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PubMed2000

An unusual insertion in intron 2 of apparently functional MHC class I alleles in rhesus macaques.

Here we describe a nucleotide insertion in intron 2 of two rhesus major histocompatibility complex (MHC) class I alleles, Mamu-A*05 and Mamu-A*07. This resulted in an intron 2 that was nearly twice the length of any other intron 2 of primate MHC class I genes sequenced to date. This insertion was most similar (93% identity) to the beginning of intron 3 of HLA-A alleles. It was also similar to intron 3 of several human MHC class I pseudogenes and MHC class I pseudogenes from cotton top tamarin and cat. The finding of this insertion in two rhesus MHC class I genes is surprising given the uniformity in length and sequence of introns of functional HLA-A, -B and -C genes.

Alleles↗

PubMed1989

Exon shuffling by recombination between self-splicing introns of bacteriophage T4.

The organization of genes into exons separated by introns may permit rapid evolution of protein-coding sequences by exon shuffling. Introns could provide non-coding targets for recombination, which would then give rise to novel combinations of exons. Evidence to support this theory is indirect and consists of examples of homologous domains of protein structure encoded in different genes, with introns in conserved positions at the boundaries of these domains. Here, we report the first direct evidence for exon shuffling. Two spontaneous deletion mutations of phage T4 have been characterized by sequencing, and they are clearly the result of recombination between homologous regions of two self-splicing group I introns. As a result of the recombination, exons of different genes are transcribed together, with a hybrid intron between them. One of these introns is proficient in self-splicing.

Exons↗

PubMed1994

Five identical intron positions in ancient duplicated genes of eubacterial origin.

In 1985 Cornish-Bowden wrote "although there is now much to suggest that introns are an ancient relic of primordial genes, convincing proof must await the discovery of clearly corresponding intron arrangements in genes that arose by duplication before the separation of prokaryotes and eukaryotes". Genes for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases of eukaryotes are descendants of an ancient gene family that existed in the common ancestor of extant eubacteria. During eukaryotic evolution, both genes were transferred to the nucleus from the antecedents of present-day chloroplasts and mitochondria, respectively. Here we report the discovery of five spliceosomal introns at positions that are precisely conserved between nuclear genes for this chloroplast/cytosol enzyme pair. These data provide strong evidence in favour of the 'introns early' hypothesis, which proposes that introns were present in the earliest cells, consistent with the idea that introns facilitated the assembly of primordial genes by accelerating the rate of exon shuffling.

Amino Acid Sequence↗

PubMed2002

Selection for short introns in highly expressed genes.

Transcription is a slow and expensive process: in eukaryotes, approximately 20 nucleotides can be transcribed per second at the expense of at least two ATP molecules per nucleotide. Thus, at least for highly expressed genes, transcription of long introns, which are particularly common in mammals, is costly. Using data on the expression of genes that encode proteins in Caenorhabditis elegans and Homo sapiens, we show that introns in highly expressed genes are substantially shorter than those in genes that are expressed at low levels. This difference is greater in humans, such that introns are, on average, 14 times shorter in highly expressed genes than in genes with low expression, whereas in C. elegans the difference in intron length is only twofold. In contrast, the density of introns in a gene does not strongly depend on the level of gene expression. Thus, natural selection appears to favor short introns in highly expressed genes to minimize the cost of transcription and other molecular processes, such as splicing.

Animals↗

PubMed2002

A conserved intronic response element mediates direct p53-dependent transcriptional activation of both the human and murine bax genes.

Both the human and the mouse bax promoters contain p53 binding sites which are sufficient to confer p53-dependent transcriptional activation in a heterologous setting. Nevertheless in the context of the bax promoter, these sites do not mediate a p53-dependent response, suggesting that bax may not be a direct transcriptional target of p53. Here, data are presented identifying a conserved p53 response element in the first intron of both the human and the murine bax genes. This element both in isolation and in the context of the first intron conferred p53-dependent transcriptional activation upon a minimal promoter. Electrophoretic mobility shift assays demonstrated that this sequence also is capable of mediating sequence specific binding to p53. p53 effectively activated transcription through both human and murine bax gene reporter constructs, whereas deletion of the intronic response element abrogated the p53-responsiveness of both reporters. Interestingly, tumor-derived mutants of p53 which are defective in inducing an apoptotic response retain the ability to activate transcription via the bax intronic p53 site. Since these mutants are transcriptionally inactive on the p53 site in the bax promoter, the ability of these mutants to up-regulating endogenous bax mRNA levels supports a role for the intronic element in p53-dependent up-regulation of bax expression. Taken together, these results show the requirement for a novel intronic element in the p53-dependent transcriptional activation of bax, and demonstrate that bax is indeed a direct and evolutionarily conserved transcriptional target of p53.

Animals↗

PubMed1995

Structure of the human aggrecan gene: exon-intron organization and association with the protein domains.

The complete exon-intron organization of the human aggrecan gene has been defined, and the exon organization has been compared with the individual domains of the protein core. A yeast artificial chromosome containing the aggrecan gene was selected from the Centre d'Etude du Polymorphisme Humaine yeast artificial chromosome library. A cosmid sulibrary was created from this, and direct sequencing of individual cosmids was used to provide the exon-intron organization. The human aggrecan gene was found to be composed of 19 exons ranging in size from 77 to 4224 bp. Exon 1 is non-coding, whereas exons 2-19 code for a protein core of 2454 amino acids with a calculated mass of 254379 Da. Intron 1 of the gene is at least 13 kb. Overall, the sizes of the 18 introns range from 0.5 to greater than 13 kb. Each intron begins with a GT and ends with an AG, thus obeying the GT/AG rule of splice-junction sequences. The entire coding region is contained in 39.4 kb of the gene. The organization of exons is strongly related to the specific domains of the protein core. The A loop of G1 and the interglobular domain are encoded by exons 3 and 7 respectively. The B and B' loops of G1 are encoded by exons 4-6, and those of G2 are encoded by exons 8-10. These sets of exons, coding for the B and B' loops, are identical in size and organization. This is supported by the intron classes associated with these exons. Exon 11 codes for the 5' half of the keratan sulphate-rich region, and exon 12 codes for the 3' half of the keratan sulphate-rich region as well as the entire chondroitin sulphate-rich region. G3 is encoded by exons 13-18, including the alternatively spliced epidermal growth factor-like and complement regulatory protein-like domains. The correspondence between the exon organization and the protein domains argues strongly for modular assembly of the aggrecan gene.

Aggrecans↗

PubMed1999

Mutations in intron 3 of GH-1 gene associated with isolated GH deficiency type II in three Japanese families.

OBJECTIVE: Isolated GH deficiency (IGHD) type II is a disorder inherited in an autosomal dominant manner. Three mutations at the donor splice site of intron 3 of the GH-I gene have been identified in five families. In this report, we describe a novel mutation also at the donor splice site of intron 3 in patients with IGHD type II. PATIENTS: Five individuals diagnosed as IGHD: two sporadic cases and one family with three affected individuals (two siblings and their father). MEASUREMENT: Genomic DNA was extracted from peripheral mononuclear cells. All the exons and introns of the GH-I gene were amplified by polymerase chain reaction (PCR) and subjected to sequence analysis. RESULTS: A guanine to adenine transition at the fifth base of intron 3 (mutE), which has not been reported, was identified in the familial case but not in unaffected members of the family including the paternal grandparents. In the other two families with sporadic cases, a guanine to adenine transition at the first base of intron 3 (mutA) was identified in the affected subjects but not in other members of the families. CONCLUSION: MutE has not been previously reported and is the fourth mutation associated with IGHD type II. The guanine residue mutated in mutA was the second nucleotide of a CpG dinucleotide, which is regarded as a hot spot for mutations by a methylation-deamination mechanism. Since mutA has previously been identified in three type II IGHD kindreds belonging to different ethnic backgrounds, this appears to be the most frequent GH-I gene mutation in IGHD with a dominant inheritance. Because de novo mutations appeared to have occurred in all three families analyzed in the present study and the presence or absence of these mutations can easily be tested by PCR and restriction enzyme digestion, not only the familial cases but also sporadic cases with IGHD should be examined for a possible mutation at the donor splice site of intron 3 in the GH-1 gene.

Adolescent↗

PubMed1997

Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1.

The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1-GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1-GUS fusions, and they act post-transcriptionally to increase the steady-state level of mRNA.

Anthranilate Phosphoribosyltransferase↗

PubMed1998

Aberrant splicing of intron 1 leads to the heterogeneous 5' UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content.

We have previously reported that three distinct patterns of waxy (Wx) gene transcript accumulation were present in 31 rice cultivars. The cultivars with high amylose content (group I) contain a 2.3 kb mature Wx mRNA, cultivars with intermediate amylose content (group II) produce both a 3.3 kb Wx pre-mRNA, which contains intron 1, and the 2.3 kb Wx mature mRNA, and cultivars with no amylose (group III) accumulate only the 3.3 kb Wx pre-mRNA. Analyses of the cDNAs reveals that four splice donor sites and three splice acceptor sites in intron 1 give rise to six splicing patterns in 2.3 kb Wx mRNA of group II cultivars. In addition, aberrant intron 1 excision causes either deletion of 4 or 5 nucleotides, or addition of 7 and 13 nucleotides at the junction of exon 1 and exon 2 of the 2.3 kb mRNA. In contrast, only one normal splicing pattern (one splice donor site and one splice acceptor site) was found in the 2.3 kb mRNA of group I cultivars. Nucleotide sequences of the Wx intron 1 in group I and group II cultivars differ by 16 individual bases. We suggest that these deletions or additions contribute to inefficient splicing of intron 1 from the 3.3 kb Wx pre-mRNA, as well as an aberrant splicing of the Wx intron 1 to produce the 2.3 kb mRNA with a heterogeneous 5' untranslated region (5'-UTR). As a consequence, the total amount of translatable Wx mRNA, and therefore the Wx protein and amylose content, are reduced in the group II cultivars compared with the group I cultivars.

Alternative Splicing↗

PubMed2002

Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii.

Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the chloroplast group I rRNA intron of Chlamydomonas reinhardtii, Cr.LSU. In vitro assays showed that these substitutions had surprisingly strong effects on Cr.LSU self-splicing; however, splicing of all but the P6 mutations could be at least partially recovered by increasing the Mg2+ concentration. The mutant constructs were transformed into chloroplasts to replace the wild-type intron; however, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-deficient P4125A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least two nuclear genes. The 7151 suppressor mutation, which defines the chloroplast-splicing suppressor (css1) gene, had no obviously altered growth phenotype with the wild-type intron, and was dominant in vegetative diploids containing the mutant intron. All three of the suppressor strains also suppressed a mutation in the P4 region of the fourth psbA intron, Cr.psbA4, indicating that these genes play a role in splicing of multiple group I introns in the chloroplast.

Animals↗

PubMed1999

Novel intronic polymorphisms in the presenilin-2 gene and a case-control association study of Alzheimer's disease.

Several alleles of introns or untranslated regions in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes have been reported to behave as risk factors for senile Alzheimer's disease (AD). On the other hand, mutations in the three presenile AD genes also have been identified in a small number of sporadic presenile AD and senile AD cases. The present study evaluated the genetic contributions of PS-2 exons and introns to 56 senile and 18 Japanese cases of presenile AD using polymerase chain reaction single-strand conformation polymorphism analysis. In the PS-2 gene, one exonic polymorphic site without amino acid substitution, 9 intronic polymorphic sites, and 2 intronic variant sites were detected. However, in all cases, amino acid substitutions in exons between 4 and 12 of the PS-2 gene were not observed. The risk factors of senile and presenile AD were evaluated using a population-based study of restriction cleavages between patients and controls in introns 3, 4, 10 and 11. Regarding PS-2, there was no association between AD and intronic polymorphisms.

Alzheimer Disease↗

PubMed2003

Bacteria and Archaea Group II introns: additional mobile genetic elements in the environment.

Self-splicing group II introns are present in the organelles of lower eukaryotes, plants and Bacteria and have been found recently in Archaea. It is generally accepted that group II introns originated in bacteria before spreading to mitochondria and chloroplasts. These introns are thought to be related to the progenitors of spliceosomal introns. Group II introns are also mobile genetic elements. In bacteria, they appear to spread using either other mobile genetic elements or low-expression regions as target sites. Bacteria and Archaea genome sequence annotations have revealed the diversity of group II intron classes and that they are involved in vertical and horizontal inheritance.

Archaea↗

PubMed2005

A bacterial group II intron-encoded reverse transcriptase localizes to cellular poles.

The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein) that binds the intron RNA to promote RNA splicing and intron mobility. Here, we used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to cellular poles in Escherichia coli and Lactococcus lactis. This polar localization occurs with or without coexpression of Ll.LtrB intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three nonoverlapping LtrA subsegments, possibly reflecting dependence on common redundant signals and/or protein physical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. The polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in the origin and terminus regions of the E. coli chromosome, may facilitate access to exposed DNA in these regions, and could potentially link group II intron mobility to the host DNA replication and/or cell division machinery.

Bacterial Proteins↗

PubMed2002

tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.

Cloning, Molecular↗

PubMed1987

Alternative processing of bovine growth hormone mRNA: nonsplicing of the final intron predicts a high molecular weight variant of bovine growth hormone.

We have detected a variant species of bovine growth hormone mRNA in bovine pituitary tissue and in a stably transfected bovine growth hormone-producing cell line. Analysis of this variant mRNA indicated that the last intervening sequence (intron D) had not been removed by splicing. Inspection of the sequence of intron D reveals an open reading frame through the entire intron, with a termination codon encountered 50 nucleotides into the fifth exon, which is shifted from the normal reading frame in this variant mRNA. If translated, this variant mRNA would encode a growth hormone-related polypeptide having 125 amino-terminal amino acids identical to wild-type growth hormone, followed by 108 carboxyl-terminal amino acids encoded by the 274 bases of intron D along with the first 50 nucleotides of exon 5. This variant polypeptide would be 42 amino acids longer than wild-type bovine growth hormone or approximately 5000 greater in molecular weight. The intron D-containing variant of bovine growth hormone mRNA was demonstrated to exist on polysomes, suggesting that this mRNA species is translated into a polypeptide. Cytosolic mRNA species containing any of the other three introns of the bovine growth hormone gene were not detectable.

Amino Acid Sequence↗

PubMed1991

I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.

Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.

Base Sequence↗

PubMed1991

Heterologous introns can enhance expression of transgenes in mice.

In a previous study we showed that genomic constructs were expressed more efficiently in transgenic mice than constructs that were identical except for the lack of introns. Using the mouse metallothionein promoter-rat growth hormone gene construct as a model, we show that the first intron of the rat growth hormone gene is essential for high-level expression, whereas the other three introns are less effective. Several heterologous introns placed 3' of the coding region of an intronless rat growth hormone gene are also ineffective. However, insertion of some heterologous introns between the metallothionein promoter and the growth hormone gene improves expression. To determine whether addition of heterologous introns would provide a general strategy for improving expression, we have tested them in conjunction with other intronless genes and with different promoters.

Animals↗

PubMed1992

A region of group I introns that contains universally conserved residues but is not essential for self-splicing.

The catalytic core of the self-splicing group I intron RNAs is composed of six paired regions together with their connecting sequences; these are thought to form two elongated domains, with paired regions P5, P4, and P6 aligned along one axis and P8, P3, and P7 along the other. Most of the very highly conserved residues of the group I introns lie in or near P7, but two occur in L4, the internal loop connecting P4 and P5. It is generally believed that such bases are conserved because they are essential for splicing. Mutants were created in a member of each of the two major subclasses of group I introns, in which P5, L4, and the distal portion of P4 were deleted. Splicing activity was still detected in these mutants, albeit substantially weakened; splicing was accurate and occurred by the normal group I mechanism, with addition of a guanosine molecule to the intron. Thus the deleted region, containing two universally conserved bases, is not essential but facilitates splicing. Another reaction characteristic of group I introns, hydrolysis of the 3' splice site, was less severely affected by the deletions. The results are discussed in terms of the prevailing three-dimensional model for the core structure of the group I introns.

Animals↗