Evidence For Alternative Splicing Research Articles

Crustacean hyperglycemic hormone of Portunus trituberculatus: evidence of alternative splicing and potential roles in osmoregulation.

The crustacean hyperglycemic hormone (CHH) gene of Portunus trituberculatus (Pt-CHH) consists of four exons and three introns spanning 3849bp in size and generating two mature mRNA, Pt-CHH1, and Pt-CHH2. The primary gene transcript produces a cDNA encoding for the putative Pt-CHH2 from exons 1, 2, 3, and 4 and an alternative transcript encodes for a putative Pt-CHH1 peptide from exons 1, 2, and 4. A promoter fragment of about 3kb was obtained by genomic walking. The tissue-specific expression pattern is examined by reverse transcriptase chain reaction, and the results show that Pt-CHH1 is detected in the eyestalk, brain, muscle, and blood. However, Pt-CHH2 is detected in the ganglia thoracalis and gill. The results indicate that the expression of Pt-CHH2 in the gill might suggest a potential role in osmoregulation. The Pt-CHH transcript level in the gill increases when the crab is exposed to low salinity. The injection of dsRNA for Pt-CHH causes a significant reduction in Pt-CHH2 transcript level and the activity of Na+/K+-ATPase, and carbonic anhydrase (CA) show a serious decrease. In conclusion, this study provides molecular evidence to support the osmoregulatory function of Pt-CHH2.

Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish.

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey.

Anti-angiogenic VEGFAxxxb transcripts are not expressed in the medio-basal hypothalamus of the seasonal sheep.

This study investigated Vegfa expression in the pars tuberalis (PT) of the pituitary and medio-basal hypothalamus (MBH) of sheep, across seasons and reproductive states. It has recently been proposed that season impacts alternative splicing of Vegfa mRNA in the PT, which shifts the balance between angiogenic VEGFAxxx and anti-angiogenic VEGFAxxxb isoforms (with xxx the number of amino acids of the mature VEGFA proteins) to modulate seasonal breeding. Here, we used various RT-PCR methodologies and analysis of RNAseq datasets to investigate seasonal variation in expression and splicing of the ovine Vegfa gene. Collectively, we identify 5 different transcripts for Vegfa within the ewe PT/MBH, which correspond to splicing events previously described in mouse and human. All identified transcripts encode angiogenic VEGFAxxx isoforms, with no evidence for alternative splicing within exon 8. These findings led us to investigate in detail how “Vegfaxxxb-like” PCR products could be generated by RT-PCR and misidentified as endogenous transcripts, in sheep and human HEK293 cells. In conclusion, our findings do not support the existence of anti-angiogenic VEGFAxxxb isoforms in the ovine PT/MBH and shed new light on the interpretation of prior studies, which claimed to identify Vegfaxxxb isoforms by RT-PCR.

QTL Mapping for Resistance to Iridovirus in Asian Seabass Using Genotyping-by-Sequencing

Identifying quantitative trait loci (QTL) for viral disease resistance is of particular importance in selective breeding programs of fish species. Genetic markers linked to QTL can be useful in marker-assisted selection (MAS) for elites resistant to specific pathogens. Here, we conducted a genome scan for QTL associated with Singapore grouper iridovirus (SGIV) resistance in an Asian seabass (Lates calcarifer) family, using a high-density linkage map generated with genotyping-by-sequencing. One genome-wide significant and three suggestive QTL were detected at LG21, LG6, LG13, and LG15, respectively. The phenotypic variation explained (PVE) by the four QTL ranged from 7.5 to 15.6%. The position of the most significant QTL at LG21 was located between 31.88 and 36.81cM. The SNP marker (SNP130416) nearest to the peak of this QTL was significantly associated with SGIV resistance in an unrelated multifamily population. One candidate gene, MECOM, close to the peak of this QTL region, was predicted. Evidence of alternative splicing was observed for MECOM and one specific category of splicing variants was differentially expressed at 5days post-SGIV infection. The QTL detected in this study are valuable resources and can be used in the selective breeding programs of Asian seabass with regard to resistance to SGIV.

Regulation of alternative splicing in human obesity loci.

Multiple obesity susceptibility loci have been identified by genome-wide association studies, yet the mechanisms by which these loci influence obesity remain unclear. Alternative splicing could contribute to obesity by regulating the transcriptomic and proteomic diversity of genes in these loci. Based on a database search, 72 of the 136 genes at the 13 obesity loci encoded multiple protein isoforms. Thus, alternative splicing of these genes in adipose tissue samples was analyzed from the Metabolic Syndrome in Men population-based study and from two weight loss intervention studies (surgical and very low calorie diet). Alternative splicing was confirmed in 11 genes with PCR capillary electrophoresis in human subcutaneous adipose tissue. Interestingly, differential splicing of TRA2B, BAG6, and MSH5 was observed between lean individuals with normoglycemia and overweight individuals with type 2 diabetes. Of these genes, we detected fat depot-dependent splicing of TRA2B and BAG6 and weight loss-induced regulation of MSH5 splicing in the intervention studies. Finally, body mass index was a major determinant of TRA2B, BAG6, and MSH5 splicing in the combined data. This study provides evidence for alternative splicing in obesity loci, suggesting that alternative splicing at least in part mediates the obesity-associated risk in these loci.

Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated.

Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5'- and alternative 3'-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes.

Alternative splicing of mini-exons in the Arabidopsis leaf rust receptor-like kinase LRK10 genes affects subcellular localisation.

AtLRK10L1.2 produces a variety of alternatively spliced variants in the region a mini-exon and skipping of the mini-exon alters the subcellular localization of the protein. We have examined expression and alternative splicing in the gene encoding Arabidopsis LRK10-like 1 (AtLRK10L1) which is most closely related to wheat leaf rust 10 disease-resistance locus receptor-like protein kinase (LRK10). AtLRK10L1 produces two different transcripts, LRK10L1.1 and 1.2 through the use of two different promoters. We found no evidence of alternative splicing for the AtLRK10L1.1 transcript but identified numerous alternative splicing variants of AtLRK10L1.2 by sequencing of cloned cDNAs prepared from RNA isolated from whole cell, nucleolar and nucleoplasmic fractions. Many of these transcripts contained unspliced introns and accumulated differentially in the nucleolus and the nucleoplasm consistent with intron retention transcripts being retained in the nucleus (Göhring et al., Plant Cell 26:754-764, 2014). We examined the fate of different alternatively spliced transcripts by fusing variants to YFP and expressing them by agroinfiltration in Nicotiana benthamiana. AtLRK10L1 contains a 45 nt mini-exon which encodes part of a putative transmembrane domain. Full-length cDNA of LRK10L1.2 fused to YFP targeted the fusion protein to the plasma membrane while expression of transcripts where the mini-exon had been deleted, altered the localization of the fusion protein to the endoplasmic reticulum. Similarly, expression of full-length and mini-exon deleted versions of three other members of the LRK10 receptor-like kinase (RLK) gene family also showed the switch in localization. Thus, the mini-exons in Arabidopsis LRK10 genes are required for localization to the plasma membrane.

Structured and disordered facets of the GPCR fold.

The seven-transmembrane (7TM) helix fold of G-protein coupled receptors (GPCRs) has been adapted for a wide variety of physiologically important signaling functions. Here, we discuss the diversity in the structured and disordered regions of GPCRs based on the recently published crystal structures and sequence analysis of all human GPCRs. A comparison of the structures of rhodopsin-like receptors (class A), secretin-like receptors (class B), metabotropic receptors (class C) and frizzled receptors (class F) shows that the relative arrangement of the transmembrane helices is conserved across all four GPCR classes although individual receptors can be activated by ligand binding at varying positions within and around the transmembrane helical bundle. A systematic analysis of GPCR sequences reveals the presence of disordered segments in the cytoplasmic side, abundant post-translational modification sites, evidence for alternative splicing and several putative linear peptide motifs that have the potential to mediate interactions with cytosolic proteins. While the structured regions permit the receptor to bind diverse ligands, the disordered regions appear to have an underappreciated role in modulating downstream signaling in response to the cellular state. An integrated paradigm combining the knowledge of structured and disordered regions is imperative for gaining a holistic understanding of the GPCR (un)structure-function relationship.

Abstract 162: Hypoxia Inducible Factor Regulates Cardiac Ribonucleic Acid Splicing

Ischemic cardiomyopathy is associated with elevated levels of hypoxia inducible factor (HIF)-1, a transcription factor that plays a key role in the response to hypoxia. We have created a transgenic mouse model with inducible cardiomyocyte-specific expression of an oxygen-stable form of HIF-1α to better understand the role of HIF-1 in ischemic cardiomyopathy. We performed RNAseq on polyA selected cardiac RNA before and after three days of transgene expression, with 10 million processed reads per sample. Genes were considered possibly differentially expressed if, following adjustment for multiple testing, p-value for the comparison to uninduced was less than 0.05. Genes having evidence of alternative splicing (those with a q-value <0.05 suggesting a change in the relative abundance of the different transcripts deriving from a single transcription start site) were identified. A total of 22 alternatively spliced genes were investigated. One transcript in particular, that for Calmodulin-dependent protein kinase II isoform gamma 2 (CaMKIIγ) was of particular interest because CaMKII homologs are involved in the phosphorylation of both RyR2 and PLB, and might thus be important in excitation-contraction coupling and cardiac contractility. The reference RNA sequence for CaMKIIγ was down regulated 11.7-fold, while variant 1 (missing Exons 7 and 8), and variant 2 (missing Exon 8) were up regulated 2.6-fold and 1.7-fold, respectively. Real-time PCR validation of the RNAseq data has been obtained in the transgenic hearts, and, to a lesser extent, in wild type mice subjected to LAD ligation. The cardiac effects of CaMKIIγ have been little studied in the response to hypoxia. Taken together with our prior observations of SERCA2a down-regulation and increased phosphorylation of PLB in the transgenic model, this new evidence suggests that another way in which HIF-1 may be modulating calcium flux is by reconfiguring the abundance of CaMKIIγ splice variants. The biochemical effect(s) of this variation in splice variants remains unknown, but seems likely to be connected to the decrement in cardiac contractility seen with HIF expression, and in ischemic cardiomyopathy. The surprising role of HIF in regulating splicing will also be a fertile area for further investigation.

Characterization of two alternative splice variants associated with the Arabidopsis rhomboid protein gene At1g25290

Rhomboid proteases are involved in various cellular activities, from development to cancer, and cellular processes and substrates associated with rhomboid proteases or rhomboid-like proteins have been identified for a range of organisms. Plant rhomboid proteases or rhomboid-like proteins are the least understood of the group. Moreover, the general phenomenon of alternative splicing and rhomboid protein genes has yet to be studied robustly. This study thus focused on the alternative splicing events associated with the Arabidopsis rhomboid protein gene At1g25290. The patterns obtained through RT-PCR and DNA sequencing provided evidence of alternative splicing in the At1g25290 transcript population, especially in the region spanning exons 5 and 6. The levels of the two splice variants involving exons 5 and 6 appear to be sufficiently abundant to possess functional significance and appear to adjust relative to each other in different contexts. Adjustments were observed in tissues of different developmental stages, in an Arabidopsis plant bearing a mutation in another rhomboid protein, and in response to transgenic manipulations affecting the levels of Tic40, a plastid translocon component. The resulting change to the protein sequence may, in turn, affect how At1g25290 proteins interact with their substrates. Collectively, the evidence suggests that alternative splicing of At1g25290 bears functional significance in Arabidopsis.

Characterization of CbCyp51 from Field Isolates of Cercospora beticola

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 μg ml(-1).

Evidence of alternative splicing of the chi2 chitinase gene from Metarhizium anisopliae

Metarhizium anisopliae is a filamentous fungus used in the biological control of arthropods and produces several chitinases in order to break the host cuticle chitin fibers. Chitinase function during fungal cell development and/or infection processes is also an important aspect when analyzing the life cycle of entomopathogens. The expression profile analysis of the endochitinase chi2 gene acquired by RT-PCR experiments indicated the presence of two different transcripts, suggesting the occurrence of alternative splicing in the chi2 gene. The presence of two transcripts, characterized by the removal or retention of the second 72 bp intron, was further confirmed by DNA sequencing, Northern blot and qRT-PCR. Furthermore, we detected the synthesis of two different proteins from the transcripts by two-dimensional Western blot and mass spectrometry analyses. This is the first reported occurrence of alternative splicing in M. anisopliae.

Abstract 5249: Identification of genome-wide alternative splicing events in high-risk MYCN amplified neuroblastomas

Abstract Introduction: High-risk neuroblastoma (H-NB) is an aggressive tumor of the sympathetic nervous system for which there are few predictors of therapeutic response. We sought to determine the association of global alternative splicing (AS) gene expression patterns with NB aggressiveness and MYCN gene status. Methods: As part of the NCI-sponsored Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, we profiled 190 primary human NBs using Affymetrix Human Exon arrays. Tumors were obtained from children who participated in Children's Oncology Group trials with informed consent. The high-risk cohort included tumors with (H-NBA n=48) and without MYCN amplification (H-NBN n=111), a key molecular determinant of clinical aggressiveness. Twenty-four samples from low-risk NB lacking MYCN amplification (L-NB) were also profiled as a comparison group. Robust multichip analysis (RMA) was used for summarization of probe selection regions (PSR) and transcript regions. Finding isoforms using RMA (FIRMA) was used to generate PSR isoform scores. After an unsupervised pre-filtering step based on coefficient of variation, ANOVA analyses were conducted comparing H-NBA or H-NBN against the L-NB group for differential transcript and isoform expression. Last, gene set enrichment analyses (GSEA) comparing high-risk to low-risk NBs were conducted using unfiltered transcript data and the Molecular Signature Database's (MsigDB) canonical gene sets. Results: 1914 out of the 22,011 core transcripts passed our strict prefiltering criteria that required a high coefficient of variation. There was a larger number of genes with significant differential expression between H-NBA vs. L-NB than H-NBN vs. L-NB (23.5% versus 4.6%, P<0.001). There was also significantly larger number of PSRs exhibiting differential FIRMA scores between H-NBA vs. L-NB compared to H-NBN vs. L-NB (14.6% versus 2.3% of PSRs; P<0.001). NTRK1, a gene with known alternative splicing pattern among neuroblastomas, was identified in our data to have lower expression in both H-NBA and H-NBN vs. the L-NB subgroup but showed significant evidence of alternative splicing only in the H-NBA group. GSEA analyses also identified more differentially expressed pathways in H-NBA vs. L-NB than in H-NBN versus L-NB neuroblastomas (66 versus 31 pathways; P<0.001). Pathways related to cell cycle and axon guidance were among the top differentially expressed pathways in the H-NBA vs. L-NB comparison. Conclusion: Significant differences in gene expression and pathway expression are identified among H-NBA compared to H-NBN tumors. In addition, we show for the first time a high degree of alternative splicing events in the H-NBA tumors. These findings could aid in understanding the regulatory networks in the two subgroups of high-risk neuroblastomas and suggest possible therapeutic targets for further validation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5249.

ProPhenolOxidase in Daphnia magna: cDNA sequencing and expression in relation to resistance to pathogens

Invertebrates utilise the innate immune system when defending against pathogenic attack. However, except for some effectors as proPhenolOxidase (proPO), the innate immune response is less well understood outside model insect species, and its role in natural host–pathogen systems is generally not well documented. We have therefore initiated studies on the immune response of the crustacean Daphnia when exposed to the specialist endobacterial pathogen, Pasteuria ramosa. This study was focused on the proPO gene of Daphnia magna. D. magna possesses a single copy of proPO (as does its congener, D. pulex), but there was some evidence of alternative splicing. Analyses of sequence similarity in a range of arthropod taxa suggested that the proPO gene in Daphnia was as dissimilar to other crustaceans as it was to insects, while analysis on intraspecific variation indicated that the gene is highly conserved. ProPO was found to be significantly up-regulated within 1–4 h following exposure to the bacteria. This is the first evidence of a Daphnia immune response, and our observations raise the possibility that the PhenolOxidase (PO) cascade is involved in the defence against pathogenic gram-positive bacteria.

Analysis of Alternatively Spliced Rice Transcripts Using Microarray Data

Abstract Alternative splicing creates a diversity of gene products in higher eukaryotes. Twenty-five percent (1,583/6,371) of predicted alternatively spliced transcripts can be detected using the NSF45K rice whole-genome oligonucleotide array. We used the NSF45K array to assess differential expression patterns of 507 loci showing at least a twofold change in expression between light- and dark-grown seedlings. At least 42% of these loci show evidence of alternative splicing in aerial seedling tissue of Oryza sativa ssp. japonica cv. Nipponbare. Most alternative splice forms display the same pattern of regulation as the primary, or most highly expressed, transcript; however, splice forms for ten loci, represented by 35 oligos, display opposite expression patterns in the light vs. dark. We found similar evidence of alternative splicing events in Affymetrix microarray data for Nipponbare rice treated with the causative agent of fungal rice blast, Magnaporthe grisea. This strategy for analyzing alternative splicing in microarray data will enable delineation of the diversity of splicing in rice.

Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

BackgroundNucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes.ResultsWe analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied.ConclusionTranscripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.

Expression pattern of the AHP gene family from Arabidopsis thaliana and organ specific alternative splicing in the AHP5 gene

Histidine-containing phosphotransmitters (AHPs) transfer a phosphoryl group from membrane receptors to effectors in the nucleus. Five AHP genes have been identified in Arabidopsis. Real-time reverse transcription polymerase chain reaction (RT-PCR) was employed to quantify contents of individual transcripts in Arabidopsis leaves, roots, stems, flowers and siliques. High organ specificity of gene expression was found in AHP1, AHP2, AHP4 while expression of AHP3 and AHP5 appears more ubiquitous. We detected two AHP5 specific PCR products (AHP5 and AHP5L). Out of the five AHP5 introns, the second one was retained unspliced in the longer product (AHP5L) while AHP5 corresponded to a completely spliced AHP5 mRNA. The ratio between alternately and completely spliced AHP5 mRNAs was highest in flowers and lowest in siliques and stems. No evidence for alternative splicing in the remaining AHP transcripts was found. Two open reading frames were identified in AHP5L. The conserved part of the phosphotransfer domain remains unaffected in the longer one as the translation initiation at an intron ATG would result in a polypeptide coded by exons 3 to 6 and a short extension encoded by the intron part of the alternative open reading frame. However, the isoelectric point would shift by about 3 units towards neutral.

TassDB: a database of alternative tandem splice sites

Subtle alternative splice events at tandem splice sites are frequent in eukaryotes and substantially increase the complexity of transcriptomes and proteomes. We have developed a relational database, TassDB (TAndem Splice Site DataBase), which stores extensive data about alternative splice events at GYNGYN donors and NAGNAG acceptors. These splice events are of subtle nature since they mostly result in the insertion/deletion of a single amino acid or the substitution of one amino acid by two others. Currently, TassDB contains 114 554 tandem splice sites of eight species, 5209 of which have EST/mRNA evidence for alternative splicing. In addition, human SNPs that affect NAGNAG acceptors are annotated. The database provides a user-friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download large datasets. This database should facilitate further experimental studies and large-scale bioinformatics analyses of tandem splice sites. The database is available at .

Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways inChlamydomonas reinhardtii

A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing.