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PubMed1990

Isolation and characterization of QCR9, a nuclear gene encoding the 7.3-kDa subunit 9 of the Saccharomyces cerevisiae ubiquinol-cytochrome c oxidoreductase complex. An intron-containing gene with a conserved sequence occurring in the intron of COX4.

A nuclear gene (QCR9) encoding the 7.3-kDa subunit 9 of the mitochondrial cytochrome bc1 complex from Saccharomyces cerevisiae has been isolated from a yeast genomic library by hybridization with a degenerate oligonucleotide corresponding to nine amino acids proximal to the N terminus of purified subunit 9. QCR9 includes a 195-base pair open reading frame capable of encoding a protein of 66 amino acids and having a predicted molecular weight of 7471. The N-terminal methionine of subunit 9 is removed posttranslationally because the N-terminal sequence of the purified protein begins with serine 2. The ATG triplet corresponding to the N-terminal methionine is separated from the open reading frame by an intron. The intron is 213 base pairs long and contains previously reported 5' donor, 3' acceptor, and TACTAAC sequences necessary for splicing. The splice junctions, as well as the 5' end of the message, were confirmed by isolation and sequencing of a cDNA copy of QCR9. In addition, the intron contains a nucleotide sequence in which 15 out of 18 nucleotides are identical with a sequence in the intron of COX4, the nuclear gene encoding cytochrome c oxidase subunit 4. The deduced amino acid sequence of the yeast subunit 9 is 39% identical with that of a protein of similar molecular weight from beef heart cytochrome bc1 complex. If conservative substitutions are allowed for, the two proteins are 56% similar. The predicted secondary structure of the 7.3-kDa protein revealed a single possible transmembrane helix, in which the amino acids conserved between beef heart and yeast are asymmetrically arranged along one face of the helix, implying that this domain of the protein is involved in a conserved interaction with another hydrophobic protein of the cytochrome bc1 complex. Two yeast strains, JDP1 and JDP2, were constructed in which QCR9 was deleted. Both strains grew very poorly, or not at all, on nonfermentable carbon sources and exhibited, at most, only 5% of wild-type ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from the deletion strains revealed slightly reduced levels of cytochrome b. When JDP1 and JDP2 were complemented with a plasmid carrying QCR9, the resulting yeast grew normally on ethanol/glycerol and exhibited normal cytochrome c reductase activities and optical spectra. These results indicate that QCR9 encodes a 7.3-kDa subunit of the bc1 complex that is required for formation of a fully functional complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1982

Critical sequences within mitochondrial introns: cis-dominant mutations of the "cytochrome-b-like" intron of the oxidase gene.

We have established the DNA sequence of two cis-dominant mutations located in the fourth intron, a14, of the yeast mitochondrial gene oxi3. These mutations prevent the synthesis of subunit I of cytochrome oxidase. Both mutations affect a very short DNA sequence located several hundred base pairs from the intron-exon junctions. An identical sequence is found in the cob-box gene; and this sequence is critical for the excision of the cytochrome b intron. Our interpretation is that this short sequence represents a common signal that must be recognized by the box7-encoded mRNA maturase, in conjunction with the mitochondrial ribosome, to splice out the introns in the two nonhomologous genes, cob-box and oxi3.

Base Sequence↗

PubMed2004

Polymorphisms and intronic structures in the 18S subunit ribosomal RNA gene of the fungi Scytalidium dimidiatum and Scytalidium hyalinum. Evidence of an IC1 intron with an His-Cys endonuclease gene.

The fungi Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are mainly encountered in (sub)tropical areas as plant pathogens and agents of human dermatomycosis. Because the classification and differentiation of these two species is unclear, we studied 22 S. dimidiatum and 15 S. hyalinum isolates in order to identify potential species-specific insertions and polymorphisms in the 18S subunit ribosomal gene. The presence of an IE intron in S. dimidiatum, together with a single polymorphism (A in S. dimidiatum, G in S. hyalinum) in the coding region, allowed us to differentiate these two species in most cases. Moreover, in one S. dimidiatum isolate we found a group IC1 intron containing a putative truncated His-Cys endonuclease gene. This enzyme shows strong similarity to the intronic homing endonuclease of Physarum polycephalum. Based on these results and our previous findings, we propose an evolutionary pathway for 18S rDNA S. dimidiatum insertions, implying independent events.

Ascomycota↗

PubMed1996

Two-dimensional graphical representation of DNA sequences and intron-exon discrimination in intron-rich sequences.

We have shown earlier that analysis of DNA sequences using a two-dimensional graphical representation provides considerable information on new global sequence patterns and homologies, repeated structures, relative base abundances, probable evolutionary paths and evolutionary divergence. We have also reported that at a more micro level the graphical representation reveals distinct differences in the features of intron and exon segments of eukaryotic sequences. In this paper, the distinguishing features of the intron and exon segments are exploited to show, through several examples of different gene structures, that an averaging procedure over the slopes of the representative maps provides an easy technique to differentiate between probable intron and exon regions. We thus expect that this method will enable a rapid search and preliminary indication of possible locations of protein coding regions in long eukaryotic sequences.

Animals↗

PubMed1999

Intron homing with limited exon homology. Illegitimate double-strand-break repair in intron acquisition by phage t4.

The td intron of bacteriophage T4 encodes a DNA endonuclease that initiates intron homing to cognate intronless alleles by a double-strand-break (DSB) repair process. A genetic assay was developed to analyze the relationship between exon homology and homing efficiency. Because models predict exonucleolytic processing of the cleaved recipient leading to homologous strand invasion of the donor allele, the assay was performed in wild-type and exonuclease-deficient (rnh or dexA) phage. Efficient homing was supported by exon lengths of 50 bp or greater, whereas more limited exon lengths led to a precipitous decline in homing levels. However, extensive homology in one exon still supported elevated homing levels when the other exon was completely absent. Analysis of these "one-sided" events revealed recombination junctions at ectopic sites of microhomology and implicated nucleolytic degradation in illegitimate DSB repair in T4. Interestingly, homing efficiency with extremely limiting exon homology was greatly elevated in phage deficient in the 3'-5' exonuclease, DexA, suggesting that the length of 3' tails is a major determinant of the efficiency of DSB repair. Together, these results suggest that illegitimate DSB repair may provide a means by which introns can invade ectopic sites.

Bacteriophage T4↗

PubMed1995

An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.

Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.

Alternative Splicing↗

PubMed1992

Constraints on the evolution of plastid introns: the group II intron in the gene encoding tRNA-Val(UAC).

The evolution of the group II intron in the plastid gene encoding tRNA(Val)UAC (trnV) from seven plant taxa was studied by aligning secondary and other structural features. Levels of evolutionary divergence between six angiosperms and a liverwort, Marchantia polymorpha, were compared for the six domains commonly demonstrated for group II introns and were shown to be statistically heterogeneous. Evolutionary rates varied substantially among various domains and other features. Domain II showed the highest evolutionary rate, approaching the synonymous substitution rate reported for cpDNA-encoded genes, while domain VI and the helix and loop region bearing EBS1 evolved at rates similar to those for nonsynonymous substitutions of a number of cpDNA-encoded genes. The minimum free-energy structure of domain I varied among the seven taxa, suggesting that possible protein-RNA or tertiary interactions are important for intron processing.

Base Sequence↗

PubMed1990

Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria.

A self-splicing group I intron has been found in the gene for a leucine transfer RNA in two species of Anabaena, a filamentous nitrogen-fixing cyanobacterium. The intron is similar to one that is found at the identical position in the same transfer RNA gene of chloroplasts of land plants. Because cyanobacteria were the progenitors of chloroplasts, it is likely that group I introns predated the endosymbiotic association of these eubacteria with eukaryotic cells.

Animals↗

PubMed1994

Insertion variant in intron 9, but not microsatellite in intron 2, of the insulin receptor gene is associated with essential hypertension.

OBJECTIVE: To determine whether a microsatellite polymorphism of the insulin receptor gene (INSR) is associated with essential hypertension, as has been demonstrated previously for an RsaI restriction fragment length polymorphism (RFLP), and to examine blood pressure and plasma lipid profiles in relation to genotype. DESIGN AND METHODS: The study involved 75 Caucasian, non-diabetic hypertensive patients whose parents were both hypertensive, and 75 age-matched normotensive subjects whose parents were each normotensive after the age of 50 years. Genotypes for the microsatellite polymorphism were determined for each subject using leucocyte DNA and a polymerase chain reaction method. Other parameters, including pretreatment blood pressure, body mass index and plasma lipids, were also determined. RESULTS: Comparison of microsatellite data for the eight genotypes and four alleles that were detected showed no significant difference by chi 2 analysis, either between the hypertensive and normotensive groups, or between obese and non-obese subgroups of hypertensives. This is in contrast to the significantly higher frequency seen for the R1- allele of an RsaI RFLP of INSR: 0.71 in the hypertensive group compared with 0.56 in the normotensive group. R1- allele frequency was elevated in all age groups of hypertensives and did not differ between obese and non-obese subgroups. The non-obese hypertensives also had different plasma lipid profiles according to genotypes of the RFLP, with higher total and low-density lipoprotein-cholesterol in patients having the hypertension-associated R1- allele of the intron 9 polymorphism. Moreover, systolic blood pressure was significantly greater in patients carrying the R1- allele and aged > or = 60 years. CONCLUSIONS: The present study allows definition of the hypertension-associated variants of INSR as those which are in linkage disequilibrium with a (CA)-repeat insertion polymorphism in intron 9 of the large, 22 exon, > 120-kb gene, but not those associated with a polymorphism in the second intron.

Adult↗

PubMed1993

Short report: insertion variant in intron 9, but not microsatellite in intron 2, of the insulin receptor gene is associated with essential hypertension.

OBJECTIVE: To determine whether a microsatellite polymorphism of the insulin receptor gene (INSR) is associated with essential hypertension, as has been demonstrated previously for an Rsal restriction fragment length polymorphism (RFLP), and to examine blood pressure and plasma lipid profiles in relation to genotype. DESIGN AND METHODS: The study involved 75 Caucasian, non-diabetic hypertensive patients whose parents were both hypertensive, and 75 age-matched normotensive subjects whose parents were each normotensive after the age of 50 years. Genotypes for the microsatellite polymorphism were determined for each subject using leucocyte DNA and a polymerase chain reaction method. Other parameters, including pretreatment blood pressure, body mass index and plasma lipids, were also determined. RESULTS: Comparison of microsatellite data for the eight genotypes and four alleles that were detected showed no significant difference by chi 2 analysis, either between the hypertensive and normotensive groups, or between obese and non-obese subgroups of hypertensives. This is in contrast to the significantly higher frequency seen for the R1- allele of an Rsal RFLP of INSR: 0.71 in the hypertensive group compared with 0.56 in the normotensive group. R1- allele frequency was elevated in all age groups of hypertensives and did not differ between obese and non-obese subgroups. The non-obese hypertensives also had different plasma lipid profiles according to genotypes of the RFLP, with higher total and low-density lipoprotein-cholesterol in patients having the hypertension-associated R1- allele of the intron 9 polymorphism. Moreover, systolic blood pressure was significantly greater in patients carrying the R1- allele and aged > or = 60 years. CONCLUSIONS: The present study allows definition of the hypertension-associated variants of INSR as those which are in linkage disequilibrium with a (CA)-repeat insertion polymorphism in intron 9 of the large, 22 exon, > 120-kb gene, but not those associated with a polymorphism in the second intron.

Adult↗

PubMed1984

Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene.

Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene. These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF. To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product. For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene. The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857. The hybrid protein was partially purified and antibodies were raised against it. These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell. These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF. The autoregulated mRNA maturase synthesis model is discussed in relation to these results.

Base Sequence↗

PubMed1985

Tobacco chloroplast tRNA(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron.

The nucleotide sequence of a tRNA(Lys)(UUU) gene on tobacco (Nicotiana tabacum) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNA(Gly)(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long.

Journal Article↗

PubMed1984

Intron bypass: a rapid procedure for eliminating introns from cloned genomic DNA and its application to a cellulase gene.

We have devised a DNA cloning procedure in which the introns present in a genomic DNA fragment can be eliminated easily and rapidly. The technique combines the methods of cDNA and genomic cloning in a way which assures full-length representation of the intron-free transcript. Moreover, plasmids made by this technique can be designed to contain flanking untranscribed regions which may play a role in the regulation of expression. One strand of a linearized plasmid containing the 3'-end of a gene is used to prime cDNA synthesis from an annealed mRNA template. A second plasmid containing the 5'-end of the gene is linearized, denatured, and annealed to the extended 3'-end molecules and the resulting circular, partial duplexes are used to transform bacterial cells. Two different recombinant plasmids which contain DNA encoding the cellulase, exocellobiohydrolase I, from Trichoderma reesei have been constructed using this method. They both contain the entire translated region of the gene uninterrupted by introns. One plasmid contains additional DNA at the 5'-end, including approximately 150 bp 5' to the start of transcription. The inserts of both plasmids can be excised in one piece.

Base Sequence↗

PubMed2000

A novel cryptic exon in intron 2 of the human dystrophin gene evolved from an intron by acquiring consensus sequences for splicing at different stages of anthropoid evolution.

The dystrophin gene, which is mutated in Duchenne muscular dystrophy, is thus the largest human gene. A full spectrum of splicing of the dystrophin transcript has not been elucidated yet, though more than 10 alternative splicings have been identified in the 5' region of the dystrophin gene. In this study, two novel dystrophin transcripts containing a 140-nucleotide insertion precisely between exons 2 and 8 or between exons 2 and 18 were identified in skeletal muscle. The genomic region corresponding to and surrounding this 140-nucleotide sequence was sequenced to reveal that the insertion possessed a branch point and both acceptor and donor splice site consensus sequences perfectly. Therefore, the 140-bp insertion sequence was considered to be a novel exon. The novel exon was mapped to intron 2 and was designated exon 2a. Reverse-transcription PCR screening for transcripts containing exon 2a in 12 human tissues revealed its presence in 3 of them, including skeletal muscle. Phylogenetic studies disclosed that exon 2a evolved from intron DNA by the progressive acquisition of nucleotide substitutions in ancestral hominoids.

Animals↗

PubMed1997

Plasticity of the mitochondrial genome in Podospora. Polymorphism for 15 optional sequences: group-I, group-II introns, intronic ORFs and an intergenic region.

The mitochondrial chromosome of 15 Podospora anserina and one Podospora comata wild-type strains have been extensively examined for the presence of optional elements and for sequence divergence. Among the P. anserina strains, nine optional sequences were found. By comparing P. anserina with the closely related and weakly interfertile P. comata species, six additional optional sequences were detected. These optional elements correspond to mitochondrial introns belonging to different groups and subgroups (11 cases), intronic open reading frames (two cases), a complex insert and an intergenic region. Although difficult to explain, the distribution of optional mitochondrial sequences among the 15 wild-type isolates of P. anserina is far from random.

Ascomycota↗

PubMed1995

A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility.

The mobility (homing) of the yeast mitochondrial DNA group II intron al2 occurs via target DNA-primed reverse transcription at a double-strand break in the recipient DNA. Here, we show that the site-specific DNA endonuclease that makes the double-strand break is a ribonucleoprotein complex containing the al2-encoded reverse transcriptase protein and excised al2 RNA. Remarkably, the al2 RNA catalyzes cleavage of the sense strand of the recipient DNA, while the al2 protein appears to cleave the antisense strand. The RNA-catalyzed sense strand cleavage occurs via a partial reverse splicing reaction in which the protein component stabilizes the active intron structure and appears to confer preference for DNA substrates. Our results demonstrate a biologically relevant ribozyme reaction with a substrate other than RNA.

Base Sequence↗

PubMed2000

Alzheimer's disease due to an intronic presenilin-1 (PSEN1 intron 4) mutation: A clinicopathological study.

We describe 21 affected individuals from a kindred with early-onset autosomal dominant familial Alzheimer's disease caused by an intronic presenilin-1 mutation (in intron 4). Mean age at onset of symptoms was 37.4 years [95% confidence interval (CI): 36.6-38.2 years], mean age at death was 44.7 years (95% CI: 43.1-46.3 years) and mean duration of illness was 7.3 years (95% CI: 5.9-8.7 years). Myoclonus and seizures were prominent features of this pedigree. In the four cases for whom neuropsychometric data were available, verbal memory impairment preceded visual memory deficits; naming was relatively preserved until late in the disease. One of these four cases underwent serial volumetric MRI scans demonstrating in vivo brain tissue loss of 3.9% (38.9 ml, annualized rate of atrophy: 1. 7%) over 22 months of follow-up. The four individuals who had necropsies demonstrated the neuropathological hallmarks of Alzheimer's disease. Apolipoprotein E (APOE) status was assessed in five individuals: the case with the youngest age at onset at 33 years of age was found to be homozygous epsilon4/epsilon4, > 1 SD below the mean age of onset for those of known APOE genotype (36.4 +/- 2.3 years, mean +/- SD), and > 2 SDs below the mean age of onset for the pedigree as a whole (37.4 +/- 1.7 years, mean +/- SD). APOE genotype may therefore modulate age at onset in this pedigree.

Adult↗

PubMed1996

Nucleotide sequences of MHC class I introns 1, 2, and 3 in humans and intron 2 in nonhuman primates.

HLA-class I genes are the most polymorphic genetic system yet known. The polymorphic substitutions are mostly located in exon 2 and 3, encoding alpha 1 and alpha 2 domains, respectively, which are involved in peptide binding and T cell receptor interaction. In this study, we present the sequences of the introns neighboring the polymorphic exons in humans with few examples from nonhuman primates. In general, intron sequences are found to be less polymorphic than the adjacent exons, displaying numerous locus-specific and group-specific sites. These sequences will provide important information for developing DNA based typing strategies for HLA-class I alleles.

Animals↗