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At least 1,657 records · Page 92Linked to original sources
PubMed2005

Fixation of conserved sequences shapes human intron size and influences transposon-insertion dynamics.

The basis for intron expansion in humans is largely unexplored. In this article, we demonstrate that intron expansion has primarily been determined by fixation of multispecies conserved sequences (MCSs) over time. The presence of MCSs has shaped intron features: the insertion of transposable elements (TEs) has been constrained as more MCSs were fixed. Analysis of TE and MCS distribution suggested an unprecedented estimate of information requirements for proper splicing of long introns with indication of sequence constraints extending up to >3 kb downstream 5' splice sites.

Alternative Splicing↗
PubMed2006

Intron size in mammals: complexity comes to terms with economy.

Different and contrasting models have been proposed to explain intron size evolution in mammals. Here, we demonstrate that intron and intergenic size per se has no adaptive role in gene expression regulation but reflects the need to preserve conserved intronic elements. Although the amount of non-coding functional elements explains the within-genome size variation of intergenic spacers, we show that an additional, additive pressure has been acting on highly expressed introns to reduce the cost of their transcription.

Animals↗
PubMed2005

The intron 1 of HPV 16 has a suboptimal branch point at a guanosine.

The branch point sequence (BPS) of intron 1 of the HPV-16 was determined via RT-PCR in a cell free system, using lariat intermediates obtained by in vitro splicing reactions. We used synthetic E6/E7 transcripts and HeLa nuclear protein extracts to obtain the splicing intermediates. Then, a divergent oligonucleotide primer set, pairing on the lariat RNA that encompassed the 2'-5' phosphodiester bond formed between the 5' end of the intron and the BPS, was used for cDNA synthesis and PCR amplification. Subsequent RT-PCR assays revealed four splicing intermediates, made up of a major intermediary corresponding to the BPS and four cryptic branched sequences. Only intermediates bound at the 5' end of the intron are probably the authentic branch point sequence, and all of them branch at guanosine 328 instead of the typical adenosine. Unusually, the BPS of intron 1 of HPV-16 is a suboptimal sequence (AGUGAGU) that differs from the eukaryotic consensus BPS, which correlates with the splicing profile observed for early transcripts of HPV-16 in tumors and tumor derived cell lines. The implications of this unusual branch point sequence for splicing of the HPV-16 pre-mRNA are discussed.

Cell Extracts↗
PubMed2006

Intron length and accelerated 3' gene evolution.

Genetic evolution depends in part upon a balance between negative selection and environmentally driven mutation. To explore whether this balance is affected by gene structure, we have used phylogenetic data mining to compare gene compositions across a range of species. Here we show that genomes of higher species exhibit a greater frequency of 5' CpG islands and of CpG-->TpG/CpA transitions. This latter mutational pattern exhibits a 5'-to-3' trend in higher species, consistent with a length-dependent effect on methylation-dependent CpG suppression. Associated strand asymmetry (TpG>CpA) declines with gene length, implying attenuation of transcription-coupled repair 3' to introns. A sharp 3' rise in coding region single-nucleotide polymorphism frequency further supports a mechanistic role for intron length in promoting genetic variation by reducing repair and/or weakening negative selection. Consistent with this, the Ka/Ks ratio of 3' exons exceeds that of centrally located exons in intron-containing, but not in intronless, genes (p<0.0003). We conclude that the efficiency of transcription-coupled repair decreases with gene length, suggesting in turn that 3' gene evolution is accelerated both by introns and by gene methylation.

3' Untranslated Regions↗
PubMed2005

A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro.

The splicing pattern of pre-mRNA is unpredictable in genes harboring a single-nucleotide change within the consensus sequence of a splice-donor site. In the dystrophin gene, a transition from G to A at the fifth position of intron-32 (4518+5G > A) has been reported as a polymorphism within the consensus sequence or a mutation identified in Duchenne muscular dystrophy (DMD). Here, we report both in vivo and in vitro evidence that shows inactivation of the splice-donor site caused by this mutation. In one Japanese DMD case, two novel dystrophin mRNAs were identified in the patient's lymphocytes, one with a 98 bp deletion of the 3' end of exon-32 (dys32-98) and the other with a 28 bp intron retained between exons 32 and 33 (dys32 + 28). Genomic sequencing disclosed a single-nucleotide change from G to A at the fifth position of intron-32 (4518+5G > A). To demonstrate in vitro the inactivation of this splice-donor site by this nucleotide change, mini-dystrophin genes comprising three exons harboring either normal or mutant intron-32 sequences were expressed in HeLa cells, and the splicing products were analyzed by reverse-transcription PCR amplification. A normal transcript consisting of three exons was obtained from the normal construct. From the mutant, we obtained one product containing a 98 bp deletion at the 3' end of exon-32, indicating complete inactivation of the native splice-donor site. Thus, both in vivo and in vitro experiments demonstrate that 4518+5G > A causes a splicing error leading to transcript termination; it did not behave like a silent polymorphism. Our results indicate that the in vitro splicing system is a powerful tool for determining the underlying mechanism of a disease-causing mutation in a splicing consensus sequence.

Base Sequence↗
PubMed2005

PMM2 intronic branch-site mutations in CDG-Ia.

Congenital Disorders of Glycosylation (CDG, OMIM#212065)-Ia is an autosomal recessive disorder, characterized by central nervous system dysfunction and multiorgan failure associated with mutations in the PMM2 gene. We report two patients who are compound heterozygotes with respect to two new intronic mutations that affect a highly conserved adenosine in a consensus branch-site sequence. The mutations, one in intron 7: c.340 -23A > G (IVS7 -23A > G) and the other in intron 2: c.179 -25A > G (IVS2 -25A > G), are associated with the c.422G > A (R141H) and c.193 G > T (D65Y) mutations, respectively. The c.179 -25A > G and the c.340 -23A > G changes cause exon 3 and exon 8 to be lost at the RNA level, respectively. This kind of mutation can cause a problem in molecular diagnosis of CDG-Ia if intronic primers are not correctly chosen, and if molecular diagnosis is not performed at both the DNA and mRNA levels.

Base Sequence↗
PubMed2004

Turtle phylogeny: insights from a novel nuclear intron.

Introns have gained considerable popularity as markers for molecular phylogenetics. However, no primers exist for a nuclear intron that amplifies across all turtles. Available data from morphology and mitochondrial DNA have not unambiguously resolved relationships within the superfamily Trionychoidea and the family Chelidae, which together form a large portion of extant turtle diversity. We tested the phylogenetic utility of a novel intron from the RNA fingerprint protein 35 (R35) as applied to these two areas of turtle systematics. We found the intron to be a single-copy locus that provides excellent resolving power for lineages among turtles, though problems with alignment made it impossible to infer deeper amniote relationships. Maximum parsimony and maximum likelihood both demonstrated the polyphyly of Trionychoidea and the reciprocal monophyly of Australian/New Guinea and South American chelid turtles. This is the first study to resolve such relationships with strong statistical support, and we suggest that R35 holds great promise for resolving additional persistent problems in the phylogeny of living turtles.

Animals↗
PubMed2005

Incongruence between primary sequence data and the distribution of a mitochondrial atp1 group II intron among ferns and horsetails.

Using DNA sequence data from multiple genes (often from more than one genome compartment) to reconstruct phylogenetic relationships has become routine. Augmenting this approach with genomic structural characters (e.g., intron gain and loss, changes in gene order) as these data become available from comparative studies already has provided critical insight into some long-standing questions about the evolution of land plants. Here we report on the presence of a group II intron located in the mitochondrial atp1 gene of leptosporangiate and marattioid ferns. Primary sequence data for the atp1 gene are newly reported for 27 taxa, and results are presented from maximum likelihood-based phylogenetic analyses using Bayesian inference for 34 land plants in three data sets: (1) single-gene mitochondrial atp1 (exon+intron sequences); (2) five combined genes (mitochondrial atp1 [exon only]; plastid rbcL, atpB, rps4; nuclear SSU rDNA); and (3) same five combined genes plus morphology. All our phylogenetic analyses corroborate results from previous fern studies that used plastid and nuclear sequence data: the monophyly of euphyllophytes, as well as of monilophytes; whisk ferns (Psilotidae) sister to ophioglossoid ferns (Ophioglossidae); horsetails (Equisetopsida) sister to marattioid ferns (Marattiidae), which together are sister to the monophyletic leptosporangiate ferns. In contrast to the results from the primary sequence data, the genomic structural data (atp1 intron distribution pattern) would seem to suggest that leptosporangiate and marattioid ferns are monophyletic, and together they are the sister group to horsetails--a topology that is rarely reconstructed using primary sequence data.

Amino Acid Sequence↗
PubMed2005

Regulation of alpha-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-beta1 control element.

Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of alpha-smooth muscle actin (alpha-SMA); however, little is known regarding molecular mechanisms that control activation of alpha-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for alpha-SMA expression in myofibroblasts in vivo and to determine whether activation of alpha-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-beta1 control element (TCE) that are required for alpha-SMA expression in smooth muscle cells. A Lac Z transgene construct from -2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous alpha-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-beta1 in cultured transgenic fibroblasts. These same elements were also critical in regulating alpha-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of alpha-SMA expression in myofibroblasts in granulation tissue.

Actins↗
PubMed1999

Exon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in Dubin-Johnson syndrome.

BACKGROUND & AIMS: The Dubin-Johnson syndrome is characterized by conjugated hyperbilirubinemia and by impaired secretion of anionic conjugates from hepatocytes into bile. Absence of the multidrug-resistance protein 2 (MRP2; symbol ABCC2), an adenosine triphosphate-dependent conjugate export pump, from the hepatocyte canalicular membrane is the molecular basis of this syndrome. The aim of this study was the elucidation of all exon-intron boundaries of the MRP2 gene as a prerequisite for the analysis of mutations in patients with Dubin-Johnson syndrome. METHODS: Exon-intron boundaries of MRP2 were determined, and the amplified exons were screened for mutations. Immunofluorescence microscopy served to localize the MRP2 protein in human liver. RESULTS: The human MRP2 gene is approximately 45 kilobases long; it contains 32 exons and a high proportion of class 0 introns. In 2 patients with Dubin-Johnson syndrome, we detected a nonsense mutation at codon 1066 and a 6-nucleotide deletion mutation affecting codons 1392-1394. The MRP2 protein was absent from the canalicular membrane of both patients. CONCLUSIONS: The mutations detected so far show that various mutations in the MRP2 gene can lead to the Dubin-Johnson syndrome. The exon-intron boundaries established in this article will facilitate the analysis of additional mutations in the MRP2 gene.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Association of apolipoprotein E genotype and intronic polymorphism of the presenilin-1 gene with Alzheimer's disease in elderly Taiwan Chinese.

We studied the apoliprotein E (apo-E) allele frequencies and intronic polymorphism of the presenilin-1 (PS-1) gene in 55 patients with late-onset Alzheimer's disease (AD) and 93 age- and sex-matched controls. The apoE epsilon4 allele frequency was significantly higher in the AD group than in the control group (0.255 versus 0.070, P<0.0001). The odds ratio for AD in individuals with either one or two epsilon4 alleles was 5.22 (95% confidence interval: 2.32-11.70). The polymorphism within the region composing intron 3' to exon 8 of the PS-1 gene showed a similar distribution between AD patients and controls. This is the first study on the intronic polymorphism of the PS-1 gene in Chinese. Our results support an association between apoE epislon4 and AD in Chinese, but not between the intronic polymorphism of the PS-1 gene and AD. However, the allele frequency of apo-E epsilon4 among Chinese is lower than that among Caucasians. The interaction between apo-E and PS-1 genotypes is still unclear.

Aged↗
PubMed1999

No association between an intronic polymorphism in the presenilin-1 gene and Alzheimer disease in a German population.

A polymorphism in intron 8 of the presenilin-1 (PS-1) gene has been demonstrated to increase the risk for developing late-onset Alzheimer disease (AD). Conflicting results exist for the association between this intronic polymorphism and AD probably due to variations in the PS-1 gene among different ethnic groups. We investigated the genetic association between this intronic polymorphism in the PS-1 gene and AD in a homogenous group of German Caucasians. The control group consisted of healthy subjects and depressed patients. There were no significant differences in the distribution of the PS-1 genotypes and allele frequencies between AD patients and controls. Our data do not support an association between the intronic polymorphism of the PS-1 gene and AD and there was no interaction between the PS-1 genotype and apolipoprotein E epsilon4 allele.

Aged↗
PubMed1988

Conserved quartets near 5' intron junctions in primate nuclear pre-mRNA.

Analysis of a 1000 nucleotide span around 664 primate 5' exon/intron junctions revealed frequent recurrences of G-rich runs downstream of the 5' splice sites. In particular, AGGG, GGGA, GGGG, GGGT and TGGG are frequent at this site. Some C-rich quarters are frequent upstream of the 5' splice site. Similar behaviour of these G- and C-rich quartets is indicated for the 587 rodent introns and for a combined eukaryotic file containing 1688 introns. (A)GGG(A) is also frequent in the introns 60 nucleotides upstream of the 3' splice site, and (A)CCC(A) is frequently found in the exons downstream of the 3' site. The same consistent behaviour of the 3' splice sites is obtained as for the 5' sites, for the primates, rodents and combined eukaryotic file. These results suggest that in addition to the well-conserved 5' and 3' splice sequences, exon as well as intron sequences may play a role in nuclear pre-mRNA splicing.

Animals↗
PubMed1996

A tyrosyl-tRNA synthetase recognizes a conserved tRNA-like structural motif in the group I intron catalytic core.

The Neurospora crassa mitochondrial (mt) tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns, in addition to aminoacylating tRNA(Tyr). Here, we compared the CYT-18 binding sites in the N. crassa mt LSU and ND1 introns with that in N. crassa mt tRNA(Tyr) by constructing three-dimensional models based on chemical modification and RNA footprinting data. Remarkably, superimposition of the CYT-18 binding sites in the model structures revealed an extended three-dimensional overlap between the tRNA and the group I intron catalytic core. Our results provide insight into how an RNA-splicing factor can evolve from a cellular RNA-binding protein. Further, the structural similarities between group I introns and tRNAs are consistent with an evolutionary relationship and suggest a general mechanism for the evolution of complex catalytic RNAs.

Base Sequence↗
PubMed1997

Mobility of yeast mitochondrial group II introns: engineering a new site specificity and retrohoming via full reverse splicing.

The mobile group II introns aI1 and aI2 of yeast mtDNA encode endonuclease activities that cleave intronless DNA target sites to initiate mobility by target DNA-primed reverse transcription. For aI2, sense-strand cleavage occurs mainly by a partial reverse splicing reaction, whereas for aI1, complete reverse splicing occurs, leading to insertion of the linear intron RNA into double-stranded DNA. Here, we show that aI1 homing and reverse splicing depend on the EBS1 (RNA)/IBS1(DNA) pairing and that target specificity can be changed by compensatory changes in the target site and the donor intron. Using well-marked strains to follow coconversion of flanking DNA, we show that homing occurs by both RT-dependent and -independent pathways. Remarkably, in most RT-dependent events, the reverse spliced intron is the initial template for first-strand cDNA synthesis.

Base Sequence↗
PubMed2002

Differential amplifying RT-PCR: a novel RT-PCR method to differentiate mRNA from its DNA lacking intron.

A major problem with reverse transcription-polymerase chain reaction (RT-PCR) is that the products amplified from RNA and DNA are not distinguishable. The use of a primer set targeted for an intron-containing sequence or RNA extract without contaminating DNA has been established to overcome this problem. However, an intron sequence does not always exist in the target region and complete removal of DNA during RNA extraction is not practical. For these reasons, we developed differential amplifying RT-PCR (DART-PCR) based on differences in reaction temperatures between RT and PCR, as well as in architectures between RNA and DNA. DART-PCR was designed to differentiate products amplified from RNA and DNA regardless of the presence of introns. DART-PCR can be readily applied for diagnosis of active infection of human cytomegalovirus by detection of glycoprotein B mRNA, which gene lacks introns. Other advantages of DART-PCR were as follows: there is no need for separation of RNA from DNA, simultaneous/differential detection in a single tube and possibility of determination of relative amounts of RNA and DNA.

Cell Line↗
PubMed2000

The chicken genome contains no HMG1 retropseudogenes but a functional HMG1 gene with long introns.

We have cloned the genomic sequence coding for the high mobility group 1 (HMG1) protein in chickens. Multiple sequence alignment shows that the chicken HMG1 gene is highly homologous to the human and the mouse HMG1 genes. The gene structure of chicken HMG1 is similar to that of the mouse and the human HMG1 genes, with the same exon-intron boundaries. However, in contrast to other avian genes that have shorter introns, the chicken HMG1 gene has introns that are twice as long as their mammalian homologues. In addition to the functional, intron-containing HMG1 gene, all mammalian genomes contain more than 50 copies of HMG1 retropseudogenes each, while in the chicken genome there are no HMG1 retropseudogenes. This finding suggests that the HMG1 retropseudogenes arose in mammals after their divergence away from the birds.

Amino Acid Sequence↗
PubMed2001

The human HMGB1 promoter is modulated by a silencer and an enhancer-containing intron.

The highly conserved, ubiquitous high mobility group protein HMGB1 (formerly named as HMG1) is an architectural transcription factor encoded by a single functional gene in human. HMGB1 is expressed in almost all cell or tissue types studied. In general, it is expressed at a basal level in most cells but at a slightly elevated level of 2--3-fold in actively proliferating tissues or estrogen stimulated breast cancer cells. To understand the regulatory mechanism controlling expression of the human HMGB1 gene, we cloned and analyzed the upstream region as well as the first intron of this gene. We found that transcription of the human HMGB1 gene in the breast cancer MCF-7 cells starts at one major site 57 nucleotides upstream from the first exon-intron boundary. Expression of the human HMGB1 gene is under the control of a very strong TATA-less promoter, which has an activity more than 18-fold that of the SV40 promoter. Immediately upstream, a silencer element is present. This silencer can repress the activity of the HMGB1 promoter down to just one-sixth. The first intron of the human HMGB1 gene contains enhancer elements, which can increase the human HMGB1 promoter activity by 2--3-fold. We postulate that the human HMGB1 gene is capable of being expressed at a very high level. The basal level of expression observed in most cells is probably a result of the strong promoter being held in check by the silencer. The 2--3-fold increase in HMGB1 expression observed in proliferating cells or breast cancer cells stimulated by estrogen may probably result from the action of the enhancer elements in intron 1.

Base Sequence↗