Transcription Of Exon Research Articles

Conservation of a pair of serpin 2 genes and their expression in Amphiesmenoptera

Silk secreted by the larvae of Hydropsyche angustipennis (Trichoptera) contains serpins HaSerp2A and HaSerp2B that are homologous to serpin 2 known from several lepidopterans and some other insects. The gene HaSerp2A is 2684bp downstream from the HaSerp2B gene. The genes possess identical exon/intron segmentation (9 exons) and their sequences are nearly identical: only 8 out of 1203nt differ in the coding region, 4 out of 567nt in the introns and 2 out of 52nt in 3′ UTR. Both genes are highly expressed in the silk glands whereas expression in larval carcass devoid of the silk glands is hard to detect. Translation products of the genes consist of 401 amino acids, are 98.8% identical, and are secreted as 45kDa proteins into silk. Homologous genes in similar tandem arrangement occur on chromosome 15 of Bombyx mori (Lepidoptera). The upstream gene BmSerp2B is modified in several exons and does not seem to produce functional mRNA. The gene BmSerp2A contains two copies of exon 9, of which only the second one is used. One kind of mRNA does and the other does not include exon 1, which encodes a signal peptide. The mRNA yielding secreted BmSerp2A is expressed in the posterior, and that encoding the cytoplasmic BmSerp2A in the middle silk gland region; both kinds are strongly expressed in the anterior region. The data indicate that (1) A duplication of serpin 2 gene occurred either before Trichoptera and Lepidoptera diverged as separate orders or independently in early phylogeny of either order; (2) In the caddisfly H. angustipennis, both genes are expressed specifically in the silk glands and generate proteins deposited in the silk; (3) Only one gene seems to be functional in B. mori and is expressed in a cytoplasmic and in a secreted forms in diverse organs, including the silk glands.

Valproic acid increases SMN2 expression and modulates SF2/ASF and hnRNPA1 expression in SMA fibroblast cell lines

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is caused by loss of the survival motor neuron gene, SMN1. SMA treatment strategies have focused on production of the SMN protein from the almost identical gene, SMN2. Valproic acid (VPA) is a histone deacetylase inhibitor that can increase SMN levels in some SMA cells or SMA patients through activation of SMN2 transcription or splicing correction of SMN2 exon 7. It remains to be clarified what concentration of VPA is required and by what mechanisms the SMN production from SMN2 is elicited. We observed that in two fibroblast cell lines from Japanese SMA patients, more than 1mM of VPA increased SMN2 expression at both the transcript and protein levels. VPA increased not only full-length (FL) transcript level but also exon 7-excluding (Δ7) transcript level in the cell lines and did not change the ratio of FL/Δ7, suggesting that SMN2 transcription was mainly activated. We also found that VPA modulated splicing factor expression: VPA increased the expression of splicing factor 2/alternative splicing factor (SF2/ASF) and decreased the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). In conclusion, more than 1mM of VPA activated SMN2 transcription and modulated the expression of splicing factors in our SMA fibroblast cell lines.

Differential Regulation and Function of 5′-Untranslated GR-Exon 1 Transcripts

Alternative splicing serves to increase biological diversity and adaptation. Many genes, including the glucocorticoid receptor (GR), contain multiple 5'-untranslated exons in their promoter regions that can give rise to various mRNA isoforms encoding the same protein. To date, information on the mouse GR promoter remains sparse. Here, we extensively characterize alternative first exons of the mouse GR to reveal homology to the rat and human. We further find that, although most promoters are broadly expressed in various tissues, transcription of individual promoters can be differentially regulated by growth factor- and depolarization-induced signaling. Moreover, in addition to selective promoter usage, the alternative first exon transcripts differentially control RNA stability and translation efficiency, indicative of their role in GR expression. In conclusion, the composite GR promoter enables multilayered adjustments in gene expression through transcriptional and posttranscriptional mechanisms that may serve varying physiological demands.

Tumor necrosis factor-α increases brain-derived neurotrophic factor expression in trigeminal ganglion neurons in an activity-dependent manner

Many chronic trigeminal pain conditions, such as migraine or temporo-mandibular disorders, are associated with inflammation within peripheral endings of trigeminal ganglion (TG) sensory neurons. A critical role in mechanisms of neuroinflammation is attributed to proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α (TNFα) that also contribute to mechanisms of persistent neuropathic pain resulting from nerve injury. However, the mechanisms of cytokine-mediated synaptic plasticity and nociceptor sensitization are not completely understood. In the present study, we examined the effects of TNFα on neuronal expression of brain-derived neurotrophic factor (BDNF), whose role in synaptic plasticity and sensitization of nociceptive pathways is well documented. We show that 4- and 24-h treatment with TNFα increases BDNF mRNA and protein, respectively, in neuron-enriched dissociated cultures of rat TG. TNFα increases the phosphorylated form of the cyclic AMP-responsive element binding protein (CREB), a transcription factor involved in regulation of BDNF expression in neurons, and activates transcription of BDNF exon IV (former exon III) and, to a lesser extent, exon VI (former exon IV), but not exon I. TNFα-mediated increase in BDNF expression is accompanied by increase in calcitonin gene-related peptide (CGRP), which is consistent with previously published studies, and indicates that both peptides are similarly regulated in TG neurons by inflammatory mediators. The effect of TNFα on BDNF expression is dependent on sodium influx through TTX-sensitive channels and on p38-mitogen-activated protein kinase. Moreover, electrical stimulation and forskolin, known to increase intracellular cAMP, potentiate the TNFα-mediated upregulation of BDNF expression. This study provides new evidence for a direct action of proinflammatory cytokines on TG primary sensory neurons, and reveals a mechanism through which TNFα stimulates de novo synthesis of BDNF in these neurons. Thus, TNFα should be considered in mechanisms of BDNF-dependent neuronal plasticity.

Epigenetic Regulation of BDNF Expression via the Scaffolding Protein RACK1

Scaffolding proteins are major contributors to the spatial and temporal orchestration of signaling cascades and hence cellular functions. RACK1 is a scaffolding protein that plays an important role in the regulation of, and cross-talk between, various signaling pathways. Here we report that RACK1 is a mediator of chromatin remodeling, resulting in an exon-specific expression of the brain-derived neurotrophic factor (BDNF) gene. Specifically, we found that following the activation of the cAMP pathway, nuclear RACK1 localizes at the promoter IV region of the BDNF gene by its association with histones H3 and H4, leading to the dissociation of the transcription repressor methyl-CpG-binding protein 2 (MeCP2) from the promoter, resulting in the acetylation of histone H4. These chromatin modifications lead to the activation of the promoter and to the subsequent promoter-controlled transcription of BDNF exon IV. Our findings expand our knowledge regarding the function of scaffolding proteins such as RACK1. Furthermore, this novel mechanism for the regulation of exon-specific expression of the BDNF gene by RACK1 could have implications on the neuronal functions of the growth factor including synaptic plasticity, learning, and memory.

The Functional Effect of R1648H, a Sodium Channel Mutation that Causes Generalized Epilepsy with Febrile Seizures Plus in Splice Variants of SCN1A

SCN1A, the gene that encodes the alpha subunit of the voltage-gated sodium channel Nav1.1, is alternatively spliced at exon 5. SCN1A contains two copies of exon 5, denoted 5N and 5A (for ‘Neonatal’ and ‘Adult’ according to their developmental expression). There are 3 amino acid substitutions between the splice variants, all within the D1:S3/S4 extracellular linker. It is unknown how exons 5N and 5A alter channel function. Because patients with Generalized Epilepsy with Febrile Seizures plus (GEFS+) frequently exhibit age-dependent changes in seizure frequency and severity, we have asked whether the GEFS+-associated SCN1A mutation R1648H differentially affects Nav1.1-5N and Nav1.1-5A.

RGS17, an Overexpressed Gene in Human Lung and Prostate Cancer, Induces Tumor Cell Proliferation Through the Cyclic AMP-PKA-CREB Pathway

We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth.

The Aldehyde Dehydrogenase Gene Superfamily Resource Center

The website http://www.aldh.org is a publicly available database for nomenclature and functional and molecular sequence information for members of the aldehyde dehydrogenase (ALDH) gene superfamily for animals, plants, fungi and bacteria. The site has organised gene-specific records. It provides synopses of ALDH gene records, marries trivial terms to correct nomenclature and links global accession identifiers with source data. Server-side alignment software characterises the integrity of each sequence relative to the latest genomic assembly and provides identifier-specific detail reports, including a graphical presentation of the transcript's exon - intron structure, its size, coding sequence, genomic strand and locus. Also included are a summary of substrates, inhibitors and enzyme kinetics. The site provides reference lists and is designed to facilitate data mining by interested investigators.

In vitro and in vivo production and purification of circular RNA aptamer

RNA aptamers are potential candidates for RNA therapeutics. They must be clinically modified for medical applications because they are vulnerable to indigenous ribonucleases. Since circular RNA molecules without any chemical modification are much more stable than linear ones in a cell extract, we report the production of a circular form of streptavidin RNA aptamer both in vitro and in vivo. Circularization was accomplished by self-splicing permuted intron–exon sequences derived from T4 bacteriophage gene td. This sequence was producible in both Escherichia coli cells and in vitro. The circularized streptavidin RNA aptamer retained its binding of streptavidin and was stabile in HeLa cell extracts compared to the linear form of the streptavidin aptamer. The self-spliced circular RNA from the transcribed permuted intron–exon transcripts in E. coli cells was purified from a total RNA fraction using the solid-phase DNA probe method following anion exchange chromatography that excluded gel electrophoresis. This study provides an alternative method for designing and purifying useful RNA aptamers.

Genetic Modulation of CD44 Expression by Intragraft Fibroblasts

This study investigated the genetic composition and the functional implication of CD44 species expressed by intragraft fibroblasts. An LEW-to-F344 heart transplant model of chronic rejection was used. Intragraft fibroblasts recovered from the chronically rejecting allografts displayed a 4.5-fold increase in expression of CD44 mRNA when compared with that of the fibroblasts isolated from non-rejecting heart allografts (P < 0.01). The intragraft fibroblasts preferentially expressed CD44 variant isoforms containing v1 exon transcript. Automated nucleotide sequence analysis revealed that the majority (90.12%) of the CD44 v1 isoforms expressed by the rejecting graft fibroblasts were encoded by a mutated CD44 mRNA, which contained two point mutations and a codon deletion in the v1 coding region. Histochemistry demonstrated a massive deposition of extracellular HA in the rejecting heart allografts. Hyaluronic acid (HA) was able to promote in vitro fibroblast adhesion, migration in a CD44-dependent manner, and survival in a serum-free culture condition. The study concludes that up-regulation of CD44 v1 isoforms expressed by the intragraft fibroblasts is associated with an increase in the deposition of extracellular HA, the principal ligand for CD44, in the allografts, suggesting that CD44-HA interaction plays an important role in regulating fibroblast recruitment and growth in allografts developing chronic rejection.

A renin transcript lacking exon 1 encodes for a non‐secretory intracellular renin that increases aldosterone production in transgenic rats

Renin transcripts lacking exon 1 and thus the signal sequence for co-translational transport to the endoplasmatic reticulum encode for a protein (exon[2-9]renin), that is confined to the cytoplasm. The function of exon(2-9)renin is currently unknown. Mitochondrial renin increases under conditions which stimulate aldosterone production. We hypothesized that exon(2-9)renin (1) is translated into a functionally active protein in vivo, (2) is not secreted but remains within the cytoplasm and (3) stimulates aldosterone production. To test these hypotheses we generated transgenic rats overexpressing exon(2-9)renin. Four transgenic lines were obtained expressing the transcript in various tissues including the heart and the adrenal gland. Renin was enriched particularly in the cytoplasm of transgenic rats. Renin was not elevated in plasma, indicating that exon(2-9)renin is produced but not secreted. The ratio of aldosterone to renin concentrations in plasma (PAC/PRC) was elevated in all transgenic lines except line 307, which also did not exhibit elevated cytoplasmatic renin levels in the adrenal gland (PAC/PRC in controls: 2.8±2.3; line 307: 1.9±0.8; n. s.; line 284: 5.8±1.9; P<0.02; line 294: 5.0±2.3; P<0.001; line 276: 10.3±5.1; P<0.001). We conclude that the exon(1A-9) renin transcript (1) is translated into a functionally active intracellular protein; (2) is targeted to the cytoplasm rather than being sorted to the secretory pathways and (3) is functionally active, regulating aldosterone production. The CX-(exon2-9)renin transgenic rat appears to be a useful model to study the role and the mechanisms of action of cytoplasmatic renin derived from exon(1A-9) transcripts.

Variant CD44 expression by human fibroblasts

CD44 is a cell surface glycoprotein with roles in tumour invasion and metastasis. CD44 is variably spliced from ten variant exons and mis-splicing is a biomarker for detection of colon, urothelial and other carcinomas. Fibroblasts are normally considered to lack variant exons and thus should not generate false-positive signals. Transcription of variant exons by fibroblasts was investigated by exon-specific reverse transcription-polymerase chain reaction (RT-PCR) for variant exons v2–v10 using normal primary fibroblasts, immortalized and experimentally transformed fibroblasts. Flow cytometry, immunocytochemistry and Western blotting were used to determine expression. All types of fibroblasts, including normal primary culture fibroblasts, transcribed low levels of variant exon mRNA. Expression could not be detected by blotting or immunocytochemistry but flow cytometry revealed minor expression of some exons by all three types of cultured fibroblast. Fibroblasts do transcribe and express small amounts of variant exon CD44. This may need to be considered when using exon splicing as a biomarker for malignancy in clinical samples containing connective tissue.

445: Preferential expression of CD44 v1 isoforms by intragraft fibroblasts from cardiac allografts with chronic rejection

Purpose: CD44 is a transmembrane glycoprotein primarily involved in cell-cell interactions and cell migration. This study investigated the genetic composition and the functional implication of CD44 species expressed by intragraft fibroblasts (IF). Methods and Materials: Intragraft fibroblasts recovered from the chronically rejecting heart allografts of rats were examined for CD44 gene expression using quantitative real time PCR. CD44 specific cDNA libraries were constructed and selected CD44 clones were analyzed in automate nucleotide sequencing. The role of CD44/hyaluronan interaction was studied using in vitro migration and proliferation assays. Results: IF displayed a 4.5-fold increase in expression of CD44 mRNA when compared with that of the fibroblasts isolated from non-rejecting heart allografts (p 0.01). The IF preferentially expressed CD44 variant isoforms containing v1 exon transcript. Automate nucleotide sequence analysis revealed that the majority (90.12%) of the CD44 v1 isoforms expressed by the rejecting graft fibroblasts were encoded by a mutated CD44 mRNA, which contained 2 point mutations and a codon deletion in the v1 coding region. Histochemistry demonstrated a massive deposition of extracellular hyaluronan in the rejecting heart allografts. Hyaluronan promoted in vitro fibroblast adhesion, migration in a CD44 dependent manner, and survival in a serum-free culture condition. Conclusions: A significant increase in CD44 expression by the rejecting graft fibroblasts was coupled with augmentation of extracellular hyaluronan synthesis in the heart allografts, indicating that CD44-HA interaction is critically involved in the pathogenesis of graft fibrosis. CD44v1 isoforms may play a role, in a way similar to other documented v exons such as v3 or v6, to provide additional signaling for enhancing or suppressing the biological activities of intragraft fibroblasts. CD44 antagonism may affect multiple cellular activities mediated by the CD44-HA pathway, leading to alleviation of allograft fibrosis and chronic inflammation.

A Protean Insect Receptor Holds the Key to Broad-Based Pathogen Recognition

Anything that's alive runs the risk of infection. How you respond to infection, however, depends on where you sit on the evolutionary tree. Humans and other vertebrates can fend off billions of pathogens by routinely recombining bits of genes for surface molecules on the cells charged with pathogen recognition. Insects and other invertebrates rely to a large degree on the pathogen recognition molecules (called pattern recognition receptors) they were born with. When a pattern recognition receptor detects a pathogen—based on what's known as its pathogen-associated molecular pattern—the receptor can launch a direct attack that either engulfs the invader, through encapsulation or phagocytosis, or triggers signaling pathways that regulate immune system genes involved in killing the pathogen. In a new study, Yuemei Dong, Harry Taylor, and George Dimopoulos found a mosquito gene that vastly boosts the ability of insect pattern recognition receptors to detect pathogens. Originally implicated in neuron development, the gene can create a plethora of receptors for the malaria vector Anopheles gambiae. The AgDscam gene—short for Anopheles gambiae Down syndrome cell adhesion molecule gene—has 101 protein-coding regions (called exons) that can be mixed and matched after transcription to produce over 31,000 possible alternative splice forms with different properties. Thus, while B cell and T cell receptor diversity is generated largely at the gene sequence level before transcription, AgDscam diversity is produced by reshuffling sections of gene transcripts before translation into protein. Dscam was first characterized in the fruitfly (Drosophila melanogaster), where it can generate about 38,000 splice forms with different recognition and binding specificities from 95 variable exons. It's been suggested that a diverse inventory of adhesion molecules may help olfactory nerves establish the proper connections during development. But the presence of high levels of Dscam in cells that function in the fly's innate immune system and evidence of involvement in phagocytosis raised the possibility that the gene also plays a diverse recognition role in immunity. Dong et al. found that AgDscam, like the fly version, has three variable regions within a portion of the immunoglobulin (Ig) gene. Each region contains different numbers of alternative splicing exons: Ig4 has 14, Ig6 has 30, and Ig10 has 38, leading to a possible 31,920 alternative splice forms. The researchers worked with a mosquito immune cell line to investigate AgDscam's response to infection. Exposure to bacteria, fungi, and parasite surface molecules caused the cells to produce different AgDscam splice-form repertoires with different interaction properties. As with the cell lines, bacterial infection of adult mosquitoes also caused alternative splicing of AgDscam. Infecting mosquitoes with two different Plasmodium malaria parasites produced completely different AgDscam splice-form repertoires. When the researchers cut AgDscam protein levels in half with a technique that “silences” a gene by degrading its transcript, they could link its function with phagocytosis of pathogens. Mosquitoes with a silenced AgDscam gene succumbed to bacterial infections (caused by two types of bacteria that produce different surface proteins) at much higher rates than did mosquitoes with a functioning AgDscam gene. Silencing AgDscam also resulted in a “profound proliferation” of opportunistic microbes, suggesting its essential role in defending the mosquito against bacterial infections. When gene-silenced mosquitoes fed on blood infected with malaria parasites, the researchers found a 65% increase of parasites on the insects' guts. The researchers confirmed the specificity of these associations between splice forms and particular pathogens by selectively silencing the exon transcripts induced by different bacteria. Disabling bacteria-specific exons reduced binding for the target bacteria but had no effect on other bacteria. Altogether, these results show that infection-induced AgDscam splicing creates receptors better equipped to recognize—and defend against—the invading pathogen. Cells generated different splice-form repertoires depending on the source of infection. Alternative splicing allows the insect to vastly increase its repertoire of pattern recognition receptors from one single gene and thereby fight infection more efficiently. This work suggests that a better understanding of how A. gambiae's hypervariable receptor AgDscam recognizes the Plasmodium parasite might suggest novel ways to control malaria by targeting the parasite inside its mosquito host.

A Novel Exon Duplication Event Leading to a Truncating Germ-line Mutation of the APC Gene in a Familial Adenomatous Polyposis Family

Familial Adenomatous Polyposis (FAP) is an autosomal dominant condition predisposing to multiple adenomatous polyps of the colon. FAP patients frequently carry heterozygous mutations of the APC tumour suppressor gene. Affected individuals from a cohort of FAP families (n=22), where no germ-line APC mutation was detected by direct sequencing, were analysed by Multiplex Ligation-dependent Probe Amplification (MLPA). MLPA identified a previously unreported APC mutation involving duplication of exon 4. Subsequent analysis of cDNA from affected family members revealed expression of mutant mRNA species containing two copies of exon 4, resulting in a frameshift and premature stop codon. Bioinformatic analysis of the relevant APC genomic segment predicted a role for homologous recombination possibly involving Alu repeats in the generation of this genotype. Our results highlight the importance of MLPA as an adjunct to exon-by-exon sequencing in identifying infrequent mutational events in cancer predisposing genes.

A New Intronic Mutation in the DPM1 Gene Is Associated With a Milder Form of CDG Ie in Two French Siblings

Congenital disorders of glycosylation (CDG) type I (CDG I) are rare autosomal recessive diseases caused by deficiencies in the assembly of the dolichol-linked oligosaccharide (DLO) that is required for N-glycoprotein biosynthesis. CDG Ie is due to a defect in the synthesis of dolichyl-phosphoryl-mannose (Dol-P-Man), which is needed for DLO biosynthesis as well as for other glycosylation pathways. Human Dol-P-Man synthase is a heterotrimeric complex composed of DPM1p, DPM2p, and DPM3p, with DPM1p being the catalytic subunit. Here, we report two new CDG Ie patients who present milder symptoms than the five other CDG Ie patients described to date. The clinical pictures of the patients MS and his sister MT are dominated by major ataxia, with no notable hepatic involvement. MS cells accumulate the immature DLO species Dol-PP-GlcNAc2Man5 and possess only residual Dol-P-Man synthase activity. One homozygous intronic mutation, g.IVS4-5T>A, was found in the DPM1 gene, leading to exon skipping and transcription of a shortened transcript. Moreover, DPM1 expression was reduced by more than 90% in MS cells, in a nonsense-mediated mRNA decay (NMD)-independent manner. Full analysis of the DPM2 and DPM3 genes revealed a decrease in DPM2 expression and normal expression of DPM3. This description emphasizes the large spectrum of symptoms characterizing CDG I patients.

Titin and Diaphragm Dysfunction in Chronic Obstructive Pulmonary Disease

Recently, we have shown that Ca2+-activated force generation in diaphragm single fibers is impaired in patients with mild to moderate chronic obstructive pulmonary disease (COPD). For optimal active-force generation, the passive elasticity provided by titin is indispensable. In the present study, we determined the passive-tension-length relations of single fibers of patients with mild to moderate COPD, hypothesizing that passive-elastic properties of diaphragm fibers are compromised. Passive-tension-length relations were determined in diaphragm fibers from patients with and without COPD (predicted mean FEV1, 76 and 102%, respectively). In diaphragm homogenates titin expression was studied at the protein level by gel electrophoresis and at the transcript level by using a novel titin exon microarray. Diaphragm fibers from patients with COPD generate less passive tension on stretch. Titin content in the diaphragm did not differ between patients with and without COPD. However, titin exon transcript studies revealed up-regulation of seven exons, which code for spring elements in the elastic segment rich in proline, glutamate, valine, and lysine. Immunofluorescence analysis indicated elevated protein expression of the up-regulated splice variant in the COPD diaphragm. Simulation studies on titin molecules including the amino acids encoded by the seven up-regulated exons predicted reduced passive-tension generation on molecule stretch. Passive-tension generation of diaphragm single fibers is reduced in patients with COPD. Our results suggest that alternative splicing of the titin gene, resulting in increased length of the elastic segment rich in proline, glutamate, valine, and lysine, is involved. Interestingly, these changes occur already in patients with mild to moderate COPD.

Semiquantitative Detection of Abnormal CD44 Transcripts in Colon Carcinomas by Reverse Transcription-Polymerase Chain Reaction Enzyme-linked Immunosorbant Assay (RT-PCR ELISA).

Background: Abnormal expression of CD44 variant RNA has been detected in a variety of human tumors and has been shown to be a potential diagnostic marker. To date, such analysis requires time-consuming gel electrophoresis, blotting, and autoradiographic procedures, and this approach may not be suitable for routine laboratory examinations. We have developed a rapid and semiquantitative reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA) method and used it to analyze CD44 expression in colon carcinoma tissues and exfoliated cancer cells in colon luminal washings. Methods and Results: RNA was extracted from sample cells and tissues and converted to cDNA. PCR amplification products, labeled by incorporation of digoxigenin-11-dUTP, were hybridized with biotinylated probes complementary to CD44 exon 12 or to exons in the standard portion (CD44s) of the gene. Hybridized DNA complexes were immobilized on streptavidin-coated microtiter plates, and the bound PCR products were detected with a peroxidase-conjugated antibody to digoxigenin. CD44-derived PCR products were quantified by absorbance of a chromogenic reaction. Elevated expression of CD44 variant exon 12 was detected initially by Southern blot analysis in all of the 9 colon carcinoma tissues, while weak expression was observed in only 3 of 9 normal mucosas. This tumor-related differential expression was confirmed by the newly developed PCR-ELISA method. Elevated expression of CD44 exon 12 was also detected in exfoliated colonic epithelial cells from 10 of 13 carcinoma cases but not in exfoliated cells from 4 patients with inflammatory bowel disease. Conclusions: Raised expression of CD44 variant exon transcripts can be detected reliably in colonic tumor tissue and in exfoliated colonic cancer cells by a semiquantitative RT-PCR ELISA method. This was shown to be as sensitive as conventional RT-PCR using chemiluminescent detection. Therefore, CD44-based RT-PCR ELISA could facilitate detection of neoplasia in clinical specimens including colon washings and naturally micturated urine.

Intergenic transcripts in genes with phase I introns

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that detects and degrades aberrant transcripts prematurely terminating translation. NMD may be elicited by intergenic transcripts that contain premature termination codons (PTCs), but chimeric mRNAs of genes that have introns of identical phase would be predicted to lack PTCs and escape NMD. We examined intron phase I-containing HLA class II genes for the presence of intergenic mRNAs and found an extraordinary diversity of correctly spliced and polyadenylated intergenic transcripts. They lacked a significant homology at the chimeric joins and had no PTCs. Their expression levels were very low and positively correlated with the expression of natural transcripts. In contrast, pair-wise mixtures of separately transcribed plasmids carrying full-length HLA-DQB1, - DQA1, - DRB1, and - DRA cDNAs produced only hybrid molecules that lacked canonical exon boundaries, had homologous chimeric joins, and occasionally contained PTCs, implicating in vitro artifacts generated by template switching of Taq polymerase and reverse transcriptase. A differential exon structure of hybrid molecules observed in vitro and in cellular RNA preparations suggests that intergenic mRNAs with canonical exon boundaries arise in vivo during exon joining and/or transcription. Since the observed intergenic mRNAs may encode mixed class II heterodimers that were previously shown to present antigens it will be interesting to determine functional properties of such molecules in future studies.

Computational analysis suggests that alternative first exons are involved in tissue-specific transcription in rice (Oryza sativa)

Transcription start site selection and alternative splicing greatly contribute to diversifying gene expression. Recent studies have revealed the existence of alternative first exons, but most have involved mammalian genes, and as yet the regulation of usage of alternative first exons has not been clarified, especially in plants. We systematically identified putative alternative first exon transcripts in rice, verified the candidates using RT-PCR, and searched for the promoter elements that might regulate the alternative first exons. As a result, we detected a number of unreported alternative first exons, some of which are regulated in a tissue-specific manner. http://www.bioinfo.sfc.keio.ac.jp/research/intron.