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At least 1,603 records · Page 89Linked to original sources
PubMed2006

Loss of introns along the evolutionary diversification pathway of snake venom disintegrins evidenced by sequence analysis of genomic DNA from Macrovipera lebetina transmediterranea and Echis ocellatus.

Analysis of cDNAs from Macrovipera lebetina transmediterranea (Mlt) and Echis ocellatus (Eo) venom gland libraries encoding disintegrins argued strongly for a common ancestry of the messengers of short disintegrins and those for precursors of dimeric disintegrin chains. We now report the sequence analysis of disintegrin-coding genes from these two vipers. Genomic DNAs for dimeric disintegrin subunits Ml_G1 and Ml_G2 (Mlt) and Eo_D3 (Eo) contain single 1-kb introns exhibiting the 5'-GTAAG (donor)/3'-AG (acceptor) consensus intron splicing signature. On the other hand, the short RTS-disintegrins Ml_G3 (Mlt) and Eo_RTS (Eo) and the short RGD-disintegrin ocellatusin (Eo) are transcribed from intronless genomic DNA sequences, indicating that the evolutionary pathway leading to the emergence of short disintegrins involved the removal of all intronic sequences. The insertion position of the intron within Ml_G1, Ml_G2, and Eo_D3 is conserved in the genes for vertebrate ADAM (A disintegrin and metalloproteinase) protein disintegrin-like domains and within the gene for the medium-size snake disintegrins halystatins 2 and 3. However, a comparative analysis of currently available disintegrin(-like) genes outlines the view that a minimization of both the gene organization and the protein structure underlies the evolution of the snake venom disintegrin family.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of the host-adapted pathogen Verticillium longisporum on the basis of a group-I intron found in the nuclear SSU-rRNA gene.

Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb. nov. With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca. 2.5 kb was generated from all isolates of V. longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates. On the contrary, all the other phytopathogenic and nonphytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V. lecanii and two of Cordyceps sp., generated a product of ca. 1.65 kb. Sequence analysis of the SSU-rRNA gene of two typical isolates of V. longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae). The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements. Primers designed from the 839-bp intron sequence amplified only the V. longisporum. Phylogenetic analysis based on SSU-rDNA sequences showed that V. longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies.

Base Sequence↗
PubMed2000

Intragenic suppressors that restore the splicing and homing activities of the protein encoded by the second intron of the Saccharomyces capensis cyt b gene.

The second (bi2) intron of the mitochondrial cyt b gene from Saccharomyces capensis encodes a bifunctional protein which acts both as a maturase, promoting intron splicing, and as a homing-endonuclease, I-ScaI, promoting intron mobility. In this work we isolated and characterized revertants from a respiratory-deficient mutant in which both functions of the protein have been lost. Intragenic revertants resulted mainly from monosubstitutions in the mutated codon and in one case from a distant second site mutation. All novel variants of the S. capensis bi2 intron-encoded protein are competent for the maturase activity but only two of them can partially complement the homing function.

Cytochrome b Group↗
PubMed1997

The actin gene of the glaucocystophyte Cyanophora paradoxa: analysis of the coding region and introns, and an actin phylogeny of eukaryotes.

We isolated the actin gene of the glaucocystophyte alga Cyanophora paradoxa and analyzed the coding region and its introns. Phylogenetic analyses of the actin coding region and the inferred protein sequence in data sets containing 47 other actin sequences show Cyanophora to be a member of the eukaryotic crown-group radiation in agreement with ribosomal DNA sequence analyses. Four of the five Cyanophora actin introns are relatively short (55-59 nt) and occupy novel positions in a catalogue of actin introns containing 56 distinct sites. The fifth intron has a length of 171 nt and occurs also in actin genes from green algae and the crustacean Artemia.

Actins↗
PubMed1997

Introns boost transgene expression in Drosophila melanogaster.

Using Drosophila as a host, we have examined the effect that the presence of an intron has on the accumulation of a processed transgene mRNA. To provide a model system that was free from major position effects, two fish antifreeze protein (AFP) transgenes were arranged in divergent transcriptional orientation from a central Drosophila yolk protein promoter/enhancer region and introduced into the flies by P-element transformation and/or mobilization. In this way the organization of both the structural genes and the promoter elements mimicked their natural arrangements. When one member of the fish AFP transgene pair had its single 180 bp intron deleted, there was a 2- to 11-fold (average 5-fold) decrease in its mRNA level compared to that generated from the control gene containing the fish AFP intervening sequence. When the deleted intron was replaced by a 70 bp intervening sequence originating from the yolk protein 1 gene, mRNA accumulation was restored to its original level. Even for the streamlined genome of Drosophila, where the intron number and size are generally reduced compared to mammals, the presence of an intervening sequence appears to facilitate mRNA accumulation.

Animals↗
PubMed1997

Mitochondrial protein synthesis is not required for efficient excision of intron aI5 beta from COX1 pre-mRNA in Saccharomyces cerevisiae.

Splicing of the group I intron aI5 beta from the yeast mitochondrial COX1 transcript requires at least four proteins, encoded by the nuclear genes PET54, MRS1/PET157, SUV3 and MSS18. These proteins either act directly to facilitate intron aI5 beta excision, or indirectly in some manner. One possible indirect mode of action of these nuclear gene products is in stimulation of expression of a mitochondrial protein, such as a maturase, that is necessary for intron aI5 beta excision. To test this possibility, splicing of intron aI5 beta was examined in a rho-strain, which is incapable of mitochondrial protein synthesis. A quantitative RT-PCR assay was set up to compare levels of spliced COX1 mRNA present in three strains: a wild-type rho + strain; the rho-strain 7-49b-11, which retains the entire COX1 transcription unit; and a strain bearing a null mutation in the nuclear PET54 gene. The results showed that excision of aI5 beta occurs relatively efficiently in the rho-strain, and therefore does not require any mitochondrial-encoded proteins.

DNA Restriction Enzymes↗
PubMed1999

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reihardtii and Volvox carteri: phylogenetic origins and recent insertion of introns.

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble "proto-splice" sequences in the Chlamydomonas gene.

Acetolactate Synthase↗
PubMed2002

Analysis of 22 deletion breakpoints in dystrophin intron 49.

Over 60% of Duchenne and Becker muscular dystrophies are caused by deletions spanning tens or hundreds of kilobases in the dystrophin gene. The molecular mechanisms underlying the loss of DNA at this genomic locus are not yet understood. By studying the distribution of deletion breakpoints at the genomic level, we have previously shown that intron 49 exhibits a higher relative density of breakpoints than most dystrophin introns. To determine whether the mechanisms leading to deletions in this intron preferentially involve specific sequence elements, we sublocalized 22 deletion endpoints along its length by a polymerase-chain-reaction-based approach and, in particular, analyzed the nucleotide sequences of five deletion junctions. Deletion breakpoints were homogeneously distributed throughout the intron length, and no extensive homology was observed between the sequences adjacent to each breakpoint. However, a short sequence able to curve the DNA molecule was found at or near three breakpoint junctions.

Base Sequence↗
PubMed2000

Identification of an intronic regulatory element in the human protein C (PROC) gene.

Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms.

Base Sequence↗
PubMed2000

No association between presenilin 1 (PS1) intronic polymorphism and sporadic Alzheimer's disease in Koreans.

To investigate the possible involvement of an intronic polymorphism in the presenilin 1 (PS1) gene and its interactions with the aplolipoprotein E (APOE) or alpha-1 antichymotrypsin (ACT) polymorphisms in the manifestation of AD, we analyzed the PS1, APOE and ACT genotypes of 100 sporadic AD patients and 199 normal elderly controls in Koreans. The genotypic (chi2= 0.92, df = 2, P > 0.1) and allelic (chi2 = 0.01, df = 1, P > 0.1) frequencies of the PS1 polymorphism in the late- and early-onset sporadic AD patients did not differ from those in the controls. And the occurrence of the APOE epsilon4 allele and ACT A allele did not influence the distribution of the PS1 intronic polymorphism. The PS1 intronic polymorphism didn't influence the age-at-onset of AD (F = 0.02, df = 2, P > 0.1). In conclusion, the PS1 intronic polymorphism did not modify the risk for sporadic AD, neither independently nor synergistically with the APOE epsilon4 allele or ACT A allele, in Koreans.

Age of Onset↗
PubMed2006

Group I intron renders differential susceptibility of Candida albicans to Bleomycin.

The alarming increase in drug resistance gained by fungal pathogens has raised an urgent need to develop drugs against novel targets. Candida albicans, an opportunistic fungal pathogen, harbors in its 25S rRNA gene, a self-splicing Group I intron, which can act as a selective drug target. We report that Bleomycin selectively inhibits the self-splicing of Group I intron of C. albicans at IC(50) = 1.2 microM, leading to accumulation of precursor RNA as evinced by Reverse Transcriptase PCR. Drug susceptibility assays including MIC determination, growth curve analysis and disc diffusion assays indicate a strong susceptibility of the intron-containing strain (4-1) than the intronless strain (62-1). These results on the preferential targeting of Group I intron of C. albicans by Bleomycin might form a basis for design of small molecules that inhibit self-splicing of RNA as a antimicrobial tool against life-threatening microorganisms.

Antifungal Agents↗
PubMed2006

Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to tomato yellow leaf curl virus infection in transgenic tomato plants.

The whitefly-transmitted Tomato Yellow Leaf Curl Virus (TYLCV) is the major pathogen of tomato crop in Cuba and one of the most outstanding viral diseases of plants worldwide. In this work, we have developed transgenic tomato plants, transformed with an intron-hairpin genetic construction to induce post- transcriptional gene silencing against the early TYLCV replication associated protein gene (C1). The intron-hairpin RNA produced involves 726 nts of the 3' end of the TYLCV C1 gene as the arms of the hairpin, and the castor bean catalase intron. Transgenic tomato plants belonging to line 126, which harbor a single transgene copy, showed immunity to TYLCV, even in extreme conditions of infection (4-leaf-stage plants and 300 to many hundreds viruliferous whiteflies per plant during 60 days). Dot blot hybridization of these plants showed no TYLCV DNA presence 60 days after inoculation. Small interfering RNA molecules were detected in both inoculated and non-inoculated plants from line 126. These transgenic tomato plants of the otherwise very TYLCV-susceptible Campbell-28 tomato cultivar, are the first report of resistance to a plant DNA virus obtained by the use of the intron-hairpin RNA approach.

Gene Silencing↗
PubMed1989

Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: receptor binding within intron I.

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the expression of cytochrome P450c were investigated using cultured fetal rat hepatocytes. Cytochrome P450c mRNA content, determined by Northern blot analysis, was induced in cells treated with 1,2-benzanthracene from levels undetectable in untreated cells. When dexamethasone was included in the culture medium together with 1,2-benzanthracene there was a further 2-fold increase in the induction of cytochrome P450c mRNA. The concentration of dexamethasone required for a half-maximal increase in cytochrome P450c mRNA content was approximately 10(-9) M. By nuclear run-on transcription assays, treatment with 1,2-benzanthracene induced cytochrome P450c transcription 5.3-fold over untreated cells. In the presence of dexamethasone and 1,2-benzanthracene, there was a further 2-fold increase in cytochrome P450c transcription. Southwestern blotting and exonuclease footprinting methods have identified binding interactions of a purified glucocorticoid receptor fraction with portions of the cytochrome P450c gene within the first intron. Using a chimeric plasmid containing the first intron, the first exon, and 824 bp of 5'-flanking region of the cytochrome P450c gene, chloramphenicol acetyltransferase activity was induced in transfected HepG2 hepatoma cells by the addition of 1,2-benzanthracene. The addition of dexamethasone induced a further 2.2-fold increase in activity. Deletion of the first intron within the chimeric plasmid abolished responsiveness to dexamethasone. It is concluded that glucocorticoids act together with polycyclic aromatic hydrocarbons to increase the levels of cytochrome P450c expressed in the fetal rat hepatocyte, and that this action is mediated by the glucocorticoid receptor. A glucocorticoid responsive element, which binds the glucocorticoid receptor, has been identified within the first intron of the cytochrome P450c gene. These results suggest that glucocorticoids play a significant role in the response of the hepatic cytochrome P450c gene to xenobiotics.

Actins↗
PubMed1996

Evolution of legumin genes: loss of an ancestral intron at the beginning of angiosperm diversification.

The polymerase chain reaction was used to survey gymnosperm legumin genes. Characterization of 46 cloned amplificates, differing in sequence and size (1.2-1.6 kb), revealed the ubiquitous occurrence of legumin genes and their organization in small subfamilies in the 22 species investigated. The 3' portions of the genes, coding for the legumin beta-polypeptides, show a highly conserved intron/exon structure divergent from those of angiosperms: an additional intron (intron IV) uniformly interrupts the region coding for the C-terminal part of the beta-polypeptides. Phylogenetic analysis of the respective coding sequences as well as the organization of the Magnolia B14 legumin gene also investigated here both indicate that intron IV is ancestral and was lost during early angiosperm evolution. Taking into account the intron/exon structures from all legumin genes known, our results suggest that legumin genes evolved by subsequent loss of introns, providing also further evidence for a common origin of legumins and vicilins.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide and encoded amino acid sequence of a mammalian myosin heavy chain gene. Evidence against intron-dependent evolution of the rod.

The complete nucleotide sequence and exon/intron structure of the rat embryonic skeletal muscle myosin heavy chain (MHC) gene has been determined. This gene comprises 24 X 10(3) bases of DNA and is split into 41 exons. The exons encode a 6035 nucleotide (nt) long mRNA consisting of 90 nt of 5' untranslated, 5820 nt of protein coding and 125 nt of 3' untranslated sequence. The rat embryonic MHC polypeptide is encoded by exons 3 to 41 and contains 1939 amino acid residues with a calculated Mr of 223,900. Its amino acid sequence displays the structural features typical for all sarcomeric MHCs, i.e. an amino-terminal "globular" head region and a carboxy-terminal alpha-helical rod portion that shows the characteristics of a coiled coil with a superimposed 28-residue repeat pattern interrupted at only four positions by "skip" residues. The complex structure of the rat embryonic MHC gene and the conservation of intron locations in this and other MHC genes are indicative of a highly split ancestral sarcomeric MHC gene. Introns in the rat embryonic gene interrupt the coding sequence at the boundaries separating the proteolytic subfragments of the head, but not at the head/rod junction or between the 28-residue repeats present within the rod. Therefore, there is little evidence for exon shuffling and intron-dependent evolution by gene duplication as a mechanism for the generation of the ancestral MHC gene. Rather, intron insertion into a previously non-split ancestral MHC rod gene consisting of multiple tandemly arranged 28-residue-encoding repeats, or convergent evolution of an originally non-repetitive ancestral MHC rod gene must account for the observed structure of the rod-encoding portion of present-day MHC genes.

Amino Acid Sequence↗
PubMed1988

Structural organization of the mouse aspartate aminotransferase isoenzyme genes. Introns antedate the divergence of cytosolic and mitochondrial isoenzyme genes.

We have cloned and characterized a mouse cytosolic aspartate aminotransferase (AspAT) (EC 2.6.1.1) gene, which is about 32,000 base-pairs long and is interrupted by eight introns. The 5' and 3'-flanking regions, and the exact sizes and boundaries of the exon blocks, including the transcription-initiation sites, were determined. The 5' end of the gene lacks the TATA and CAAT boxes characteristic of eukaryotic promoters, but contains G + C-rich sequences, three putative binding sites for a cellular transcription factor, Sp1, and multiple transcription-initiation sites. The sequences around the transcription-initiation sites are compatible with the formation of a number of potentially stable stem-loop structures. We compared the structural organization of the mouse cytosolic AspAT gene with that of the mouse mitochondrial AspAT gene, which has nine introns. We found that the promoter regions share a high level of homology and five of the introns are at identical places. This close matching leads to the tentative conclusion that the introns were in place before the divergence of cytosolic and mitochondrial isoenzyme genes.

Amino Acid Sequence↗
PubMed1990

Effect of deletions at structural domains of group II intron bI1 on self-splicing in vitro.

Some group II introns can undergo a protein-independent splicing reaction with the basic reaction pathway similar to nuclear pre-mRNA splicing and the catalytic functions of some of the structural components have been determined. To identify further functional domains, we have generated an ensemble of partial and complete deletions of domains I, II, III and IV of the self-splicing group II intron bI1 from yeast mitochondria and studied their effects on the splicing reaction in vitro. Our results indicate that domains II and IV, which vary considerably in length and structure among group II introns, do not play a direct role in catalysis but mainly help to ensure the proper interaction between upstream and downstream catalytically active structural elements. Deletions of sub-domains of domain I and domain III indicate that these elements are involved in 5' cleavage by hydrolysis and in a reaction in trans (exon reopening), and that this function can be inhibited without affecting the normal 5' cleavage by transesterification. Yet, we infer that the helical structures affected by the mutational alterations might not contribute to this reaction mode per se but that changes within local secondary structures perturb the internal conformation of the ribozyme. Furthermore, we have designed an abbreviated version of intron bI1, with a length of 542 nucleotides, which is still catalytically active.

Base Sequence↗
PubMed1990

Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis.

Alignment of the 87 available sequences of group I self-splicing introns reveals numerous instances of covariation between distant sites. Some of these covariations cannot be ascribed to historical coincidences or the known secondary structure of group I introns, and are, therefore, best explained as reflecting tertiary contacts. With the help of stereochemical modelling, we have taken advantage of these novel interactions to derive a three-dimensional model of the conserved core of group I introns. Two noteworthy features of that model are its extreme compactness and the fact that all of the most evolutionarily conserved residues happen to converge around the two helices that constitute the substrate of the core ribozyme and the site that binds the guanosine cofactor necessary for self-splicing. Specific functional implications are discussed, both with regard to the way the substrate helices are recognized by the core and possible rearrangements of the introns during the self-splicing process. Concerning potential long-range interactions, emphasis is put on the possible recognition of two consecutive purines in the minor groove of a helix by a GAAA or related terminal loop.

Animals↗