Kb Of Genomic Sequence Research Articles

Genome‐wide genetic diversity of ‘Nici’, the DNA source for the CHORI‐260 turkey BAC library and candidate for whole genome sequencing

Vertebrate whole genome sequence assembly can benefit from a priori knowledge of variability in the target genome, with researchers often selecting highly inbred individuals for sequencing. However, for most species highly inbred research lines are lacking, requiring the use of an outbred individual(s). Here we examined the source DNA [Nicholas inbred (Nici)] of the CHORI-260 turkey bacterial artificial chromosome (BAC) library through analysis of microsatellites and BAC sequences. Heterozygosity of Nici was compared with that of individuals from several breeder lines. Seventy-eight microsatellites were screened for polymorphism in a total of 43 birds, identifying an average individual heterozygosity of 0.39, with Nici at 0.35. Additional loci (total of 147) were examined on a subset of individuals to obtain better genome coverage. The mean heterozygosity for this subset was 0.33 with Nici at 0.31. Examination of approximately 200 kb of genome sequence identified SNPs in the order of one per 200 bp in Nici. These data suggest that the heterozygosity of Nici is comparable to other birds of selected breeder lines and that whole genome sequencing would result in an abundant resource of genome-wide polymorphisms.

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

BackgroundCampylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes.ResultsEighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp.ConclusionThe comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus.

EBV LMP-2A employs a novel mechanism to transactivate the HERV-K18 superantigen through its ITAM

EBV encodes latent membrane protein (LMP)-2A that functions as a homologue of the activated BCR. We have previously shown that LMP-2A transactivates a human endogenous retrovirus, HERV-K18, in infected B-lymphocytes. The Env protein of HERV-K18 encodes a superantigen that strongly stimulates a large number of T cells. To delineate the mechanism through which LMP-2A transactivates HERV-K18 env, we tested a panel of tyrosine mutants of LMP-2A in a murine B lymphoma that stably harbors HERV-K18. Our analysis revealed that the immunoreceptor tyrosine-based activation motif (ITAM) of LMP-2A is important for HERV-K18 env transactivation. ITAM contains 2 tyrosines that initiate signaling cascades when both residues are phosphorylated. However, in our study, single-tyrosine mutants of ITAM still retained full induction of HERV-K18 env. After truncating 25 kb of genomic sequence downstream of HERV-K18, LMP-2A failed to transactivate HERV-K18 env. Thus, an LMP-2A-inducible element is located downstream of HERV-K18.

Genotyping of Bacillus cereus Strains by Microarray-Based Resequencing

The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a “one reaction” genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.

TRAIL in the mandarin fish Siniperca chuatsi: Gene and its apoptotic effect in HeLa cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the Cys 231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPalp, NF-κB and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis.

Alu Recombination-Mediated Structural Deletions in the Chimpanzee Genome

With more than 1.2 million copies, Alu elements are one of the most important sources of structural variation in primate genomes. Here, we compare the chimpanzee and human genomes to determine the extent of Alu recombination-mediated deletion (ARMD) in the chimpanzee genome since the divergence of the chimpanzee and human lineages (∼6 million y ago). Combining computational data analysis and experimental verification, we have identified 663 chimpanzee lineage-specific deletions (involving a total of ∼771 kb of genomic sequence) attributable to this process. The ARMD events essentially counteract the genomic expansion caused by chimpanzee-specific Alu inserts. The RefSeq databases indicate that 13 exons in six genes, annotated as either demonstrably or putatively functional in the human genome, and 299 intronic regions have been deleted through ARMDs in the chimpanzee lineage. Therefore, our data suggest that this process may contribute to the genomic and phenotypic diversity between chimpanzees and humans. In addition, we found four independent ARMD events at orthologous loci in the gorilla or orangutan genomes. This suggests that human orthologs of loci at which ARMD events have already occurred in other nonhuman primate genomes may be “at-risk” motifs for future deletions, which may subsequently contribute to human lineage-specific genetic rearrangements and disorders.

Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI-1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response.

Identification and characterization of bovine regulator of telomere length elongation helicase gene (RTEL): molecular cloning, expression distribution, splice variants and DNA methylation profile

BackgroundThe genetic basis of telomere length heterogeneity among mammalian species is still not well understood. Recently, a gene named regulator of telomere length elongation helicase (RTEL) was identified and predicted to be an essential participant in species-specific telomere length regulation in two murine species. To obtain broader insights into its structure and biological functions and to ascertain whether RTEL is also a candidate gene in the regulation of telomere length diversity in other mammalian species, data from other mammals may be helpful.ResultsHere we report the cDNA cloning, genomic structure, chromosomal location, alternative splicing pattern, expression distribution and DNA methylation profile of the bovine homolog of RTEL. The longest transcript of bovine RTEL is 4440 nt, encompassing 24.8 kb of genomic sequence that was mapped to chromosome 13q2.2. It encodes a conserved helicase-like protein containing seven characterized helicase motifs in the first 750 aa and a PIP box in the C-terminus. Four splice variants were identified within the transcripts in both the coding and 5'-untranslated regions; Western blot revealed that the most abundant splice variant SV-1 was translated to a truncated isoform of RTEL. The different 5'UTRs imply alternative transcription start sites in the promoter; Bovine RTEL was transcribed at the blastocyst stage, and expression levels were highest in adult testis, liver and ovary. DNA methylation analysis of tissues that differed significantly in expression level indicated that relatively low DNA methylation is associated with higher expression.ConclusionIn this study, we have identified and characterized a bovine RTEL homolog and obtained basic information about it, including gene structure, expression distribution, splice variants and profile of DNA methylation around two putative transcription start sites. These data may be helpful for further comparative and functional analysis of RTEL in mammals.

Comparative genomics of xenobiotic metabolism: a porcine-human PXR gene comparison

The pregnane X receptor (PXR) plays a crucial role in xenobiotic and drug metabolism, being the major transcriptional regulator of cytochrome P-450 monooxygenase 3A4, which metabolizes more than 50% of all clinically used drugs. Recent pharmacodynamic studies have shown that the mouse is not an ideal model for predicting human clinical drug study outcomes. Therefore, we characterized the porcine PXR (pPXR) gene to evaluate the utility of the pig as an alternate preclinical animal model. The complete sequence of pPXR mRNA and 11 kb of genomic sequence were obtained. Similar to the human PXR gene, the pPXR gene revealed multiple splice variants in the ligand-binding domain. All pPXR splice variants (SV) were porcine-specific. The pPXR mRNAs varied in 3'-UTR length due to differential termination and specific deletions. Northern blot analyses identified high levels of pPXR mRNA expression in the liver, small intestine, heart, kidney, and colon. RT-PCR amplification detected lower levels of pPXR expression in multiple tissues. Ninety-three pigs representing eight breeds were analyzed for single nucleotide polymorphisms (SNPs). Only one nonsynonymous SNP (S178L) was found in the pPXR ligand-binding domain. This characterization of the pPXR gene contributes to the development of a porcine model for human drug metabolic studies.

Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks

The Drosophila element Mos1 is a class II transposon, which moves by a 'cut-and-paste' mechanism and can be experimentally mobilized in the Caenorhabditis elegans germ line. Here, we triggered the excision of identified Mos1 insertions to create chromosomal breaks at given sites and further manipulate the broken loci. Double-strand break (DSB) repair could be achieved by gene conversion using a transgene containing sequences homologous to the broken chromosomal region as a repair template. Consequently, mutations engineered in the transgene could be copied to a specific locus at high frequency. This pathway was further characterized to develop an efficient tool--called MosTIC--to manipulate the C. elegans genome. Analysis of DSB repair during MosTIC experiments demonstrated that DSBs could also be sealed by end-joining in the germ line, independently from the evolutionarily conserved Ku80 and ligase IV factors. In conjunction with a publicly available Mos1 insertion library currently being generated, MosTIC will provide a general tool to customize the C. elegans genome.

Emergence of primate genes by retrotransposon-mediated sequence transduction.

Gene duplication is one of the most important mechanisms for creating new genes and generating genomic novelty. Retrotransposon-mediated sequence transduction (i.e., the process by which a retrotransposon carries flanking sequence during its mobilization) has been proposed as a gene duplication mechanism. L1 exon shuffling potential has been reported in cell culture assays, and two potential L1-mediated exon shuffling events have been identified in the genome. SVA is the youngest retrotransposon family in primates and is capable of 3' flanking sequence transduction during retrotransposition. In this study, we examined all of the full-length SVA elements in the human genome to assess the frequency and impact of SVA-mediated 3' sequence transduction. Our results showed that approximately 53 kb of genomic sequences have been duplicated by 143 different SVA-mediated transduction events. In particular, we identified one group of SVA elements that duplicated the entire AMAC gene three times in the human genome through SVA-mediated transduction events, which happened before the divergence of humans and African great apes. In addition to the original AMAC gene, the three transduced AMAC copies contain intact ORFs in the human genome, and at least two are actively transcribed in different human tissues. The duplication of entire genes and the creation of previously undescribed gene families through retrotransposon-mediated sequence transduction represent an important mechanism by which mobile elements impact their host genomes.

Intelectin gene from the grass carp Ctenopharyngodon idella: cDNA cloning, tissue expression, and immunohistochemical localization

The cDNA encoding grass carp intelectin was isolated from a head kidney cDNA library, and termed gcIntL. The deduced amino acid sequence of gcIntL consists of 318 amino acids, and about 55% identical and 74% similar to human intelectin, which is a new type of lectin recognizing galactofuranose, and plays a role in the recognition of bacteria-specific components in animal hosts. The gcIntL gene consists of seven exons and six introns, spacing over approximately 3 kb of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcIntL formed a clade with Danio rerio intelectin and 35 kDa serum lectin. By real-time quantitative RT-PCR analysis, gcIntL transcripts were significantly induced in head kidney, trunk kidney, spleen, and intestine from LPS-stimulated fish. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcIntL gene have the same expression pattern, and both were detected in brain, gill, intestine, head kidney, trunk kidney, spleen, and heart. Furthermore, gcIntL protein could be detected in gill, intestine, trunk kidney, head kidney, spleen, heart, and brain including medulla oblongata and optic lobe, as determined by immunohistochemistry. This is the first report of intelectin expression pattern in fish, and of recombinant gcIntL and polyclonal antibody against gcIntL.

The first non-mammalian CXCR3 in a teleost fish: Gene and expression in blood cells and central nervous system in the grass carp ( Ctenopharyngodon idella)

A novel fish chemokine receptor gene, chemokine (C–X–C motif) receptor 3 (CXCR3)-like was isolated from the grass carp Ctenopharyngodon idella, with its full-length genomic sequence. The cDNA of grass carp CXCR3-like (gcCXCR3-like) consists of 1261 bp with a 49 bp 5′-UTR and a 189 bp 3′-UTR. An open reading frame of 1023 bp encodes a 341-amino acid peptide, with seven transmembrane helices. The deduced amino acid sequence showed the same sequence identities (37.8%) with its counterparts in goat and human. The gcCXCR3-like gene consists of two exons, with one intervening intron, spaced over approximately 2 kb of genomic sequence. Phylogenetic analyses clearly demonstrated that the gcCXCR3-like resembles the CXCR3s of other vertebrates. Real-time PCR analysis showed that gcCXCR3-like was expressed in all tested organs except heart and the expression level of gcCXCR3-like was highest in brain. Flow cytometric analyses showed the positive rate of labelled leukocytes from the healthy grass carp was 17.3%, and the labelled leukocytes were divided into three types by cell sorting. Immunohistochemical localization revealed that gcCXCR3-like expressed in whole brain regions including cerebel, diencephalon, medulla oblongata, optic lobe, and rhinencephalon, and that the labelled leukocytes are actually populations of monocyte and/or phagocyte, lymphocyte and the granulocyte. It is considered that fish CXCR expression and their function may need to be investigated in both nervous and immune systems.

Irinogenetics: What Is the Right Star?

Irinogenetics: What Is the Right Star?

Characterization of the canine CLCN3 gene and evaluation as candidate for late-onset NCL

BackgroundThe neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of inherited progressive neurodegenerative diseases in different mammalian species. Tibetan Terrier and Polish Owczarek Nizinny (PON) dogs show rare late-onset NCL variants with autosomal recessive inheritance, which can not be explained by mutations of known human NCL genes. These dog breeds represent animal models for human late-onset NCL. In mice the chloride channel 3 gene (Clcn3) encoding an intracellular chloride channel was described to cause a phenotype similar to NCL.ResultsTwo full-length cDNA splice variants of the canine CLCN3 gene are reported. The current canine whole genome sequence assembly was used for gene structure analyses and revealed 13 coding CLCN3 exons in 52 kb of genomic sequence. Sequence analysis of the coding exons and flanking intron regions of CLCN3 using six NCL-affected Tibetan terrier dogs and an NCL-affected Polish Owczarek Nizinny (PON) dog, as well as eight healthy Tibetan terrier dogs revealed 13 SNPs. No consistent CLCN3 haplotype was associated with NCL.ConclusionFor the examined animals we excluded the complete coding region and adjacent intronic regions of canine CLCN3 to harbor disease-causing mutations. Therefore it seems to be unlikely that a mutation in this gene is responsible for the late-onset NCL phenotype in these two dog breeds.

Isolation and Characterization of cDNA Encoding Three Dehydrins Expressed During Coffea canephora (Robusta) Grain Development

Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized. The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT-PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2). The CcDH1 and CcDH2 genes encode Y(3)SK(2) dehydrins and the CcDH3 gene encodes an SK(3) dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized. cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.

Identification of the microdeletion breakpoint in aGLRA1null allele of Turkish hyperekplexia patients

Hyperekplexia (startle disease) is a hereditary motor disease caused by mutations within the GLRA1 gene (Chr. 5q33.1), which encodes the alpha1 subunit of the inhibitory glycine receptor (GlyR). While most patients are diagnosed with dominant hyperekplexia associated with point mutations within or adjacent to the channel pore, recessive hyperekplexia is less frequent. Here, we report five new pedigrees of recessive hyperekplexia in apparently unrelated families of Kurdish origin associated with a deletion of exons 1-7 of the GLRA1 gene. The deletion was identical in all families, encompassing 329 Kb of genomic sequence. No other known functional genes were involved, indicating that the GLRA1null allele is distinct from the 5q syndrome. Analysis of the DNA sequence flanking the proximal and distal breakpoint revealed no significant homology of sequences immediately adjacent to the breaks. Consensus sites for Toposiomerase II were detected close to the breakpoint compatible with an illegitimate recombination event. No heterozygous carriers of the deletion allele were detected by screening of 500 individuals from the southeastern Mediterranean region belonging to four different ethnic groups. Hence, the identical nature of the breakpoint junction in all patients and carriers suggests a founder mutation in an ethnic population originating from Turkey.

Characterization of 23 CC chemokine genes and analysis of their expression in channel catfish ( Ictalurus punctatus)

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. CC chemokines constitute the largest subfamily of chemokines, with 28 CC chemokines identified from mammalian species. However, the status of CC chemokines in teleosts is yet to be determined. We previously identified 26 catfish CC chemokine cDNAs from catfish. In this study, we isolated and sequenced 23 channel catfish CC chemokine genes amounting to a total of over 56 kb of genomic sequences. Genomic organization of the 23 CC chemokine genes was determined by comparing the generated genomic sequences with the previously identified cDNA sequences. Microsatellites were identified from 16 catfish CC chemokine genes allowing them to be utilized for genome mapping. Structural analysis indicated conservation of genomic organization of CC chemokine genes, which may facilitate the establishment of orthologies. Expression of all known catfish CC chemokine transcripts was assessed in nine important tissues. Of the 26 catfish CC chemokine genes, 14 were universally expressed, six were widely expressed in many tissues, while six were highly tissue-specific.

Polymorphism discovery in 51 chemotherapy pathway genes

Candidate gene pharmacogenetic studies offer a strategy for the rapid assessment of putative predictive markers. As a first step toward studying the pharmacogenetics of cancer chemotherapy, 51 candidate genes from the pathways of antineoplastic agents were resequenced to identify common genetic polymorphisms that might alter therapeutic response or toxicity. Forty DNA samples were screened from each of three population groups: African-Americans, Asian-Americans and European-Americans. Nearly 378 kb of genomic sequence was obtained from each sample. Nine hundred and four variants were identified, including 139 coding single nucleotide polymorphisms (cSNPs). Three hundred and fifty-six (40%) polymorphisms were common to all three populations and 366 (41%) were population specific. Three hundred and forty-six (38%) variants were novel polymorphisms that were not present in the three public databases that were examined. One hundred and eleven (35%) of the 319 non-synonymous cSNPs that were identified by either resequencing or database mining were predicted by PolyPhen to be either possibly or probably damaging. For the non-synonymous cSNPs identified by resequencing, both the number of cSNPs found and the maximum estimated allele frequency decreased with increasing predicted severity. These results provide experimental validation and estimated allele frequencies for polymorphisms in three common ethnic groups and facilitate applied pharmacogenetic studies of anticancer drugs.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrane protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAIL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRF1 sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish ( Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen.