Exon Microarray Research Articles

Platelet Gene Expression as a Biomarker Risk Stratification Tool in Acute Myocardial Infarction: A Pilot Investigation

Platelets play a major role in the pathophysiology of acute myocardial infarction (AMI). Recent evidence reveals megakaryocyte-derived platelet pre-mRNA is spliced to mRNA and then translated into functional proteins in response to external stimulation. An exon microarray analyzes pre-mRNA alternative splicing and is thus applicable for studying gene expression in the anucleate platelet. We hypothesized a subset of megakaryocyte/platelet genes exists that are significantly over or underexpressed in AMI compared with stable coronary artery disease (CAD), yielding a gene expression profile for further study. Microarray analysis employing platelet mRNA was used to generate gene expression data in the above two patient groups. Unsupervised hierarchical clustering has revealed an expression profile that includes 95 over- or under-expressed genes depicted in a heat map where separation of both sets takes place. This preliminary study reveals a platelet-based gene expression signature that differentiates between AMI and stable CAD, and further study may yield a prognostic tool for a future AMI event in atherosclerosis risk factor-based subsets of CAD patients.

Effect of Excision of Titin's PEVK Exons 219-225 on Skeletal Muscle Structure and Function

We recently published a PEVK KO mouse model in which all exons that constitute the PEVK region of cardiac titin (N2B cardiac isoform), exons 219-225, had been excised, and reported cardiac hypertrophy and increased passive stress (Granzier et al., 2009 Circ Res., 11, 557). Here we investigated the phenotype of EDL (fast twitch) and soleus (slow twitch) skeletal muscle of wildtype and homozygous PEVK KO mice. Muscle mass was significantly increased in both soleus (51±5%) and EDL (21±6%) muscles; we are currently studying whether the underlying hypertrophy mechanisms are similar to those previously found in the heart. Because the excised exons make up a small portion of the PEVK segment of skeletal muscle titins we expected modest differences, if any at all, in passive stress. Unexpectedly, passive stress was significantly increased in soleus and EDL muscles, both when measurements (SL 3.0μm) were made at the whole muscle level (40±7% and 67±16%, respectively) and at the fiber bundle level (50±14% and 81±1%). Gel electrophoresis revealed, in both EDL and soleus, expression of a single titin isoform in wildtype muscle, but surprisingly, co-expression of two isoforms in the KO muscles. The larger isoform co-migrated with the isoform expressed in wt muscle and thus is likely to represent KO titin (exons 219-225 represent ∼30kDa, which is insufficient to cause a mobility difference). The smaller isoform, found in both EDL and soleus KO muscle, is likely to represent a splicing adaption that correlates with increased passive tension. We are currently investigating the identity of these exons using titin exon microarray analysis. In summary, excision of titin exons 219-225 in skeletal muscle results in elevated passive stress with the underlying cause likely being due to an adaption in titin splicing.

Alternative splicing is frequent during early embryonic development in mouse

BackgroundAlternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development.ResultsA genome-wide analysis of alternative isoform expression in embryonic day 8.5, 9.5 and 11.5 mouse embryos and placenta was carried out using a splicing-sensitive exon microarray. We show that alternative splicing and isoform expression is frequent across developmental stages and tissues, and is comparable in frequency to the variation in whole-transcript expression. The genes that are alternatively spliced across our samples are disproportionately involved in important developmental processes. Finally, we find that a number of RNA binding proteins, including putative splicing factors, are differentially expressed and spliced across our samples suggesting that such proteins may be involved in regulating tissue and temporal variation in isoform expression. Using an example of a well characterized splicing factor, Fox2, we demonstrate that changes in Fox2 expression levels can be used to predict changes in inclusion levels of alternative exons that are flanked by Fox2 binding sites.ConclusionsWe propose that alternative splicing is an important developmental regulatory mechanism. We further propose that gene expression should routinely be monitored at both the whole transcript and the isoform level in developmental studies

Genome-Wide Analysis of Alternative Splicing Points to Novel Leukemia Relevant Genes in Acute Myeloid Leukemia.

Abstract 2391Poster Board II-368Alternative mRNA splicing represents an effective mechanism of regulating gene function as well as a key element to increase the coding capacity of the human genome. Today, an increasing number of reports illustrates that aberrant splicing events can contribute to human disease and that alterations in the splicing machinery are common and functionally important for cancer development. Aberrant splice forms can for example have genome-wide effects by deregulating key signaling pathways. However, for most of the aberrant mRNA transcripts detected it remains unclear whether they directly contribute to the malignant phenotype or just represent a by-product of cellular transformation. Thus, more comprehensive analyses of the transcriptome splicing are warranted in order to get novel insights into the biology underlying malignancies like, e.g., acute myeloid leukemia (AML).Here, we performed a genome-wide screening of splicing events in AML using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). We analyzed forty AML cases with complex karyotypes and twenty Core Binding Factor (CBF) AML cases (entered on a multicenter trial for patients <60 years, AMLSG 07-04) using this microarray approach allowing the detection of splice variants. In order to detect alternative splicing events distinguishing different leukemia subgroups we applied a commercial and an open source software tool: XRAY version 3.9 (Biotique Systems) and the OneChannelGUI package for R (version 1.10.7 available at http://www.bioinformatica.unito.it/oneChannelGUI/). Using XRAY supervised analysis comparing subgroups of CBF and complex karyotype AML we identified 1120 transcripts to be potentially alternatively spliced. In parallel, the analysis of the same AML subgroups using the OneChannelGUI package in R revealed 1439 candidates with an overlap of only 211 genes. Of these transcripts, that have been indicated by both programs as potentially alternatively spliced, selected candidates were further investigated by RT-PCR, quantitative RT-PCR and sequence analysis for the presence of splice-variants. Of 26 candidate genes studied, we could confirm alternative splice forms for 5 genes that might potentially be involved in driving leukemogenesis, such as the protein coding gene arginine methyltransferase 1 (PRMT1), which regulates transcription through histone methylation and participates in DNA damage response. Furthermore, we could confirm differential exon usage in the protein tyrosine phosphatase non-receptor type (PTPN6) transcript, which encodes for a negative regulator of numerous signaling pathways involved in cell cycle control and apoptosis. Similarly, the mRNA of the protein Rho GTPase activating protein 4 (ARHGAP4), which has been shown to regulate cell motility, was alternatively spliced between CBF and complex karyotype subgroups.In summary, these first gene expression data demonstrate that the attempt to elucidate the splicing of transcriptome in AML by applying Exon microarray technology is challenging in particular with regard to the currently available software solutions. Nevertheless, our results show that this approach offers the ability to detect novel alternatively spliced candidate genes. Being involved in cell cycle control, regulation of transcription or remodeling of the cytoskeleton, alternative splicing of these genes might play a potential role in the pathomechanism of distinct AML subgroups. Thus, in the future more extensive Exon array profiling with more sophisticated software solutions at hand is likely to provide additional insights into the molecular mechanisms of leukemogenesis and might reveal novel targets for refined therapeutic strategies in AML. Disclosures:No relevant conflicts of interest to declare.

Tuning Passive Mechanics through Differential Splicing of Titin during Skeletal Muscle Development

During postnatal development, major changes in mechanical properties of skeletal muscle occur. We investigated passive properties of skeletal muscle in mice and rabbits that varied in age from 1 day to ∼1 year. Neonatal skeletal muscle expressed large titin isoforms directly after birth, followed by a gradual switch toward progressively smaller isoforms that required weeks-to-months to be completed. This suggests an extremely high plasticity of titin splicing during skeletal muscle development. Titin exon microarray analysis showed increased expression of a large group of exons in neonatal muscle, when compared to adult muscle transcripts, with the majority of upregulated exons coding for the elastic proline-glutamate-valine-lysine (PEVK) region of titin. Protein analysis supported expression of a significantly larger PEVK segment in neonatal muscle. In line with these findings, we found >50% lower titin-based passive stiffness of neonatal muscle when compared to adult muscle. Inhibiting 3,5,3′-tri-iodo-L-thyronine and 3,5,3′,5′-tetra-iodo-L-thyronine secretion did not alter isoform switching, suggesting no major role for thyroid hormones in regulating differential titin splicing during postnatal development. In summary, our work shows that stiffening of skeletal muscle during postnatal development occurs through a decrease in titin isoform size, due mainly to a marked restructuring of the PEVK region of titin.

Low-risk susceptibility alleles in 40 human breast cancer cell lines

BackgroundLow-risk breast cancer susceptibility alleles or SNPs confer only modest breast cancer risks ranging from just over 1.0 to1.3 fold. Yet, they are common among most populations and therefore are involved in the development of essentially all breast cancers. The mechanism by which the low-risk SNPs confer breast cancer risks is currently unclear. The breast cancer association consortium BCAC has hypothesized that the low-risk SNPs modulate expression levels of nearby located genes.MethodsGenotypes of five low-risk SNPs were determined for 40 human breast cancer cell lines, by direct sequencing of PCR-amplified genomic templates. We have analyzed expression of the four genes that are located nearby the low-risk SNPs, by using real-time RT-PCR and Human Exon microarrays.ResultsThe SNP genotypes and additional phenotypic data on the breast cancer cell lines are presented. We did not detect any effect of the SNP genotypes on expression levels of the nearby-located genes MAP3K1, FGFR2, TNRC9 and LSP1.ConclusionThe SNP genotypes provide a base line for functional studies in a well-characterized cohort of 40 human breast cancer cell lines. Our expression analyses suggest that a putative disease mechanism through gene expression modulation is not operative in breast cancer cell lines.

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.

Titin Isoform Transitions and Passive Stiffness During Skeletal Muscle Development

During postnatal striated muscle development, multiple changes in active and passive properties occur, reflecting an altered mechanical demand. For instance, during postnatal cardiac development titin isoform expression switches from large isoforms to small stiffer isoforms, likely affecting diastolic filling behavior. In the present study, we investigated whether titin isoform transitions also take place during skeletal muscle development. We used gel-electrophoresis to determine changes in titin isoform size in mice and rabbits of various ages. A titin exon microarray was used to evaluate transcript expression of all of titin's exons. To investigate the mechanical effect of titin isoform transitions, passive properties of neonatal and adult skeletal muscles were determined.Neonatal mice were found to express large titin isoforms, which are gradually replaced by smaller isoforms during skeletal muscle development. The half transition time of the isoform for tibialis cranialis (TC), soleus, extensor digitorum longus, gastrocnemius and diaphragm were 6, 17, 17, 12, and 10 days, respectively. Essentially similar findings were obtained from NZW rabbits, with the exception that the half-life of the isoform transitions was slightly longer compared to murine skeletal muscles. Titin exon analysis in neonatal murine gastrocnemius muscle revealed increased expression of a large group of exons when compared to adult muscle transcripts, with all upregulated exons coding for exons of the elastic PEVK region of titin. In line with these observations, we found ∼50% lower titin-based passive stiffness of murine neonatal soleus and TC when compared to adult muscle.These data demonstrate that during skeletal muscle development titin isoform transitions occur from large compliant isoforms to smaller and stiffer isoforms in adult muscle, likely due to changed expression of PEVK exons.

Probe-level estimation improves the detection of differential splicing in Affymetrix exon array studies

A novel statistical procedure is presented that uses probe-level information on Affymetrix exon arrays to detect differential splicing.

Stage-Specific Switches in Alternative Pre-mRNA Splicing during Late Erythropoiesis Are Conserved from Mouse to Human

Spatial and temporal regulation of alternative pre-mRNA splicing determines which exons are incorporated into mature mRNA, modulating mRNA coding capacity to ensure synthesis of appropriate protein isoforms throughout normal differentiation and development. During erythropoiesis, a stage-specific switch in pre-mRNA splicing activates incorporation of protein 4.1R exon 16, thereby increasing 4.1R affinity for spectrin and actin and mechanically strengthening red blood cell membranes. We are exploring the hypothesis that stage-specific changes in pre-mRNA splicing regulate expression of other critical genes during terminal erythropoiesis. Last year we described exon microarray and RT-PCR studies that revealed several novel pre-mRNA splicing switches in terminally differentiating human erythroid progenitors. These alternative splicing events involved well-annotated exons with consensus exon-intron boundaries, supporting a model in which these events represent a regulated alternative splicing program rather than a breakdown of splicing integrity in late erythropoiesis. Here we report additional evidence for this model by showing that several erythroid stage-specific switches in alternative pre-mRNA splicing are conserved between human and mouse. Primary mouse splenic erythroblasts from FVA-infected mice were cultured in vitro under differentiation conditions and used as the source of RNA for analysis of murine erythroid splicing events. From a total of seven internal cassette exons whose splicing was activated in late human erythroblasts, five exhibited an analogous splicing switch in murine erythroblasts. Comparative genomic analysis showed that these alternative exons are embedded in regions of unusually high sequence conservation among vertebrate species, suggesting that important regulatory signals are contained within the adjacent introns. Indeed, the flanking introns for several of these exons contain binding motifs for Fox2, an RNA binding protein and known splicing regulator for many tissue-specific splicing events. Further analysis of the conserved erythroid splicing events revealed the following:three splicing switches occur in transcripts encoding RNA binding proteins (MBNL2, HNRPLL, and SNRP70), suggesting significant changes in the RNA processing machinery of late erythroblasts; andthree of these alternative exons encode premature stop codons that could induce nonsense mediated decay (NMD) and contribute to down-regulation of these genes during terminal erythropoiesis.Consistent with the latter hypothesis, inhibition of NMD in murine erythroblast cultures led to increased accumulation of mRNA isoforms containing the premature stop codons. Together these results suggest the existence of a highly regulated alternative splicing program that is critical for late erythroid differentiation.

FIRMA: a method for detection of alternative splicing from exon array data.

Motivation: Analyses of EST data show that alternative splicing is much more widespread than once thought. The advent of exon and tiling microarrays means that researchers now have the capacity to experimentally measure alternative splicing on a genome wide level. New methods are needed to analyze the data from these arrays.Results: We present a method, finding isoforms using robust multichip analysis (FIRMA), for detecting differential alternative splicing in exon array data. FIRMA has been developed for Affymetrix exon arrays, but could in principle be extended to other exon arrays, tiling arrays or splice junction arrays. We have evaluated the method using simulated data, and have also applied it to two datasets: a panel of 11 human tissues and a set of 10 pairs of matched normal and tumor colon tissue. FIRMA is able to detect exons in several genes confirmed by reverse transcriptase PCR.Availability: R code implementing our methods is contributed to the package aroma.affymetrix.Contact: epurdom@stat.berkeley.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Exon and junction microarrays detect widespread mouse strain- and sex-bias expression differences

BackgroundStudies have shown that genetic and sex differences strongly influence gene expression in mice. Given the diversity and complexity of transcripts produced by alternative splicing, we sought to use microarrays to establish the extent of variation found in mouse strains and genders. Here, we surveyed the effect of strain and sex on liver gene and exon expression using male and female mice from three different inbred strains.Results71 liver RNA samples from three mouse strains – DBA/2J, C57BL/6J and C3H/HeJ – were profiled using a custom-designed microarray monitoring exon and exon-junction expression of 1,020 genes representing 9,406 exons. Gene expression was calculated via two different methods, using the 3'-most exon probe ("3' gene expression profiling") and using all probes associated with the gene ("whole-transcript gene expression profiling"), while exon expression was determined using exon probes and flanking junction probes that spanned across the neighboring exons ("exon expression profiling"). Widespread strain and sex influences were detected using a two-way Analysis of Variance (ANOVA) regardless of the profiling method used. However, over 90% of the genes identified in 3' gene expression profiling or whole transcript profiling were identified in exon profiling, along with 75% and 38% more genes, respectively, showing evidence of differential isoform expression. Overall, 55% and 32% of genes, respectively, exhibited strain- and sex-bias differential gene or exon expression.ConclusionExon expression profiling identifies significantly more variation than both 3' gene expression profiling and whole-transcript gene expression profiling. A large percentage of genes that are not differentially expressed at the gene level demonstrate exon expression variation suggesting an influence of strain and sex on alternative splicing and a need to profile expression changes at sub-gene resolution.

Eight-channel iTRAQ Enables Comparison of the Activity of Six Leukemogenic Tyrosine Kinases

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRbeta, FIP1/PDGFRalpha, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach.

Discovery of tissue-specific exons using comprehensive human exon microarrays

Comprehensive exon microarrays with a simple intra-gene normalization algorithm were used to detect human tissue-specific alternative splicing events, suggesting significant expression outside of known exons and well annotated genes and a high frequency of alternative splicing events.

Combinatorial Regulation of Protein 4.1R Exon 16 Alternative Splicing: Modulation of Fox-2 Activated Splicing by Other Intronic and Exonic Motifs.

During erythroid differentiation, stage-specific activation of protein 4.1R exon 16 splicing is critical for the mechanical stability of the erythrocyte plasma membrane. The molecular mechanism of this erythroid splicing switch involves multiple factors including stimulation by Fox proteins acting at splicing enhancers in the proximal downstream intron, and inhibition by hnRNPA1 protein acting at silencer elements in exon 16. To explore how the dynamic interplay among these and other factors can fine tune splicing efficiency, we created a series of exon 16-containing minigenes in which splicing efficiency was measured as a function of variation in exon and intron regulatory elements. In the context of wild type exon 16 with intact hnRNP A1 silencer elements and a weak 5′ splice site, an enhancerless construct with no Fox binding sites exhibited little or no exon 16 inclusion in transfected HeLa cells, and over-expression of Fox-2 did not significantly promote inclusion. Insertion of two wild type UGCAUG elements enhanced splicing substantially in a Fox-2-dependent manner and four elements gave even stronger inclusion. Since another study identified the pentamer GCAUG as the Fox binding site, we tested binding site sequence as a potential source of variation in splicing efficiency. Mutation of the first U residue in UGCAUG yielded weaker, but still Fox-2 dependent, activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. To investigate whether enhancer activity of Fox binding sites can be modulated by adjacent sequence motifs, we compared exon 16 splicing efficiency in constructs having Fox sites flanked either by neutral sequence or by an A1 silencer element UAGGG. Introduction of the A1 binding site led to a dramatically reduced enhancer activity including its responsiveness to Fox-2 overexpression. These results indicated that efficiency of splicing of Fox-regulated exons is strongly influenced by the number and sequence of intron enhancer elements and by the presence of adjacent antagonistic elements. In further experiments, we demonstrated that the efficiency of splicing is also strongly dependent on exon 16 and its splice sites. Constructs lacking the major exon 16 silencer element for hnRNP A1 binding exhibited partial exon 16 inclusion in the absence, and very strong inclusion in the presence, of a Fox intron enhancer. Finally, strengthening the weak 5′ splice site of exon 16 abrogated many of these regulatory effects and led to strong inclusion of exon 16 independent of other variables. These findings are consistent with previous data showing antagonism between A1 and Fox in their effects on exon 16 splicing, and suggest that Fox proteins primarily function to overcome the weak 5′ splice site and its repression by hnRNP A1 bound at nearby exonic site(s). We propose that the erythroid alternative splicing program can activate splicing a number of alternative exons with variable efficiency based on each exon's individual complement of exonic and intronic splicing regulatory elements. Modulation of splicing factor expression, typified by the stage-specific down-regulation of hnRNP A1 during erythroblast differentiation, can further alter splicing efficiency of these exons in a selective, motif-dependent manner. Future experiments with exon microarrays will be aimed at identifying some of the alternative exons that are regulated by that program, and determine its importance to the erythroid differentiation process.

How Many New Genes Are There?

How Many New Genes Are There?

Physiological genomics in PG and beyond: October to December 2005

physiological genomics ( PG ) publishes, on a quarterly basis, brief editorial perspectives entitled “Physiological genomics in PG and beyond.” These editorial perspectives provide brief, accessible highlights of the studies published in PG in the most recent quarter, and relate those studies to

A statistical framework for genome-wide discovery of biomarker splice variations with GeneChip Human Exon 1.0 ST Arrays.

Alternative splicing is an important regulatory mechanism that generates multiple mRNA transcripts which are transcribed into functionally diverse proteins. According to the current studies, aberrant transcripts due to splicing mutations are known to cause for 15% of genetic diseases. Therefore understanding regulatory mechanism of alternative splicing is essential for identifying potential biomarkers for several types of human diseases. Most recently, advent of GeneChip Human Exon 1.0 ST Array enables us to measure genome-wide expression profiles of over one million exons. With this new microarray platform, analysis of functional gene expressions could be extended to detect not only differentially expressed genes, but also a set of specific-splicing events that are differentially observed between one or more experimental conditions, e.g. tumor or normal control cells. In this study, we address the statistical problems to identify differentially observed splicing variations from exon expression profiles. The proposed method is organized according to the following process: (1) Data preprocessing for removing systematic biases from the probe intensities. (2) Whole transcript analysis with the analysis of variance (ANOVA) to identify a set of loci that cause the alternative splicing-related to a certain disease. We test the proposed statistical approach on exon expression profiles of colorectal carcinoma. The applicability is verified and discussed in relation to the existing biological knowledge. This paper intends to highlight the potential role of statistical analysis of all exon microarray data. Our work is an important first step toward development of more advanced statistical technology. Supplementary information and materials are available from http://bonsai.ims.u-tokyo.ac.jp/~yoshidar/IBSB2006_ExonArray.htm.

Genome-wide analysis of mouse transcripts using exon microarrays and factor graphs

Recent mammalian microarray experiments detected widespread transcription and indicated that there may be many undiscovered multiple-exon protein-coding genes. To explore this possibility, we labeled cDNA from unamplified, polyadenylation-selected RNA samples from 37 mouse tissues to microarrays encompassing 1.14 million exon probes. We analyzed these data using GenRate, a Bayesian algorithm that uses a genome-wide scoring function in a factor graph to infer genes. At a stringent exon false detection rate of 2.7%, GenRate detected 12,145 gene-length transcripts and confirmed 81% of the 10,000 most highly expressed known genes. Notably, our analysis showed that most of the 155,839 exons detected by GenRate were associated with known genes, providing microarray-based evidence that most multiple-exon genes have already been identified. GenRate also detected tens of thousands of potential new exons and reconciled discrepancies in current cDNA databases by 'stitching' new transcribed regions into previously annotated genes.