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PubMed2001

PCR primers for an aldolase-B intron in acanthopterygian fishes.

BACKGROUND: Nuclear DNA sequences provide genetic information that complements studies using mitochondrial DNA. Some 'universal' primer sets have been developed that target introns within protein-coding loci, but many simultaneously amplify introns from paralogous loci. Refining existing primer sets to target a single locus could circumvent this problem. RESULTS: Aldolase intron 'G' was amplified from four fish species using previously described primer sets that target several loci indiscriminately. Phylogenetic analyses were used to group these fragments and other full-length aldolase proteins from teleost fishes into orthologous clades and a primer set was designed to target specifically an intron within the aldolase-B locus in acanthopterygian fishes. DNA amplifications were tried in a variety of acanthopterygian fishes and amplification products, identifiable as aldolase-B intron 'G', were observed in all atherinomorph and percomorph taxa examined. Sequence variation within this locus was found within and among several species examined. CONCLUSIONS: Using 'universal' primer sets coupled with phylogenetic analyses it was possible to develop a genetic assay to target a specific locus in a variety of fish taxa. Sequence variation was observed within and among species suggesting that this targeted assay might facilitate interspecific and intraspecific comparisons.

Amino Acid Sequence↗
PubMed2003

Two reactions of Haloferax volcanii RNA splicing enzymes: joining of exons and circularization of introns.

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon-intron junctions, producing 2',3'-cyclic phosphate and 5'-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3' terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5' and 3' flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3',5'-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.

Exons↗
PubMed2004

Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase.

Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein's back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.

Amino Acid Sequence↗
PubMed2006

Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons.

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I-VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I-III), or five (I-V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter-lipc-terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.

Base Sequence↗
PubMed2004

Intronic hormone response elements mediate regulation of FKBP5 by progestins and glucocorticoids.

Expression of FKBP51, a large molecular weight immunophilin, is strongly enhanced by glucocorticoids, progestins, and androgens. However, the activity of a 3.4-kb fragment of the FKBP51 gene (FKBP5) promoter was only weakly increased by progestin and we show here that it is unresponsive to glucocorticoids and androgens. The entire FKBP5 was scanned for consensus hormone response elements (HREs) using MatInspector. We found that 2 regions of intron E, which are conserved in rat and mouse FKBP5, contain HRE-like sequences with high match scores. Deoxyribonucleic acid fragments (approximately 1 kb in length) containing these regions were amplified and tested in reporter gene assays for steroid responsiveness. One region of intron E of FKBP5 (pIE2) conferred both glucocorticoid and progestin responsiveness to 2 heterologous reporter genes, whereas the other, less-conserved region of intron E (pIE1) was responsive only to progestins. The inclusion of pIE1 upstream of pIE2 (pIE1IE2) enhanced progestin but not glucocorticoid responsiveness. None of the constructs containing intronic sequences was responsive to androgens. Mutation of the putative HREs within pIE1 and pIE2 eliminated hormone responsiveness. Electrophoretic mobility shift assays demonstrated that progesterone receptors (PR) bound to the HRE in pIE1, whereas both PR and glucocorticoid receptors interacted with the HRE in pIE2. These data suggest that distal intronic elements significantly contribute to transcriptional regulation of FKBP5 by glucocorticoids and progestins.

Androgens↗
PubMed2006

Gene silencing in vitro and in vivo using intronic microRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a posttranscriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest clinical and circumstantial malfunction. Type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of nonproteincoding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Animals↗
PubMed1998

Sequence analysis of thyroid transcription factor-1 gene reveals absence of mutations in patients with thyroid dysgenesis but presence of polymorphisms in the 5' flanking region and intron.

Congenital hypothyroidism is caused by several mechanisms. The most common cause worldwide is iodine deficiency, but in iodine-sufficient regions thyroid dysgenesis is the most common cause of congenital hypothyroidism. In the present study we analyzed the thyroid transcription factor-1 (TTF-1) gene in patients with congenital hypothyroidism due to thyroid dysgenesis: three patients with athyrosis, five with ectopy, and one with hypoplasia. Genomic DNA was isolated from peripheral leukocytes, and the TTF-1 gene, including a 5' flanking region, two exons and one intron was amplified by polymerase chain reaction (PCR) with 4 pairs of primers. The PCR products were directly sequenced by the Dye Terminator Cycle Sequencing method. We could not find any mutations specific for the thyroid dysgenesis in the 5' flanking region, two exons and one intron in the TTF-1 gene, but two heterozygous nucleotide substitutions were detected in the intron: a G to A transition at nucleotide 469 (G469A) and a C to A transversion at nucleotide 866 (C866A). The same nucleotide changes were detected in some normal subjects. Allelic frequencies of the polymorphisms G469A and C866A were 23% and 10%, respectively. Another normal polymorphism in the 5' flanking region was a G to T transversion at nucleotide -845 from the transcription start site (G-845T). The allelic frequency of the polymorphism G-845T was 28%. We also found 12 polymorphisms in the 5' flanking region, two in the intron and one in the 3' untranslated region. These polymorphisms were detected in 100% chromosomes. These results suggest that congenital hypothyroidism associated with thyroid dysgenesis is unlikely to be caused by mutations in the TTF-1 gene in which, however, were detected normal polymorphisms in the 5' flanking region, intron and 3' untranslated region.

Adolescent↗
PubMed1994

Short introns interrupting the Oct-2 POU domain may prevent recombination between POU family genes without interfering with potential POU domain 'shuffling' in evolution.

Transcription factors are often encoded by gene families that share the same type of DNA binding domain. The POU domain genes are one such paradigm. We compared the genomic DNA encoding the POU domain of the Oct-2 genes in human and mouse. In both species this domain is split into a cluster of four exons by short, highly diverged introns. We postulate that the main role of these introns is to prevent ectopic homologous recombination with other members of the POU gene family, with its potentially deleterious effects in somatic and germline cells. Such rapidly diverging introns may generally promote evolution by facilitating the maintenance of duplicated genes. The use of different codons for the same protein domain among members of a gene family may be a slower process that serves a similar purpose. Introns that split conserved domains such as the POU domain do not conform to the exon shuffling hypothesis originally put forward by W. Gilbert (1978). However, we note that the introns flanking the POU domain are in the same phase, i.e. interrupt codons in the same reading frame. Thus we propose that the entire POU domain, which is encoded by a tight cluster of exons, has been shuffled together during evolution as a functional unit, or 'shufflon'.

Animals↗
PubMed2004

Lack of association between perlecan gene intron 6 BamHI polymorphism and risk of mitral valve prolapse in Taiwan Chinese.

Abnormalities of proteoglycan, collagen, and elastic fibers were found in floppy mitral valves. Perlecan is one of the three major classes of heparan sulfate proteoglycans within the cardiovascular system. The role of perlecan genetic variant in mitral valve prolapse (MVP) has not been studied. We therefore performed a case-controlled study investigating the possible relation between the perlecan gene intron 6 BamHI polymorphism and MVP among the Chinese population in Taiwan. We studied 100 patients with MVP diagnosed by echocardiography and 100 age- and sex-matched normal control subjects. The perlecan gene intron 6 BamHI polymorphism was identified by polymerase chain reaction-based restriction analysis. There were no significant differences in either the genotype distribution or allelic frequencies between MVP cases and controls for perlecan gene intron 6 BamHI polymorphism (P = 0.20 and 0.76, respectively). Further categorization of the MVP patients into mild and severe subgroups also revealed no statistical difference from controls for perlecan gene intron 6 BamHI polymorphism. It is concluded that perlecan gene intron 6 BamHI polymorphism is not a suitable genetic marker of MVP in Taiwan Chinese.

Adolescent↗
PubMed2003

Insertional mutation in the intron 1 of Unc5h3 gene induces ataxic, lean and hyperactive phenotype in mice.

Mice carrying a mutation in the first intron of Unc5h3 were accidentally produced by transgenic insertion and characterized for their homozygous mutant phenotypes. Morphological and histological analysis revealed cerebellar and midbrain abnormalities, which are similar to the previously reported phenotypes of the Unc5h3 mutant. Behavioral analysis showed higher ambulatory activity and circling, and defects in habituation in a novel environment. Their body weights were 10-30% less than wildtype mice from 2-3 weeks of age to 22 months possibly due to reduced accumulation of adipose tissues. The transgenic insertion site was identified and mapped to the intron 1 of Unc5h3 gene with approximately 50 kb deletion of the intron sequence. This intronic mutation interfered with the mRNA expression of the Unc5h3 gene not in testes, but in many tissues including the brain, implying that this intronic region may play a role in regulating tissue-specific transcription of Unc5h3.

Animals↗
PubMed2006

Micro-RNA-like effects of complete intronic sequences.

MicroRNAs (miRNAs) have been suggested as suppressors of numerous target genes in human cells. In this report, we present gene chip array data indicating that in the absence of miRNA sequences, complete human introns are similarly capable of coordinating expression of large numbers of gene products at spatially diverse sites within the genome. The expression of selected intronic sequences (6a, 14b and 23) derived from the cystic fibrosis transmembrane conductance regulator (CFTR) gene caused extensive and specific transcriptional changes in epithelial cells (HeLa) that do not normally express this gene product. Each intron initiated a distinctive pattern of gene transcription. Affected genes such as FOXF1, sucrase-isomaltase, collagen, interferon, complement and thrombospondin 1 have previously been linked to CFTR function or are known to contribute to the related processes of epithelial differentiation and repair. A possible regulatory function of this nature has not been demonstrated previously for non-coding sequences within eukaryotic DNA. The results are consistent with the observation that splicesomal introns are found only in eukaryotic organisms and that the number of introns increases with phylogenetic complexity.

Cell Differentiation↗
PubMed2000

Cell-specific expression of the murine CD21 gene depends on accessibility of promoter and intronic elements.

The murine complement receptor type 2 gene (Cr2/CD21) is transcriptionally active in murine B and follicular dendritic cells, but not in murine T cells. We have previously shown that altering chromatin structure via histone deacetylase inhibitors results in CD21 expression in murine T cells, and that the minimal CD21 promoter provided appropriate cell-specific expression of luciferase reporter constructs only in the presence of the first third of intron 1, fragment A. We extend this work by showing that replacing the CD21 gene promoter with the SV40 promoter resulted in the loss of this cell-specific control. Further delineation of intronic regulatory elements by fragmentation also resulted in the loss of cell-specific gene expression, suggesting that multiple CD21 promoter and intronic elements interact for appropriate CD21 gene expression. To assess this model, we performed EMSAs to define protein binding sites within promoter and intronic regions and DNase I hypersensitivity assays to determine chromatin accessibility. Multiple DNA binding factors were shown to be present in B and T cell extracts; a minority demonstrated B cell specificity. However, the DNase I sensitivity of T cell CD21 regulatory elements was not comparable to that of B cells until the histone acetylation status of the gene was altered. Taken together, these data suggest that chromatin remodeling facilitates cell-specific CD21 gene expression by modulating access of transcription factors to regulatory elements in the promoter and intron.

Acetylation↗
PubMed2002

Yin Yang 1, Oct1, and NFAT-4 form repeating, cyclosporin-sensitive regulatory modules within the murine CD21 intronic control region.

The murine complement receptor type 2 gene (Cr2/CD21) is expressed by murine B and follicular dendritic cells, but not murine T cells. We have previously shown that appropriate transcriptional control of the CD21 gene requires the CD21 promoter as well as intronic sequences. We have also demonstrated that altering chromatin structure by inhibiting histone deacetylases induces CD21 expression in murine T cells by increasing the accessibility of promoter and intronic regulatory elements. In this report, we identify seven distinct regulatory areas within the first intron of the murine CD21 gene that are conserved between mouse and human CD21 intronic sequences. EMSA competition and supershift analyses reveal the formation of multiple DNA-protein complexes at these sites that include Yin Yang 1, Oct1, and NFAT-4. NFAT-containing complexes were altered in B cells treated with the NFAT inhibitor cyclosporin A and correlated with a repression of CD21 gene transcription implicating NFAT transcriptional control. Functional data revealed that no single region conferred cell-specific reporter gene expression, but rather the entire CD21 regulatory element was required to confer cell-specific gene expression. Taken together, these data demonstrate the formation of repeating, overlapping regulatory modules, all of which are required to coordinately control the cell-specific expression of the murine CD21 gene. We propose a model in which Yin Yang 1 and Oct1 may recruit histone deacetylase to multiple sites in the CD21 intronic regulatory element in nonexpressing cells and NFAT either displaces this histone deacetylase or recruits a histone acetylase to allow the formation of a functional transcriptional complex in expressing cells.

Animals↗
PubMedDate not supplied

[Statistical analysis of the exon-intron structure of higher eukaryote genes].

The exon-intron structure of human, insect (Drosophila sp.), and dicot plant (Arabidopsis thaliana) genes was considered. In each genome there exists a characteristic intron length. Anomalously long introns was usually the first introns in genes. In each sample there are correlations between the lengths of neighboring exons and between exon lengths and closeness to the consensus of the sites at exon boundaries. Exons and exon pairs containing an integer number of triplets are preferred. These results are relevant to the study of splicing mechanism and evolution of introns, as well as construction of gene recognition algorithms.

Algorithms↗
PubMed2000

Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice.

Alternative splicing plays a major role in regulating tissue-specific expression of cytoskeletal protein 4.1R isoforms. In particular, expression of the protein's functionally critical spectrin-actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre-messenger RNA (pre-mRNA), we explored the sequence requirements for excision of the introns flanking exon 16. These studies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron. Constructs designed to test the converse pathway were spliced less efficiently and with less fidelity, in part due to activation of a cryptic 5' splice site in exon 16. This downstream-first model for ordered splicing is consistent with the hypothesis that regulated alternative splicing requires cooperation between multiple exonic and/or intronic regulatory elements whose spatial organization is critical for recruitment of appropriate splicing factors. Our results predict that exon 16 splicing is regulated at the first step-excision of the downstream intron-and that cells unable to catalyze this step will exhibit exon 16 skipping. In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain. (Blood. 2000;95:692-699)

Alternative Splicing↗
PubMed1999

[Molecular evolution and structure of eukaryotic nuclear RNA polymerase subunits in light of the exon-intron organization of their genes].

Analysis of literary data (for Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, Homo sapiens, and some other Eucarya) and our data (for Schizosaccharomyces pombe) on the exon-intron organization of the genes encoding subunits of nuclear RNA polymerases showed that introns in the orthologous genes from different organisms are arranged nonrandomly, namely, their positions, if projected on the map of the comparison of the amino acid sequences of the orthologous subunits, not infrequently coincide in evolutionarily distant species. As a rule, intron positions correspond to the boundaries of the structurally conserved regions (domains) or to the sites of possible turns of the polypeptide chain. For example, introns flank the secondary structure elements in the Rpb8 subunit with the known three-dimensional structure or the structure-function modules in subunits Rpb10 and Rpc10. These facts are in agreement with the idea of the ancient origin of introns, and with the notion of evolution of ancient protein sequences through the assembly of their genes from short protoexons selected by the nature as far back as the RNA world times. Comparative analysis of the primary structures of the subunits of eukaryotic RNA polymerases allowed us to reveal a nuclear localization signal in subunit Rpb10 and some hypothetical archaeal homologues of subunit Rpc10.

Amino Acid Sequence↗
PubMed2001

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression.

The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene.

3T3 Cells↗
PubMed1992

The MRS1 gene of S. douglasii: co-evolution of mitochondrial introns and specific splicing proteins encoded by nuclear genes.

We have developed a rapid and simple methodology to locate yeast genes within cloned inserts, obtain partial sequence information, and construct chromosomal disruptions of these genes. This methodology has been used to study a nuclear gene from the yeast S. douglasii (a close relative of S. cerevisiae), which is essential for the excision of the mitochondrial intron aI1 of S. douglasii (the first intron in the gene encoding subunit I of cytochrome oxidase), an intron which is not present in the mitochondrial genome of S. cerevisiae. We have shown that this gene is the homologue of the S. cerevisiae MRS1 gene, which is essential for the excision of the mitochondrial introns bI3 and aI5 beta of S. cerevisiae, but is unable to assure the excision of the intron aI1 from the coxI gene of S. douglasii. The two genes are very similar, with only 13% nucleotide substitutions in the coding region, transitions being 2.5 times more frequent than transvertions. At the protein level there are 86% identical residues and 7% conservative substitutions. The divergence of the MRS1 genes of S. cerevisiae and S. douglasii, and the concomitant changes in the structure of their mitochondrial genomes is an interesting example of the co-evolution of nuclear and mitochondrial genomes.

Amino Acid Sequence↗