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PubMed1998

Prolactin-inducible enhancer activity of the first intron of the bovine beta-casein gene.

We have analyzed the regulatory roles of the first intron (intron-1) of the bovine beta-casein gene in the bovine beta-casein/CAT expression system using a mouse mammary epithelial cell line, HC11. After a combined treatment of HC11 cells with insulin, dexamethasone and prolactin, the induced expression of p beta c1.8/+ICAT vector including 2 kb intron-1 and 1.8 kb promoter was greatly increased to 23.5 folds, while that of p beta ca.8CAT basic vector with 1.8 kb promoter only, was 6.5. A classical enhancer activity was shown in the 2 kb intron fragment from the experiment in which the orientation and the position of the intron-1 on the vectors were changed. The enhancer activity was largely dependent on the lactogenic hormones, especially prolactin. A stepwise reduction of the inducibility in the 5' to 3' deletion analysis of the intron-1 indicates the existence of several functional elements in the region. In particular, an internal fragment (+1071 to +1490) was important for the prolactin-dependent enhancing activity of the intron-1. These results suggest that several elements in the intron-1 of the bovine beta-casein gene cooperatively interact not only with each other but also with its promoter for hormonal induction.

Animals↗

PubMed1985

Evolution and structure of the fibrinogen genes. Random insertion of introns or selective loss?

Chromosomal linkage as well as sequence homologies provide unequivocal evidence that the genes for the alpha, beta and gamma chains of fibrinogen arose by successive duplication of a single ancestral gene. Yet, when the three fibrinogen chains are aligned by amino acid homology, the positions of intervening sequences coincide at only two positions for all three chains. While one additional intron occurs at a homologous site in the beta and gamma chains, none of the positions of the remaining 11 introns in the three genes is shared. This arrangement of introns in the three fibrinogen genes suggests that either introns were selectively lost, implying that there is essential information in the retained introns, or the common introns were present in the ancestral fibrinogen gene and introns have been randomly inserted since the triplication of the original gene. The more likely possibility of selective loss of introns implies that the ancestral gene, as it existed about one billion years ago, must have been composed of numerous small exons.

Amino Acid Sequence↗

PubMed1980

Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the omega and rib-1 loci.

The complete nucleotide sequence has been determined for the intron, its junctions and the flanking exon regions of the 21S rRNA gene in three genetically characterized strains differing by their omega alleles (omega+, omega- and omega n) and by their chloramphenicol-resistant mutations at the rib-1 locus. Comparison of these DNA sequences shows that: --omega+ differs from omega- and omega n by the presence of the intron (1143 bp), as well as by a second and unexpected mini-insert (66 bp) located 156 bp upstream within the exon, whose nature and functions are still unknown but whose striking palindromic structure may suggest a mitochondrial transposable element. --The two mutations C321R and C323R correspond to two different monosubstitutions, 56 bp apart in the omega- and omega n strains but separated by the intron in the omega+ strains. In relation to previous genetic results, a model is discussed assuming that the interactions of two different regions or genetic loci determine the chloramphenicol resistance, one of which contains the omega n mutations. --A long uninterrupted coding sequence able to specify a 235 amino acid polypeptide exists within the intron. This remarkable observation gives new insight into the origin of the mitochondrial introns and raises the question of the possible functions of intron-encoded polypeptides. Finally, sequence comparisons with evolutionarily distant organisms, showing that different rRNA introns are inserted at different positions of an otherwise highly conserved region of the gene, suggest a recent insertion of these introns and a mechanism for splicing after the assembly of the large ribosomal subunit.

Alleles↗

PubMed1986

Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease.

The intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (r1 intron) possesses a 235 codon long internal open reading frame (r1 ORF) whose translation product determines the duplicative transposition of that intron during crosses between intron-plus strains (omega+) and intron-minus ones (omega-). Using site-directed mutagenesis, we have constructed a universal code equivalent of the r1 ORF that, under appropriate promoter control, allows the overexpression in E. coli of a protein identical to the mitochondrial intron encoded "transposase". This protein exhibits a double strand endonuclease activity specific for the omega- site. This finding demonstrates, for the first time, the enzymatic activity of an intron encoded protein whose function is to promote the spreading of that intron by generating double strand breaks at a specific sequence within a gene.

Amino Acid Sequence↗

PubMed1986

A self-splicing RNA excises an intron lariat.

We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.

DNA, Mitochondrial↗

PubMed1986

Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns.

A functional cDNA from Aspergillus nidulans encoding triosephosphate isomerase (TPI) was isolated by its ability to complement a tpi1 mutation in Saccharomyces cerevisiae. This cDNA was used to obtain the corresponding gene, tpiA. Alignment of the cDNA and genomic DNA nucleotide sequences indicated that tpiA contains five introns. The intron positions in the tpiA gene were compared with those in the TPI genes of human, chicken, and maize. One intron is present at an identical position in all four organisms, two other introns are located in similar positions in A. nidulans and maize, and the remaining two introns are unique to A. nidulans. These Aspergillus-specific introns are located in regions of the protein that were predicted to be interrupted by introns based on analysis of a Go plot of chicken TPI. These comparisons are discussed in relation to the evolution of introns within TPI genes.

Amino Acid Sequence↗

PubMed1986

Self-splicing of group II introns in vitro: mapping of the branch point and mutational inhibition of lariat formation.

Group II intron bl1 from yeast mitochondria can undergo self-splicing in vitro. Exons become correctly ligated, and the excised intron has a lariat structure similar to that of introns from nuclear mRNA. The branch point of the bl1 lariat is located eight or nine nucleotides upstream of the 3' end of the intron and is part of a hairpin structure that is well conserved among group II introns. Several mutations next to the branch point and in other parts of the core structure of group II introns are shown to affect lariat formation. One of them, carried by strain M4873, abolishes splicing in vivo and in vitro, apparently by changing the architecture of the hairpin structure containing the branch point. Similarities between group II introns and nuclear pre-mRNA introns are discussed in terms of evolutionary relatedness.

Base Sequence↗

PubMed2004

Exon junction sequences as cryptic splice sites: implications for intron origin.

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns. To do this, we experimentally identified nine cryptic splice sites within the coding sequence of actin genes from humans, Arabidopsis, and Physarum by inactivating their normal intron splice sites. We found that seven of these cryptic splice sites correspond exactly to the positions of exon junctions in actin genes from other species. Because actin genes are highly conserved, we could conclude that at least seven actin introns are flanked by cryptic splice sites, and from the phylogenetic evidence, we could also conclude that actin introns were inserted into these cryptic splice sites during evolution. Furthermore, our results indicate that these insertion events were dependent upon the splicing machinery. Because most introns are flanked by similar sequences, our results are likely to be of general relevance.

Actins↗

PubMed1983

Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast.

The temperature-sensitive mutation rna2 causes the accumulation of higher molecular weight transcripts from the ribosomal protein 51 (rp51) gene of yeast and many other yeast ribosomal protein genes. We have determined the DNA sequence of the rp51 gene, confirming that it contains an intron and that the higher molecular weight transcript is an intron-containing precursor RNA. These data and other experiments suggest that the rna2 mutation affects mRNA processing (splicing) and that the presence of an intron is sufficient to render expression of a gene sensitive to the rna2 mutation. To test these hypotheses, we have inserted the rp51 intron into the coding region of a hybrid Escherichia coli beta-galactosidase gene, thereby interrupting the open reading frame subsequent to the initiating methionine codon. Despite the presence of the intron, the beta-galactosidase gene is expressed in yeast. Thus, the rp51 intron is properly excised from the normally intronless gene. The presence of the rp51 intron causes the beta-galactosidase activity to be sensitive to the rna2 mutation, consistent with the notion that this mutation affects gene expression at the level of splicing. The experiments suggest that an intron-containing beta-galactosidase gene can be used in a general way to study mRNA splicing.

Amino Acid Sequence↗

PubMed1992

Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage.

The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids. We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function. Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii. Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion. Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting. Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns. Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron.

Amino Acid Sequence↗

PubMed1987

Cytochrome Oxidase Subunit II Gene from Carrot Contains an Intron.

Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map.

Journal Article↗

PubMed1996

Analysis of a hotspot for deletion formation within the intron of the chloroplast trnI gene.

The chloroplast genomes of higher plants encode several tRNA genes that contain highly conserved type II introns. Using primers specific to conserved 5' and 3' regions within the introns of the genes trnA (tRNA-ala) and trnI (tRNA-ile) we have PCR amplified parts of these introns from 36 plant species representing a wide range of plant families. Deletions were found in the introns of both tRNA genes. Fourteen species had detectable deletions within the intron of trnI and four species within the intron of trnA. The occurrence of these deletions among the various plant families suggests that the events leading to the formation of these deletions occurred independently many times during the evolution of higher plants. Analysis of the amplified PCR products from the trnI intron suggests that these independent deletions may not be random but appear to fall into two size classes. Several members of each class were cloned and sequenced and the end points of the deletions were mapped. The 3' ends of all deletions studied terminate within the same short region. The 5' ends of the deletions map to two different regions, giving rise to the two size classes. These two 5' deletion endpoint regions show some sequence similarity. Only two of the identified deletions contain directly repeated sequences at the deletion endpoints, a feature associated with homologous recombination. Our results suggest that within the trnI intron, there are preferred sites or "hotspots" for deletion formation involving a novel imprecise recombination mechanism. The significance of these sequences and possible mechanisms for deletion formation are discussed.

Base Sequence↗

PubMed1995

Homozygous deletion of exon 9 causes lipoprotein lipase deficiency: possible intron-Alu recombination.

We studied a homozygous deletion in the lipoprotein lipase gene at the molecular level. Comprising the end of intron 8, the whole of exon 9, and about two-thirds of intron 9, this 2.136-kb deletion caused complete lipoprotein lipase deficiency and severe hypertriglyceridemia (type I hyperlipoproteinemia). Intron 9 of a normal control subject was also sequenced in order to define the exact borders of the deletion. Up to now, only the first 0.721 kb of intron 9 had been sequenced. Thus the complete sequence of intron 9 (3.090 kb) is now available. Three Alu sequences were characterized in the normal intron 9, while the proband had only the third complete Alu sequence. The first Alu sequence was located in the deleted region, and only the left arm of the second was present, as the deletion began near its center. A stem-loop structure involving a 14-nt region towards the end of intron 8 and an Alu sequence in intron 9 might have led to the deletion. Sequence analysis showed that the three Alu sequences belonged to the 40-million-year-old Alu-Sa subclass.

Adult↗

PubMed2026

Suppression of HIV-1 replication in CEM-A cell cultures by trans-splicing group I introns targeting PAS/PBS sequences and conditionally expressing ΔN-Bax.

Anti-HIV group I introns containing antisense guide sequences directed against the HIV-1 primer activation signal and primer-binding site (PAS/PBS) were designed and evaluated. Because PAS/PBS sequences are present in the viral RNA species examined, these RNAs can serve as trans-splicing substrates. The introns were active against both artificial target RNAs and viral RNA generated during infection. Cleavage and degradation of targeted viral RNA may have contributed to suppression, whereas inclusion of a 3' exon encoding the proapoptotic protein ΔN-Bax was associated with increased programmed cell death and may have augmented suppression of viral replication. In cultured CEM-A cells, transgene expression of these introns markedly suppressed HIV-1 replication, with p24 levels falling below the assay detection limit in selected clones. RESULTS: RT-PCR and sequence analysis detected splice products containing the expected PAS/PBS junctions. In the dual-luciferase assay, intron expression reduced normalized Gaussia luciferase signal by approximately 70% relative to the negative control. Qualitative Annexin V imaging and caspase-3 assays were consistent with infection-dependent apoptosis after ΔN-Bax splice-product formation. Transient expression of each intron in HEK293T cells followed by infection with VSV-G-pseudotyped HIV-1NL4-3 at an MOI of 2 reduced p24 levels by approximately 50% at 4 days post-infection. Construct 128L produced the strongest RT-PCR band under the tested conditions and was selected for subsequent experiments. A canonical splice product and a low-abundance noncanonical splice product were detected; both involved the intended HIV-derived target RNA, although transcriptome-wide off-target splicing was not assessed. Heterogeneous transformed HEK293T populations showed an approximately 2-log10 reduction in p24. In selected clonal HEK293T and CEM-A lines, p24 was below the assay detection limit at the measured endpoints, including up to 90 days after infection in some CEM-A clones. CONCLUSIONS: PAS/PBS-targeting group I introns suppressed HIV-1-associated p24 production in the tested cell-culture models. Linking the introns to a ΔN-Bax 3' exon was associated with infection-dependent apoptosis and may further limit viral replication and spread. The use of highly conserved, functionally constrained target sequences may reduce the likelihood of escape, but viral evolution and transcriptome-wide off-target effects were not assessed. This conditional death-upon-infection strategy warrants further evaluation in primary-cell and in vivo models.

Humans↗

PubMed1983

Construction of novel cytochrome b genes in yeast mitochondria by subtraction or addition of introns.

The mitochondrial cob-box gene coding for apocytochrome b in yeast has five introns and six exons or two introns and three exons depending on the wild-type strain considered. Some intron mutations in this gene affect not only its expression but also that of another mitochondrial gene: oxi3. To understand better the function of introns in gene expression, we have constructed a series of new strains that differ only by the presence or absence of one of the five wild-type introns in the cytochrome b gene, the rest of the mitochondrial and nuclear genome remaining unchanged. All constructions result from in vivo recombination events between rho- donor and rho+ recipient mtDNA. The following genes have been constructed: [see text]. Interestingly, all the genes lead to the synthesis of cytochrome b, while only the genes having the intron bI4 allow the expression of oxi3. A nuclear gene, when mutated, can compensate for the absence of the intron bI4.

Cytochrome b Group↗

PubMed1987

Self-splicing of a group II intron in yeast mitochondria: dependence on 5' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.

Base Sequence↗

PubMed1989

Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5 beta from pre-mRNA for cytochrome c oxidase subunit I.

The nuclear PET54 gene of Saccharomyces cerevisiae was cloned and a pet54::LEU2 gene disruption strain was constructed. Analysis of the phenotype of this strain revealed a defect in expression of two mitochondrial genes: COX1, which encodes cytochrome c oxidase subunit I, and COX3, which encodes cytochrome c oxidase subunit III. The defect in COX1 gene expression in the pet54 mutant was shown to be the result of inefficient excision of COX1 intron aI5 beta. Two lines of evidence indicate that inefficient excision of intron aI5 beta is the sole defect in COX1 gene expression. First, a pet54::LEU2 cytoductant bearing the 'short' mitochondrial genome that lacks both COX1 introns aI5 alpha and aI5 beta is defective only in COX3 gene expression and not in COX1 mRNA splicing or mRNA translation. Second, Northern analysis of COX1 transcipts from the pet54 mutant showed that a 3.8 kb COX1 transcript containing unexcised intron aI5 beta and lacking intron aI5 alpha is accumulated while the amount of 2.2 kb mature COX1 mRNA is diminished. In an effort to relate the role of the PET54 gene product in splicing of COX1 pre-mRNA to the previously characterized role for PET54 in translation of mitochondrial COX3 mRNA, the sequence of the PET54-responsive portion of the COX3 5' untranslated leader region was compared to the COX1 intron aI5 beta sequence. Two blocks of RNA sequence present in COX3 have similar counterparts within intron aI5 beta of COX1. The possibility that the PET54 protein binds to one or the other of these blocks of RNA sequence and the potential consequences of this interaction are discussed.

Base Sequence↗

PubMed1990

A yeast tRNA precursor containing a pre-mRNA intron is spliced via the pre-mRNA splicing mechanism.

We have replaced the 14 nucleotide long intervening sequence of the Saccharomyces cerevisiae SUP6 (ochre) tRNA(Tyr) gene by the 52 nucleotide long second intron of the S. cerevisiae MATa1 gene. Yeast cells containing this modified pre-tRNA showed the typical suppressor phenotype indicating that the MATa1 pre-mRNA intron was exactly excised in vivo from the primary tRNA transcript and a mature and functional tRNA was formed. Several lines of evidence show that the splicing reaction proceeded via the pre-mRNA splicing mechanism: the reaction yielded a lariat shaped excised intron with a lariat shaped intron-exon 2 molecule as intermediate; point mutations in the conserved UAC-UAAC box of the intron impaired splicing of the precursor RNA; in a temperature sensitive rna2 strain splicing of this tRNA precursor was inhibited at the restrictive temperature. Our results imply that in yeast the excision of a pre-mRNA intron is not dependent on the transcription apparatus by which it was generated and that transcription and splicing are uncoupled processes in vivo, too. Furthermore these data demonstrate that recognition of an RNA as a substrate for a pre-mRNA splicing reaction is, at least qualitatively, only intron dependent.

Base Sequence↗