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PubMed1990

The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns.

The Saccharomyces cerevisiae nuclear gene NAM2 codes for mitochondrial leucyl-tRNA synthetase (mLRS). Herbert et al. (1988, EMBO J 7:473-483) proposed that this protein is involved in mitochondrial RNA splicing. Here we present the construction and analyses of nine mutations obtained by creating two-codon insertions within the NAM2 gene. Three of these prevent respiration while maintaining the mitochondrial genome. These three mutants: (1) display in vitro a mLRS activity ranging from 0%-50% that of the wild type: (2) allow in vivo the synthesis of several mitochondrially encoded proteins; (3) prevent the synthesis of the COXII protein but not of its mRNA; (4) abolish the splicing of the group I introns bI4 and aI4; and (5) affect significantly the excision of the group I introns bI2, bI3 and aI3. Importation of the bI4 maturase from the cytoplasm into mitochondria in a nam2- mutant strain does not restore the excision of the introns bI4 and aI4 implying that the splicing deficiency does not result from the absence of the bI4 maturase. We conclude that the mLRS is a splicing factor essential for the excision of the group I introns bI4 and aI4 and probably important for the excision of other group I introns.

Cell Nucleus↗
PubMed1990

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina.

In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron alpha) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron alpha, was detected in five senescent subclones which all showed strong amplification of the intronic alpha sequence (Sen DNA alpha).

Cloning, Molecular↗
PubMed1991

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome.

In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain anar-14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.

Amino Acid Sequence↗
PubMed1995

RNA editing of a group II intron in Oenothera as a prerequisite for splicing.

The trans-splicing group II intron c/d in the Oenothera mitochondrial nad1 gene is modified by RNA editing in domain 6. This C-to-U conversion generates the typical domain 6 structure, which prompted us to speculate that this RNA editing event might be essential for splicing. To test this hypothesis, we investigated the influence of unedited and edited sequences of the Oenothera intron on splicing in vitro. The stem of domain 6 of intron nad1-c/d was transplanted into the autocatalytic yeast intron aI5c, yielding chimeras with the genomic C and the edited U, respectively, 5' of the branchpoint A. When incubated under self-splicing conditions, only the edited chimera was released as a lariat, while the precursor with the genomically coded C remained inactive. Our results support the hypothesis that Oenothera group II intron nad1-c/d cannot be spliced from the primary transcript without previous editing in domain 6.

Base Sequence↗
PubMedDate not supplied

Frequent site-specific mit- deletions at cryptic exon-intron junctions in the COX1 gene of yeast mtDNA.

A class of large site-specific deletions (del-B) occurs with exceptionally-high frequencies of 10(-3) in the mitochondrial COX1 gene of Mn(2+)-treated yeast cells. This work shows that del-B deletions are associated with COX1 intron aI1. All five deletion mutants studied have their upstream end at the authentic 3' splice site of this intron. The deletion ends 8.2 kb downstream in intron aI5b. This downstream deletion-end constitutes a potentially-cryptic 5' splice site for intron aI1. The coincidences of the del-B deletion-ends with authentic and cryptic RNA splice sites suggest that the group-II intron aI1, and/or the RNA maturase encoded in it, plays an active role in this exceptionally-frequent, site-specific deletion process.

Base Sequence↗
PubMed1991

The group IIB intron from the green alga Scenedesmus obliquus mitochondrion: molecular characterization of the in vitro splicing products.

In the presence of high molar salt concentrations, the mitochondrial group IIB intron (rI1) from the green alga Scenedesmus obliquus is capable of splicing in vitro. After establishing the optimal conditions for RNA processing the in vitro splicing products were unequivocally identified in self-splicing experiments by Northern hybridization analysis employing 3'end-labelled RNAs or exon- and/or intron-specific probes. Finally, two trans-esterification products were identified by sequencing of the spliced RNA. From our data we conclude that the processing of group II introns from both algal and yeast mitochondria is preceded by identical consecutive trans-esterification steps. The predicted secondary and tertiary structure of intron rI1 of S. obliquus contains all the motifs necessary for optimal self-splicing and which are characteristic of other group IIB introns from different species.

Base Sequence↗
PubMed1993

A multitude of suppressors of group II intron-splicing defects in yeast.

Disruption of the nuclear MRS2 gene (mrs2-1 mutation) causes a strong pet- phenotype in strains with mitochondrial group II introns, and a leaky pet- phenotype in strains without group II introns. MRS3 and MRS4, the genes for two mitochondrial-solute carrier proteins, can suppress both phenotypes when present in high-copy-number plasmids. In order to search for further multicopy suppressors of the mrs2-1 mutant phenotype, an yeast genomic DNA library, MW90, was constructed in YEp351 from a strain deleted for the MRS2, MRS3 and MRS4 genes. Ten different Sau3A DNA fragments that act as multicopy suppressors of the mrs2-1 respiratory-deficient phenotype were isolated from this library. Some of the newly isolated genes suppress the pet- phenotypes of mrs2-1 cells in strains with and without mitochondrial group II introns. Other genes, however, are suppressors only for the mitochondrial intron-less strains. This supports the notion that the MRS2 gene product is bifunctional i.e., it is essential for the splicing of group II introns and is also involved in processes of mitochondrial biogenesis other than RNA splicing.

Genes, Fungal↗
PubMed1993

Splicing of group II introns in spinach chloroplasts (in vivo): analysis of lariat formation.

To investigate the mechanism of chloroplast mRNA splicing in vivo, RNAs from four spinach chloroplast group II intron-containing genes were analyzed. For each of these genes, atpF, rpoC1, petD, and petB, Northern analysis of chloroplast RNAs detected putative lariat-intron/3' exon-splicing intermediates. Treatment of these RNAs with HeLa cell-debranching extract caused the putative splicing intermediates to disappear, thereby confirming their identities. The lariat-splicing intermediates were further examined by reverse transcriptase extension to determine the branch point location. The in vivo branch points of the atpF and petD introns were found to be eight bases upstream of their respective 3' intron/exon boundaries. In contrast, no splicing intermediates could be detected by primer-extension analysis of petB and rpoC1. This unexpected result served to demonstrate that the quantity of lariat-intron/3' exon-splicing intermediates present in the chloroplast RNA population is considerably less in the cases of rpoC1 and petB compared to atpF and petD. The steady-state level of any splicing intermediate is the result of a balance between the splicing kinetics of a particular RNA and the susceptibility of the splicing intermediate to degradation. We conclude that the balance between these two factors varies significantly for chloroplast introns, even for those, such as petB and petD, that are transcribed from the same promoter.

Base Sequence↗
PubMed1986

An additional class II intron with homology to reverse transcriptase in rapidly senescing Podospora anserina.

Senescence in Podospora anserina is maternally inherited and the parameters of senescence are race specific. We have compared the restriction enzyme fragment maps of race A, the most rapidly senescing race, with race s and have found three inserts in race A which are not present in race s mitochondrial DNA. Fragment A was mapped and found to be located downstream of the so-called alpha senDNA, a class II intron, near the 5' end of the COI gene, separated from alpha senDNA by two class I introns. DNA sequence analysis showed that fragment A is also a class II intron, but with only 10% DNA sequence homology to alpha senDNA. Like alpha senDNA, intron A contains significant amino acid homology with known reverse transcriptases. The importance of this additional class II intron in the mitochondrial genome with the relative rate of senescence in race A is discussed.

Amino Acid Sequence↗
PubMed1989

Synthesis and function of the mitochondrial intron--encoded bI4 RNA maturase from Saccharomyces cerevisiae. Effects of upstream frame-shift mutations.

We have analyzed the expression and function of the intron-encoded bI4 maturase when frame-shift mutations in the upstream exon alter the translational process. By constructing secondary cis-acting mutations within the bI4 intron, we observed (1) that the bI4 maturase is still translated in the presence of the upstream mutation, albeit in very low amounts, and (2) that the limited amounts of bI4 maturase made under these conditions is no longer able to promote the splicing process of the aI4 intron. These observations, which further strengthen the maturase model, strongly suggest that bI4 maturase acts sequentially on the bI4 intron and then on the aI4 intron.

Base Sequence↗
PubMed1992

Self-splicing of a mitochondrial group I intron from the cytochrome b gene of the ascomycete Podospora anserina.

We have shown that the second intron of the Podospora mitochondrial gene coding for cytochrome b (Cytb 12) splices autocatalytically, using in vitro transcripts generated from the T7 promoter. The reaction takes place at 37 degrees C in the presence of 50 mM TRIS-HCl pH 7.5, 60 mM MgCl2 and 1 mM GTP but shows a low efficiency even at high KCl concentrations of up to 1.2 M. Under these conditions, intron bI2 follows the conventional pathway of group I splicing, and all characteristic products, with regard to both transesterification and hydrolysis, could be identified. Moreover, the intron is capable of undergoing cyclization, thereby releasing the noncoded G and one additional nucleotide (U) from the 5' end. The 5' cleavage site is preceded by the same two nucleotides, indicating a base-pairing at the same site of the internal guide sequence (IGS) for both splicing and cyclization ("one-binding-site model"). In addition, products resulting from site-specific hydrolysis 138 nucleotides downstream of the 5' splice site were detected. Unusually, the shortened intron is also able to form a circular RNA and an alternative sequence that aligns the cyclization site to the catalytic core of the intron must be assumed.

Ascomycota↗
PubMed1994

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice.

A new series of expression vectors, each comprised of the beta-lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSA in vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levels in vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml-1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml-1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trend in vitro with the trend of expression in vivo (% which express) at detectable levels (p = 0.0015) and at the level of greater than 0.1 mg ml-1 (p = 0.0156) was demonstrated. A weak correlation existed (p = 0.0526) at in vivo levels of 1 mg ml-1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.

Animals↗
PubMed1987

The analysis of intron data and their use in the detection of short signals.

In order to examine whether certain short DNA sequences (putative splice signals) occurred in a certain region of an intron more often than would be expected by chance, intron data were examined to see what structure they took. There were significant departures from equal nucleotide frequency, and successive nucleotides clearly did not occur independently in the rat and mouse introns examined. The nonindependence was mainly due to a CG shortage and a less marked TA shortage. However the pairwise frequencies explained almost all the variability in triplet frequencies in the data and so the data could be approximately modeled by using nucleotide frequencies conditional on what the previous nucleotide was. Some coding DNA was also examined and the pairs in second and third positions, and third and first positions in a codon, showed similar departures from independence to those of the intron data. Using the probability model derived for intron data, expected frequencies of putative signals were derived and compared with the observed frequencies.

Base Sequence↗
PubMed1996

A small insertion in the SSU rDNA of the lichen fungus Arthonia lapidicola is a degenerate group-I intron.

Insertions of less than 100 nt occurring in highly conserved regions of the small subunit ribosomal DNA (SSU rDNA) may represent degenerate forms of the group-I introns observed at the same positions in other organisms. A 63-nt insertion at SSU rDNA position 1512 (relative to the Escherichia coli SSU rDNA) of the lichen-forming fungus Arthonia lapidicola can be folded into a secondary structure with two stem loops and a pairing of the insertion and flanking sequences. The two stem loops may correspond to the P1 and P2, and the insertion-flanking pairing to the P10, of a group-I intron. Considering these small insertions as degenerate introns provides important clues to the evolution and catalytic function of group-I introns. Keywords Ribosomal DNA middle dot Small subunit middle dot 18s middle dot Degenerate introns middle dot Ascomycetes

Ascomycota↗
PubMed1997

Evolution of orthologous intronless and intron-bearing globin genes in two insect species.

While globin genes ctt-2beta and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2beta and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species.

Amino Acid Sequence↗
PubMed2002

The influence of adjacent nucleotides on the pattern of nucleotide substitution in mitochondrial introns of angiosperms.

It has been known that in noncoding regions of the chloroplast genome, the pattern of nucleotide substitution is influenced by the two nucleotides flanking the substitution site. In a GC-rich environment, a bias toward transition was observed, whereas in an AT-rich environment, a bias toward transversion was observed. In this study, the influence of the two adjacent neighbors on the substitution pattern was observed in the first intron of the mitochondrial nad4 gene, although the AT content of this intron is only 48%. The proportion of transversions increases from 0.32 to 0.75 as the A + T content (number of A's + T's) of the two nearest neighbors increases from 0 to 2. This trend was also observed in another mitochondrial group I intron with an AT content of 64%. In addition, a similar, though weaker, effect was observed in vertebrate pseudogenes. So this effect is present in all three types of genomes. Furthermore, in contrast to the situation in the noncoding regions of chloroplast DNA, where most nucleotide substitutions occurred in the categories with an A + T content of either 1 or 2, nucleotide substitutions in the mitochondrial first nad4 intron occurred more evenly in three categories of different A + T contents. This might be due largely to the difference in the AT content (0.48 vs. 0.72) between the mitochondrial first nad4 intron and the chloroplast DNA regions studied.

Base Composition↗
PubMed2005

Actin phylogeny and intron distribution in bangiophyte red algae(rhodoplantae).

The molecular phylogeny of red algal actin genes, with emphasis on the paraphyletic "Bangiophyceae," was examined and compared to the rhodophyte SSU rDNA phylogeny. Nineteen new genomic actin sequences and seven SSU rDNA sequences were obtained and subjected to diverse phylogenetic analyses (maximum likelihood, distance/neighbor-joining, maximum parsimony, Bayesian analyses, and, with respect to protein sequences, also quartet puzzling). The actin trees confirmed most of the major clades found in the SSU rDNA phylogenies, although with a lower resolution. An actin gene duplication in the florideophycean lineage is reported, presumably related to an increased complexity of sexual reproduction. In addition, the distribution and characteristics of spliceosomal introns found in some of the actin sequences were examined. Introns were found in almost all florideophycean actin genes, whereas only two bangiophyte sequences contained introns. One intron in the florideophycean actin genes was also found in metazoan, and, shifted by one or two nucleotides, in a glaucocystophyte, a cryptophyte, and two fungal actin genes, and thus may be an ancient intron.

Actins↗
PubMed2003

Extensive polymorphism and different evolutionary patterns of intron 2 were identified in the HLA-DQB1 gene.

Exon 2 and intron 2 of the HLA DQB1 gene from 20 individuals were cloned and sequenced and eight alleles were obtained. Based on our analysis, the nucleotide diversity of the 5' end of intron 2 was higher than the synonymous nucleotide diversity of exon 2, which may be due to the lower GC content and the 'hitch-hiking effect'. In contrast, the opposite phenomenon was observed for the 3' end of intron 2, which may be the result of the recombination between the 3' end and 5' end of intron 2 and the subsequent genetic drift. The results indicated that different regions of intron 2 in the DQB1 gene had different evolutionary patterns.

Alleles↗