Kb Of Genomic Sequence Research Articles

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies

Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total. Rapid heme panel's average coverage is 1500× with <5% of the amplicons with <50× coverage, and it reproducibly detects single nucleotide variants and small insertions/deletions at allele frequencies of ≥5%. Comparison with a capture-based next-generation sequencing assay showed that there is >95% concordance among a wide array of variants across a range of allele frequencies. Read count analyses that used rapid heme panel showed high concordance with karyotypic results when tumor content was >30%. The average turnaround time was 7 days over a 6-month span with an average volume of ≥40 specimens per week and a low sample fail rate (<1%), demonstrating its suitability for clinical application.

The draft genome sequence of Xanthomonas species strain Nyagatare, isolated from diseased bean in Rwanda.

We announce the genome sequence for Xanthomonas species strain Nyagatare, isolated from beans showing unusual disease symptoms in Rwanda. This strain represents the first sequenced genome belonging to an as-yet undescribed Xanthomonas species known as species-level clade 1. It has at least 100 kb of genomic sequence that shows little or no sequence similarity to other xanthomonads, including a unique lipopolysaccharide synthesis gene cluster. At least one genomic region appears to have been acquired from relatives of Agrobacterium or Rhizobium species. The genome encodes homologues of only three known type-three secretion system effectors: AvrBs2, XopF1 and AvrXv4. Availability of the genome sequence will facilitate development of molecular tools for detection and diagnostics for this newly discovered pathogen of beans and facilitate epidemiological investigations of a potential causal link between this pathogen and the disease outbreak.

Generation of BAC reporter cell lines for drug discovery.

Bacterial artificial chromosome (BAC) reporter cell lines are generated through stable transfection of a BAC reporter construct wherein the gene of interest is tagged with a reporter gene such as eGFP. The large capacity of BACs (up to 350 kb of genomic sequence) enables the inclusion of all regulatory elements that ensure appropriate regulation of the gene of interest. Furthermore, the reporter gene allows the expression of the gene of interest to be readily detected by flow cytometry. Cell lines can also be easily cultured for extended periods with minimal cost. These features of BAC reporter cell lines make them highly amenable for use in high-throughput screening of large drug libraries for compounds that induce the expression of the gene of interest. This chapter describes a method for generation of BAC reporter cell lines that are suitable as cellular assay systems in high-throughput screening. Briefly, this method involves (A) generation of cell clones stably transfected with a BAC reporter construct, (B) selection of "candidate" cell clones based on the responsiveness to known inducers, (C) confirmation of the integrity of the BAC reporter construct integrated within the candidate clones, and (D) assessment of the developmental regulation of the BAC reporter construct. As an example, we describe the generation of a BAC reporter cell line containing the human β-globin locus modified to express γ-globin as eGFP for use as a cellular reporter assay for screening of drugs that can reactivate expression of developmentally silenced γ-globin for the treatment of β-hemoglobin disorders.

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-κB and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-κB, as well as in regulating innate immune genes.

Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.

Generalized bacterial genome editing using mobile group II introns and Cre-lox.

Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns ('targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%.

Transgene-Mediated Rescue of Spermatogenesis in Cldn11-Null Mice1

Claudins comprise a large family of tight junction (TJ) proteins that are often expressed broadly during development and in adult tissues and constitute the physical barriers that occlude the paracellular space in polarized epithelia. In mouse testis, the integrity of TJs is critical to normal spermatogenesis and is dependent on CLDN11 expression. In the current study, we have generated multiple transgenic mouse lines in which steady-state levels of transgene-derived Cldn11 mRNA are up to fourfold greater than endogenous gene expression. Spermatogenesis in all founder mice harboring two copies of the endogenous Cldn11 gene is normal. These animals breed well, indicating that transgene overexpression, at least at the level of mRNA, is well tolerated by Sertoli cells. In addition, we demonstrate that the promoter/enhancer of the transgene, comprising 5 kb of genomic sequence upstream of exon 1 of the mouse Cldn11 gene, is sufficient to rescue azoospermia in Cldn11-null mice. Finally, using transient transgenic mice, we narrow the location of Sertoli cell-specific cis regulatory elements to a 2-kb region upstream of the Cldn11 transcription start site. Together, these data provide essential information for further investigation of the biological regulation of CLDN11 TJs in the testis.

Focus on Molecules: ADAMTSL4

The ADAMTSL4 gene (MIM:610113) is a member of the ADAMTSL (A Disintegrin AndMetalloproteinase with Thrombospondin Motifs Like) gene family. It is found on chromosome 1q21 and encodes the 1074 amino acid ADAMTS-Like 4 protein (NP_061905). ADAMTSL4 consists of 17 coding exons and two untranslated exons, which span 11.5 kb of genomic sequence. ADAMTS-Like 4 is one of the family of 7 ADAMTS-Like proteins, themselves part of the Thrombospondin type 1 repeat (TSR) superfamily of proteins. These 7 proteins are divided into two distinct clades, of which ADAMTS-Like 4 and ADAMTS-Like 6 form part of one clade, differing fromADAMTS-Like 1, ADAMTS-Like 3 and ADAMTS-Like 7 by lacking immunoglobulin repeat regions (Apte, 2009). The function and disease associations of these proteins are as yet unknown, and thus comparisons between structure and function remain challenging. ADAMTS proteins comprise a protease domain and an ancillary domain, the latter of which determines substrate recognition and function in tissue specificity. ADAMTS-Like proteins are structurally similar to the ADAMTS ancillary domain, including at least one TSR module. Although the TSR repeats are very closely related, ADAMTS-Like proteins lack the proteolytic domains found in ADAMTS proteins. This homology with the ancillary domain may suggest an inhibitory or enhancer relationship between the ADAMTS-Like and ADAMTS proteins. Alternatively this relationship

Genetic variation and linkage disequilibrium in Bacillus anthracis

We performed whole-genome amplification followed by hybridization of custom-designed resequencing arrays to resequence 303 kb of genomic sequence from a worldwide panel of 39 Bacillus anthracis strains. We used an efficient algorithm contained within a custom software program, UniqueMER, to identify and mask repetitive sequences on the resequencing array to reduce false-positive identification of genetic variation, which can arise from cross-hybridization. We discovered a total of 240 single nucleotide variants (SNVs) and showed that B. anthracis strains have an average of 2.25 differences per 10,000 bases in the region we resequenced. Common SNVs in this region are found to be in complete linkage disequilibrium. These patterns of variation suggest there has been little if any historical recombination among B. anthracis strains since the origin of the pathogen. This pattern of common genetic variation suggests a framework for recognizing new or genetically engineered strains.

Foxf2: A Novel Locus for Anterior Segment Dysgenesis Adjacent to the Foxc1 Gene

Anterior segment dysgenesis (ASD) is characterised by an abnormal migration of neural crest cells or an aberrant differentiation of the mesenchymal cells during the formation of the eye's anterior segment. These abnormalities result in multiple tissue defects affecting the iris, cornea and drainage structures of the iridocorneal angle including the ciliary body, trabecular meshwork and Schlemm's canal. In some cases, abnormal ASD development leads to glaucoma, which is usually associated with increased intraocular pressure. Haploinsufficiency through mutation or chromosomal deletion of the human FOXC1 transcription factor gene or duplications of the 6p25 region is associated with a spectrum of ocular abnormalities including ASD. However, mapping data and phenotype analysis of human deletions suggests that an additional locus for this condition may be present in the same chromosomal region as FOXC1. DHPLC screening of ENU mutagenised mouse archival tissue revealed five novel mouse Foxf2 mutations. Re-derivation of one of these (the Foxf2 W174R mouse lineage) resulted in heterozygote mice that exhibited thinning of the iris stroma, hyperplasia of the trabecular meshwork, small or absent Schlemm's canal and a reduction in the iridocorneal angle. Homozygous E18.5 mice showed absence of ciliary body projections, demonstrating a critical role for Foxf2 in the developing eye. These data provide evidence that the Foxf2 gene, separated from Foxc1 by less than 70 kb of genomic sequence (250 kb in human DNA), may explain human abnormalities in some cases of ASD where FOXC1 has been excluded genetically.

Regulation of POU4F3 gene expression in hair cells by 5′ DNA in mice

The POU-domain transcription POU4F3 is expressed in the sensory cells of the inner ear. Expression begins shortly after commitment to the hair cell (HC) fate, and continues throughout life. It is required for terminal HC differentiation and survival. To explore regulation of the murine Pou4f3 gene, we linked enhanced green fluorescent protein (eGFP) to 8.5 kb of genomic sequence 5' to the start codon in transgenic mice. eGFP was uniformly present in all embryonic and neonatal HCs. Expression of eGFP was also observed in developing Merkel cells and olfactory neurons as well as adult inner and vestibular HCs, mimicking the normal expression pattern of POU4F3 protein, with the exception of adult outer HCs. Apparently ectopic expression was observed in developing inner ear neurons. On a Pou4f3 null background, the transgene produced expression in embryonic HCs which faded soon after birth both in vivo and in vitro. Pou4f3 null HCs treated with caspase 3 and 9 inhibitors survived longer than untreated HCs, but still showed reduced expression of eGFP. The results suggest the existence of separate enhancers for different HC types, as well as strong autoregulation of the Pou4f3 gene. Bioinformatic analysis of four divergent mammalian species revealed three highly conserved regions within the transgene: 400 bp immediately 5' to the Pou4f3 ATG, a short sequence at -1.3 kb, and a longer region at -8.2 to -8.5 kb. The latter contained E-box motifs that bind basic helix-loop-helix (bHLH) transcription factors, including motifs activated by ATOH1. Cotransfection of HEK293 or VOT-E36 cells with ATOH1 and the transgene as a reporter enhanced eGFP expression when compared with the transgene alone. Chromatin immunoprecipitation of the three highly conserved regions revealed binding of ATOH1 to the distal-most conserved region. The results are consistent with regulation of Pou4f3 in HCs by ATOH1 at a distal enhancer.

Establishment of transgenic lines that report nervous system specific Notch activity based on nort gene regulatory sequence

Notch signaling is important in development of the vertebrate retina. During zebrafish retinal neurogenesis Notch activity is polarized as demonstrated by the Tg(−3.4 kb her4.3:dRFP)knu2 transgenic line (Del Bene, 2008). Destabilized fluorescent protein (FP) expression driven by the her4 regulatory sequence was elevated in retinal progenitors with apical nuclei. We are interested in determining if her4.3 FP expression is unique to this transgene or general to other Notch target genes. To investigate this phenomenon we generated additional Notch reporter lines using the previously characterized Tp1 promoter, which consists of 12 Notch responsive RBP-Jκ binding sites driving FP (Parsons, 2009). In addition, we established other Notch reporter transgenic lines based on regulatory sequence for the notch-regulated transcript (nort) gene. Expression of nort is augmented by Notch and enriched in neural progenitors, including those of the retina (Tsutsmi and Itoh, 2007). We generated a construct in which FP was expressed from 3.5 kb of genomic sequence directly upstream of nort. Expression of FP from the nort transgene overlapped with endogenous nort expression. Like endogenous nort, we found that FP from the −3.5 kb nort transgene was partially dependent on Notch activity, as demonstrated by morpholino knockdown of Notch receptors or RBP-Jκ. Similar to the her4.3:FP transgene, nort:FP and Tp1bglob:FP were elevated in a subset of retinal cells with apical nuclei. Interestingly, while each transgene showed overlapping expression, distinct temporal patterns were noted. We are currently assessing how each expression pattern relates to cell fate within the retina.

A multi gene sequence-based phylogeny of the Musaceae (banana) family

BackgroundThe classification of the Musaceae (banana) family species and their phylogenetic inter-relationships remain controversial, in part due to limited nucleotide information to complement the morphological and physiological characters. In this work the evolutionary relationships within the Musaceae family were studied using 13 species and DNA sequences obtained from a set of 19 unlinked nuclear genes.ResultsThe 19 gene sequences represented a sample of ~16 kb of genome sequence (~73% intronic). The sequence data were also used to obtain estimates for the divergence times of the Musaceae genera and Musa sections. Nucleotide variation within the sample confirmed the close relationship of Australimusa and Callimusa sections and showed that Eumusa and Rhodochlamys sections are not reciprocally monophyletic, which supports the previous claims for the merger between the two latter sections. Divergence time analysis supported the previous dating of the Musaceae crown age to the Cretaceous/Tertiary boundary (~ 69 Mya), and the evolution of Musa to ~50 Mya. The first estimates for the divergence times of the four Musa sections were also obtained.ConclusionsThe gene sequence-based phylogeny presented here provides a substantial insight into the course of speciation within the Musaceae. An understanding of the main phylogenetic relationships between banana species will help to fine-tune the taxonomy of Musaceae.

Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase

BackgroundThe CHD7 (Chromodomain Helicase DNA binding protein 7) gene encodes a member of the chromodomain family of ATP-dependent chromatin remodeling enzymes. Mutations in the CHD7 gene are found in individuals with CHARGE, a syndrome characterized by multiple birth malformations in several tissues. CHD7 was identified as a binding partner of PBAF complex (Polybromo and BRG Associated Factor containing complex) playing a central role in the transcriptional reprogramming process associated to the formation of multipotent migratory neural crest, a transient cell population associated with the genesis of various tissues. CHD7 is a large gene containing 38 annotated exons and spanning 200 kb of genomic sequence. Although genes containing such number of exons are expected to have several alternative transcripts, there are very few evidences of alternative transcripts associated to CHD7 to date indicating that alternative splicing associated to this gene is poorly characterized.FindingsHere, we report the cloning and characterization by experimental and computational studies of a novel alternative transcript of the human CHD7 (named CHD7 CRA_e), which lacks most of its coding exons. We confirmed by overexpression of CHD7 CRA_e alternative transcript that it is translated into a protein isoform lacking most of the domains displayed by the canonical isoform. Expression of the CHD7 CRA_e transcript was detected in normal liver, in addition to the DU145 human prostate carcinoma cell line from which it was originally isolated.ConclusionsOur findings indicate that the splicing event associated to the CHD7 CRA_e alternative transcript is functional. The characterization of the CHD7 CRA_e novel isoform presented here not only sets the basis for more detailed functional studies of this isoform, but, also, contributes to the alternative splicing annotation of the CHD7 gene and the design of future functional studies aimed at the elucidation of the molecular functions of its gene products.

A novel missense mutation in the EVC gene underlies Ellis‐van Creveld syndrome in a Pakistani family

Ellis-van Creveld (EVC) syndrome is a rare autosomal recessive disorder characterized by skeletal, ectodermal and cardiac defects. This syndrome is caused by mutations in EVC and EVC2 genes, which are separated by 2.6 kb of genomic sequence on chromosome 4p16. In the present study we ascertained a four-generation pedigree of Pakistani origin with features of EVC. Linkage was searched by genotyping microsatellite markers linked to chromosome 4p16. Affected individuals showed homozygosity to the microsatellite markers tightly linked to EVC and EVC2 genes on chromosome 4p16. It was then subjected to direct sequencing of the EVC and EVC2 genes. Mutation analysis of the EVC and EVC2 genes identified a novel missense change (c.617G>A; p.S206N) in the EVC gene. We herein report on the first family from Pakistan with a large number of individuals affected by EVC. DNA sequence analysis led to the identification of the fifth missense mutation in the EVC gene.

BAC transgenic mice reveal distal cis‐regulatory elements governing BDNF gene expression

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of neurotrophic factors, has important functions in the peripheral and central nervous system of vertebrates. We have generated bacterial artificial chromosome (BAC) transgenic mice harboring 207 kb of the rat BDNF (rBDNF) locus containing the gene, 13 kb of genomic sequences upstream of BDNF exon I, and 144 kb downstream of protein encoding exon IX, in which protein coding region was replaced with the lacZ reporter gene. This BDNF-BAC drove transgene expression in the brain, heart, and lung, recapitulating endogenous BDNF expression to a larger extent than shorter rat BDNF transgenes employed previously. Moreover, kainic acid induced the expression of the transgenic BDNF mRNA in the cerebral cortex and hippocampus through preferential activation of promoters I and IV, thus recapitulating neuronal activity-dependent transcription of the endogenous BDNF gene. genesis 48:214–219, 2010. © 2010 Wiley-Liss, Inc.

Molecular characterization and expression analysis of the IFN-gamma related gene (IFN-γrel) in grass carp Ctenopharyngodon idella

Interferon gamma (IFN-γ), the only member of the type II class of interferons, has been identified in teleost fish. In addition to the IFN-γ gene, fish possess an IFN-γ related gene (IFN-γrel) neighbouring the authentic IFN-γ gene in the genome. In the present study, the cDNA sequence encoding 167 amino acids of IFN-γrel and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted protein sequence of grass carp IFN-γrel (gcIFN-γrel) showed 63% and 50% identities to zebrafish and common carp IFN-γrel (previously termed as IFN-γ1), respectively. The IFN-γrel gene consists of 4 exons, with 3 intervening introns, spanning approximately 2 kb of genomic sequence. The gcIFN-γrel gene did not contain any polymorphic DNA repeats in the introns. Realtime PCR analysis showed that grass carp reovirus induced a high and long lasting (from days 1 to 7) expression of gcIFN-γrel in spleen. The expression of gcIFN-γrel in blood, head kidney, trunk kidney and spleen was also increased by bacterial peptidoglycan (PGN), lipopolysaccharide (LPS) and the interferon inducer polyI:C. The highest induction of gcIFN-γrel expression by PGN was observed in spleen, then in blood and head kidney. Further analysis of the expression patterns of gcIFN-γrel and PGN receptors, nucleotide oligomerization domains (NOD) 1 and 2, may suggest that IFN-γrel was possibly activated in a NOD2-dependent mechanism.

Molecular characterization and expression analysis of nuclear oligomerization domain proteins NOD1 and NOD2 in grass carp Ctenopharyngodon idella

Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs) to detect bacterial component. In this study, the molecular cloning and genomic characterization of grass carp NOD1 (gcNOD1) and grass carp NOD2 (gcNOD2) were reported. The complete open reading frame of gcNOD1 contains 2814 bp, encoding a 937-amino acid polypeptide. The gcNOD2 cDNA sequence encodes 982-amino acid polypeptide. Both gcNOD1 and gcNOD2 possess three conserved domains: carboxy terminal leucine rich repeat (LRR) domains, a central NOD, NBS or NACHT domain, and an amino terminal CARD domain (two in the case of NOD2). At the genomic level, gcNOD1 consists of 11 exons, with 10 intervening introns, spanning approximately 9 kb of genomic sequence. Whereas gcNOD2 has a length of approximately 5 kb with 9 intervening introns. Real time PCR analysis showed gcNOD1 and gcNOD2 were ubiquitously expressed in adult tissues. The highest transcript level of gcNOD1 was detected in brain, but in head kidney for gcNOD2. Grass carp reovirus significantly induced the expression of gcNOD1 and gcNOD2 in spleen (from days 1 to 6). However, expression profiles differed in time course response. Induction experiments with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyI:C revealed the differential expression and regulation of gcNOD1 and gcNOD2 in blood, head kidney, trunk kidney and spleen. All these data suggest a potential role of NOD1 and NOD2 in fish innate immune protection to bacterial and virus infections.

Mutations at the BLK locus linked to maturity onset diabetes of the young and beta-cell dysfunction.

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal pattern of inheritance and a young age at onset, often before age 25. MODY is genetically heterogeneous, with 8 distinct MODY genes identified to date and more believed to exist. We resequenced 732 kb of genomic sequence at 8p23 in 6 MODY families unlinked to known MODY genes that showed evidence of linkage at that location. Of the 410 sequence differences that we identified, 5 had a frequency <1% in the general population and segregated with diabetes in 3 of the families, including the 2 showing the strongest support for linkage at this location. The 5 mutations were all placed within 100 kb corresponding to the BLK gene. One resulted in an Ala71Thr substitution; the other 4 were noncoding and determined decreased in vitro promoter activity in reporter gene experiments. We found that BLK--a nonreceptor tyrosine-kinase of the src family of proto-oncogenes--is expressed in beta-cells where it enhances insulin synthesis and secretion in response to glucose by up-regulating transcription factors Pdx1 and Nkx6.1. These actions are greatly attenuated by the Ala71Thr mutation. These findings point to BLK as a previously unrecognized modulator of beta-cell function, the deficit of which may lead to the development of diabetes.

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (Epinephelus coioides)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5'-terminal untranslated region (5'-UTR) of 153 bp, and a 3'-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5'-UTR sequence contains an exon and an intron, and the 3'-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic-polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.