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PubMed1992

Rodent and human beta 3-adrenergic receptor genes contain an intron within the protein-coding block.

DNA blot analysis of the cloned rat beta 3-adrenergic receptor gene revealed unexpected restriction enzyme cleavage sites that suggested the presence of one or more introns near the end of the coding block. This region of the rat gene was mapped and sequenced and was found to contain two introns. The first intron occurs 12 amino acids from the end of the coding block, as deduced by comparison with the beta 3 receptor cDNA. Sequence analysis of the first intron indicates that it might contain enhancer elements that could be important in the adipose tissue-specific expression of this gene. The mouse and human beta 3 receptor genes have been assumed to be intronless; however, these genes contain potential splice sites that are homologous to those present in the rat gene. The relevant regions of the mouse and human beta 3 receptor cDNAs were cloned and, by comparing them to the respective genomic sequences, it was concluded that these genes also contain one or more introns. Sequence analysis of the mouse and human beta 3 receptor cDNAs indicates that they code for proteins that are, respectively, 12 and 6 amino acids larger than previously deduced from genomic clones.

Adipose Tissue↗
PubMed1992

Elements in the first intron of the alpha 1(I) collagen gene interact with Sp1 to regulate gene expression.

Sequences within the first intron of the alpha 1(I) collagen gene act both positively and negatively to regulate expression of the gene. We have further characterized a 274 bp intronic sequence that contains an orientation-specific inhibitory activity and represents a constitutive DNase I-hypersensitive site in the gene. We show that this sequence contains two tandem, unique binding elements for the transcription factor Sp1. In addition, an Sp1-like site, capable of competing for protein binding to the intronic elements, resides in the distal promoter of the collagen gene. The results of experiments with site-directed mutations that abolish binding to the intronic elements indicate that these protein-DNA interactions have an inhibitory effect on the transcriptional efficiency of alpha 1(I) collagen-reporter gene constructs in transient transfection analysis. These data support our conclusion that the first intron plays a complex role, involving multiple protein-DNA binding interactions, in the regulation of expression of the alpha 1(I) collagen gene.

Animals↗
PubMed2004

[Role of intron and 5' untranslated region in human thrombopoietin gene expression].

OBJECTIVE: To study the role of 5' untranslated region (UTR) and intron in the expression of human thrombopoietin (TPO) gene. METHODS: A number of expression vectors containing TPO mini-gene fused to the regulatory elements of cytomegalovirus (CMV) were constructed and transfected via lipofectin into cultured cos-1 cells for transient expression of TPO gene. The cell culture media were analyzed with highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) 48 h after the transfection. RESULTS: The expression levels of the TPO gene elements followed the order of TPO intron v> TPOcDNA> TPO intron I> TPO intron I> TPO gDNA in cos-1 cells. CONCLUSION: The last intron of TPO gene obviously enhances the expression level of TPO gene, which can be inhibited by 5'UTR of TPO gene.

5' Untranslated Regions↗
PubMed2004

[Variation in lengths of introns and exons in genes of the Arabidopsis thaliana nuclear genome].

In the nuclear genes of Arabidopsis thaliana, the length of introns and exons was shown to vary depending on the number of introns. With increasing number of introns per gene, the proportion of introns composed of 80-100 nucleotides increases whereas the proportion of introns with 400-nucleotide length decreases. Similar changes in exon length in genes result in predominance of exons of 60-120-nucleotides in length.

Arabidopsis↗
PubMed2004

[Expression of OsBP-73 gene requires involvement of its intron in rice].

It has been demonstrated in our previous work that rice OsBP-73 gene contains two exons interrupted by a 2471 bp intron. Here it was reported that the 5' flanking region of the ATG translation start codon in the first exon of OsBP-73 gene (from -1818 to +215) can not direct GUS gene expression in resistant rice calli or transgenic rice. When the complete OsBP-73 intron and its flanking region (from -1818 to +2844) are constructed in frame with GUS coding region, GUS activity can be detected in the resistant rice calli and transgenic rice. Experimental data also show that the complete OsBP-73 intron itself has no promoter activity. These results suggest that the OsBP-73 intron sequence is involved in directing the GUS gene expression. Another experiment demonstrates that the complete intron sequence can enhance the activity of OsEBP-89 gene promoter.

Gene Expression Regulation, Plant↗
PubMed1991

High levels of expression of a minigene version of the human pro alpha 1 (I) collagen gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron.

A minigene version of the human gene for the pro alpha(I) chain of type I procollagen (COL1A1) was prepared that contained -2.3 kilobases of the 5'-flanking sequence, the first 5 exons and introns, the last 6 exons and introns, and about 2 kilobases of the 3'-flanking sequence. The gene was then used for stable transfection experiments with mouse NIH 3T3 fibroblasts. Because the products of the minigene were shorter, it was possible to compare expression of the minigene with expression of the endogenous pro alpha 1 (I) gene by Northern and Western blot analyses. The results demonstrated that the construct contained enough of the gene to obtain high levels of expression in many of the stably transfected cells. Since previous observations suggested that the first intron of the pro alpha 1 (I) gene contained important cis-regulatory elements, two versions of the minigene were prepared in which most of the first intron was deleted. Comparison of expression of the minigene with expression of two deleted versions of the same gene established that 85% of the total sequences in the first intron are not essential for high levels of expression of the gene in stably transfected mouse fibroblasts.

Animals↗
PubMed1991

Intron phylogeny: a new hypothesis.

The three major classes of intron are clearly of unequal antiquity. Structured (often self-splicing and sometimes mobile) introns are the most ancient, probably dating (at least for group I) from the ancestral (eubacterial) cell 3500 million years ago, and were originally restricted to tRNA. Protein-spliced introns (usually in tRNA) probably evolved from them by a radical change in splicing mechanism in the common ancestor of eukaryotes and archaebacteria, perhaps only about 1700 million years ago. Spliceosomal introns probably evolved from group-II-like self-splicing introns after the origin of the nucleus between 1700 and 1000 million years ago, and were probably mostly inserted into previously unsplit protein-coding genes after the origin of mitochondria 1000 million years ago.

Animals↗
PubMed1990

Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression.

We have isolated the mouse thrombospondin (TS) gene and determined the DNA sequence of the first nine exons and eight introns. Comparison with the human cDNA sequence reveals a high degree of conservation in coding sequences. Exon 3 of the mouse gene, which encodes the heparin-binding domain of TS, has a higher degree of nucleotide substitution than the other exons, but the distribution of charged and hydrophobic amino acids found in the human protein is generally conserved. DNA and protein sequences in exons 6-9, which encode a procollagen homology and motifs very similar to those found in at least two malarial parasite proteins, are highly conserved. The first two of the three malarial homologies in TS, which are also found in properdin and in components C6-9 of the lytic complement complex, are each encoded by a separate exon (8 and 9) in the mouse gene. Since the sequence data did not reveal substantial similarity in sequence between intron I in the human and mouse genes, we have reexamined the role of the first intron in the transcriptional regulation of the human TS gene. In accord with published studies (Laherty, C.D., Gierman, T.M., and Dixit, V.M. (1989) J. Biol. Chem. 264, 11222-11227), we find that deletion of some intronic segments from TS-chloramphenicol acetyltransferase (CAT) constructs reduces CAT activity in NIH 3T3 cells. However, deletion of the same sequences from TS-bovine growth hormone constructs does not affect the expression of bovine growth hormone in these cells. We conclude that differences in the activity of TS-CAT constructs reflect post-transcriptional differences that are peculiar to the resulting chimeric transcripts and that there is currently no evidence for a transcriptional enhancer in the first intron of the human TS gene.

Amino Acid Sequence↗
PubMed1987

5-Fluorouracil augmentation of dihydrofolate reductase RNA containing contiguous exon and intron sequences in KB7B cells.

Quantitative S1 nuclease mapping studies were performed with uniformly labeled RNA probes, containing contiguous dihydrofolate reductase exon and intron sequences, and total RNA isolated from KB7B cells exposed to 5-fluorouracil for 5 days. Dihydrofolate reductase RNA containing both exon 1 and intron I, or exon 5 and a portion of intron V, increased up to 5-fold in cells grown in the presence of 2.0 to 3.0 microM 5-fluorouracil. Dihydrofolate reductase RNA containing exon 1 or exon 5, but lacking intron I or intron V, respectively, increased 2-fold in cells grown in the presence of 0.65 to 3.0 microM 5-fluorouracil. Primer extension analysis and S1 mapping studies revealed two major transcriptional start sites at positions -72 and -69 and minor start sites upstream from position -183, for dihydrofolate reductase RNA isolated from methotrexate-resistant KB7B cells. The results of these studies demonstrate that 5-fluorouracil alters the metabolism of dihydrofolate reductase precursor mRNA and/or processing intermediates.

Endonucleases↗
PubMedDate not supplied

Possible role of the first intron of c-H-ras in gene expression: anti-cancer elements in oncogenes.

When members of ras gene family, which encode structurally related proteins of approximately 21,000 daltons (p21), are activated by either structural mutations or enhanced promoter activities, immortalized mammalian cells can be transformed into malignant forms. The ras genes are classified as "housekeeping genes" which express relatively constantly at low levels in all tissues and throughout all developmental stages. It is therefore important to understand how regulatory, cis-acting in particular, elements of the genes control their expression. We have previously reported that the principal promoter, 51 bp long or less, located around 1370 bp upstream from the first coding ATG, plays the most important role for expression, though there seem to be residual promoter activities further downstream as well. The most upstream mRNA start site is located at the 3'-end of the principal promoter, which lies directly adjacent to the homologous sequence between human and rat c-H-ras 5'-flanking regions. It is therefore proposed that the c-H-ras gene may have a dispersed promoter in which the principal promoter and subpromoters may be distributed. On the other hand, the first intron region seems to contain two sets of positive and negative elements which appear to have, respectively, a positive and negative influence on the efficiency of focus formation with the activated c-H-ras oncogene. From testing done in our laboratory, it was further revealed that these cis-acting elements in the intron affect c-H-ras gene expression at the post-transcriptional level. The middle part of the first intron was in fact shown unusually conserved between human and rat DNA sequences. Moreover, computer analyses revealed that the intron sequences of various oncogenes (both human and rodent sequences available from GenBank DNA sequence data-bank) such as ras, fos, c-myc, N-myc and int-1, are in general significantly more highly conserved when compared with sequences of non-oncogene introns. The significance of this oncogene-related conserved sequence is discussed and the proposal is made that it may function as an "anti-cancer element" or "safety valve" against gene truncation/translocational activation of oncogenes.

Base Sequence↗
PubMed1995

U12 snRNA in vertebrates: evolutionary conservation of 5' sequences implicated in splicing of pre-mRNAs containing a minor class of introns.

A minor class of introns with noncanonical splice sites has been identified in both vertebrate and invertebrate genomes. The divergent consensus sequences within these introns suggest that splicing might be via a mechanism distinct from that used by the major class of introns. The low abundance U12 snRNA has been proposed to base pair with the predicted branch site sequence of these minor class introns, probably bulging out an adenosine to act as the nucleophile in the first step of splicing. We have identified homologues of the previously characterized human U12 snRNA in both mouse and chicken, where the minor class of introns has also been found. The U12 sequences that potentially base pair with the putative branch site are invariant. Additional conserved sequences at the 5' end of U12 snRNA could dynamically base pair with U6 snRNA sequences flanking the hexanucleotide ACAGAG to form structures analogous to those of three U2-U6 interactions genetically defined as important in the major class of spliceosome. We have also isolated two human U12 snRNA genes. One gene is functional for transcription of U12 snRNA, whereas the other appears to be a pseudogene. Sequences of the 3' box in both U12 snRNA genes are strikingly similar and bear high resemblance to those of U1 and U2 genes. Upstream elements, including the PSE and the DSE, have been identified and characterized in the functional gene. These features indicate that transcription of U12 snRNA is driven by RNA polymerase II.

Animals↗
PubMed1994

Organization of the transcription factor binding sites in the kappa Ig intron enhancer. Effects of position, orientation, and spacing.

The kappa Ig intron enhancer is comprised of multiple sequence motifs known to bind trans-acting factors that activate gene expression. A species comparison reveals a high level of conservation of the organization of the transcription factor binding sites within the enhancer. The importance of the conserved organization of the kappa intron enhancer was examined by using topologic mutations that disrupt the position, orientation, and spacing of individual binding sites within the enhancer. The effects of these changes were monitored by their effects on reporter gene activity at two distinct stages of B cell development. Previously, mutational analysis indicated the kappa B and kappa E2 sequence motifs to be the most crucial sites for intron enhancer function. We have demonstrated that intron enhancer activity is dependent on the position of the kappa B and kappa E2 sequence motifs within the enhancer. Intron enhancer function is, however, independent of kappa B and kappa E2 binding site orientation and is flexible in spacing requirements among binding sites.

Animals↗
PubMed1994

A germ line-specific sequence element in an intron in Tetrahymena thermophila.

In the ciliated protozoa, the germ line micronucleus carries DNA sequences that are eliminated during the developmental program that produces the somatic macronucleus. We have identified such a micronuclear element in an intron of a gene in the holotrichous ciliate, Tetrahymena thermophila. The macronuclear version of this gene, numbered from the transcription initiation site to the polyadenylation site, is 1569 nucleotides long and contains two introns: intron 1 from nucleotides 206 to 382 and intron 2 from nucleotides 665 to 898. The gene is transcribed into a stable polyadenylated mRNA which is predicted to encode a polypeptide with a highly repetitive structure and an unknown function. The micronuclear version of the gene contains a 2.9-kb sequence element, mse2.9, in intron 2. This element was characterized by sequencing two micronuclear genomic clones that contain 909 and 764 base pairs of micronucleus-specific sequence from its left and right ends, respectively. This sequence has an (A+T) content of 81% and is present as a single copy. The termini of mse2.9 are located within a 4-nucleotide direct repeat, TTAT, one copy of which is retained in the macronuclear gene, between positions 755 and 759. Other features near the termini of mse2.9 include a GC-rich sequence motif, CCAACCCGTTG, 31 nucleotides outside the left terminus and a 20-nucleotide palindrome 6 nucleotides outside the right terminus.

Amino Acid Sequence↗
PubMed1995

[Introns differ from exons by their redundancy].

This paper is devoted to an analysis of the intrinsic structure of the gene from the point of view of the redundancy of different structural units--exons and introns. Human genes for which the exon-intron structure has been clearly established were studied. The redundancy of each exon and intron in the gene was determined. It was shown that, in human genes, introns are more redundant than exons. Redundancy is determined as the smallest length of a word (oligonucleotide) beginning with which all words in the studied nucleotide sequence are found exactly once. Mechanisms leading to the disruption of the general pattern of ratios of redundancy of exons and introns are studied.

Base Sequence↗
PubMed1996

IFN-gamma induces the kappa intron enhancer via an IFN-stimulated response element.

IFN-gamma is a potent inducer of Ig kappa light chain gene transcription in 70Z/3 pre-B cells, but the mechanism of this induction has not been elucidated. The kappa intron enhancer contains a sequence closely resembling the IFN-stimulated response element (ISRE). We have determined that the kappa intron enhancer is IFN-gamma inducible in 70Z/3 cells and that the ISRE is required for this induction. The kappa intron ISRE specifically bound IFN response factor-1 (IRF-1) and the constitutively expressed IRF-2. This ISRE is a multifunctional motif that also binds the LPS-inducible factor kappaBF-A and is located within the kappaBS region, which confers B cell specific activity to this enhancer. However, since the expression of IRF-1 is not restricted to B cells, it must not be sufficient for the induction of kappa transcription. Furthermore, in the pre-B cell line 38B9, which is representative of an earlier stage in pre-B cell development than the 70Z/3 cell line, the kappa intron enhancer was not induced by IFN-gamma despite the activation of IRF-1. These findings suggest that IFN-gamma activation of kappa gene transcription during B cell maturation may be developmentally controlled by elements that restrict the activity of the ISRE within the context of the intron enhancer.

Animals↗
PubMed1996

[The sequences of intron 50 and 51 of DMD gene and the deletion mechanism of 51st exon].

OBJECTIVE: To investigate the relationship between the structural characters of 50th and 51st introns and deletion mechanisms of 51st exon of DMD gene. METHODS: We cloned the 3.1 Kb-Hind I human genome DNA fragment containing the 50 and 51 introns of DMD gene, sequenced the fragment completely, and analysed the character of the sequences with computer. RESULTS: The length of the fragment was 3179, including the entire 51st exon and partial 50th and 51st intron sequences. There were 36 short tandom repeats, 22 direct and inverted repeats, and 8 homologous sequences in the 50th and 51st introns. A TATA box was found in the intron 50 and it may be another new promotor of DMD gene. CONCLUSION: The hypothesis, which the recombination of repeat sequences may result in the deletion of 51st exon, is put forward.

Base Sequence↗
PubMed1997

The diversity of the HLA class I introns reflects the serological relationship of the coding regions.

The introduction of PCR-based HLA typing techniques has uncovered that the HLA system is much more variable than it has been expected from the conventional typing methods. More and more new alleles are detected which are not characterized by new sequence motifs, but by new combinations of already existing sequence motifs. This variability, reflecting the immunological necessity to have the greatest capacity for presenting antigenic peptides, is increasingly complicating DNA typing techniques based on the diversity of the coding region. We have determined the sequence of the 1st through 3rd intron of the majority of HLA-A and HLA-B alleles in 48 well-defined cell lines and 195 PCR-typed clinical samples. The few published sequences emerged to contain substantial errors. The introns turned out to be highly polymorphic. Besides extensive homologies, numerous locus- and group-specific sites could be identified. The most intriguing finding was that most of the polymorphic motifs were related to serological families. These sequence motifs were extremely beneficial for setting up PCR-based typing systems. In particular, sequencing-based typing strategies benefited from intron-restricted priming for amplification and sequencing by enabling complete analysis of the polymorphic exons. The determination of cis/trans linkages of sequence motifs was substantially facilitated. Apart from these advantages, the intron sequences were useful for evolutionary studies, delivering more insights into the genetic relationship between different alleles and the mechanisms involved in the development of the diversity of HLA. Moreover, the variability of the introns may provide a structural basis for the identification of regulatory elements acting on the level of transcription.

Cell Line↗
PubMed1996

Transcriptional activation of the alpha 1(I) procollagen gene in systemic sclerosis dermal fibroblasts. Role of intronic sequences.

OBJECTIVE: To investigate the transcriptional regulation of the alpha 1(I) procollagen gene (COL1A1) in cultured dermal fibroblasts from patients with diffuse systemic sclerosis (SSc) of recent onset and to evaluate the role that intronic sequences may play in the upregulated expression of COL1A1 in SSc dermal fibroblasts. METHODS: Dermal fibroblasts from 6 patients with diffuse SSc of recent onset and from 3 healthy individuals were studied. The steady-state levels of alpha 1(I) procollagen messenger RNA were evaluated by Northern hybridization analysis, and the transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of COL1A1 promoter (with 5' end points at -5.3 kb, -2.3 kb, and -804 bp and 3' end point at +42 bp) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. To examine the role of intronic sequences, constructs containing, in addition to the COL1A1 promoter, a portion of the first intron (+380 bp to +1,440 bp) cloned in front of the CAT gene were transfected. The efficiency of transfections was normalized relative to the net amount of CAT plasmid actually transfected into recipient cells, determined by a modified Southern hybridization procedure. RESULTS: Maximal CAT activity was observed with constructs extending from -804 bp to +42 bp in both normal and SSc fibroblasts. However, the activity driven by this construct was 80-110% higher in SSc fibroblasts. The CAT activity driven by a construct with a 5' end point at -5.3 kb was only 15-20% higher in SSc cells, and the CAT activity driven by a construct with a 5' end point -2.3 kb was 35-45% higher in SSc fibroblasts. The CAT activity driven by the -804-bp promoter construct was increased up to 4-fold in SSc fibroblasts in comparison with normal cells when the intronic segment spanning +380 bp to +1,440 bp was included in the transfected construct. CONCLUSION: The results directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from SSc patients. The data also indicate that first-intron sequences of COL1A1 are required for maximal transcriptional activity of the collagen gene and may play an important role in the up-regulation of its expression in SSc fibroblasts.

Adult↗