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PubMed1983

A seven-base-pair deletion in an intron of the albumin gene of analbuminemic rats.

Analbuminemic rats, which genetically lack serum albumin, have a mutation affecting albumin mRNA processing. Serum albumin genes were cloned from analbuminemic and normal parental Sprague-Dawley rats. Structural analyses of the two albumin genes showed that the gene from analbuminemic rats had a seven-base-pair deletion in an intron. The deletion extended from base 5 to base 11 from the 5' end of intron HI of the albumin gene. This deletion converted the sequence, G-T-A-G-G-T, which is normally located at the 5' end of intron HI, to G-T-A-G-C-G. RNA blot hybridization of analbuminemic and normal rat liver nuclear RNA using a DNA fragment containing the intron HI as a probe showed that this intron sequence persisted in albumin mRNA precursors of analbuminemic rats.

Animals↗
PubMed1985

Structure of the human phosphoglycerate kinase gene and the intron-mediated evolution and dispersal of the nucleotide-binding domain.

The human X-linked phosphoglycerate kinase (PGK) gene, which is expressed in all somatic cells, was cloned and its structure was determined. The gene is interrupted by 10 introns and spans 23 kilobases. When projected on the three-dimensional structure of the PGK protein molecule, splice junctions are located between established peptide domains. In particular, an intron separates the two mononucleotide subdomains of the ATP-binding region, and additional introns divide each of these subdomains between their characteristic beta-strands. Similar correlations are found in the bipartite NAD-binding domains of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, in each case the nucleotide-binding domain is separated from the catalytic domain by at least one intron. The homology of the exon organization in structurally similar regions of these three enzymes suggests that a nucleotide-binding domain evolved by gene duplication and was subsequently dispersed to different proteins through a process of intron-mediated recombination.

Amino Acid Sequence↗
PubMed1986

Intron-dependent evolution of the nucleotide-binding domains within alcohol dehydrogenase and related enzymes.

It has been suggested that the intron/exon structure of a gene corresponds to its evolutionary history. Accordingly, early in evolution DNA segments encoding short functional polypeptides may have been rearranged (exon-shuffling) to create full-length genes and RNA splicing may have been developed to remove intervening sequences (introns) in order to preserve translational reading frames. A conflicting viewpoint would be that introns were randomly inserted into previously uninterrupted genes after their initial evolutionary development. If so, the sites of introns would be unlikely to consistently reflect the domain structure of the protein. To address this question, the intron/exon structure of the gene encoding human alcohol dehydrogenase (ADH) was determined and compared to the gene structures for other ADHs and related proteins, all of which possess nucleotide-binding domains. Our results indicate that the introns in the nucleotide-binding domains of all the genes examined do indeed fall at positions which separate the short functional polypeptides (i.e. beta strands) which are believed to comprise this domain. We argue that our data is most easily explained by the hypothesis that introns were present in an ancestral nucleotide-binding domain which was later rearranged by exon-shuffling to form the various dehydrogenases and kinases which utilize such a domain.

Alcohol Dehydrogenase↗
PubMed1980

Nuclear RNA transcripts from Drosophila melanogaster ribosomal RNA genes containing introns.

The transcription of ribosomal genes in a cell line (Kc) derived from female D.melanogaster, has been investigated using hybridization probes prepared from restriction fragments of a cloned rDNA repeat with a 5 kb type I [Wellauer et al. (1978) Cell 14, 269-278] intron. Gels, of nuclear RNA that have been transferred to diazotized paper and hybridized with labelled intron sequences, reveal both large (1-10 kb) transcripts and a discrete 325 base species. Berk-Sharp experiments [(1977) Cell 12, 721-732] reveal large transcripts that are homologous to intron sequences and extend into 28S sequences as well. However, while the abundance of 28S transcripts is about 50,000 copies per nucleus [Clark et al. (1977) Genetics 86, 789-800], these long transcripts are only present at 1-2 copies per nucleus and the 325 base species is only 10 times more abundant. In view of the fact that female cells have about 400 rDNA genes, 49% of which have type I introns, one must conclude either that transcription rarely occurs from the genes containing introns (the majority) or these transcripts are processed unusually rapidly. Transcripts homologous to the "non-transcribed spacer" region have been found, but their abundance is no higher.

Animals↗
PubMed1985

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.

Animals↗
PubMed1985

Nuclei of adenovirus 2-infected cells contain an RNA species that corresponds to an intron excised intact from mRNA precursors.

We used Northern and S1 nuclease analyses to identify a nuclear RNA species of the structure predicted for an intron excised from the precursor of adenovirus 2 E2A early mRNA. The structure of this excised intron demonstrated that the splicing of E2A mRNA can involve the removal of the intron sequences as single intact molecules. The concentration of the excised intron is 30 copies per nuclei, comparable to the levels of E2A polyadenylated splicing precursors, but 30-fold less than nuclear E2A mRNA. Late in infection, when the production of E2A early mRNA is dramatically elevated (Goldenberg et al., J. Virol. 38:932-939, 1981), the excess intron was not detected, indicating that either its stability or the mechanism of splicing is altered. We also identified a spliced nonpolyadenylated E2A nuclear RNA species with a structure similar to the mRNA, indicating that splicing may normally occur in the absence of polyadenylation.

Adenoviruses, Human↗
PubMed2006

Intronic microRNA (miRNA).

Nearly 97% of the human genome is composed of noncoding DNA, which varies from one species to another. Changes in these sequences often manifest themselves in clinical and circumstantial malfunction. Numerous genes in these non-protein-coding regions encode microRNAs, which are responsible for RNA-mediated gene silencing through RNA interference (RNAi)-like pathways. MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) with complete or partial complementarity, are useful for the design of new therapies against cancer polymorphisms and viral mutations. Currently, many varieties of miRNA are widely reported in plants, animals, and even microbes. Intron-derived microRNA (Id-miRNA) is a new class of miRNA derived from the processing of gene introns. The intronic miRNA requires type-II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of Id-miRNA 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 miRNAs are able to induce RNA interference in not only human and mouse cells, but in also zebrafish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of intron-mediated gene silencing via functional miRNA in cell and in vivo. These findings suggest an intracellular miRNA-mediated gene regulatory system, fine-tuning the degradation of protein-coding messenger RNAs.

Journal Article↗
PubMed1990

Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer.

The p53 gene initially was thought to be an oncogene, but recent evidence suggests that wild-type p53 can function as a tumor suppressor gene in lung, colon, and breast cancer as well as less common malignancies. This study reports the first identification of intronic point mutations as a mechanism for inactivation of the p53 tumor suppressor gene. Abnormally sized p53 mRNAs found in a small cell and a non-small cell lung cancer cell line were characterized by sequence analysis of cDNA/PCR products, the RNase protection assay and immunoprecipitation. These mRNAs were found to represent aberrant splicing leading to the production of abnormal or no p53 protein. Sequence analysis of genomic DNA revealed that a point mutation at the splice acceptor site in the third intron or the splice donor site in the seventh intron accounts for the abnormal mRNA splicing. In one patient the same intronic point mutation was found in the tumor cell line derived from a bone marrow metastasis and in multiple liver metastases but not in normal DNA, indicating that it occurred as a somatic event before the development of these metastases. These findings further support the role of inactivation of the p53 gene in the pathogenesis of lung cancer and indicate the role of intronic point mutation in this process.

Amino Acid Sequence↗
PubMed2002

Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo.

BACKGROUND: Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested. RESULTS: The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1-2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (DeltaORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404-459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent. CONCLUSIONS: The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1-2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.

Journal Article↗
PubMed2000

A phylogeny of the flowering plant family Apiaceae based on chloroplast DNA rpl16 and rpoC1 intron sequences: towards a suprageneric classification of subfamily Apioideae.

The higher level relationships within Apiaceae (Umbelliferae) subfamily Apioideae are controversial, with no widely acceptable modern classification available. Comparative sequencing of the intron in chloroplast ribosomal protein gene rpl16 was carried out in order to examine evolutionary relationships among 119 species (99 genera) of subfamily Apioideae and 28 species from Apiaceae subfamilies Saniculoideae and Hydrocotyloideae, and putatively allied families Araliaceae and Pittosporaceae. Phylogenetic analyses of these intron sequences alone, or in conjunction with plastid rpoC1 intron sequences for a subset of the taxa, using maximum parsimony and neighbor-joining methods, reveal a pattern of relationships within Apioideae consistent with previously published chloroplast DNA and nuclear ribosomal DNA ITS based phylogenies. Based on consensus of relationship, seven major lineages within the subfamily are recognized at the tribal level. These are referred to as tribes Heteromorpheae M. F. Watson & S. R. Downie Trib. Nov., Bupleureae Spreng. (1820), Oenantheae Dumort. (1827), Pleurospermeae M. F. Watson & S. R. Downie Trib. Nov., Smyrnieae Spreng. (1820), Aciphylleae M. F. Watson & S. R. Downie Trib. Nov., and Scandiceae Spreng. (1820). Scandiceae comprises subtribes Daucinae Dumort. (1827), Scandicinae Tausch (1834), and Torilidinae Dumort. (1827). Rpl16 intron sequences provide valuable characters for inferring high-level relationships within Apiaceae but, like the rpoC1 intron, are insufficient to resolve relationships among closely related taxa.

Journal Article↗
PubMed1998

Evidence of Limited Structural Organization in Globin Intron Sequences of Messenger RNA.

The structural significance of introns in alpha and beta globin genes were evaluated in terms of the folding (or stacking) free energy of the nascent RNA sequences as obtained from secondary structure calculations. From calculations of the folding free energy of exons and introns, it was found that introns tend to have a more folded (globular) structure compared to the exon counterparts segments. The results suggest that significant biological information is present in all intronic sequences including spliceosome type introns.

Journal Article↗
PubMed2003

[Role of immunomodulatory treatment with Iscador QuS and Intron A of women with CIN1 with concurrent HPV infection].

OBJECTIVE: The paper presents the role of immunomodulatory treatment with Iscador QuS and Intron A of women with CIN1 and CIN2 with concurrent HPV infection. MATERIAL AND METHODS: Clinical material consisted of 96 women aged 18-52 years of life. The women were divided into three groups. Group A (35 women) treated with Iscador QuS administered s.c. twice a week for 3 months, group B (30 women) treated with Intron A, administered twice a week in the cervical injections for 3 months and control group K (31 women) without treatment followed up with cytology and colposcopy. RESULTS: In the group A (Iscador QuS) CIN remission was observed in slightly higher percentage (non significant) comparing to the control group. In the group B (Intron A) remission CIN was observed in 24 (80%) cases which was statistically significant comparing to the control and A groups. There were no progression of CIN in the group B and the stationery process was observed statistically more frequent comparing to the control and A groups. There was observed statistically higher percentage of cases without HPV infection in all groups during the experiment. The remission concerned both high and low oncogenic potency viruses. In the highest percentage CIN with concurrent HPV infection remission was observed in the B (Intron A) group. CONCLUSIONS: 1/Iscador QuS and specially Intron A increases the CIN1 and CIN2 remission rate. 2/These two agents may also affect the HPV remission.

Adjuvants, Immunologic↗
PubMed1991

Heterozygous mutation in the G+5 position of intron 33 of the pro-alpha 2(I) gene (COL1A2) that causes aberrant RNA splicing and lethal osteogenesis imperfecta. Use of carbodiimide methods that decrease the extent of DNA sequencing necessary to define an unusual mutation.

Cultured skin fibroblasts from a proband with osteogenesis imperfecta were found to synthesize normal and shortened alpha 2(I) chains of type I procollagen. A cDNA library was prepared using mRNA isolated from the proband's fibroblasts. Partial nucleotide sequencing of five clones demonstrated that two clones lacked the 54 base pairs (bp) of coding sequences found in exon 33 of the pro-alpha 2(I) gene (COL1A2). To reduce the amount of nucleotide sequencing required, heteroduplexes were prepared from two of the clones, one normal and the other lacking exon 33, and reacted with a water-soluble carbodiimide under conditions in which nonbase-paired G and T nucleotides are specifically modified by the reagent. Analysis of the heteroduplexes by immunoelectron microscopy suggested that the sequence variation near the codons of exon 33 was the only sequence difference in the cDNA clones. Amplification of cDNA from the proband by polymerase chain reaction gave products of two sizes, one of the expected size for the normal sequence and the other of the expected size for a product lacking the 54 bp in exon 33. To define the mutation in genomic DNA, a 1.6-kilobase region spanning exons 32 and 34 was amplified by the polymerase chain reaction and DNA heteroduplexes were prepared from the products. The heteroduplexes were treated with a water-soluble carbodiimide and then used as templates for primer extension under conditions in which extension terminates at the site of a carbodiimide-modified base. The results suggested a mismatch near the exon-intron boundary of exon 33 and a second mismatch near the 3' end of intron 33. Nucleotide sequencing of the polymerase chain reaction products revealed a single-base substitution in one allele that changed the moderately conserved G at position +5 of the 5' splice site of intron 33 to an A. In addition, there was an apparently neutral single-base substitution that placed both a G and T at position +661 of intron 33. The results provide only the third example of a mutation in the G at the +5 position of an intron that causes aberrant RNA splicing. Also, the results demonstrate that use of techniques involving carbodiimide modification of DNA heteroduplexes can reduce the amount of nucleotide sequencing necessary to define mutations in large and complex genes.

Adult↗
PubMed1983

RNA splicing in Neurospora mitochondria. The large rRNA intron contains a noncoded, 5'-terminal guanosine residue.

The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3' end. The intron RNA is excised in a single cleavage-ligation reaction and is detectable in mitochondria by Northern hybridization experiments. We now show (i) that the free intron RNA is a full-length linear molecule, and (ii) that it, like the Tetrahymena nuclear rRNA intron, contains an extra, noncoded guanosine residue at its 5' end. The latter finding suggests that the Neurospora mitochondrial large rRNA may be spliced via a phosphoester transfer mechanism similar to that proposed for the "self-splicing" Tetrahymena intron.

Base Sequence↗
PubMed1995

Maintenance therapy for remission in myeloma with Intron A following high-dose melphalan and either an autologous bone marrow transplantation or peripheral stem cell rescue.

Eighty-four patients with myeloma were randomized to receive maintenance Intron A (Schering-Plough, Suffolk, UK), 3 mega units/m2 s.c. three times weekly or no treatment following induction therapy with cyclophosphomide, vincristine, doxorubicin, methyl prednisolone (C-VAMP), consolidated with high-dose melphalan (HDM) 200 mg/m2 + autologous bone marrow transplantation (ABMT). The patients have been followed up for a median of 52 months. Overall, median progression-free survival (PFS) from HDM was 27 months in the control group and 46 months in the Intron A group (< 0.025). For the 65 patients who achieved complete remission (CR) with HDM, there was a significant prolongation of remission (p = 0.02) for those who received Intron A and 49% of these patients remained in remission four years after high-dose treatment. However, for partial responders (PR) and nonresponders to HDM there was no significant prolongation of PFS. Overall, survival was also significantly better for the Intron A group, with 5 deaths versus 14 deaths in the control group (p = 0.006). Subsequently, 54 consecutive patients received the same HDM followed by rescue with peripheral blood stem cells after induction chemotherapy which included C-VAMP. Intron A was started in 45 of these patients at a median of 62 days which was comparable to the ABMT arm. The overall response rate in these patients was 79.62% (43/54-CR+PR) and the probability of survival at 18 months was 74.2% by the Kaplan Meier method.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D.

Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processing. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc7O pre-mRNA intron. Competition studies indicate that a trans-acting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this factor is common to both intronic and non-intronic snoRNAs possessing nucleotide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region containing boxes C and D does not bind fibrillarin. Collectively, our results indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, thereby stabilizing the intronic snoRNA sequence in an RNP complex during processing.

Animals↗
PubMed1996

Mutation analysis in Duchenne and Becker muscular dystrophy patients from Bulgaria shows a peculiar distribution of breakpoints by intron.

For the first time in Bulgaria, a deletion/duplication screening was performed on a group of 84 unrelated Duchenne/Becker muscular dystrophy patients, and the breakpoint distribution in the dystrophin gene was analyzed. Intragenic deletions were detected in 67.8% of patients, and intragenic duplications in 2.4%. A peculiar distribution of deletion breakpoints was found. Only 13.2% of the deletion breakpoints fell in the "classical" hot spot in intron 44, whereas the majority (> 54%) were located within the segment encompassing introns 45-51, which includes intron 50, the richest in breakpoints (16%) in the Bulgarian sample. Comparison with data from Greece and Turkey points at the probable existence of a deletion hot spot within intron 50, which might be a characteristic of populations of the Balkan region.

Bulgaria↗
PubMed1996

Removal of an intron with unique 3' branch site creates an amino-terminal protein sequence directing the scERV1 gene product to mitochondria.

The yeast scERV1 gene is of special interest because it has a dual function in mitochondrial biogenesis and in the regulation of the cell cycle. The recent discovery that the yeast scERV1 gene has a structural and functional human homologue initiated a detailed comparison of the genes and their structures. In addition the homologous ALR (augmenter of liver regeneration) genes from rat and mouse have just been identified and it has been found that the mammalian proteins have a specific function in liver regeneration and in spermatogenesis. It now turns out that the organization of the 5' regions of these genes is much more complicated than expected. The latest research has discovered an additional intron in the 5' region of the mouse gene and possible amino-terminal extensions of the reading frames. In this work, reinvestigation of the 5' region of the yeast gene identifies a putative intron with an unusual 3' branch site. It is shown that a small intron of 83 nucleotides is present in this genomic region. Analysis of cDNA clones demonstrates that the intron is correctly removed from the messenger RNA and that therefore the unusual 3' branch site is probably functional. Furthermore, studies with antibodies directed against recombinant scERV1 protein demonstrate that the gene product is associated with mitochondria, in agreement with its involvement in mitochondrial biogenesis. Complementation experiments with mutants and different 5' deletions of the gene identify the corresponding promotor for transcription and the start codon for translation.

Amino Acid Sequence↗