EST Analysis Research Articles

Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP9) gene in the reproduction seasons of Chinese mitten crab, Eriocheir sinensis

Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP9) was cloned based upon EST analysis of a testis cDNA library. The full length cDNA was 898 bp and encoded a 136 aa polypeptide that was highly homologous to related genes reported in shrimp. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP9 transcripts was widely distributed with high and detectable expression levels observed in intestine, ovary, testis and heart, while expression were comparable among hepatopancreas, hemolymphe, gills, muscle, stomach and brain. Real-time quantitative RT-PCR analysis revealed that Es-FABP9 expression in testis, hemolymphe, hepatopancreas and ovarian was dependent on the status of testis development. Evidence provided in the present report supports a role of Es-FABP9 in lipid transport during the period of rapid testis growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, testis, hemolymphe and ovarian in lipid nutrient absorption and utilization processes.

Cloning and functional characterization of the p65 subunit of NF-κB from olive flounder ( Paralichthys olivaceus)

NF-κB is a master transcription factor found in almost all cell types that responds to diverse cellular stimuli by activating the expression of stress response genes, including immune-related genes. cDNA encoding the p65 subunit of olive flounder ( Paralichthys olivaceus) NF-κB (Po-p65) was isolated through an EST analysis of an olive flounder cDNA library, a screen of BAC library, and rapid amplification of cDNA ends (RACE). The cDNA for Po-p65 encodes a polypeptide 626 amino acids in length containing a well-conserved Rel-homology domain (RHD). The primary sequence of Po-p65 showed strong homology with p65 from perch and zebrafish (82.7 and 64.4%, respectively), and shared 43.4–42.1% homology with p65 from other species, including mammals, while the N-terminal RHD of Po-p65 showed strong identity (95.6–67.8%) with that of other species. Po-p65 mRNA expression was detected in all flounder tissues examined. The over-expression of full-length Po-p65 (Po-p65f), but not of a Po-p65 C-terminus deletion mutant (Po-p65ΔC), stimulated κB element-driven reporter (κB-luc) activity in a dose-dependent manner and regulated the expression of p65 target genes, including TNF-α and IκB-α, in HINAE olive flounder cells. Po-p65f translocated to the nucleus following stimulation with poly I:C in HINAE cells. Together, these results suggest that Po-p65 is evolutionarily and functionally conserved in flounder and mammals and may provide clues to the detailed molecular mechanism(s) underlying immune response regulation in flounder.

Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak.

BackgroundThe Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.ResultsWe generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html.ConclusionsThis genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.

Alternation of Venerupis philippinarum Hsp40 gene expression in response to pathogen challenge and heavy metal exposure

HSP40 was an understudied protein family with co-chaperone activity. In the present study, a HSP40 homology was cloned from Venerupis philippinarum haemocytes (designated as VpHSP40) by EST analysis and RACE approaches. The expression profiles of VpHSP40 under Vibrio anguillarum challenge and heavy metal exposure were investigated by quantitative real-time RT-PCR. The bacterial challenge could significantly up-regulate the mRNA expression, and the highest expression level was detected at 24 h post-infection with 6.0-fold increase compared with that in the control group. During heavy metal exposure experiment, the expression of VpHSP40 could also be induced by Cu 2+ and Cd 2+ at different concentration, where Cu 2+ displayed more toxic effect on clam than that of Cd 2+. Concerning the same heavy metal, varied effect on VpHSP40 expression was detected at different concentration of heavy metal. Taking together, these results suggested that VpHSP40 was perhaps involved in mediating the immune responses and environmental stresses in V. philippinarum.

Identification and Expression Analysis of Sex-Specific Expression Markers of Thai AbaloneHaliotis asinina, Linneaus, 1758

ABSTRACT Expressed sequence tag analysis of normal and subtractive complementary DNA (cDNA) libraries of Thai abalone (Haliotis asinina Linneaus) was carried out. Several reproduction-related transcripts—for example, vitelline envelope zona pellucida domain 2 (Ha-VEZPD2), vitellogenin (Ha-VTG1), sperm lysin (Ha-SL), fertilization protein (Ha-FP), tektin A1 (Ha-TekA1), axonemal protein 66.0 (Ha-Axp66.0), and double-sex male abnormal-3-related transcription factor 1—(Ha-DMRT1)—were identified. The full-length cDNA of Ha-SL (665 bp containing an open reading frame (ORF) of 453 bp corresponding to a polypeptide of 150 amino acids) was obtained. In addition, that of Ha-VEZPD2.1 (1,094 bp with an ORF of 942 bp, 313 aa) and Ha-VEZPD2.7 (1,130 bp with an ORF of 1,002 bp, 333 aa) were also deduced after cluster analyses of ESTs encoding Ha-VEZPD2. Tissue distribution analysis indicated that Ha-Axp66.0 and Ha-DMRT1 were detected in testes but not in other tissues. Conversely, Ha-VEZPD2.1, Ha-VEZPD2.3, Ha-VEZPD2.17,...

Generation and Analysis of ESTs from the Grass Carp, Ctenopharyngodon idellus

Grass carp, Ctenopharyngodon idellus (Valenciennes, 1844), is an economically important species widely cultured in the world, but its genome research resources are largely lacking. The objectives of this study were to construct normalized cDNA libraries for efficient EST analysis, to generate ESTs from these libraries, and to identify EST-related molecular markers such as microsatellites and single nucleotide polymorphisms (SNPs) for genetic analysis of this species. A total of 6,269 ESTs were generated representing 4,815 unique sequences, from which 105 putative microsatellites and 5,228 SNPs were identified. These genome resources provide the material basis for future genetic and functional analyses in this species.

Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP) gene in female Chinese mitten crab (Eriocheir sinensis)

BackgroundFatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity.ResultsTherefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP) was cloned based upon EST analysis of a hepatopancreas cDNA library. The full length cDNA was 750 bp and encoded a 131 aa polypeptide that was highly homologous to related genes reported in shrimp. The 9108 bp Es-FABP gene contained four exons that were interrupted by three introns, a genomic organization common among FABP multigene family members in vertebrates. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP transcripts in hepatopancreas, hemocytes, ovary, gills, muscle, thoracic ganglia, heart, and intestine, but not stomach or eyestalk. Real-time quantitative RT-PCR analysis revealed that Es-FABP expression in ovary, hemocytes, and hepatopancreas was dependent on the status of ovarian development, with peak expression observed in January.ConclusionsEvidence provided in the present report supports a role of Es-FABP in lipid transport during the period of rapid ovarian growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, ovary, and hemocytes in lipid nutrient absorption and utilization processes.

Gene Expression in Wheat Induced by Inoculation with Puccinia striiformis West

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat (Triticum aestivum L.) worldwide. A suppression subtractive hybridization (SSH) cDNA library was constructed from Pst-infected seedling leaves of an immune resistant germplasm Shaanmai 139. A total of 84 expressed sequence tags with high quality were obtained and alignment of the sequences identified in 56 Unigenes. Blast EST analysis of these Pst-induced genes showed that they were mostly homologous to genes that are induced by cold or heat stress. BlastX searches identified 45 of the Unigenes as similar to those encoding proteins of known function (e value < 1.00E-5), and eight of unknown function. The genes of known function include those with potential biological roles in signal transmission, energy and metabolism, transcription regulation, phenylpropanoid pathway, and defense response. Semi-quantitative reverse transcription-polymerase chain reaction studies confirmed that some of the cDNAs genes were differentially expressed in Pst-infected plants, with 24 and 72 hpi as the two critical time points for the induction of these genes by inoculation with P. striiformis West race CY32 in wheat. Bimodal patterns were presented in Pst-induced genes, such as senescence-associated genes, omega-6 fatty acid desaturase, and acyl-CoA synthetase. This suggests complex patterns of defense-related gene expression in multi-resistance gene cultivars in response to stripe rust infection.

Identification of tumor-associated cassette exons in human cancer through EST-based computational prediction and experimental validation

BackgroundMany evidences report that alternative splicing, the mechanism which produces mRNAs and proteins with different structures and functions from the same gene, is altered in cancer cells. Thus, the identification and characterization of cancer-specific splice variants may give large impulse to the discovery of novel diagnostic and prognostic tumour biomarkers, as well as of new targets for more selective and effective therapies.ResultsWe present here a genome-wide analysis of the alternative splicing pattern of human genes through a computational analysis of normal and cancer-specific ESTs from seventeen anatomical groups, using data available in AspicDB, a database resource for the analysis of alternative splicing in human. By using a statistical methodology, normal and cancer-specific genes, splice sites and cassette exons were predicted in silico. The condition association of some of the novel normal/tumoral cassette exons was experimentally verified by RT-qPCR assays in the same anatomical system where they were predicted. Remarkably, the presence in vivo of the predicted alternative transcripts, specific for the nervous system, was confirmed in patients affected by glioblastoma.ConclusionThis study presents a novel computational methodology for the identification of tumor-associated transcript variants to be used as cancer molecular biomarkers, provides its experimental validation, and reports specific biomarkers for glioblastoma.

AmphiEST: Enabling comparative analysis of ESTs from five developmental stages of amphioxus

Amphioxus has been an important model for understanding the evolution of chordates and origin of vertebrates. Comparative transcriptome analysis can facilitate delineation of gene expression patterns of amphioxus at different developmental stages. So far, however, few such analyses have been performed. Here we have systematically compared amphioxus ESTs from five developmental stages. For the egg, gastrula, neurula, larva and adult stages, amphioxus ESTs were assembled, respectively, into 3364, 3230, 10,299, 4052 and 3866 contigs, and 193, 183, 933, 178 and 151 singlets. 25,796 ORFs were identified, of which 6,529 predicted ORFs (egg, 922; gastrula, 997; neurula, 2,159; larva, 1,014; adult, 1,437) have found matches from the UniProt database. For all five stages, more GO terms were assigned to stage-specific contigs than to repertoire ones. Respectively, 2, 5, 11, 4 and 2 miRNAs were matched with ESTs from the five stages, and 12,232, 10,147, 43,170, 8,049 and 12,811 miRNA target sites were predicted from the 3′ UTR sequences. A web interface was developed for visualization of the contigs, miRNAs and annotations ( http://www.rich.yunda.org/test/amphioxusest/). The constructed AmphiEST database may serve as an essential resource for future studies of the development and evolution of amphioxus.

Identification of immune genes of the miiuy croaker ( Miichthys miiuy) by sequencing and bioinformatic analysis of ESTs

Miiuy croaker ( Miichthys miiuy) is an economically important fish in China. However, genomic research on this species is still in its infancy, and genomic resources are largely unavailable. In order to isolate functional genes involved in immunity, a normalized cDNA library was constructed from the spleen of the miiuy croaker. A total of 5053 ESTs from the library were sequenced and compared with sequences in the GenBank database. The 4609 high-quality ESTs were assembled into 3221 unigenes. Based on sequence similarities, 193 immune genes were identified such as major histocompatibility complex, cytokines and cytokine receptors, adhesive proteins, stress proteins, transcription factors for immune response, immunoglobulin and coagulation factors. Our study thus provides both a detailed annotation of immune genes in miiuy croaker and a collection of novel transcripts of Fc receptor-like 5 in teleost for the first time.

Development of specific and degenerate primers for CesA genes encoding cellulose synthase in flax (Linum usitatissimum L.)

Representatives of the CesA multigene family that control the synthesis of the catalytic subunits of the cellulose synthase complex were described for a number of higher plants. It has been established that the HVR2 region of these genes is class-specific and determines the involvement of the gene product in the synthesis of either the primary or secondary cell wall. The purpose of the current research was to develop degenerate and specific primers for parts of the CesA genes to allow the construction of molecular markers for the class-specific HVR2 region. Two pairs of specific primers for the CesA-1 and CesA-6 genes as well as a pair of degenerate primers for the HVR2 region of all flax CesA genes were developed, based on analysis of the CesA ESTs as well as the full-length cDNA sequences of the CesA genes in Arabidopsis, poplar, maize, and cotton that are available in the GenBank. Fragments of the expected size were amplified using flax cDNA as a template (201 bp for CesA-1, 300 bp for CesA-6, and 600 bp for HVR2). The markers developed in this research can be used for CesA gene cloning and sequencing, analysis of gene copy numbers as well as characterization of tissue- and development specific gene expression.

Generation, annotation, and analysis of ESTs from hemocyte of the bloody clam, Tegillarca granosa

Tegillarca granosa, bloody clam are valuable resource and extensive culture bivalve in China. In contrast to other cultured mollusca, very few genomic resources are available in this species. In an effort to identify clam genes involved in immunity, growth and hemoglobins, a normalized full-length cDNA library were constructed from hemocytes of the bloody clam. 2278 ESTs from 5′ ends of the cDNA clones representing 1501 unigenes with an average length of 459 bp were obtained from library. Cluster analysis of these ESTs identified 1501 unique sequences containing 173 contigs and 1328 singletons. Among the sequences that matched known genes, based on comparisons with the NCBI non-redundant database, the unigene were clustered into six categories, 142 (29.0%) were related to metabolism, 128 (26.3%) were related to cell structure/motility, 94 (19.2%) were related to gene/protein expression, 61 (12.5%) were related to cell signaling/communication, 50 (10.2%) were related to cell/organism immune, and 14 (2.8%) were related to cell division. All contigs and singlets were compared to the public protein database using BLASTx, and annotations and descriptions of interested genes related to immunity, growth or hemoglobin. These genome resources provide a significant number of ESTs for gene discovery and development of microarrays for gene expression.

Is a metabolic enzyme complex involved in the efficient and accurate control of Ipecac alkaloid biosynthesis in Psychotria ipecacuanha?

Ipecac alkaloids produced in the medicinal plant Psychotria ipecacuanha such as emetine and cephaeline possess a monoterpenoid-tetrahydroisoquinoline skeleton, which is formed by condensation of dopamine and secologanin. The condensation products are deglucosylated, and the resulting aglycon is further processed to protoemetine, which is condensed with the second molecule of dopamine, followed by conversion into cephaeline and emetine. Although four hydroxy groups derived from two molecules of dopamine need to be methylated to form emetine, the order of O-methylation reactions had been veiled. We recently identified three Ipecac alkaloid O-methyltransferases (IpeOMT1-IpeOMT3) that are sufficient for catalyzing O-methylations of all four hydroxy groups. Detailed characterization of their catalytic properties with integration of that of the previously identified Ipecac alkaloid β-glucosidase (IpeGlu1) revealed a large portion of the biosynthetic pathway of Ipecac alkaloids. The results provide proof-of-concept to the significance and the usefulness of the biosynthetic pathway strategy by EST analysis coupled with recombinant enzyme characterization. At the same time, however, the results raised an intriguing question about the subcellular network between the biosynthetic enzymes and intermediates. Here, we provide additional discussion about this point, and indicate what remains to be elucidated.

Molecular cloning and expression of progestin membrane receptor component 1 ( Pgmrc1) of the giant tiger shrimp Penaeus monodon

Knowledge on molecular mechanisms of steroid hormonal induction on oocyte development may lead to the possible ways to effectively induce ovarian maturation in shrimp. In this study, progestin membrane receptor component 1 ( Pgmrc1) of the giant tiger shrimp ( Penaeus monodon) initially identified by EST analysis was further characterized. The full-length cDNA of Pgmrc1 was 2015 bp in length containing an ORF of 573 bp corresponding to a polypeptide of 190 amino acids. Northern blot analysis revealed a single form of Pgmrc1 in ovaries of P. monodon. Quantitative real-time PCR indicated that the expression level of Pgmrc1 mRNA in ovaries of both intact and eyestalk-ablated broodstock was greater than that of juveniles ( P < 0.05). Pgmrc1 was up-regulated in mature (stage IV) ovaries of intact broodstock ( P < 0.05). Unilateral eyestalk ablation resulted in an earlier up-regulation of Pgmrc1 since the vitellogenic (II) ovarian stage. Moreover, the expression level of Pgmrc1 in vitellogenic, early cortical rod and mature (II–IV) ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock ( P < 0.05). Pgmrc1 mRNA was clearly localized in the cytoplasm of follicular cells, previtellogenic and early vitellogenic oocytes. Immunohistochemistry revealed the positive signals of the Pgmrc1 protein in the follicular layers and cell membrane of follicular cells and various stages of oocytes. Taken the information together, Pgmrc1 gene products seem to play the important role on ovarian development and may be used as the bioindicator for monitoring progression of oocyte maturation of P. monodon.

Prevalence of alternative splicing choices in Arabidopsis thaliana

BackgroundAround 14% of protein-coding genes of Arabidopsis thaliana genes from the TAIR9 genome release are annotated as producing multiple transcript variants through alternative splicing. However, for most alternatively spliced genes in Arabidopsis, the relative expression level of individual splicing variants is unknown.ResultsWe investigated prevalence of alternative splicing (AS) events in Arabidopsis thaliana using ESTs. We found that for most AS events with ample EST coverage, the majority of overlapping ESTs strongly supported one major splicing choice, with less than 10% of ESTs supporting the minor form. Analysis of ESTs also revealed a small but noteworthy subset of genes for which alternative choices appeared with about equal prevalence, suggesting that for these genes the variant splicing forms co-occur in the same cell types. Of the AS events in which both forms were about equally prevalent, more than 80% affected untranslated regions or involved small changes to the encoded protein sequence.ConclusionsCurrently available evidence from ESTs indicates that alternative splicing in Arabidopsis occurs and affects many genes, but for most genes with documented alternative splicing, one AS choice predominates. To aid investigation of the role AS may play in modulating function of Arabidopsis genes, we provide an on-line resource (ArabiTag) that supports searching AS events by gene, by EST library keyword search, and by relative prevalence of minor and major forms.

Sequencing, Analysis, and Annotation of Expressed Sequence Tags for Camelus dromedarius

Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF) analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and ∼40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (http://camel.kacst.edu.sa/), hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism.

Expression of the 26S proteasome subunit RPN10 is upregulated by salt stress in Dunaliella viridis

Green algae of the genus Dunaliella can adapt to hypersaline environments and are considered model organisms for salinity tolerance. In an EST analysis in Dunaliella viridis under salt stress, we isolated a salt-inducible cDNA coding for the 26S proteasome subunit RPN10, designated DvRPN10. The DvRPN10 cDNA is 1472 bp and encodes a polypeptide of 377 amino acids. The DvRPN10 protein shares a high similarity to orthologs from other species. The function of DvRPN10 was confirmed by complementation of the yeast Deltarpn10 mutant. Q-PCR analysis of D. viridis cells grown in different salinities revealed that the transcript level of DvRPN10 increased in proportion to the external salinity within a range of 0.5-3 M NaCl, but decreased significantly at extremely high salinities (4-5 M NaCl). When a salinity shock of 1-3 M NaCl was applied to D. viridis cells, DvRPN10 mRNA levels remained steady during the first 36 h, and then gradually elevated to the level observed at 3 M NaCl. The gene structure of DvRPN10 was revealed by sequencing of a BAC clone containing this gene. Possible transcription factor binding sites related to stress tolerance were found in the promoter region of DvRPN10. The expression of DvRPN10 in response to the external salinity suggests that RPN10-mediated protein degradation plays a role in the salinity tolerance of D. viridis.

Promoter polymorphism -119C/G in MYG1 (C12orf10) gene is related to vitiligo susceptibility and Arg4Gln affects mitochondrial entrance of Myg1

BackgroundMYG1 (Melanocyte proliferating gene 1, also C12orf10 in human) is a ubiquitous nucleo-mitochondrial protein, involved in early developmental processes and in adult stress/illness conditions. We recently showed that MYG1 mRNA expression is elevated in the skin of vitiligo patients. Our aim was to examine nine known polymorphisms in the MYG1 gene, to investigate their functionality, and to study their association with vitiligo susceptibility.MethodsNine single nucleotide polymorphisms (SNPs) in the MYG1 locus were investigated by SNPlex assay and/or sequencing in vitiligo patients (n = 124) and controls (n = 325). MYG1 expression in skin biopsies was detected by quantitative-real time PCR (Q-RT-PCR) and polymorphisms were further analysed using luciferase and YFP reporters in the cell culture.ResultsControl subjects with -119G promoter allele (rs1465073) exhibited significantly higher MYG1 mRNA levels than controls with -119C allele (P = 0.01). Higher activity of -119G promoter was confirmed by luciferase assay. Single marker association analysis showed that the -119G allele was more frequent in vitiligo patients (47.1%) compared to controls (39.3%, P < 0.05, OR 1.37, 95%CI 1.02-1.85). Analysis based on the stage of progression of the vitiligo revealed that the increased frequency of -119G allele occurred prevalently in the group of patients with active vitiligo (n = 86) compared to the control group (48.2% versus 39.3%, P < 0.05; OR 1.44, 95%CI 1.02-2.03). Additionally, we showed that glutamine in the fourth position (in Arg4Gln polymorphism) completely eliminated mitochondrial entrance of YFP-tagged Myg1 protein in cell culture. The analysis of available EST, cDNA and genomic DNA sequences revealed that Myg1 4Gln allele is remarkably present in human populations but is never detected in homozygous state according to the HapMap database.ConclusionsOur study demonstrated that both MYG1 promoter polymorphism -119C/G and Arg4Gln polymorphism in the mitochondrial signal of Myg1 have a functional impact on the regulation of the MYG1 gene and promoter polymorphism (-119C/G) is related with suspectibility for actively progressing vitiligo.

Combined EST and Proteomic Analysis Identifies Rapidly Evolving Seminal Fluid Proteins in Heliconius Butterflies

Seminal fluid proteins (SFPs) directly influence a wide range of reproductive processes, including fertilization, sperm storage, egg production, and immune response. Like many other reproductive proteins, the molecular evolution of SFPs is generally characterized by rapid and frequently adaptive evolution. However, the evolutionary processes underlying this often-documented pattern have not yet been confidently determined. A robust understanding of the processes governing SFP evolution will ultimately require identifying SFPs and characterizing their evolution in many different taxa, often where only limited genomic resources are available. Here, we report the first comprehensive molecular genetic and evolutionary analysis of SFPs conducted in Lepidoptera (moths and butterflies). We have identified 51 novel SFPs from two species of Heliconius butterflies (Heliconius erato and Heliconius melpomene) by combining "indirect" bioinformatic and expression analyses of expressed sequence tags from male accessory gland and wing tissues with "direct" proteomic analyses of spermatophores. Proteomic analyses identified fewer SFPs than the indirect criteria but gave consistent results. Of 51 SFPs, 40 were identified in both species but fewer than half could be functionally annotated via similarity searches (Blast, IPRscan, etc.). The majority of annotated Heliconius SFPs were predicted to be chymotrypsins. Comparisons of Heliconius SFPs with those from fruit fly, mosquito, honeybee, and cricket suggest that gene turnover is high among these proteins and that SFPs are rarely conserved across insect orders. Pairwise estimates of evolutionary rates between SFPs and nonreproductive proteins show that, on average, Heliconius SFPs are evolving rapidly. At least one of these SFPs is evolving adaptively (dN/dS > 1), implicating a role for positive selection in this rapid evolution. This work establishes a strong precedent for future research on the causes and consequences of reproductive protein evolution in the Lepidoptera. Butterflies and moths have an extremely rich history of organismal research, which will provide an informative ecological context for further molecular evolutionary investigations.