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PubMed2000

The cyanobacterial origin and vertical transmission of the plastid tRNA(Leu) group-I intron.

We have surveyed the distribution and reconstructed the phylogeny of the group-I intron that is positioned in the anticodon loop of the tRNA(Leu) gene in cyanobacteria and several plastid genomes. Southern-blot and PCR analyses showed that the tRNA(Leu) intron is found in all 330 land plants that were examined. The intron was also found, and sequenced, in all but one of nine charophycean algae examined. Conversely, PCR analyses showed that the tRNA(Leu) group-I intron is absent from the red, cryptophyte and haptophyte algae, although it is present in three members of the heterokont lineage. Phylogenetic analyses of the intron indicate that it was present in the cyanobacterial ancestor of the three primary plastid lineages, the Rhodophyta, Chlorophyta, and Glaucocystophyta. Its present-day distribution in plastids is consistent with a history of strictly vertical transmission, with no losses in land plants, several losses among green algae, and nearly pervasive loss in the Rhodophyta and its secondary derivatives.

Anticodon↗
PubMed1997

Association of transcripts from a group-I intron-containing gene with high sedimentation coefficient particles.

The mitochondrial gene coding for the large rRNA contains a self-splicing optional group-I intron (Sc-LSU.1) in some Saccharomyces cerevisiae strains. Although the mechanisms of splicing have been extensively studied, little is known about the possible interactions of this intron with other mitochondrial molecules such as proteins. Using glycerol gradients, we have compared the sedimentation coefficients of mitochondrial transcripts containing the Sc-LSU.1 intron in native yeast extracts and in purified RNA preparations. By comparing extracts from rho+ and rho- cells we have found that at least three RNA species containing the Sc-LSU.1 intron (4.5 kb, 2.7 kb and 1.2 kb respectively) are associated in vivo with a multimolecular complex of sedimentation coefficient 50S made up of nuclearly encoded proteins. Another RNA species of 2.7 kb, which may correspond to a cleavage at the dodecamer sequence of the intron, is not associated with the same particle. The possibility that the 50S particle corresponds to the mitochondrial ribosome or its precursor form(s) is discussed.

Centrifugation, Density Gradient↗
PubMed1998

Group-I introns with unusual sequences occur at three sites in nuclear 18S rRNA genes of Acanthamoeba lenticulata.

Seven of eleven isolates of Acanthamoeba lenticulata were found to have group-I introns located at one of three positions within the 18S rRNA gene. The introns are 636-721-bp long and are absent from mature rRNA. They lack open reading frames that could encode any known endonucleases. Sequences of introns from the same site in different isolates are 86.0-98.9% identical, while from different sites they are 24.2-29.8% identical. The most closely related introns from other organisms are in the 18S rRNA genes of several green algae where the 17.0-23.6% identity is mostly limited to a highly conserved core of base pairs including P, Q, R and S. Because the A. lenticulata introns only occur in one Acanthamoeba lineage, they were probably acquired after the divergence of this species.

Acanthamoeba↗
PubMed1999

Variable sizes of introns in the SSU rDNA in three species of Roccella (Arthoniales, Euascomycetes).

Three species of the lichenized fungus Roccella (Arthoniales, Euascomycetes), R. canariensis, R. tuberculata and R. montagnei, were found to possess insertions in the first half of the nuclear SSU rDNA. Both the number and the type of these insertions varied between and within species. At position 516 the insertions belonged to group-IC1 introns with a characteristic secondary structure and conserved sequences. At three positions, 300, 330 and 393, the insertions were short and lacked the characteristic features of group-I introns. The splicing-site junctions of these short introns resembled those of mRNA introns. Furthermore, the conserved sequence flanking the branch-point A of mRNA introns was also present. Extraction of RNA and sequencing of cDNA confirmed that the insertions from positions 300, 330 and 516 had been spliced from the pre-rRNA during maturation. At position 393 the insertion had been spliced from R. canariensis, whereas in R. montagnei the insertion was still present in the rRNA.

Ascomycota↗
PubMed2001

Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5' splice site.

An intron-containing beta-glucuronidase (GUS) gene has been used widely in promoter analyses and as a plant transformation marker. Maximal plant gene expression requires accurate and efficient removal of the intron from the expressed pre-mRNA transcripts by splicing. Detailed analysis of splicing of potato ST-LS1 and pea legumin introns from GUS constructs revealed the activation of a cryptic 5' splice site in the GUS coding sequence 4 nt upstream from the authentic intron 5' splice site. About 40% of transcripts utilised the cryptic 5' splice site in tobacco protoplasts, reducing the translational potential of expressed pre-mRNA. The same cryptic splicing event was evident in transgenic tobacco leaves but at reduced levels. Mutations that removed the cryptic 5' splice site are associated with a two-fold enhancement in GUS activity in tobacco protoplasts, highlighting the need for careful examination of introns and their sites of insertion into gene constructs to minimise variability in gene activity and maximise gene expression.

Glucuronidase↗
PubMed1997

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron.

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1+ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron.

Ascomycota↗
PubMed2001

Mosses share mitochondrial group II introns with flowering plants, not with liverworts.

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.

Amino Acid Sequence↗
PubMed1997

Two partial deletion mutations involving the same Alu sequence within intron 8 of the LDL receptor gene in Korean patients with familial hypercholesterolemia.

Twenty-eight unrelated persons heterozygous for familial hypercholesterolemia (FH) were screened to assess the frequency and nature of major structural rearrangements at the low-density lipoprotein (LDL) receptor gene in Korean FH patients. Genomic DNA was analyzed by Southern blot hybridization with probes encompassing exons 1-18 of the LDL receptor gene. Two different deletion mutations (FH29 and FH110) were detected in three FH patients (10.7%). Each of the mutations was characterized by the use of exon-specific probes and detailed restriction mapping mediated by long-PCR (polymerase chain reaction). Mutation FH29 was a 3.83-kb deletion extending from intron 6 to intron 8 and FH110 was a 5.71-kb deletion extending from intron 8 to intron 12. In FH29, the translational reading frame was preserved and the deducible result was a cysteine-rich A and B repeat truncated protein that might be unable to bind LDL but would continue to bind beta-VLDL. FH110 is presumed to be a null allele, since the deletion shifts the reading frame and results in a truncated protein that terminates in exon 13. Sequence analysis revealed that both deletions have occurred between two Alu-repetitive sequences that are in the same orientation. This suggested that in these patients the deletions were caused by an unequal crossing over event following mispairing of two Alu sequences on different chromatids during meiosis. Moreover, in both deletions, the recombinations were related to an Alu sequence in intron 8 and the deletion breakpoints are found within a specific sequence, 27 bp in length. This supports the hypothesis that this region might have some intrinsic instability, and act as one of the important factors in large recombinational rearrangements.

Adolescent↗
PubMed2004

Whole exon 5 and intron 5 replaced by RHCE in DVa(Hus).

The DVa(Hus) was previously investigated through cDNA analysis, which revealed an RHD-CE(5)-D hybrid allele. However, the 5' and 3' breakpoints remain unknown. In this article, gene recombinations between the RHD and RHCE alleles were investigated by a combination approach of a sequence-specific primer PCR (PCR-SSP) and an RHD full-length coding region sequencing method on two Chinese subjects with weak D phenotypes. The hybrid Rhesus box of each individual was also investigated through an established PCR-based method. As a result, two partial D phenotypes, DVa(Hus) and DVI type III, were identified, each carrying one hybrid RHD-CE-D allele. The two samples were also serotyped with Rh phontypes of DccEe and DCcee, respectively. Other sequencing analyses of the DVaHus sample showed that the sequence of intron 4 is identical with RHD, whereas the whole sequence of exon 5 and intron 5 is identical with RHCE except for seven polymorphisms in the intron 5. We may concluded that in the case of this Chinese DVa(Hus), the whole exon 5 and complete intron 5 of a total segment of 1801 nucleotides were replaced by RHCE suggesting that the breakpoints of the replaced region are the 5' end of the exon 5 and the 3' end of the intron 5.

Alleles↗
PubMed1989

Mapping introns by exon excision.

A direct method for mapping introns has been devised. The technique makes use of a radioactive synthetic RNA transcript of the gene and a complementary, single-stranded DNA copy of mRNA-derived sequences. Upon hybridization of the cDNA to RNA and cleavage with ribonuclease H, only exonic RNA sequences are degraded. The surviving RNA fragments are the introns. Electrophoretic analysis in denaturing agarose gels reveals the number and size of the introns. The order of the introns is determined separately using unlabeled RNA transcripts; surviving RNA fragments are transferred to a solid support and the blot is probed sequentially with a nested set of genomic RNA transcripts of the opposite strand. Using the human prothymosin alpha gene as an example, four introns were identified which from 5' to 3' were 2.6, 0.47, 0.47, and 0.28 kb in size. From mapping and sequencing experiments the sizes are 2.6, 0.465, 0.459, and 0.295 kb, respectively. Similarly, the presence of two 300-bp insertions in a human prothymosin alpha pseudogene was established; the inserts were later identified as 295-bp Alu repetitive elements.

Blotting, Northern↗
PubMed1989

Selection of the 3'-splice site in group I introns.

A model for selection of 3'-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3'-splice site. This tertiary pairing would bring the 3'-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3'-exon sequences and the 'internal guide sequence' near the 5'-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3'-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.

Introns↗
PubMed1992

Exon-intron organization of the Arabidopsis thaliana protein kinase genes CDC2a and CDC2b.

We have previously shown by cDNA cloning that a higher plant, Arabidopsis thaliana, possesses at least two CDC2 genes (CDC2a and CDC2b) similar to the cell-cycle-controlling cdc2 gene of Schizosaccharomyces pombe. To understand the exon-intron organization of these genes, genomic clones were isolated and their nucleotide sequences determined. The coding and 5'-untranslated regions of CDC2a were interrupted by seven and one introns, respectively, whilst CDC2b contained three introns within the coding portion. These intron positions partly overlapped with each other and with those of the yeast cdc2 gene, nevertheless the lengths and sequences of the corresponding introns were diverse.

Amino Acid Sequence↗
PubMed1989

A proposal for a possible role of nucleosome positioning in the evolutionary adjustment of introns.

1. Prokaryotes and yeast have mostly intronless genes, whereas the presence of a large number of extended introns are characteristic of the genes of of multicellular eukaryotic organisms which, however, as an exception also have a few intronless genes. 2. According to the current view, the lack of introns in prokaryotic organisms and yeast is due to the selective pressure of a short cell division time. On the other hand, the presence of introns in multicellular eukaryotic organisms is explained by the lack of selective forces against them. 3. In the present hypothesis it is proposed that introns were used as tools in the course of evolution for the organization of eukaryotic genes within the repeating units of nucleosomes, since the distinct DNA conformations of the nucleosome core particle and of the linker region, respectively, represent a constraint for the positioning of genes. 4. Recently it was shown that initiation of transcription is inhibited when the promoter sequence is within a nucleosome. 5. Since the nucleosomal organization of DNA leads to a severely deformed DNA helix and recognition of sequences by regulatory proteins is likely to depend on the conformation of the double helix, it is postulated that for the different sizes of eukaryotic genes which have to be organized within repeating units of nucleosomes, introns provided the flexibility of adjustment for the positioning of regulatory sequences, by drifting in length, sequence and position.

Biological Evolution↗
PubMed1988

Testing for intron function in the essential Saccharomyces cerevisiae tRNA(SerUCG) gene.

The gene sup61+, which codes for the essential Saccharomyces cerevisiae tRNA(SerUCG), is the only single-copy tRNA gene in this organism know to contain an intron. To assess the role of this intron in tRNA gene expression, an intron-deleted sup61+ gene was constructed in vitro and introduced into the yeast genome. Isogenic intron- and intron+ strains were found to be indistinguishable by criteria that include growth rates, ability to undergo meiosis, levels of mature tRNA(SerUCG) transcribed in vivo, and the suppressor efficiency of amber- and ochre-specific alleles of this gene.

Genes, Fungal↗
PubMed1991

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae.

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

Adenosine Triphosphatases↗
PubMed1991

Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing.

Retroviruses splice only a fraction of their primary RNA transcripts to subgenomic mRNA. The unspliced RNA is transported to the cytoplasm, where it serves as genomic RNA as well as mRNA for the gag and pol genes. Deletion of sequences from the Rous sarcoma virus gag gene, which is part of the intron of the subgenomic mRNAs, was previously observed to result in an increase in the ratio of spliced to unspliced RNA. These sequences, which we termed a negative regulator of splicing (NRS), can be moved to the intron of a heterologous gene resulting in an accumulation of unspliced RNA in the nucleus. We have used such constructs, assayed by transient expression in chicken embryo fibroblasts, to define the minimal sequences necessary to inhibit splicing. Maximal NRS activity was observed with a 300-nt fragment containing RSV nts 707-1006; two noncontiguous domains within this fragment, one of which contains a polypyrimidine tract, were both found to be essential. The NRS element was active exclusively in the sense orientation in two heterologous introns tested and in both avian and mammalian cells. Position dependence was also observed, with highest activity when the NRS was inserted in the intron near the 5' splice site. The NRS element was also active at an exon position 136 nts upstream of the 5' splice site but not at sites further upstream. In addition, it did not affect the splicing of a downstream intron.

Animals↗
PubMed1990

Integration of group II intron bI1 into a foreign RNA by reversal of the self-splicing reaction in vitro.

Group II intron bI1, the first intron of the COB gene in the mitochondria of S. cerevisiae, is able to self-splice in vitro with the basic pathway similar to nuclear pre-mRNA splicing. We show that incubation of the intron lariat with ligated exons bE1 and bE2 leads to a complete reversal of the splicing reaction. The integration of the intron into the ligated exons is correct; the reconstituted preRNA of the reverse reaction can undergo a self-splicing reaction anew. When incubated with a foreign RNA species bearing a sequence motif that is complementary to exon binding site 1, the lariat can integrate into this RNA with the position of insertion immediately downstream of this sequence. This result implies that transposition of group II introns on the RNA level by reversal of the splicing reaction is, in principle, conceivable.

Base Sequence↗
PubMed1991

Trans splicing in Oenothera mitochondria: nad1 mRNAs are edited in exon and trans-splicing group II intron sequences.

The complete NADH dehydrogenase subunit 1 (nad1) ORF in Oenothera mitochondria is encoded by five exons. These exons are located in three distant locations of the mitochondrial genome. One genomic region encodes exon a, the second encodes exons b and c, and the third specifies exons d and e. Cis-splicing group II introns separate exons b and c and d and e, while trans-splicing reactions are required to link exons a and b and c and d. The two parts of the group II intron sequences involved in these trans-splicing events can be aligned in domain IV. Exon sequences and the maturase-related ORF in intron d/e are edited by numerous C to U alterations in the mRNA. Two RNA editing events in the trans-splicing intron a/b improve conservation of the secondary structure in the stem of domain VI. RNA editing in intron sequences may thus be required for the trans-splicing reaction.

Amino Acid Sequence↗