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PubMed1991

Splicing of the Drosophila P element ORF2-ORF3 intron is inhibited in a human cell extract.

P element transposition in Drosophila melanogaster is regulated by germline-specific splicing of the P element ORF2-ORF3 intron. This regulation has been shown to depend on a cis-acting sequence located in the exon 12-31 bases from the 5' splice site. Mutations within this sequence disrupt the regulation and result in splicing of the ORF2-ORF3 intron in all tissues, indicating that the sequence is required to inhibit splicing of this intron in the soma. We now show that a trans-acting factor in a human (HeLa) cell extract can inhibit splicing of the intron, suggesting that this regulatory mechanism is conserved from flies to humans.

Animals↗
PubMed1990

Self-splicing group II and nuclear pre-mRNA introns: how similar are they?

The splicing pathway of pre-mRNA introns bears similarities to that of the group II introns, some members of which undergo self-splicing. The snRNAs may provide the pre-mRNA introns with RNA structures in trans comparable to those available in cis in group II introns. This article examines the available evidence for the hypothesis that the catalysis of these two splicing pathways is fundamentally equivalent.

Animals↗
PubMed1990

Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing.

Group I and group II introns catalyse their own splicing, but depend on protein factors for efficient splicing in vivo. Some of these proteins, termed maturases, are encoded by the introns themselves and may also function in intron mobility. Other proteins are encoded by host chromosomal genes and include aminoacyl-tRNA synthetases and various proteins that function in protein synthesis. The splicing factors identified thus far appear to be idiosyncratic, even in closely related organisms. We suggest that some of these protein-assisted splicing reactions evolved relatively recently, possibly reflecting the recent dispersal of the introns themselves.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Intron-less processed Pcdhalpha genes in the central nervous system.

The genomic organization of the Pcdhalpha is remarkably similar to those of T-cell receptor and immunoglobulin genes. To elucidate the somatic rearrangements of the genomic DNAs of Pcdhalphas in the central nervous system, we screened a genomic brain library of the C57BL/6 mouse strain. From this screening we isolated an unusual, rearranged genomic Pcdhalpha DNA. This clone contained an intron-less Pcdhalpha v6 gene resembling the cDNA generated from its mRNA, inserted into the 28S rDNA gene locus. The intron-less Pcdhalpha v6 gene possessed a putative promoter region and 10 nucleotide substitutions but no poly(A) signal. Both edges of the integration site had additional 5-bp duplicated sequences. PCRs that were performed using primers for intron-less Pcdhalphas amplified products from genomic DNAs only in the brain; moreover, using the size-fractionated genomic DNA by sucrose density gradient centrifugation, intron-less Pcdhalpha is mainly amplified in the small size fraction of the genomic DNA. Inverted PCR for this small size fraction also amplifies the Pcdhalphas connecting the both ends with repeat sequences. These features suggest the somatic reverse-transcription, circularization, and rare occasion of integration into the genome of Pcdhalpha in the brain.

Animals↗
PubMed2005

An intronic enhancer regulates cyclooxygenase-1 gene expression.

To identify cis-elements regulating PMA-induced prostaglandin H synthase-1 (PGHS-1) gene expression in the human megakaryoblast cell line, MEG-01, we performed promoter reporter assays with a luciferase reporter vector containing the -2030/-22 region of the human PGHS-1 gene. PMA treatment for 24 h increased PGHS-1 promoter activity by twofold. Mutagenesis studies of the promoter revealed a single Sp1 site essential for PMA-inducible transcription. Insertion of a highly conserved 100 bp sequence cloned from intron 8 into the -2030/-22 reporter plasmid enhanced PMA-dependent transcription 10-fold. Mutation of either a consensus AP-1 site within intron 8 or the Sp1 site in the promoter reduced PMA-induced activity by 80-100%. Gel shift assays using the intron 8 AP-1 sequence demonstrated the formation of an AP-1-specific DNA-protein complex. Our results suggest that inducible PGHS-1 gene expression involves the coordinate functioning of a Sp1 site in the promoter and an AP-1 site in intron 8.

Animals↗
PubMed2005

Use of Intron 1 and 22 inversions and linkage analysis in carrier detection of hemophilia A in Indians.

BACKGROUND: Hemophilia A is an X-linked recessively inherited bleeding disorder characterized by deficiency of procoagulant factor VIII (FVIII). METHODS: Sixty unrelated hemophilia A patients and their family members have undergone tests for carrier detection by linkage analysis using the polymorphic markers Bcl I, Xba I and Intron 13 or 22 VNTRs. In families of sporadic hemophiliacs, the carrier status of female subjects was ascertained by linkage analysis along with FVIII:C/VWD Ag estimation. RESULTS: Of the 33 families with positive family history, the defective X chromosome was tracked in 28 mothers. The carrier status of females from hemophilia A families with positive family history, ascertained by linkage analysis and Intron 22 and 1 inversion, was made out in 85% cases. FVIII:C/VWF Ag ratio was evaluated in 36 females from 9 sporadic hemophilic families. Using the FVIII:C/VWF Ag ratio along with linkage analysis, carrier status was determined in 9 (25%) of the 36 females studied. Using Intron 22 inversion along with linkage analysis and FVIII:C/VWF Ag estimation, the informativity in female subjects from families of sporadic hemophiliacs increased from 25% to 52%. CONCLUSION: In the West, linkage analysis with Bcl I, Xba I and Intron 13 or 22 VNTR markers and inversion 22 offers a good tool for carrier detection of hemophilia A in India.

Chromosome Inversion↗
PubMed2005

eNOS gene intron 4 a/b VNTR polymorphism is a risk factor for coronary artery disease in Southern Turkey.

BACKGROUND: Nitric oxide (NO) plays a major role in the regulation of vascular tone Associations between NO genotypes, coronary artery disease (CAD) and other risk factors have been described by many authors. The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) gene intron 4 a/b variable number of tandem repeats (VNTR) polymorphism and other risk factors in the development of CAD in subjects living in Southern Turkey. METHODS: Two-hundred and sixty-six patients (154 males and 112 females, aged between 30 and 80 years, mean 52.4+/-10.3) whose coronary arteries were evaluated by means of coronary angiography were enrolled in the study. Of the total, 133 had CAD (Group I) and the remaining had normal coronary arteries (Group II). The eNOS gene intron 4 a/b VNTR polymorphism was analyzed by polymerase chain reaction. The plasma lipid levels and other risk factors were also determined in all subjects. RESULTS: The a allele frequencies and genotypes carrying a allele were significantly higher in Group I. Plasma lipids, except HDL-C, were also increased in this group. We found that hypertension (HT), diabetes mellitus (DM), male gender, age and smoking were the independent predictors of CAD. CONCLUSION: a allele of eNOS intron 4 a/b VNTR polymorphism is not an independent predictor of CAD. eNOS intron 4 a/b polymorphism (presence of a allele) is a risk factor in addition to HT, DM, male gender, age and smoking for the development of CAD in Southern Turkey.

Adult↗
PubMed2004

Receptor binding specificities of Herstatin and its intron 8-encoded domain.

Retention of intron 8 in alternative HER-2 mRNA generates an inhibitory secreted ligand, Herstatin, with a novel receptor-binding domain (RBD) encoded by the intron. This study examines binding interactions with several receptors and investigates sequence variations in intron 8. The RBD, expressed as a peptide, binds at nM concentrations to HER-2, the EGFR, DeltaEGFR, HER-4 and to the IGF-1 receptor, but not to HER-3 nor to the FGF-3 receptor, whereas a rare mutation in the RBD (Arg to Ile) eliminates receptor binding. The full-length Herstatin binds with 3-4-fold higher affinity than its RBD, but with approximately 10-fold lower affinity to the IGF-IR. Sequence conservation in rhesus monkey but not in rat suggests that intron 8 recently diverged as a receptor-binding module critical for the function of Herstatin.

3T3 Cells↗
PubMed2003

Mapping and characterization of polymorphism in mtDNA of Cryphonectria parasitica: evidence of the presence of an optional intron.

The mitochondrial DNA (mtDNA) of the filamentous ascomycete Cryphonectria parasitica is large and polymorphic so, to better understand the nature of the polymorphisms within populations, a small collection of Italian strains of the fungus was examined. Known mtDNA polymorphisms were mapped and found to cluster in four regions of the mtDNA molecule, particularly in the RFLP region 2 where five different mtDNA haplotypes out of 13 strains were identified. This region included an area of 8.4kbp which was entirely sequenced in strain Ep155 showing the presence of two introns. An internal 3.2kbp portion was sequenced also in six additional strains. Sequence comparison of the C. parasitica mitochondrial intronic ORFs revealed similarities to known endonucleases such as those of Podospora anserina and Neurospora crassa. DNA sequence analysis showed that three polymorphisms of this mtDNA region within this population of 12 strains were due to the optional presence in the ND5 gene of an intron and of an intervening sequence within the intron. Evidence was also found within this population of mixed mitochondrial types within a single strain.

Base Sequence↗
PubMed2006

The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi.

The Agrocybe chaxingu and Agrocybe aegerita mitochondrial apocytochrome b coding sequences are highly similar (97% of nt identity), but have highly different sizes (2312 and 4867nt, respectively), due to the presence of three large group IB introns: two (iAae1 and iAae2) in A. aegerita, one (iAch1) in A. chaxingu. All these introns encode a homing endonuclease (HE) similar to those described in introns of mitochondrial genes (cob, cox1, and nad5) from various organisms. Phylogenetic trees were built with these HE sequences. From these trees, the Agrocybe coding introns argue for recent lateral transfers, i.e., occurring after the separation of the two Agrocybe species, involving phylogenetically distant fungi such as members of the Ascomycota phylum (for iAch1 and iAae2) and, for the first time to our knowledge, a member of the Chytridiomycota phylum (for iAae1). The grouping of the HE gene (HEG) sequences according to the mitochondrial gene (cob, cox1, and nad5) where they are inserted, suggests modifications of the interactions between the HE and the recognized sequences, leading to new target genes. The largest distribution of the iAch1 HE, shared by several cob and cox1 mitochondrial genes from Ascomycota, Basidiomycota, and Chytridiomycota phyla, suggests a higher target flexibility of this HE, perhaps related to the presence of two different LAGLIDADG motifs in the catalytic site of the enzyme.

Agaricales↗
PubMed2004

A novel mitochondrial DNA-like sequence insertion polymorphism in Intron I of the FOXO1A gene.

The human forkhead box O1A (FOXO1A) gene belongs to the human forkhead gene family and acts downstream of the human insulin signalling pathway. In this study, polymorphisms of the Intron I of FOXO1A gene were studied in Italian healthy people and insulin resistant subjects. No significant association between the germ-line variability in the Intron I of FOXO1A and insulin resistance was observed. Interestingly, during the study, a new 39-bp sequence insertion polymorphism in Intron I of FOXO1A gene was described. The polymorphism was found to co-segregate in a co-dominant Mendelian fashion and to be present in an ethnically distinct population (Greeks). A BLAST search showed that the sequence shares 100% identity with a mtDNA (mitochondrial DNA) sequence coding for the ATP synthase 8 (ATPase8) and ATP synthase 6 (ATPase6) genes. Hence, FOXO1A Intron I is a polymorphic nuclear region involved in the exchange of DNA material between mitochondrial and genomic DNA, which is a well-established mechanism of evolutionary change in eukaryotes.

Adenosine Triphosphatases↗
PubMed2004

Novel cis-element in intron 1 represses somite expression of zebrafish myf-5.

Myf-5 is a basic helix-loop-helix (bHLH) transcription factor that controls muscle differentiation. During early embryogenesis, myf-5 expression is transient, somite- and stage-specific. However, the negative regulation of myf-5 is poorly understood. We constructed a plasmid [(-9977/-1)/E1/I1/E2/GFP] that contains the sequence -9977 to -1, exon 1 (E1), intron 1 (I1), and exon 2 (E2) of zebrafish (Danio rerio) myf-5 and a reporter GFP gene. This plasmid was microinjected into zebrafish zygotes. Surprisingly, the somite-specific expression rate of reporter GFP in the transgenic embryos was extremely low (2%, n=392), compared to that of (-9977/-1)/GFP (92%, n=210). Dramatic repression of myf-5 expression was also observed in embryos microinjected with plasmids in which the sequence -8600/-1, -2937/-1 or -290/-1 was linked to E1/I1/E2/GFP. Thus, intron 1 contains a silencer that specifically represses the activity of myf-5. Functional analysis of intron 1 showed a strong, negative, cis-regulatory element was located at +502/+835. Its function was orientation- and position-dependent. The repressive capability of this silencer was completely dependent on two core motifs, IE1 (+502/+527) and IE2 (+816/+835), and a 156-bp spanning sequence that lies between them. This is the first study to identify a novel, cis-acting silencer in intron 1 that is crucial to negatively regulating zebrafish myf-5 expression.

Animals↗
PubMed2005

The evolution of introns in human duplicated genes.

In previous work [Jabbari, K., Rayko, E., Bernardi, G., 2003. The major shifts of human duplicated genes. Gene 317, 203-208], we investigated the fate of ancient duplicated genes after the compositional transitions that occurred between the genomes of cold- and warm-blooded vertebrates. We found that the majority of duplicated copies were transposed to the "ancestral genome core", the gene-dense genome compartment that underwent a GC enrichment at the compositional transitions. Here, we studied the consequences of the events just outlined on the introns of duplicated genes. We found that, while intron number was highly conserved, total intron size (the sum of intron sizes within any given gene) was smaller in the GC-rich copies compared to the GC-poor copies, especially in dispersed copies (i.e., copies located on different chromosomes or chromosome arms). GC-rich copies also showed higher densities of CpG islands and Alus, whereas GC-poor copies were characterized by higher densities of LINEs. The features of the copies that underwent the compositional transition and became GC-richer are suggestive of, or related to, functional changes.

Alu Elements↗
PubMed2004

Association of estrogen receptor 1 intron 1 C/T polymorphism in Korean vitiligo patients.

BACKGROUND: Vitiligo is a common disease characterized by cutaneous white maculae due to loss of melanocytes. It is a polygenic disease, however, the exact pathogenesis of vitiligo is not yet known. The estrogen receptor (ESR) 1 gene was selected as a candidate gene because some researchers treated vitiligo successfully with the steroid-thyroid hormone mixture containing estrogen. Furthermore ESR was expressed in the melanocytes which have an important role in the pigmentation. The polymorphisms of ESR1 gene in vitiligo patients was not reported yet. OBJECTIVE: To determine whether polymorphisms of ESR1 gene were associated with susceptibility to vitiligo patients in Korean population. METHODS: We conducted case-control association study of vitiligo patients (120) and healthy controls (254). Genotypes of ESR1 gene (intron 1 C/T, exon 4 C/G, and exon 8 A/G) were determined by polymerase chain reaction followed by restriction enzyme digestion. RESULTS: Intron 1 T/C allele frequency was significantly different between patients and controls (P = 0.034). Intron 1 T/C genotype distribution (P = 0.021) and allele frequency (P = 0.013) were different between female vitiligo patients and female controls. Intron 1 T/C allele frequency showed significantly difference between generalized type of vitiligo patients and controls (P = 0.044). Genotype distributions and allele frequencies of exon 4 C/G and exon 8 A/G polymorphisms were not different between patients and controls. CONCLUSION: The present study suggests that ESR1 may be a possible risk factor for female or generalized type of vitiligo patients.

Adolescent↗
PubMed2005

Transcriptional regulation of the sbeIIb genes in sorghum (Sorghum bicolor) and barley (Hordeum vulgare): importance of the barley sbeIIb second intron.

The transcriptional activity of the sorghum sbeIIb gene, encoding starch branching enzyme IIb, is seed specific, with expression in both the endosperm and the embryo. In comparison, expression of barley sbeIIb is confined to the endosperm, whereas that of barley sbeIIa occurs in endosperm, embryonic and vegetative tissues. It has been suggested that the second intron of barley sbeIIb may be instrumental in conferring endosperm-specific expression. Therefore, to further investigate the regulatory mechanisms of barley and sorghum sbe, we examined the tissue-specific activity of the sorghum sbe promoter in transient assays of green fluorescent protein (gfp) reporter constructs. We found that, when linked to the barley sbeIIb second intron, the sorghum sbeIIb promoter could not drive gfp transcription in sorghum or barley embryonic cells. Similar results were obtained for the barley sbeIIa promoter. Database searches showed that sequences homologous to the barley sbeIIb intron also exist in introns and flanking regions of some other grass genes. Deletion mutagenesis of the sorghum sbeIIb promoter identified the minimal promoters required for high- and low-level expression, respectively, but did not reveal any putative promoter elements crucial for expression. A sequence with similarity to the SURE element, implicated in sugar signaling, was located in the distal promoter region of sorghum sbeIIb, upstream of the minimal promoters. SURE elements are present in the proximal promoter regions of the sugar-regulated barley iso1 gene, and barley sbeIIb. In keeping in line with these observations, RNA-gel blot analyses demonstrated that expression of barley sbeIIb was sugar inducible, whereas that of sorghum sbeIIb was not.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2004

Increased protein expression from adenoviral shuttle plasmids and vectors by insertion of a small chimeric intron sequence.

Adenoviruses are widely used as gene transfer vehicles because they can be produced at high titers, they have a large transgene capacity, and can transduce both dividing and non-dividing cells. One disadvantage of adenoviral vectors is the narrow therapeutic window due to a dose-dependent humoral as well as a T-cell dependent host immune response directed against the transduced cells, that leads to a reduction of transgene expression with time. By increasing the levels of protein expression from transcription units, vector titres may be decreased without a significant loss of transgene expression. Introns are required for efficient expression of many protein coding genes. In addition, splicing signals are required for some genes in order to be translated. Therefore, a chimeric intron sequence was introduced at the 3' end of the transgenes to study its effect on protein expression from adenoviral vector constructs. Transfection of 293 cells with the adenoviral shuttle plasmids pMH4-EGFP-Int, pMH4-E314.7-Int, pMH4-BclX(L)-Int and pMH4-Luc-Int lead to a 1.8-20-fold increase in protein expression as compared to constructs lacking an intron. Injection of Ad-CMV-Luc-Int into the brain of C57Bl/6 mice results in an approximately three-fold increase of luciferase activity as compared to Ad-CMV-Luc. In conclusion, insertion of an intron sequence leads to a significant increase in transgene expression both in vitro and in vivo, thus allowing for reduction of the adenoviral vector dose used.

Adenoviridae↗
PubMed2005

Expression, purification, and biochemical characterization of the intron-encoded endonuclease, I-CreII.

The ORF of the Cr.psbA4 intron of Chlamydomonas reinhardtii mediates efficient intron homing, and contains an H-N-H and possibly a GIY-YIG motif. The ORF was over-expressed in Escherichia coli without non-native amino acids, but was mostly insoluble. However, co-over-expression of E. coli chaperonins GroEL/GroES solubilized approximately 50% of the protein, which was purified by ion-exchange and heparin-affinity chromatography. Biochemical characterization showed that the protein is a double-strand-specific endonuclease that cleaves fused psbA exon 4-exon 5 DNA, and was named I-CreII. I-CreII has a relatively relaxed divalent metal ion requirement (Mg(2+), Mn(2+), Ca(2+), and Fe(2+) supported cleavage), is insensitive to salt <350 mM, and is stabilized by DNA. Cleavage of target DNA occurs close (4 nt on the top strand) to the intron-insertion site, and leaves 2-nt 3'-OH overhangs, similar to GIY-YIG endonucleases. The boundaries of the recognition sequence span approximately 30 bp, and encompass the cleavage and intron-insertion sites. Cleavage of heterologous psbA DNAs indicates the enzyme can tolerate multiple, but not all, substitutions in the recognition site. This work will facilitate further study of this novel endonuclease, which may also find use in site-specific manipulation of chloroplast DNA.

Algal Proteins↗
PubMed2005

Atomic level architecture of group I introns revealed.

Twenty-two years after their discovery as ribozymes, the self-splicing group I introns are finally disclosing their architecture at the atomic level. The crystal structures of three group I introns solved at moderately high resolution (3.1-3.8A) reveal a remarkably conserved catalytic core bound to the metal ions required for activity. The structure of the core is stabilized by an intron-specific set of long-range interactions that involves peripheral elements. Group I intron structures thus provide much awaited and extremely valuable snapshots of how these ribozymes coordinate substrate binding and catalysis.

Binding Sites↗