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At least 433 records · Page 24Linked to original sources

PubMed2005

Elucidation of exon 1, 4, and 5 sequences of 39 infrequent HLA-B alleles.

More than 590 human leukocyte antigen (HLA)-B alleles have been identified by sequence analysis. Although the polymorphic exon 2 and 3 sequences of all HLA-B alleles are described, the sequences of the other exons of a number of infrequent B-alleles are unknown. In this study, the exon 1, 4, and 5 sequences of 39 different HLA-B alleles were elucidated by allele-specific sequencing. Overall, these exon sequences showed identity with the majority of the known sequences from the corresponding allele groups, except for four alleles B*4010, B*4415, B*4416, and B*5606. The exon 1 sequence of B*4010 had nucleotide differences with all B*40 alleles, but was identical to the B*54, *55, *56, and *59 allele groups. B*4416 differed from B*440201 at position 988, which was previously considered a conserved position. B*4415 showed exon 1, 4, and 5 sequences deviating from the other B*44 alleles, but identical to B*4501. The exon 1 and 4 sequences of B*5606 differed from other B*56 alleles, but were in complete agreement with B*7801. The deviating exon sequences of B*4415 and B*5606 confirmed the evolutionary origin of these alleles suggested by the sequences of exons 2 and 3. The polymorphism observed in exons 1, 4, and 5 merely reflects the lineage-specificity of HLA-B.

Alleles↗

PubMed2006

Antidepressants reverse corticosterone-mediated decrease in brain-derived neurotrophic factor expression: differential regulation of specific exons by antidepressants and corticosterone.

Earlier studies have implicated brain-derived neurotrophic factor in stress and in the mechanism of action of antidepressants. It has been shown that antidepressants upregulate, whereas corticosterone downregulates, brain-derived neurotrophic factor expression in rat brain. Whether various classes of antidepressants reverse corticosterone-mediated downregulation of brain-derived neurotrophic factor is unclear. Also not known is how antidepressants or corticosterone regulates brain-derived neurotrophic factor expression. To clarify this, we examined the effects of various classes of antidepressants and corticosterone, alone and in combination, on the mRNA expression of total brain-derived neurotrophic factor and of individual brain-derived neurotrophic factor exons, in rat brain. Normal or corticosterone pellet-implanted (100 mg, 21 days) rats were injected with different classes of antidepressants, fluoxetine, desipramine, or phenelzine, intraperitoneally for 21 days and killed 2 h after the last injection. mRNA expression of total brain-derived neurotrophic factor and of exons I-IV was measured in frontal cortex and hippocampus. Given to normal rats, fluoxetine increased total brain-derived neurotrophic factor mRNA only in hippocampus, whereas desipramine or phenelzine increased brain-derived neurotrophic factor mRNA in both frontal cortex and hippocampus. When specific exons were examined, desipramine increased expression of exons I and III in both brain areas, whereas phenelzine increased exon I in both frontal cortex and hippocampus but exon IV only in hippocampus. On the other hand, fluoxetine increased only exon II in hippocampus. Corticosterone treatment of normal rats decreased expression of total brain-derived neurotrophic factor mRNA in both brain areas, specifically decreasing exons II and IV. Treatment with desipramine or phenelzine of corticosterone pellet-implanted rats reversed the corticosterone-induced decrease in total brain-derived neurotrophic factor expression in both brain areas; however, fluoxetine reversed the decrease only partially in hippocampus. Interestingly, antidepressant treatment of corticosterone pellet-implanted rats increased only those specific exons that are increased during treatment of normal rats with each particular antidepressant. We found that although corticosterone and antidepressants both modulate brain-derived neurotrophic factor expression, and antidepressants reverse the corticosterone-induced brain-derived neurotrophic factor decrease, antidepressants and corticosterone differ in how they regulate the expression of brain-derived neurotrophic factor exon(s).

Animals↗

PubMed2006

Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells.

The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells.

Cell Differentiation↗

PubMed1991

Transcription initiation in the two leader exons of the rat IGF-I gene occurs from disperse versus localized sites.

Rat IGF-I mRNAs contain two different 5'-UTR sequences as a result of alternate splicing of leader exons. Using a combination of solution hybridization/RNase protection and primer extension assays, we have mapped the transcriptional start sites in these leader exons. There appear to be three putative transcription start sites in exon 1 spread over a 140-bp region, the most upstream of which defines a 381 bp-long exon 1. There appear to be three distinct start sites in exon 2, the most upstream of which defines a greater than 770 bp-long exon 2. The two downstream start sites in exon 1 together account for approximately 70% of IGF-I gene expression in adult rat liver. Essentially all of the remaining IGF-I gene expression comes from the second start site in exon 2. Rat IGF-I gene transcription may therefore be regulated by two distinct promoter regions, a disperse promoter for exon 1, with several transcription initiation sites, and a more typical promoter region for exon 2, which controls transcription initiation from a discrete region.

Animals↗

PubMed2002

Spontaneous frequency of exon skipping in the human HPRT gene.

In order to elucidate the mechanisms of mRNA splicing fidelity and the mutagenic potential of aberrant mis-spliced transcripts we have investigated the frequency of spontaneous exon skipping in the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in well characterized human primary fibroblasts isolated from two different individuals. In these cells, coexisting with the WT species, we also detected three aberrant HPRT transcripts missing exon IV, VII, or VIII. We were unable to detect transcripts missing exon II, III, V, or VI. Significantly, all the exons affected by skipping do not generate new stop codons more than 50 nucleotides upstream from the 3' most exon-exon junction. Exon VIII was the most prone to skipping with a relative frequency to WT of 0.019+/-0.004 (approximately one aberrant transcript per 50 WT transcripts). Exon IV exhibited a relative frequency of skipping of 0.006+/-0.002 ((approximately one aberrant transcript per 150 WT transcripts) and exon VII exhibited a relative frequency of skipping of 0.003+/-0.002 ((approximately one aberrant transcript per 300 WT transcripts). These data demonstrate that aberrant transcripts with exon skipped are generated spontaneously in humans and some appear to persist in the cell.

Exons↗

PubMed1998

Gene structure of chick lumican and identification of the first exon.

Three overlapping genomic clones to chick lumican were isolated and then characterized using restriction enzyme analyses, Southern blot analyses with cDNA probes, and by DNA sequencing. The results showed chick lumican gene to consist of 3 exons with a 2.9-kb first intron and a 4.2-kb second intron. Transcription initiation sites, identified by S1 nuclease experiments using genomic fragments containing exon 1 and by primer extension analysis of RNA, indicated the first exon to be 303 b. Two TATA sequences were 31 and 49 bases upstream of the first exon. The first exon contained all 5' untranslated sequence. The second exon was 896 b and contains 20 b of untranslated sequence, and codes for the start methionine to the end of the 10th leucine rich repeat. The third exon is 880 b and codes for the remainder of the core protein, and 724 b of untranslated 3' sequence. A 1-kb genomic fragment containing a portion of exon 1 and upstream sequence in a luciferase reporter sector showed specific promotor activity in the forward, but not the reverse direction when transfected into corneal fibroblasts. These results show the chick lumican gene to consist of three exons, and that regulatory elements are present within 1 kb upstream of the first exon.

Animals↗

PubMed2002

Modulation of the membrane-binding projection domain of tau protein: splicing regulation of exon 3.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. The N-terminal domain of the protein interacts with the axonal membrane, and is modulated by differential inclusion of exons 2 and 3. These two tau exons are alternatively spliced cassettes, in which exon 3 never appears independently of exon 2. Previous work with tau minigene constructs indicated that exon 3 is intrinsically suboptimal and its primary regulator is a weak branch point. In this study, we confirm the role of the weak branch point in the regulation of exon 3 but also show that the exon is additionally regulated by a combination of exonic enhancers and silencers. Furthermore, we demonstrate that known splicing regulators affect the ratio of exon 3 isoforms, Lastly, we tentatively pinpoint the site of action of several splicing factors which regulate tau exon 3.

Animals↗

PubMed2003

Modulation of the membrane-binding domain of tau protein: splicing regulation of exon 2.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. The N-terminal domain of the protein interacts with the axonal membrane, and is modulated by regulated inclusion of exons 2 and 3. These two tau exons are alternatively spliced cassettes, in which exon 3 never appears independently of exon 2. Previous work with tau minigene constructs indicated that exon 2 resembles a constitutive exon. In this study, we show that exon 2 is regulated by a combination of exonic and intronic enhancers and silencers. Furthermore, we demonstrate that known splicing regulators affect the ratio of exon 2 isoforms. Lastly, we tentatively pinpoint the site of action of several splicing factors which regulate tau exon 2.

Amino Acid Sequence↗

PubMed2001

Analysis of exonic splicing enhancers in the mouse gonadotropin-releasing hormone (GnRH) gene.

We have previously reported that efficient removal of the first intron (intron A) of gonadotropin-releasing hormone (GnRH) pre-mRNA is the prerequisite event for post-transcriptional regulation. In the present study, using an in vitro HeLa splicing system, we examined the enhancing activities of exonic elements for GnRH pre-mRNA splicing. While not excised by exon 2 alone, intron A was efficiently excised when exon 3 and/or exon 4 was combined with exon 2, suggesting the presence of exonic splicing enhancers (ESEs) in exons 3 and 4. Purine-rich sequences located in the border of exons 2 and 3 (denoted ESE3) and in exon 4 (ESE4) revealed strong splicing enhancing activities. Mutation in ESE3 decreased pre-mRNA splicing, while mutation in purine-rich sequences in exon 2 did not. We further analyzed the functional activity of ESE4 by mutations or deletions of the ESE4 sequence that consists of three purine-repeats separated by two spacers and a putative hairpin constructing sequence. An UV cross-linking assay using the RNA sequence of ESE4 examined the presence of ESE4-specific binding proteins in the nuclear extracts from GT1 hypothalamic GnRH neurons. Collectively, this study indicates that a sequence context of ESE4 and its binding proteins may be crucially involved in enhanced GnRH pre-mRNA splicing. However, it should be further clarified as to which splicing factor(s) is responsible for ESE4-dependent GnRH pre-mRNA splicing.

Animals↗

PubMed1998

The association of nonsense codons with exon skipping.

Some genes that contain premature nonsense codons express alternatively-spliced mRNA that has skipped the exon containing the nonsense codon. This paradoxical association of translation signals (nonsense codons) and RNA splicing has inspired numerous explanations. The first is based on the fact that premature nonsense codons often reduce mRNA abundance. The reduction in abundance of full-length mRNA then allows more efficient amplification during PCR of normal, minor, exon-deleted products. This mechanism has been demonstrated to explain an extensive correlation between nonsense codons and exon-skipping for the hamster Hprt gene. The second explanation is that the mutation producing an in-frame nonsense codon has an effect on exon definition. This has been demonstrated for the Mup and hamster Hprt gene by virtue of the fact that missense mutations at the same sites also are associated with the same exon-deleted mRNA. The third general explanation is that a hypothetical process takes place in the nucleus that recognizes nonsense codons, termed 'nuclear scanning', which then has an effect on mRNA splicing. Definitive evidence for nuclear scanning is lacking. My analysis of both nonsense and missense mutations associated with exon skipping in a large number of genes revealed that both types of mutations frequently introduce a T into a purine-rich DNA sequence and are often within 30 base pairs of the nearest exon boundary. This is intriguing given that purine-rich splicing enhancers are known to be inhibited by the introduction of a T. Almost all mutations associated with exon skipping occur in purine-rich or A/C-rich sequences, also characteristics of splicing enhancers. I conclude that most cases of exon skipping associated with premature termination codons may be adequately explained either by a structural effect on exon definition or by nonquantitative methods to measure mRNA, rather than an effect on a putative nuclear scanning mechanism.

Animals↗

PubMed2002

Sequence analysis of exons 1, 2, 3, 4 and 5 of the HLA-B5/35 cross-reacting group.

The HLA-B5/35 cross-reacting group (CREG) is a set of closely related antigens including HLA-B35, B51, B52, B53 and B78. The nucleotide sequences of exon 1 through 5 of the B5/35 CREG were determined to assess the level of polymorphism. For exons 2 and 3, the previously described sequence-based typing (SBT) strategy was applied, the nucleotide sequences of exon 1, 4 and 5 were determined by allele-specific sequencing. A total of 225 unrelated individuals were HLA-B typed by heterozygous sequencing of exons 2 and 3. In the B5/35 CREG, 26 different alleles were identified, whereas 63 non-B5/35 CREG alleles were sequenced. The SBT strategy was proven to be reliable and efficient for high resolution typing of the B5/35 CREG. The nucleotide sequences of exon 1, 4 and 5 were determined for the 26 different B5/35 CREG alleles to establish the level of polymorphism. For seven different alleles, of which the exon 1, 4 and 5 sequences were hitherto unknown, the sequences were elucidated and in agreement with the known B5/35 sequences. Nineteen HLA-B5/35 CREG alleles with previously published exon 1, 4 and 5 sequences were sequenced in at least two individuals. Three new alleles were identified. The first, B*5204, showed a difference at position 200 compared to B*52011, which was previously considered a conserved position. The other two alleles, B*3542 and B*51015, showed exon 2 and 3 sequences identical to B*35011 and B*51011, but differences in exons 1 and 4, respectively. B*3542 had differences at position 25 and 72 and B*51015 showed a difference at position 636. More polymorphism might be present outside exons 2 and 3 than previously thought.

Alleles↗

PubMed2003

Trapping and sequence analysis of 1138 putative exons from human chromosome 18.

In a search for novel genes on chromosome 18 (HC18), on which several regions have been linked to bipolar disorder, we applied exon trapping to HC18-specific cosmids. Among the 1138 exons trapped, 1052 of them have been mapped to HC18, and the remaining 86 have not been localized. No exons were localized to genomic regions other than HC18. BLAST database search revealed that 190 exons were identical to 98 Unigenes on HC18; 98 identical to additional 82 clusters of ESTs not present in the HC18 Unigene set; 39 homologous to genes from human and other species (e<10(-3)); and the remaining 811 exons had no significant homology to transcripts in public databases. The mapped exons were compared to the 867 annotated genes on HC18 in the Celera databases; 216 exons were identical to 104 Celera 'genes' and the remaining 836 exons were not found in the Celera databases. On average, there were two exons for a matched transcript (known genes and ESTs). Therefore, the 850 novel exons may represent hundreds of novel genes on chromosome 18.

Bipolar Disorder↗

PubMed1993

Phosphoglucomutase 1: a gene with two promoters and a duplicated first exon.

In view of its central role in glycolysis and gluconeogenesis and its polymorphic genetic variability, the phosphoglucomutase 1 (PGM1) gene in man has been the target of protein structural studies and genetic analysis for more than 25 years. We have now isolated genomic clones containing the complete PGM1 gene and have shown that it spans over 65 kb and contains 11 exons. We have also shown that the sites of the two mutations which form the molecular basis for the common PGM1 protein polymorphism lie in exons 4 and 8 and are 18 kb apart. Within this region there is a site of intragenic recombination. We have discovered two alternatively spliced first exons, one of which, exon 1A, is transcribed in a wide variety of cell types; the other, exon 1B, is transcribed in fast muscle. Exon 1A is transcribed from a promoter which has the structural hallmarks of a housekeeping promoter but lies more than 35 kb upstream of exon 2. Exon 1B lies 6 kb upstream of exon 2 within the large first intron of the ubiquitously expressed PGM1 transcript. The fast-muscle form of PGM1 is characterized by 18 extra amino acid residues at its N-terminal end. Sequence comparisons show that exons 1A and 1B are structurally related and have arisen by duplication.

Amino Acid Sequence↗

PubMed1996

Structural motifs encoded by individual exons of the human neurokinin-1 receptor gene interact differentially with selective agonists and antagonists.

Three chimeric receptors were constructed by exchanging exons between human neurokinin NK1 and NK3 receptor genes. The N-terminal sequences of these chimeric receptors are encoded by exon 1, exon 1-2, or exon 1-3 of the NK1 receptor gene, whereas the remaining C-terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK3 receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK1 receptor gene may also play an important role for substance P binding. On the contrary, potent NK1 antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK1 agonists, [Sar9, Met(O2)11] substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK1 receptor. These results show that the exon-exchanging technique can be a useful tool for studying structure-function relationships of receptors in which exon-intron junctions are fully conserved among receptor subtypes.

Amino Acid Sequence↗

PubMed1987

Organization of the neural cell adhesion molecule (N-CAM) gene: alternative exon usage as the basis for different membrane-associated domains.

The neural cell adhesion molecule, N-CAM, is expressed as at least three polypeptide chain, (ld, sd, and ssd chains) specified by a single gene and derived by alternative splicing and polyadenylation-site selection during RNA processing. We describe here the characterization of seven overlapping genomic phage clones reactive with N-CAM cDNA, indicating that the chicken N-CAM gene is more than 50 kilobases long. Analysis of the gene shows that there are at least 19 exons and that the coding sequences for the ld, sd, and ssd chains are assembled from 18, 17, and 15 exons, respectively. The first 14 exons appear to be common to all three chains and encode the amino-terminal portion of N-CAM, which contains five tandem homologous repeats resembling those seen in the immunoglobulin gene superfamily. In contrast to other genes containing such domains, each of these segments in N-CAM is specified by two exons. The carboxyl-terminal portion of each N-CAM chain is different as a result of the alternative use of exons. A single exon encodes the carboxyl-terminal 26 amino acids of the ssd chain and the 3' untranslated region of its mRNA, ending with a poly(A)-addition site. Two exons encode the transmembrane and cytoplasmic sequences common to the ld and sd chains, and another exon encodes the additional 261 amino acids found in the cytoplasmic domain of the ld chain. The carboxyl-terminal 21 amino acids common to the ld and sd chains and the 3' untranslated region common to their mRNAs are encoded by a single large exon of 3475 base pairs that ends with a second poly(A)-addition site. Sequences from the 13-kilobase intron that separates the exons encoding the amino-terminal and carboxyl-terminal regions of the molecule hybridize to a 2-kilobase poly(A)+ RNA transcript of unknown identity. This description of the chicken N-CAM gene provides a basis for determining the mechanisms that regulate the differential expression of the N-CAM polypeptide chains during development.

Amino Acid Sequence↗

PubMed1997

Sequence requirements for regulated RNA splicing of the human fibroblast growth factor receptor-1 alpha exon.

Progression of astrocytes from a benign to a malignant phenotype is accompanied by a change in the RNA processing of the fibroblast growth factor receptor 1 (FGFR-1) gene. The level of a high affinity form of the FGFR-1 is dramatically elevated as a result of alpha-exon skipping during RNA splicing. In this paper we have been able to duplicate this tumor-specific RNA processing pathway by transfection of a chimeric minigene containing a 4-kilobase fragment of the human FGFR-1 gene (including the alpha-exon) into a variety of cell lines. In a transfected human astrocytoma cell line, alpha-exon skipping was consistently observed for RNA transcripts derived from both the chimeric minigene and endogenous gene expression. This exon skipping phenotype was dependent on the size of the flanking intron as deletions which reduced the introns to less than approximately 350 base pairs resulted in enhanced alpha-exon inclusion. Increased exon inclusion was not sequence-specific as exon skipping could be restored with insertion of nonspecific sequence. Cell-specific exon recognition was maintained with a 375-nucleotide sequence inclusive and flanking the alpha-exon, provided that intron size was maintained. These results identify the minimal cis-regulatory sequence requirements for exclusion of FGFR-1 alpha-exon in astrocytomas.

Base Sequence↗

PubMed1998

Identification of a novel type of alternatively spliced exon from the acetylcholinesterase gene of Bungarus fasciatus. Molecular forms of acetylcholinesterase in the snake liver and muscle.

The venom of the snake Bungarus fasciatus contains a hydrophilic, monomeric species of acetylcholinesterase (AChE), characterized by a C-terminal region that does not resemble the alternative T- or H-peptides. Here, we show that the snake contains a single gene for AChE, possessing a novel alternative exon (S) that encodes the C-terminal region of the venom enzyme, located downstream of the T exon. Alternative splicing generates S mRNA in the venom gland and S and T mRNAs in muscle and liver. We found no evidence for the presence of an H exon between the last common "catalytic" exon and the T exon, where H exons are located in Torpedo and in mammals. Moreover, COS cells that were transfected with AChE expression vectors containing the T exon with or without the preceding genomic region produced exclusively AChET subunits. In the snake tissues, we could not detect any glycophosphatidylinositol-anchored AChE form that would have derived from H subunits. In the liver, the cholinesterase activity comprises both AChE and butyrylcholinesterase components; butyrylcholinesterase corresponds essentially to nonamphiphilic tetramers and AChE to nonamphiphilic monomers (G1na). In muscle, AChE is largely predominant: it consists of globular forms (G1a and G4a) and trace amounts of asymmetric forms (A8 and A12), which derive from AChET subunits. Thus, the Bungarus AChE gene possesses alternatively spliced T and S exons but no H exon; the absence of an H exon may be a common feature of AChE genes in reptiles and birds.

Acetylcholinesterase↗

PubMed1998

Identification of intron and exon sequences involved in alternative splicing of insulin receptor pre-mRNA.

The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (- exon 11) and B-like (+ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5' end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3' end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3' end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively.

Alternative Splicing↗