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At least 1,423 records · Page 79Linked to original sources
PubMed1994

Intron creation and polyadenylation in maize are directed by AU-rich RNA.

Intron recognition in Angiosperms is hypothesized to require AU-rich motifs within introns. In this report we examined the role of AU-rich motifs in pre-mRNA processing. AU-rich segments of maize introns inserted near the single intron of the maize Bronze-2(Bz2) gene result in alternative splicing. Other insertions of AU-rich sequence in the Bz2 cDNA resulted in de novo intron creation using splice junctions at the edges of the AU-rich region. Surprisingly, the five AU-rich inserts that we tested also caused polyadenylation, even though none had been selected for that function in plants. Insertions of GC-rich sequence into Bz2 did not cause either splicing or polyadenylation. We propose that AU-rich motifs are a general signal for RNA processing in maize and that in the absence of a 5' splice site, polyadenylation is the default pathway.

Alternative Splicing↗
PubMed2000

Conservation, regulation, synteny, and introns in a large-scale C. briggsae-C. elegans genomic alignment.

A new algorithm, WABA, was developed for doing large-scale alignments between genomic DNA of different species. WABA was used to align 8 million bases of Caenorhabditis briggsae genomic DNA against the entire 97-million-base Caenorhabditis elegans genome. The alignment, including C. briggsae homologs of 154 genetically characterized C. elegans genes and many times this number of largely uncharacterized ORFs, can be browsed and searched on the Web (http://www.cse.ucsc.edu/ approximately kent/intronerator). The alignment confirms that patterns of conservation can be useful in identifying regulatory regions and rarely expressed coding regions. Conserved regulatory elements can be identified inside coding exons by examining the level of divergence at the wobble position of codons. The alignment reveals a bimodal size distribution of syntenic regions. Over 250 introns are present in one species but not the other. The 3' and 5' intron splice sites have more similarity to each other in introns unique to one species than in C. elegans introns as a whole, suggesting a possible mechanism for intron removal.

Algorithms↗
PubMed2004

A functional analysis of ACP-20, an adult-specific cuticular protein gene from the beetle Tenebrio: role of an intronic sequence in transcriptional activation during the late metamorphic period.

A gene encoding the adult cuticular protein ACP-20 was isolated in Tenebrio. It consists of three exons interspersed by two introns, intron 1 interrupting the signal peptide. To understand the regulatory mechanisms of ACP-20 expression, ACP-20 promoter-luciferase reporter gene constructs were transfected into cultured pharate adult wing epidermis. Transfection assays needed the presence of 20-hydroxyecdysone, confirming that ACP-20 is up-regulated by ecdysteroids. Analysis of 5' deletion constructs revealed that three regions are necessary for high levels of transcription. Interaction experiments between intronic fragments and epidermal nuclear proteins confirmed the importance of intron 1 in ACP-20 transcriptional control, which results from the combined activity of regulatory cis-acting elements of the promoter and those of intron 1.

Amino Acid Sequence↗
PubMed1997

High-degree sequence conservation in LPA kringle IV-type 2 exons and introns.

In the search for factors contributing to the regulation of the Lp(a) lipoprotein concentration, we have sequenced the kringle IV-type 2 encoding exons 1 and 2 together with the flanking intron sequences of the LPA gene in individuals with different serum concentrations of Lp(a) lipoprotein. The high degree of sequence identity between the kringle IV-type 2 repeats made it possible to analyse all the 3-42 kringles simultaneously by polymerase chain reaction and direct DNA sequencing. The strategy used allowed us to determine approximately 700 bp from each kringle IV-type 2 repeat, resulting in a rapid screen of on average 28,000 bp of the LPA gene from each individual. Comparing these bipartite kringle IV-type 2 repeat sequences from 12 individuals with high and 11 individuals with low Lp(a) lipoprotein level revealed that: 1. no sequence polymorphism could be detected in the exons examined; 2. no sequence polymorphism could be detected in the consensus GT/AG splicing signals of exon/intron junctions; and 3. the proximal intron sequences seemed almost completely conserved in the 76-135 bp analysed. Only one position in the intron sequences exhibited the pattern of a G/A polymorphism. We observed no differences between the group with high and the group with low Lp(a) lipoprotein level. The very high conservation of intron sequences could support the hypothesis that the LPA gene evolved relatively recently. The contradictory finding of a corresponding sequence conservation between the human LPA and the plasminogen gene suggests that an evolutionary pressure has preserved these intron sequences over the last 40-90 million years.

Conserved Sequence↗
PubMed2004

The phylogenies of introns 4-7 demonstrate an inconsistent pattern between human leukocyte antigen-C group topologies.

In this study, we have sequenced introns 4-7 in 31 human leukocyte antigen-C (HLA-C) alleles representing all allelic groups. Intron sequences show a patchwork pattern of polymorphism. Bootstrap support for phylogenetic lineages and for differentiation between groups is limited due to the high homology of intron sequences, where the substitution of a single nucleotide may lead to the assignment to different clusters. The intron data suggest the idea of a Cw*06/Cw*12 family, which is closely related to a hypothetical Cw*05/Cw*08 family. Moreover, a third family consisting of Cw*01/Cw*04/Cw*18 may exist. The intron data compiled in our study may be the basis for further sequencing studies. The detection of three novel alleles (Cw*0401new, Cw*140201new, and Cw*1701new) suggests that the HLA-C polymorphism might have been strongly underestimated to date.

Alleles↗
PubMed2004

Hydrolytic cleavage by a group I intron ribozyme is dependent on RNA structures not important for splicing.

DiGIR2 is the group I splicing-ribozyme of the mobile twin-ribozyme intron Dir.S956-1, present in Didymium nuclear ribosomal DNA. DiGIR2 is responsible for intron excision, exon ligation, 3'-splice site hydrolysis, and full-length intron RNA circle formation. We recently reported that DiGIR2 splicing (intron excision and exon ligation) competes with hydrolysis and subsequent full-length intron circularization. Here we present experimental evidence that hydrolysis at the 3'-splice site in DiGIR2 is dependent on structural elements within the P9 subdomain not involved in splicing. Whereas the GCGA tetra-loop in P9b was found to be important in hydrolytic cleavage, probably due to tertiary RNA-RNA interactions, the P9.2 hairpin structure was found to be essential for hydrolysis. The most important positions in P9.2 include three adenosines in the terminal loop (L9.2) and a consensus kink-turn motif in the proximal stem. We suggest that the L9.2 adenosines and the kink-motif represent key regulatory elements in the splicing and hydrolytic reaction pathways.

Hydrolysis↗
PubMedDate not supplied

A group I intron in the SSUrDNA of some Naegleria spp. demonstrated by polymerase chain reaction amplification.

The small subunit ribosomal DNA (SSUrDNA) of all described Naegleria spp. was amplified by polymerase chain reaction with universal primers. In all strains of N. andersoni andersoni, N. andersoni jamiesoni, N. australiensis italica and two related strains, and one out of four clusters of N. gruberi, a band of approximately 3.3 kb was obtained. All other strains displayed a band with the expected DNA length of 2.0 kb. This means the former have a 1.3 kb intron in the SSUrDNA. Restriction analysis demonstrated that the intron is between two conserved Pst I sites at the 5' end of the SSUrDNA. It also suggested the introns might not be identical in each species or subspecies. The Pst I fragment of SSUrDNA containing the 1.3 kb insert in N. andersoni andersoni was cloned and sequenced. The 1,296-nucleotide insert is situated in helix 19 of the SSUrDNA, which is an area of conserved primary and secondary structure. Sequence and secondary structure analyses of the insert revealed it it is a group I intron. This group I intron is very large and contains an open reading frame that could serve to encode a polypeptide of 139 amino acids in size.

Amino Acid Sequence↗
PubMedDate not supplied

Evidence for the ancestral origin of group I introns in the SSUrDNA of Naegleria spp.

The sequence variation within the group I intron in five Naegleria spp. was studied and compared with the sequence variation within the flanking small subunit ribosomal DNA. Considerable sequence divergence was observed in the introns as well as in the rDNA. In the intron deletions and insertions are only detected in the sequence contributing to the secondary structure, not in the open reading frame. Most of the sequence variation is detected in the unpaired loops. In the case of nucleotide substitution in helices, compensating base pair changes were observed. The sequence variation does not induce variation in the secondary structure model. The phylogenetic tree based on the intron sequences is similar to the tree based on the flanking rDNA sequences. This observation indicates that the intron might have been acquired at an early stage in evolution, and lost in the majority of Naegleria spp.

Animals↗
PubMed1999

Phylogenetic history of hominoid DRB loci and alleles inferred from intron sequences.

The evolutionary relationships among the MHC class II DRB4, DRB5 and DRB6 loci as well as the allelic lineages and alleles of the DRB1 locus were studied based on intron 1 and intron 2 sequences from humans, chimpanzee (Pan troglodytes), bonobo (Pan paniscus) and gorilla (Gorilla gorilla). The phylogenetic trees for these sequences indicate that most of the DRB1 allelic lineages predate the separation of the hominoid species studied, consistent with previous analysis of the coding sequences of these lineages. However, the intron sequence variation among alleles within DRB1 allelic lineages is very limited, consistent with the notion that the majority of the contemporary alleles have been generated within the last 250,000 years. The clustering of the DRB1 allelic lineages *08 and *12 with *03 supports a common ancestry for the DR8 and DR52 haplotypes. Similarly, the clustering of DRB1 allelic lineages *15 and *01 with the DRB3 locus is consistent with a common ancestry for the DR1 and DR51 haplotypes. Two cases of recombination around the second exon were observed: 1) the HLA-DRB6 locus appears to have been generated through a recombination between a DRB5 allele and an ancestral DRB6 allele, and 2) the gorilla sequence Gogo-DRB1 *03 appears to have been generated through a recombination between the DRB3 locus and an allele from the DRB1 *03 allelic lineage. The nucleotide substitution rate of DRB introns was estimated to 0.85-1.63 x 10(-9) per site per year, based on comparisons between the most closely related sequences from different hominoid species. This estimate is similar to the substitution rate for other intronic regions of the primate genome.

Alleles↗
PubMed2005

Cloning, expression, and functional characterization of human cyclooxygenase-1 splicing variants: evidence for intron 1 retention.

Recently, a splicing variant of cyclooxygenase (COX)-1, arising via the retention of its intron 1, was identified in canine. It was called COX-3 and was reported to be differentially sensitive to inhibition by various nonsteroidal anti-inflammatory drugs (NSAIDs) as well as acetaminophen (Chandrasekharan et al., 2002). However, the existence of an orthologous splicing variant in human tissues has been questioned due to a reading frame shift and premature termination. In this study, we first confirmed the existence of intron 1-retained COX-1 in certain human tissues at both the mRNA and protein levels. Molecular biology studies revealed that three distinct COX-1 splicing variants exist in human tissues. The most prevalent of these variants, called COX-1b1, arises via retention of the entire 94 base pair (bp) of intron 1, leading to a shift in the reading frame and termination at bp 249. However, the other two variant types, called COX-1b2 and COX-1b3, retain entire intron 1, but they are missing a nucleotide in one of two different positions, thereby encoding predicted full-length and likely COX-active proteins. Functional studies revealed that the COX-1b2 is able to catalyze the synthesis of prostaglandin F2alpha from arachidonic acid with Km and Vmax values of 0.54 microM and 3.07 pmol/mg/min, respectively. However, no significant differential selectivity for inhibition by selected NSAIDs was observed. Accordingly, we conclude that intron 1-retained human COX-1 is not likely to be the therapeutic target of acetaminophen or a candidate of COX-3.

Acetaminophen↗
PubMed1990

Localization of an acetylcholine receptor intron to the nuclear membrane.

The first intron of the RNA for the acetylcholine receptor (AChR) alpha subunit shows a ringlike distribution around nuclei in multinucleated myotubes by in situ hybridization. This pattern is not observed for an actin intron or U1 RNA. Quantitation of the intron sequences reveals large variations in the amount of both the AChR and actin introns between nuclei within the same myotube, although all nuclei express equivalent amounts of U1 RNA. This differential RNA expression indicates that nuclei can individually control expression of messenger RNAs. The restricted distribution of the AChR intron RNA suggests a previously unknown step in RNA processing.

Actins↗
PubMed1996

Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns.

A conserved sequence element in a minor class of eukaryotic pre-messenger RNA (pre-mRNA) introns was previously proposed to base pair with a complementary sequence in the U12 small nuclear RNA (snRNA) in a manner analogous to the pairing of US snRNA with the branch site sequence of the major class of introns. Here, mutations generated in this conserved sequence element block the splicing of a member of this minor intron class in vivo. The block was relieved by coexpression of a U12 snRNA containing compensatory mutations that restore the proposed base pairing interaction. These results show that this minor class of pre-mRNA introns is a distinct class existing alongside the major class of introns in animal genomes, and these results also establish an in vivo function for U12 snRNA.

Animals↗
PubMed1996

U1-mediated exon definition interactions between AT-AC and GT-AG introns.

A minor class of metazoan introns has well-conserved splice sites with 5'-AU-AC-3' boundaries, compared to the 5'-GU-AG-3' boundaries and degenerate splice sites of conventional introns. Splicing of the AT-AC intron 2 of a sodium channel (SCN4A) precursor messenger RNA in vitro did not require inhibition of conventional splicing and required adenosine triphosphate, magnesium, and U12 small nuclear RNA (snRNA). When exon 3 was followed by the 5' splice site from the downstream conventional intron, splicing of intron 2 was greatly stimulated. This effect was U1 snRNA-dependent, unlike the basal AT-AC splicing reaction. Therefore, U1-mediated exon definition interactions can coordinate the activities of major and minor spliceosomes.

Adenosine Triphosphate↗
PubMed1988

Exon-intron organization in genes of earthworm and vertebrate globins.

The structure of an invertebrate, intron-containing globin gene has been determined as part of a study of the evolution of hemoglobin. The gene encoding chain c of Lumbricus terrestris hemoglobin has the two-intron, three-exon structure characteristic of vertebrate globin genes, and the exact positions of the splice junctions are conserved. The two introns interrupting the coding sequence are longer than those of known hemoglobins but shorter than myoglobin introns. The gene encodes a secretory preglobin containing a 16-residue signal peptide, as expected for an extracellular hemoglobin. However, no intron separates the DNA encoding the signal sequence from that of the globin sequence. The 3' untranslated region of the Lumbricus gene is much longer than those of the genes for other hemoglobins and is similar to those found for myoglobins.

Animals↗
PubMed2002

Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion.

We analyzed the role of integrons in the dissemination of antibiotic resistance in a recent multiresistant clinical isolate, Serratia marcescens SCH88050909 (SCH909). This isolate harbors three integrons, all on a 60-kb conjugative plasmid. By PCR, hybridization, and sequencing analyses, we found that integron 1 has the dfrA1 and ant(3")-Ia cassettes. The first cassette in integron 2 contains the ant(2")-Ia gene, separated from its attC site (59-base element) by a 1,971-bp insert containing a group II intron; this intron codes for a putative maturase-reverse transcriptase on the complementary strand and is the first such intron to be found associated with an integron. The attC site is followed by a novel aminoglycoside resistance gene, ant(3")-Ii-aac(6')-IId, which has been characterized for its bifunctional ANT(3")-I and AAC(6')-II activities. DNA sequence analysis of this fused cassette suggests that insertion and excision due to the integrase activity could have an important role in the evolution of aminoglycoside resistance genes. This gene is followed by an unknown open reading frame with a typical attC site and a partial cassette composed of the beginning of the bla(OXA-10) cassette interrupted by IS1. The sequence downstream of IS1 revealed that the bla(OXA-10) cassette is incomplete and that the 3' conserved segment of this integron is absent. Integron 3 is in a Tn1696-like transposon with the aac(3)-Ia cassette followed by three unknown cassettes and ant(3")-Ia. The presence of the group II intron and the relationship of group II introns in eubacteria with mobile elements suggest a possible role of this element in events such as cassette formation and/or plasmid evolution.

Amino Acid Sequence↗
PubMed1999

Analysis of Pneumocystis carinii introns.

Pneumocystis carinii is an ascomycete phylogenetically related to Schizosaccharomyces pombe. Little is known about gene regulation in P. carinii. The removal of introns from pre-mRNA requires spliceosomal recognition of the intron-exon boundary. In S. pombe and higher eukaryotes, this boundary and a branch site within the intron are conserved. We recently demonstrated that P. carinii cdc2 cDNA can complement S. pombe containing conditional mutations of cdc2, an essential gene involved in cell cycle regulation. We next tested whether P. carinii genomic cdc2 (with six introns) could also complement S. pombe cdc2 mutants and found genomic sequences incapable of this activity. Reverse transcriptase PCR confirmed the inability of the S. pombe cdc2 mutants to splice the P. carinii genomic cdc2. Analysis of 83 introns from 19 P. carinii protein-encoding genes demonstrated that the sequence GTWWDW functions as a donor consensus in P. carinii, whereas YAG serves as an acceptor consensus. These sequences are similar in S. pombe; however, a branch site sequence was not found in the P. carinii genes studied.

Genetic Complementation Test↗
PubMed2002

Group I self-splicing intron in the recA gene of Bacillus anthracis.

Self-splicing introns are rarely found in bacteria and bacteriophages. They are classified into group I and II according to their structural features and splicing mechanisms. While the group I introns are occasionally found in protein-coding regions of phage genomes and in several tRNA genes of cyanobacteria and proteobacteria, they had not been found in protein-coding regions of bacterial genomes. Here we report a group I intron in the recA gene of Bacillus anthracis which was initially found by DNA sequencing as an intervening sequence (IVS). By using reverse transcriptase PCR, the IVS was shown to be removable from the recA precursor mRNA for RecA that was being translated in E. coli. The splicing was visualized in vitro with labeled free GTP, indicating that it is a group I intron, which is also implied by its predicted secondary structure. The RecA protein of B. anthracis expressed in E. coli was functional in its ability to complement a recA defect. When recA-negative E. coli cells were irradiated with UV, the Bacillus RecA reduced the UV susceptibility of the recA mutant, regardless of the presence of intron.

Bacillus anthracis↗
PubMed2005

Introns in the cytolethal distending toxin gene of Actinobacillus actinomycetemcomitans.

In eukaryotic cells, genes are interrupted by intervening sequences called introns. Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA. However, introns are rarely found in bacteria. Actinobacillus actinomycetemcomitans is a periodontal pathogen implicated in aggressive forms of periodontal disease. This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G2/M phase of the cell cycle. In this study, we report the presence of introns within the cdt gene of A. actinomycetemcomitans. By use of reverse transcription-PCR, cdt transcripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected. In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA. Similar results were obtained when a plasmid carrying cdt was cloned into Escherichia coli. Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3. Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1. Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur. The catalytic region of the cdt RNA was located within the cdtC gene. This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in both cis and trans configurations.

Aggregatibacter actinomycetemcomitans↗