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PubMed1987

Two spliceosomes can form simultaneously and independently on synthetic double-intron messenger RNA precursors.

We have investigated the formation of splicing complexes in vitro on mRNA precursors (pre-mRNAs) containing two introns. Sucrose gradient sedimentation analysis revealed that the double-intron substrate becomes associated with 60S structures, which are larger than the 50S splicing complexes we previously observed with single-intron pre-mRNA precursors. We have demonstrated that the 60S complex represents the assembly of two single splicing complexes on the individual introns by conversion of the 60S double splicing complexes into single 50S spliceosomes by oligodeoxynucleotide directed RNase H cleavage of the double-intron pre-mRNAs within the middle exon. In addition, we have observed by native gel electrophoresis a transient double 'pre-splicing' complex analogous to the 35S 'pre-splicing' complex previously found with single-intron pre-mRNAs. Our results indicate that splicing complexes can form independently and simultaneously on the individual introns of multi-intron pre-mRNAs and that the assembly of these multiple spliceosomes proceeds with the same stepwise pathway observed for single-intron RNAs.

Cell Nucleus↗

PubMed1994

Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers.

Group II introns ai1 and ai2 of the Saccharomyces cerevisiae mitochondrial COXI gene encode proteins having a dual function (maturase and reverse transcriptase) and are mobile genetic elements. By construction of adequate donor genomes, we demonstrate that each of them is self-sufficient and practises homing in the absence of homing-type endonucleases encoded by either group I introns or the ENS2 gene. Each of the S. cerevisiae group II self-mobile introns was tested for its ability to invade mitochondrial DNA (mtDNA) from two related Saccharomyces species. Surprisingly, only ai2 was observed to integrate into both genomes. The non-mobility of ai1 was clearly correlated with some polymorphic changes occurring in sequences flanking its insertion sites in the recipient mtDNAs. Importantly, studies of the behaviour of these introns in interspecific crosses demonstrate that flanking marker co-conversion accompanying group II intron homing is unidirectional and efficient only in the 3' to 5' direction towards the upstream exon. Thus, the polar co-conversion and dependence of the splicing proficiency of the intron reported previously by us are hallmarks of group II intron homing, which significantly distinguish it from the strictly DNA-based group I intron homing and strictly RNA-based group II intron transposition.

Base Sequence↗

PubMed1993

Analysis of the chloroplast large subunit ribosomal RNA gene from 17 Chlamydomonas taxa. Three internal transcribed spacers and 12 group I intron insertion sites.

Previous reports on the chloroplast large subunit rRNA genes of the two distantly related green algae Chlamydomonas eugametos and Chlamydomonas reinhardtii indicate differences in the distribution of group I introns and suggest a different arrangement of internal transcribed spacers. To provide insights into the origin of these two types of intervening sequences, we have undertaken the sequencing of the chloroplast rrnL genes of 15 additional Chlamydomonas taxa and have characterized the mature large subunit rRNA species they encode in addition to those specified by the C. reinhardtii rrnL. These analyses disclosed the presence of three internal transcribed spacers sharing the same positions in all of the 17 taxa as well as the presence of a total of 39 group I introns representing 12 insertion sites. Of these insertion sites, only one has been identified in non-Chlamydomonas taxa. The distribution of Chlamydomonas introns is highly variable and, in many respects, is not consistent with the phylogeny deduced from chloroplast rRNA sequence comparisons. This phylogeny features two main lineages of Chlamydomonas taxa forming sister groups. Because earlier branching organisms in the green algal/land plant lineage display no chloroplast rDNA introns, it appears that all of the intron insertion positions in Chlamydomonas are of recent origins, with some of the positions having arisen subsequent to the divergence of the two main Chlamydomonas lineages. Remarkably, the rRNA regions corresponding to most of the group I intron insertion positions in rRNA genes have been assigned functional roles suggesting that they lie in exposed regions of the ribosome. On the basis of this striking correlation between exposed rRNA regions and intron insertion sites, we speculate that the reversal of the self-splicing reaction has played a major role in the creation of the multiple intron insertion positions found in rRNA genes as well as in the proliferation of group I introns elsewhere in the Chlamydomonas chloroplast genome.

Animals↗

PubMed2000

Split gene origin and periodic introns.

The introns-early view has been challenged for several genes; prominent instances are triose phosphate isomerase (TPI), aldolase, pyruvate kinase (PK), alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and myosin heavy chain. While some of their introns appear to be phylogenetically ancient and/or to delineate exons corresponding to protein modules, a considerable number seemingly do not. But it is argued here that many of these anomalous introns are periodic, that is, relics of internal sequence repetitions within the ancestral gene. Some of these periodic-intron patterns are shared between related genes, as in the alphabeta -barrels of TPI, aldolase and PK, or the Rossmann nucleotide-binding domain common to PK, ADH and GAPDH. This is further evidence for the ancestral status of these introns. The myosin heavy chain C-terminal rod region is paradoxical in that its sequence is clearly periodic but its intron placements are not; however, they exhibit a remarkable coherence of intron translational phases, suggesting that these introns may also have originally had a periodic arrangement now obscured by intron slipping.

Alcohol Dehydrogenase↗

PubMed1999

Effect of rev on the cytoplasmic localization of intron-containing human immunodeficiency virus type 1 RNA.

Human immunodeficiency virus type 1 (HIV-1) proteins are expressed from both intron-containing and completely spliced RNAs. Rev, an HIV-1 regulatory protein, is necessary for the expression of intron-containing RNAs. The effect of Rev on the subcellular localization of intron-containing HIV-1 RNA was examined by in situ RNA hybridization. In the presence of Rev, intron-containing HIV-1 RNA accumulated at the nuclear membrane and within the cytoplasm of transfected cells. In the absence of Rev, intron-containing HIV-1 RNA accumulated within the nucleus. In approximately 20% of the cells transfected in the absence of Rev, intron-containing HIV-1 RNA was also found in the cytoplasm. Differences in the subcytoplasmic localization of intron-containing HIV-1 RNA in the presence and absence of Rev were not observed using in situ RNA hybridization. To determine the effect of Rev on RNA localization within the cytoplasm, an extensive fractionation protocol involving both hypotonic and detergent lysis was used. In the presence of Rev, 40.9 +/- 4.6% of the cytoplasmic intron-containing HIV-1 RNA was released by hypotonic lysis. A similar fractionation profile was seen for several other translated viral and cellular RNAs. However, in the absence of Rev, only 16.5 +/- 5.1% of the cytoplasmic intron-containing HIV-1 RNA was released on hypotonic lysis (P < 0. 005). Thus the cytoplasmic fractionation pattern of this RNA was altered in the absence of Rev.

Cell Line↗

PubMed1994

In vivo analysis of intron processing using splicing-dependent reporter gene assays.

The mechanisms of intron recognition and processing have been well-studied in mammals and yeast, but in plants the biochemistry of splicing is not known and the rules for intron recognition are not clearly defined. To increase understanding of intron processing in plants, we have constructed new pairs of vectors, pSuccess and pFail, to assess the efficiency of splicing in maize cells. In the pFail series we use translation of pre-mRNA to monitor the amount of unspliced RNA. We inserted an ATG codon in the Bz2 (Bronze-2) intron in frame with luciferase: this construct will express luciferase activity only when splicing fails. In the pSuccess series the spliced message is monitored by inserting an ATG upstream of the Bz2 intron in frame with luciferase: this construct will express luciferase activity only when splicing succeeds. We show here, using both the wild-type Bz2 intron and the same intron with splice site mutations, that the efficiency of splicing can be estimated by the ratio between the luciferase activities of the vector pairs. We also show that mutations in the unique U-rich motif inside the intron can modulate splicing. In addition, a GC-rich insertion in the first exon increases the efficiency of splicing, suggesting that exons also play an important role in intron recognition and/or processing.

Base Sequence↗

PubMed1995

Intron-dependent transient expression of the maize GapA1 gene.

Transient expression experiments show that the maize GapA1 promoter exhibits a requirement for sequences contained within intron 1 and surrounding exon border regions for expression in maize Black Mexican Sweet cells. Maize GapA1-promoter constructs lacking intron 1 are inactive. Intron 1 and its exon border sequences, when reintroduced into constructs lacking introns, restore gene activity whereas intron 2 and its exon borders to not. The minimal promoter so defined encompasses roughly 250 bp upstream of the in vivo transcription start and appears also to include intron 1. An octameric sequence was identified in intron 1 of maize GapA1 which is similar to sequence motifs found in other maize introns known to increase transient expression. Partial restoration of gene expression in GapA1 constructs lacking intron 1 was achieved through insertion of the identified octameric sequence.

Base Sequence↗

PubMedDate not supplied

Transmission, recombination and conversion of mitochondrial markers in relation to the mobility of a group I intron in Chlamydomonas.

Mitochondrial DNA transmission has been analyzed in diploids produced from sexual crosses or artificial fusions between Chlamydomonas strains which differ by several genetic markers: a group I intron (Cs cob. 1 or alpha intron), three restriction sites (Nh, Nc and H markers) located 0.5-5 kb from the insertion site of the intron, and a MUD2 point mutation (27 bp from the insertion site) conferring resistance to myxothiazol. Recombination between mitochondrial markers is a general property of all crosses and fusions analyzed. In crosses between two intron-containing (alpha+) strains or two intron-less (alpha-) strains, the transmission is preferentially paternal (mt-), with a preponderance depending on the nature of the parental genomes. In crosses between alpha+ and alpha- strains, the conversion of intron-less molecules intron+ is frequent when the alpha+ parent is maternal (mt+) and nearly absolute when the alpha+ parent is paternal (mt-). In 94% of cases, the conversion is accompanied by the co-conversion of the MUD2 marker. In both crosses and artificial fusions, the conversion of alpha- into alpha+ also influences the transmission of the more distant Nh, Nc and H markers. It is hypothesized that the more frequent transmission of the genome containing the intron results from the elimination of alpha- molecules, as a result of a double-strand cut which is induced by an endonuclease encoded by the intron.

Animals↗

PubMed1990

The absence of introns in yeast mitochondria does not abolish mitochondrial recombination.

The respiratory competency of a yeast strain devoid of mitochondrial introns is quite normal. However, it may be asked whether intron-encoded proteins participate in metabolisms other than those of mitochondrial introns. Using strains without mitochondrial introns we have answered two questions. The first was: does the absence of intron-encoded proteins abolish mitochondrial recombination? The second was: do mitochondrial introns and intron-encoded proteins play a part in mitochondrial DNA rearrangements induced by ethidium bromide (rho- production)? We have shown that the introns and intron-encoded proteins are not essential components of either phenomenon.

DNA, Fungal↗

PubMed1991

Distribution of mitochondrial intron sequences among 21 yeast species.

The mitochondrial and nuclear genomes of 21 yeast species belonging to 12 genera have been tested for the presence of sequences similar to seven S. cerevisiae mitochondrial introns (Se cox1.1,2,3,4,5c, Sc cob.4 and Sc LSU.1) and one K. lactis mitochondrial intron (K1 cox1.2). Some introns, (Sc cox1.4, Sc cob.4, Sc LSU.1 and Kl cox1.2-all group I type), are widely distributed and are found in species with either basidiomycete or ascomycete affinities. This distribution is suggestive of recent sequence transfer between species. The remaining S. cerevisiae introns cross react with an additional species but with no set pattern. Pulsed field gel electrophoretic studies confirm that none of the tested mitochondrial introns cross react with nuclear DNA. These introns are, therefore, mitochondria-specific. Seven strains of K. lactis exhibit striking variability in intron content. In contrast to all mitochondrial introns tested, two introns of nuclear genes (the K. lactis actin gene and the S. cerevisiae RP29B gene) are not detected beyond their source species.

DNA, Fungal↗

PubMed1988

RNA splicing in yeast mitochondria: DNA sequence analysis of mit- mutants deficient in the excision of introns aI1 and aI2 of the gene for subunit I of cytochrome c oxidase.

We have characterized two yeast mutants deficient in the splicing of transcripts of the mitochondrial gene for cytochrome c oxidase subunit I (coxI). Both map to the first intron (aI1). RNA blot analysis shows that in addition to a reduced (mutant M15-190) or blocked (mutant M12-193) excision of the mutated intron aI1, the mutants are unable to excise the adjacent aI2 intron, the reading frame of which displays an amino acid sequence similarity to aI1. Splicing of the downstream introns is not affected, however. Sequence analysis of the first mutant DNA (M12-193) reveals a premature termination of the intron-encoded open reading frame, followed by two alterations at a short distance downstream. The other (M15-190) contains 11 separate changes. Although these occur in the intron reading frame, their main effect on RNA splicing may be exerted through the disturbance of intron secondary structure proposed for the 5' end of several group II introns. The implications of these findings in relation to maturase function and structure of intron aI1 are discussed.

Base Sequence↗

PubMed1989

An optional group I intron between the chloroplast small subunit rRNA genes of Chlamydomonas moewusii and C. eugametos.

We report the presence of a 402 bp group I intron in the chloroplast small subunit (SSU) rRNA gene of Chlamydomonas moewusii. The intron in inserted within the highly conserved '530 loop', at a site corresponding to positions 531-532 of the E. coli 16rRNA. Residues surrounding the insertion site almost certainly play an important role in ribosomal proofreading function as they proved to be protected by tRNAs in E. coli 16S rRNA (Moazed and Noller 1986; Stern et al. 1986). The C. moewusii intron revealed a secondary structure model which differs substantially from those of the typical subgroup IA and IB introns. This model, however, shows striking similarities with the structures of the C. reinhardtii chloroplast 23S rRNA gene intron (Rochaix et al. 1985), the S. cerevisiae mitochondrial COB3 intron (Holl et al. 1985) and the three introns of phage T4 in the nrdB, td and sunY genes (Shub et al. 1988). The SSU rRNA gene intron is absent from C. eugametos, an alga that is interfertile with C. moewusii. The presence/absence of the intron account for a 390 bp restriction fragment length polymorphism between the two algal SSU rRNA genes, a polymorphic locus that is strictly co-inherited with a tightly linked streptomycin resistance mutation (sr-2) in interspecific hybrids between the two algae.

Base Sequence↗

PubMed1990

Conservation of intron position indicates separation of major and variant H2As is an early event in the evolution of eukaryotes.

Genomic clones of Drosophila and Tetrahymena histone H2A variants were isolated using the corresponding cDNA clones (van Daal et al. 1988; White et al. 1988). The site corresponding to the initiation of transcription was defined by primer extension for both Drosophila and Tetrahymena genomic sequences. The sequences of the genomic clones revealed the presence of introns in each of the genes. The Drosophila gene has three introns: one immediately following the initiation codon, one between amino acids 26 and 27 (gln and phe), and one between amino acids 64 and 65 (glu and val). The Tetrahymena gene has two introns, the positions of which are identical to the first two introns of the Drosophila gene. The chicken H2A.F variant gene has been recently sequenced and it contains four introns (Dalton et al. 1989). The first three of these are in the same positions as the introns in the Drosophila gene. The fourth intron interrupts amino acid 108 (gly). In all cases the sizes and the sequences of the introns are divergent. However, the fact that they are in conserved positions suggests that at least two of the introns were present in the ancestral gene. A phylogenetic tree constructed from the sequences of the variant and major cell cycle-regulated histone H2A proteins from several species indicates that the H2A variant proteins are evolutionarily separate and distinct from the major cell cycle-regulated histone H2A proteins. The ancestral H2A gene must have duplicated and diverged before fungi and ciliates diverged from the rest of the eukaryote lineage. In addition, it appears that the variant histone H2A proteins analyzed here are more conserved than the major histone H2A proteins.

Amino Acid Sequence↗

PubMed1996

Intron-specific stimulation of anaerobic gene expression and splicing efficiency in maize cells.

Most of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes characterized in plants and algae to date have one intron very close to the 5' end of the gene. To study the functional relevance of some of these introns for gene expression we have analysed the influence of three 5' introns on transient gene expression of the anaerobically inducible maize GapC4 promoter in maize cells. Under aerobic conditions, reporter gene expression is increased in the presence of the first introns of the GapC4 and GapC1 genes, and the first intron of the nuclear encoded chloroplast-specific GapA1 gene. In contrast, the GapC4 intron increases anaerobic gene expression above the level obtained for the intronless construct, while anaerobic expression of constructs harboring the GapA1 and GapC1 introns was similar to the anaerobic expression level of the intronless construct. Splicing analysis revealed that the GapC4 intron is processed more efficiently under anaerobic conditions, while no change in splicing efficiency is observed for the GapC1 and the GapA1 introns when subjected to anaerobic conditions. These results suggest that an increase in splicing efficiency contributes to the anaerobic induction of the maize GapC4 gene.

Base Sequence↗

PubMed1996

Group-I intron family in the nuclear ribosomal RNA small subunit genes of Cenococcum geophilum isolates.

A family of optional group-I introns was found near the 3' end of the nuclear small subunit rRNA genes in 61 out of 70 isolates of the deuteromycete mycorrhizal fungus Cenococcum geophilum. DNA sequence polymorphisms among the introns (termed CgSSU introns) from ten of the isolates were studied. The sequences, ranging in size from 488 to 514 nucleotides, were from 93.2% to 99.6% similar to each other. Mutations were less common in predicted base-paired regions (33% of all mutations) than in free-standing regions (67%). The introns were self-spliced in vitro and were closest to subgroup IC1 according to sequence and predicted secondary structure. Group-I intron pairing regions P1 through P10, including core regions P, Q, R and S, were present in all ten CgSSU introns studied. No lengthy open reading frames were found in any of the introns, indicating that the introns do not encode a protein, and therefore may not be mobile. It is likely that a single intron entered a progenote of C. geophilum and changed as the species evolved.

Base Sequence↗

PubMed1999

Intron-genome size relationship on a large evolutionary scale.

The intron-genome size relationship was studied across a wide evolutionary range (from slime mold and yeast to human and maize), as well as the relationship between genome size and the ratio of intervening/coding sequence size. The average intron size is scaled to genome size with a slope of about one-fourth for the log-transformed values; i.e., on the global scale its increase in evolution is lower than the increase in genome size by four orders of magnitude. There are exceptions to the general trend. In baker's yeast introns are extraordinarily long for its genome size. Tetrapods also have longer introns than expected for their genome sizes. In teleost fish the mean intron size does not differ significantly, notwithstanding the differences in genome size. In contrast to previous reports, avian introns were not found to be significantly shorter than introns of mammals, although avian genomes are smaller than genomes of mammals on average by about a factor of 2.5. The extra-/intragenic ratio of noncoding DNA can be higher in fungi than in animals, notwithstanding the smaller fungal genomes. In vertebrates and invertebrates taken separately, this ratio is increasing as the increase in genome size. Two hypotheses are proposed to explain the variation in the extra-/intragenic ratio of noncoding DNA in organisms with similar numbers of genes: transition (dynamic) and equilibrium (static). According to the transition model, this variation arises with the rapid shift of genome size because the bulk of extragenic DNA can be changed more rapidly than the finely interspersed intron sequences. The equilibrium model assumes that this variation is a result of selective adjustment of genome size with constraints imposed on the intron size due to its putative link to chromatin structure (and constraints of the splicing machinery).

Animals↗

PubMed2002

Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease.

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.

Cloning, Molecular↗

PubMed2002

The evolution of single-copy Drosophila nuclear 4f-rnp genes: spliceosomal intron losses create polymorphic alleles.

This study provides the first report in which spliceosomal intron losses within a single-copy gene create functional polymorphic alleles in a population. 4f-rnp has previously been shown to be a nuclear gene that is localized on the X chromosome in D. melanogaster and to have eight short spliceosomal introns. An insect species survey was done via polymerase chain reaction (PCR) amplification and sequencing of a 1028-bp gene fragment spanning introns 4-8, which are located in the 3' half of the gene. The results show that 4f-rnp and (thus far) introns 7 and 8 are at least as old as order Odonata (dragonflies), an early-diverging insect line. Unexpectedly, several species within the dipteran family Drosophilidae were found to contain two differently sized 4f-rnp gene sequence variants, owing to precise in-frame intron losses. Results of single-male D. melanogaster PCR analyses show that the two gene size variants are allelic and that the intron loss mechanism appears to be biased toward the 3' end of the gene. A stable potential stem-loop has been identified in D. melanogaster, predicted to fold the 4f-rnp mRNA 3' terminus into a natural primer for subsequent reverse transcription into cDNA. When results are displayed in a phylogenetic context, multiple independent intron loss events are identified. These observations support a model in which frequently occurring cDNAs have led to numerous independent intron losses via homologous recombination/gene conversion during 4f-rnp gene evolution. The results provide insights into the evolution of intron loss and may lead to improved understanding of the dynamics of this process in natural populations.

Animals↗