3' splice site. 5' Truncated forms of the wild-type intron can cleave RNA in trans. Cleavage takes place downstream of sequences similar to the last few bases of the 5' exon. The ligated exons would therefore be a potential substrate. We believe that the intron uses the terminal guanosine to compete with exogenous GTP until the ligated exons have dissociated from their binding site. Other interactions between intron sequences which are known to aid in 3' splice-site recognition may assist in this process.

Animals↗
PubMed1994

The ribosomal RNA gene region in Acanthamoeba castellanii mitochondrial DNA. A case of evolutionary transfer of introns between mitochondria and plastids?

Acanthamoeba castellanii, an amoeboid protozoan, occupies an intriguing position in phylogenetic trees based on nuclear rRNA sequences, branching together with or near (as an outgroup to) green algae and land plants. To gain insight into the organization, expression and evolutionary affiliations of the mtDNA of this non-photosynthetic protist, we determined the sequence of a 7778 base-pair region containing the single-copy large subunit (LSU) and small subunit (SSU) rRNA genes (rnl and rns, respectively) of the approximately 40 kilobase-pair A. castellanii mitochondrial genome. We also sequenced the 5'- and 3'-terminal portions of the corresponding LSU and SSU rRNAs. In A. castellanii mtDNA, rnl is flanked both upstream and downstream by a cluster of five tRNA genes, with rns and then cox1 (the cytochrome oxidase subunit 1 gene) following immediately further downstream. These genes are all in the same transcriptional orientation and are separated by only short non-coding spacers. Although rnl and rns are organized in a novel way in A. castellanii mtDNA, their SSU and LSU rRNA products are strikingly similar to their eubacterial homologs in primary sequence, secondary structure and post-transcriptional modification. In these characteristics, the A. castellanii mitochondrial rRNAs much more closely resemble their counterparts in land plants than do the corresponding mitochondrial rRNAs in the green alga, Chlamydomonas reinhardtii. Although no intervening sequences have so far been found in the mitochondrial rnl of angiosperms (flowering plants), A. castellanii mitochondrial rnl contains three group I introns, all located within highly conserved regions in the 3'-half of the gene and each possessing a free-standing open reading frame (ORF). The insertion site of one of these introns is identical to that of the single group I intron in the chloroplast rnl of C. reinhardtii, and sequence comparison reveals that these two introns (one mitochondrial, the other chloroplast) are structurally homologous both within the core region and within the ORFs they encode. These observations are indicative of intron movement between mitochondria and chloroplasts, either intracellularly in a photosynthetic, remote common ancestor of A. castellanii and C. reinhardtii or, more recently, as a result of an intercellular exchange of genetic information.

Acanthamoeba↗
PubMed1994

On the identification of group II introns in nucleotide sequence data.

Four different consensus sequences (GTI, group II identifiers) have been derived from domains V of known group II introns and are used as query input sequences for sensitive database screenings with the FASTA and LFASTA programs. The set of four GTI sequences can identify all domains V of the 96 known group II introns in the completely sequenced chloroplast genomes of Marchantia polymorpha, Epifagus virginiana, Oryza sativa, Nicotiana tabacum and the completely sequenced mitochondrial genomes of Saccharomyces cerevisiae, Podospora anserina, Schizosaccharomyces pombe and Marchantia polymorpha. Seven moderately high-scoring hits can easily be rejected as false-positives since they do not fulfil secondary structure requirements. Large FASTA outputs obtained after screening the entire nucleotide sequence database are evaluated in a second step by a program (D5SCAN) that allows the assignment of variable selection criteria for potential domain V secondary structures. Database searches with these routines yield evidence for several group II intron sequences previously unrecognized. These include novel intron structures in the cyanobacterium Synechocystis and in the mitochondrial genomes of Marchantia, soybean, pea, broad bean, sugar beet and a heterobasidiomycete. Potential intron remnants are found contributing to the secondary structure of rRNAs in several trypanosome species. At a given sensitivity of 95% positively identified true domains V, the search routine produces one false positive hit per 10,000 kb.

Amino Acid Sequence↗
PubMed1996

Analysis of the CYT-18 protein binding site at the junction of stacked helices in a group I intron RNA by quantitative binding assays and in vitro selection.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. Previous studies showed that CYT-18 binds with high affinity to the P4-P6 domain of the catalytic core and that there is some additional contribution to binding from the P3-P9 domain. Here, quantitative binding assays with deletion derivatives of the N. crassa mitochondrial large rRNA intron showed that at least 70% of the binding energy can be accounted for by the interaction of CYT-18 with the P4-P6 domain. Within this domain, P4 and P6 are required for high affinity CYT-18 binding, while the distal elements P5 and P6a may contribute indirectly by stabilizing the correct structure of the binding site in P4 and P6. CYT-18 binds to a small RNA corresponding to the isolated P4-P6 domain, but not to a permuted version of this RNA in which P4-P6 is a continuous rather than a stacked helix. Iterative in vitro selection experiments with the isolated P4-P6 domain showed a requirement for base-pairing to maintain helices P4, P6 and P6a, but indicate that P5 is subject to fewer constraints. The most strongly conserved nucleotides in the selections were clustered around the junction of the P4-P6 stacked helix, with ten nucleotides (J3/4-2,3, P4 bp -1 and 3, and P6 bp -1 and 2) found invariant in the context of the wild-type RNA structure. In vitro mutagenesis confirmed that replacement of the wild-type nucleotides at J3/4-2 and 3 or P4 bp-3 markedly decreased CYT-18 binding, reflecting either base specific contacts or indirect readout of RNA structure by the protein. Our results suggest that a major function of CYT-18 is to promote assembly of the P4-P6 domain by stabilizing the correct geometry at the junction of the P4-P6 stacked helix. The relatively large number of conserved nucleotides at the binding site suggests that the interaction of CYT-18 with group I introns is unlikely to have arisen by chance and could reflect either an evolutionary relationship between group I introns and tRNAs or interaction with a common stacked-helical structural motif that evolved separately in these RNAs.

Base Sequence↗
PubMed1997

Suppressors of cis-acting splicing-deficient mutations that affect the ribozyme core of a group II intron.

Many of the cis-dominant mutations that lead to respiratory deficiency by preventing maturation of specific yeast mitochondrial transcripts are found to affect the ribozyme core of group I and group II introns. We have searched for suppressors of mutations in the ribozyme-encoding sections of a group II intron, the first intron in the COX1 gene of Saccharomyces cerevisiae, which was independently subjected to in vitro site-directed mutagenesis. Three of the original mutants bore multiple mutations, which act synergistically, since for most individual mutations, suppressors could be obtained that ensured at least partial recovery of respiratory competence and splicing. Out of a total of ten suppressor mutations that were identified, three were second-site substitutions that restored postulated base-pairings in the ribozyme core. Remarkably, and as is observed for group I introns, at least half of the cis-dominant mutations in the first two group II introns of the COX1 gene affect sites that have been shown to participate in RNA tertiary interactions. We propose that this bias reflects cooperativity in the formation of ribozyme tertiary but not secondary structure, on the one hand, and the need for synergistic effects in order to generate a respiratory-deficient phenotype in the laboratory on the other. Finally, a novel in vivo splicing product of mutant cells is attributed to bimolecular splicing at high concentrations of defective transcripts.

Base Sequence↗
PubMed1997

Splicing and intron-internal RNA editing of trnK-matK transcripts in barley plastids: support for MatK as an essential splice factor.

Group II introns frequently require assistance by specific factors, maturases, for folding and effective splicing in vivo. The only putative maturase of higher plant chloroplasts is encoded by matK, located in the intron of trnK. We show that in barley matK transcripts are modified at a first codon base by C-to-U RNA editing. The resulting H --> Y substitution restores a sequence motif that is present in maturases of yeast and plant mitochondria and of Lactococcus ltrA and that is positioned within the X domain. Processing of trnK-matK transcripts was further investigated in plastids lacking functional ribosomes due to a mutation. Absence of the intron-encoded matK gene product in these plastids is correlated with the accumulation of precursor transcripts for tRNALys(UUU)-matK, processed to different degrees, and by the lack of mature and spliced tRNA molecules. These results suggest an essential role of MatK for splicing of its own transcript in vivo. Processing of the 5' end of trnK exon 1 was found to proceed efficiently also in the mutant plastids although the two tRNA exons were separated by the 2481 nt intron. Consequently, presence of the intron does not interfere with the formation of mature 5' termini.

Amino Acid Sequence↗
PubMed1999

Antagonistic substrate binding by a group II intron ribozyme.

In this study, the thermodynamic properties of substrate-ribozyme recognition were explored using a system derived from group II intron ai5gamma. Substrate recognition by group II intron ribozymes is of interest because any nucleic ac?id sequence can be targeted, the recognition sequence can be quite long (>/=13 bp), and reaction can proceed with a very high degree of sequence specificity. Group II introns target their substrates throug?h the formation of base-pairing interactions with two regions of the intron (EBS1 and EBS2), which are usually located far apart in the secondary structure. These structures pair with adjacent, corresponding sites (IBS1 and IBS2) on the substrate. In order to understand the relative energetic contribution of each base-pairing interaction (EBS1-IBS1 or EBS2-IBS2) to substrate binding energy, the free energy of each helix was measured. The individual helices were found to have base-pairing free energies similar to those calculated for regular RNA duplexes of the same sequence, suggesting that each recognition helix derives its binding energy from base-pairing interactions alone and that each helix can form independently. Most interestingly, it was found that the sum of the measured individual free energies (approximately 20 kcal/mol) was much higher than the known free energy for substrate binding (approximately 12 kcal/mol). This indicates that certain group II intron ribozymes can bind their substrates in an antagonistic fashion, paying a net energetic penalty upon binding the full-length substrate. This loss of binding energy is not due to weakening of individual helices, but appears to be linked to ribozyme conformational changes induced by substrate binding. This coupling between substrate binding and ribozyme conformational rearrangement may provide a mechanism for lowering overall substrate binding energy while retaining the full information content of 13 bp, thus resulting in a mechanism for ensuring sequence specificity.

Binding Sites↗
PubMed2001

The evolutionary split of Pinaceae from other conifers: evidence from an intron loss and a multigene phylogeny.

The second intron in the mitochondrial gene nad1 was surveyed using PCR, DNA sequencing, or Southern hybridization in 323 species (313 genera, 212 families) of seed plants. The intron was absent in all 22 species (22 genera, 8 families) of non-Pinaceae conifers studied, in Welwitschia mirabilis, and in seven angiosperms. Whereas absence of the intron in seven angiosperms and Welwitschia is likely due to seven independent losses when evaluated against the recently published multigene phylogenies, the lack of the intron in all non-Pinaceae conifers can be best explained by a single loss. These data suggest that the non-Pinaceae conifers represent a monophyletic group. We also conducted a phylogenetic analysis of seed plants using a combined data set of the partial exon and intron sequences of nad1 generated from this study and published sequences of mitochondrial cox1 and small subunit (SSU) rDNA, chloroplast rbcL, and nuclear 18S rDNA. The results supported the split of conifers into two groups: Pinaceae and non-Pinaceae conifers. The Gnetales were sister to Pinaceae, in agreement with the conclusion from other recent molecular phylogenetic studies that refute the anthophyte hypothesis.

Base Sequence↗
PubMed2002

A test of a mitochondrial gene-based phylogeny of woodpeckers (genus Picoides) using an independent nuclear gene, beta-fibrinogen intron 7.

A conservative estimate of the species tree for the woodpecker genus Picoides based on two mitochondrial protein-coding genes is tested using sequences of an independently evolving nuclear intron, beta-fibrinogen intron 7. The mitochondrial gene-based topology and the intron-based topology are concordant, and a partition-homogeneity statistical test did not detect phylogenetic heterogeneity. The intron evolves more slowly than the mitochondrial sequences and tends not to resolve relationships among recently evolved species. However, the intron is superior over mitochondrial genes in resolving older bifurcations in the phylogeny. The two data sets were combined resulting in a robust estimate of the Picoides species tree in which most every node is statistically supported by bootstrap proportions. The Picoides species tree clearly shows that many morphological and behavioral characters used to lump species into this single genus have evolved by convergent evolution. Picoides is considered the largest genus of woodpeckers, but the molecular-based species tree suggests that Picoides is actually a conglomerate of several smaller groups.

Animals↗
PubMed1996

Plant nuclear tRNA(Met) genes are ubiquitously interrupted by introns.

We have isolated three independent clones for nuclear elongator tRNA(Met) genes from an Arabidopsis DNA library using a tRNA(Met)-specific probe generated by PCR. Each of the coding sequences for tRNAMet in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA(Met) is dispersed at at least eight loci in the genome. The unspliced precursor tRNA(Met) intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA(Met) genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5'-distal end of each intron.

Arabidopsis↗
PubMed1995

The marine red alga Chondrus crispus has a highly divergent beta-tubulin gene with a characteristic 5' intron: functional and evolutionary implications.

We characterized a nuclear gene and its corresponding cDNA encoding beta-tubulin (gene TubB1) of the marine red alga Chondrus crispus. The deduced TubB1 protein is the most divergent beta-tubulin so far reported with only 64 to 69% amino acid identity relative to other beta-tubulins from higher and lower eukaryotes. Our analysis reveals that TubB1 has an accelerated evolutionary rate probably due to a release of functional constraints in connexion with a specialization of microtubular structures in rhodophytes. It further indicates that isoform diversity and functional differentiation of tubulins in eukaryotic cells may be controlled by independent selective constraints. TubB1 has a short spliceosomal intron at its 5' end which seems to be a characteristic feature of nuclear protein-coding genes from rhodophytes. The splice junctions of the four known rhodophyte introns comply well with the corresponding consensus sequences of higher plants in agreement with previous suggestions from phylogenetic inference that red algae and green plants may be sister groups. The paucity and asymmetrical location of introns in rhodophyte genes can be explained by differential intron loss due to conversion of genes by homologous recombination with cDNAs corresponding to reverse transcribed mRNAs or partially spliced pre-mRNAs, respectively. The identification of an intron containing TubB1 cDNA in C. crispus confirms that pre-mRNAs can escape both splicing and degradation in the nucleus prior to transport into the cytoplasm. Differential Southern hybridizations under non-stringent conditions with homologous and heterologous probes suggest that C. crispus contains a second degenerate beta-tubulin gene (or pseudogene?) which, however, is only distantly related to TubB1 as it is to the more conserved homologues of other organisms.

Amino Acid Sequence↗
PubMed1994

Similarity landscapes: a way to detect many structural and sequence motifs in both introns and exons.

When investigators undertake searches of DNA databases, they normally discard large numbers of alignments that demonstrate very weak resemblances to each other, retaining only those that show statistically significant levels of resemblance. We show here that a great deal of information can be extracted from these weak alignments by examining them en masse. This is done by building three-dimensional similarity landscapes from the alignments, landscapes that reveal whether an unusual number of individually nonsignificant alignments tend to match up to a particular region of the query sequence being searched. The power of the search is increased by the use of libraries consisting entirely of introns or of exons. We show that (1) similarity landscapes with a variety of features can be generated from both intron and exon libraries, using introns or exons as query sequences; (2) the landscape features are real and not a statistical artifact; (3) well-known protein motifs used as query sequences can generate various landscape features; and (4) there is some evidence for resemblances between short regions of sequence carried by introns and exons. One possible interpretation of these results is that both introns and exons may have been built up during their evolution from short regions of sequence that as a result are now widely distributed throughout eukaryotic genomes. Such an interpretation would imply that these short regions have common ancestry. Alternatively, the wide sharing of short pieces of DNA may reflect regions with particular structural properties that have arisen through convergent evolution. The similarity-landscape approach can be used to detect such widespread structural motifs and sequence motifs in the genome that might be missed by less-global searches. It can also be used in conjunction with algorithms developed for detecting significant multiple alignments by isolating promising subsets of the databases that can be examined in more detail.

Animals↗