Zebrafish Protein Research Articles

A Novel Monoclonal Antibody Specific to DIXDC1 Protein

DIXDC1, a novel type of DIX domain-containing protein in zebrafish, is the human homolog of Ccd1. It upregulates the Wnt signaling pathway functioning downstream of Wnt and upstream of axin. Here we aimed to efficiently generate a monoclonal antibody against DIXDC1 protein, using it as a tool to study the role of DIXDC1 in colonic tumorigenesis. We transformed the plasmids of PGEX-DIXDC1-C and PGEX-DIXDC1-N separately into Escherichia coli BL21 (DE3) for expression, and then injected the purified GST-DIXDC1 fusion protein into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay with purified fusion protein. Eight hybridoma clones effective in detecting the DIXDC1 protein were characterized by immunohistochemistry and Western immunoblotting. By using the MAbs, we found that the DIXDC1 protein was overexpressed in human colorectal cancer tissues. Taken together, the MAbs would be helpful for understanding the distribution and functions of DIXDC1.

Xenopus and Zebrafish Annotation in the UniProt Knowledgebase (UniProtKB)

AbstractThe African clawed frog Xenopus laevis and the zebrafish Danio rerio have both proved to be good model organisms for studying early vertebrate cellular and developmental biology. More recently, the related western clawed frog Xenopus tropicalis has become a popular choice in the laboratory, since its shorter life style and diploid genome make it more amenable to genetic analysis. Ongoing sequencing of the X. tropicalis and D. rerio genomes, together with the growing number of EST/cDNA projects, is generating large amounts of sequence data and revealing many human developmental and disease genes that have counterparts in fish and frog.UniProtKB/Swiss-Prot curates Xenopus and zebrafish proteins with functional and sequence annotation from the literature and sequence analysis tools, using both controlled vocabularies (including GO terms) and free text. The tetraploid nature of the X. laevis and D. rerio genomes complicates annotation since the protein copies need to be identified and curated as separate UniProtKB/Swiss-Prot entries. The recent addition of Xenbase cross-references in Xenopus UniProtKB entries has been the result of cross-talk with Xenbase, and we continue to collaborate with ZFIN to ensure consistency between databases. UniProt is mainly supported by the NIH, European Commission FELICS, Swiss Federal Government, PATRIC BRC and NSF grants.

A Novel Transcription Factor, zbtb11 Is Critical for Neutrophil Development in Zebrafish

Zinc finger and BTB domain containing proteins (BTB-ZF) are transcriptional repressors from a family including members with critical roles in hematopoiesis and oncogenesis (BCL6, PLZF, HIC). In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen for defects in myeloid development, we identified a neutropenic zebrafish mutant with a mutation in zbtb11, a novel BTB-ZF transcription factor, suggesting that zbtb11 is critical for normal neutrophil development.The mutant was found in a gynogenetic haploid ENU screen for defective expression of genes along the developmental pathway from mesoderm to mature neutrophil (deltaC, spi1 and myeloperoxidase/mpx), undertaken to search for novel genetic regulators of myelopoiesis in an unbiased fashion. Since zebrafish are poikilothermic, embryos were screened at 33°C to maximise recovery of temperature dependant alleles; marsanne (man) was one mutant pedigree recovered. man is an early embryonic lethal mutant with normal scl and spi1 expression but markedly reduced numbers of mpx expressing cells. Erythropoiesis is unaffected. Reduced numbers of Sudan Black stained cells demonstrate that man is deficient in neutrophils themselves, not just mpx transcripts. man mutants also develop brain and spinal cord degeneration with hydrocephalus due to apoptosis. man is a temperature sensitive allele i.e. numbers of mpx expressing cells per embryo decrease at higher (restrictive) temperatures and are partially restored at lower (permissive) temperatures:Water temperatureNumber of mpx expressing cells/embryo (mean ± SD, 48hpf)man/man+/?22°C75±1589±1428°C42±1081±1233°C27±1181±22Hence man serves as a conditional allele, allowing manipulation of protein function via manipulation of water temperature. man was mapped to a 40kb region on chromosome 6 containing a single gene, zbtb11. Sequencing of man and WT zbtb11 cDNA revealed a missense mutation (346T->A, 116Cys->Ser) affecting a cysteine residue conserved throughout vertebrate evolution. Pedigree analysis proved this substitution was introduced by ENU mutagenesis and segregated absolutely with the man phenotype in 3863 mutant embryos. Genetic tests implicated this zbtb11 mutation as causing the man phenotype, zbtb11 knockdown with an antisense morpholino oligonucleotide recapitulated the mpx deficiency and other features of the man phenotype. Injection of WT but not mutant zbtb11 mRNA into man embryos completely rescued mpx expression (125 +/−38 mpx positive cells per embryo vs. 67+/−35, p<0.001; uninjected WT 109 +/19, mean +/− SD, 48hpf) and all other aspects of the man phenotype. Since the mutant Zbtb11 protein is inactive in this assay, we hypothesise the amino acid substitution in the N-terminus of the protein compromises function. In view of the temperature sensitive nature of man, this may result from an effect on weak bonding, differentially affecting protein structure at permissive vs. restrictive temperatures. Zebrafish zbtb11 encodes a 1146 amino acid protein with a predicted N-terminal BTB (also known as POZ) domain and 12 C-terminal C2H2 (Kruppel-type) zinc fingers. The BTB domain is a highly conserved, approximately 100 amino acid domain, mediating protein-protein interaction and dimerization. The zebrafish protein is approximately 75% identical to mouse and human ZBTB11 across its BTB domain and zinc fingers. ZBTB11 is expressed physiologically in human hematopoietic cells (Ferrari F et al. BMC Genomics. 2007) and mouse embryonic brain (Gray PA et al. Science. 2004). It appears to be over-expressed in K562 CML cells compared to other acute leukemia and lymphoma lines (Su AI et al. PNAS 2002), suggesting a possible role in oncogenesis. The function of mammalian ZBTB11 is unknown and there are no mammalian models of ZBTB11 dysfunction. The rescue of man by WT zbtb11 forms the basis of a bioassay to test modified Zbtb11 proteins, and a whole animal assay for chemical modifiers of Zbtb11 function. Rescue of man with human ZBTB11 is being attempted to assess functional conservation and to provide a heterologous mammalian bioassay. Temperature-shift experiments exploiting the temperature sensitivity of man should define the time-dependant requirement for Zbtb11 during development. man provides a unique reagent with which to advance understanding of Zbtb11 biochemistry and biology in vivo.

Human Nucleophosmin (NPM1) Perturbs Myeloid Development in Zebrafish in Vivo

The most frequent genetic alterations in adult cases of Acute Myeloid Leukemia (AML) are mutations in the human nucleophosmin (hNPM1) gene. In about 30% of AMLs this nucleolar phosphoprotein is aberrantly localized to the cytoplasm (hNPMc) due to these mutations affecting the protein's nuclear shuttling. hNPMc AMLs exhibit distinctive clinical and biological features that have led to its WHO classification as a distinct myeloid neoplasm. Despite its prominent association with AML, in vitro and in vivo models of hNPMc transformation in myeloid cells are lacking and its role in this process remains poorly understood. To further our understanding of hNPM function we are using the zebrafish vertebrate model system that is ideally suited for the in vivo analysis of cellular function and development during embryonic hematopoiesis. Importantly, the wide variety of blood cell types and key cellular factors regulating hematopoiesis are highly conserved between mammals and zebrafish. To investigate the in vivo role of hNPMc in hematopoiesis, we injected mRNAs encoding hNPM1wt and the leukemia-associated mutant hNPMc into one-cell stage zebrafish embryos. By fusing GFP to these hNPM proteins, we were able to follow the developmental expression of hNPM1 and its subcellular localization during embryogenesis. Analysis using confocal microscopy showed that NPMc is localized to the cell cytoplasm, while NPM1wt is found in nucleoli, as in human and mouse cells. These studies demonstrate the conservation of the nuclear-cytoplasmic transport functions of the human proteins in the zebrafish and further support its validity as a model to analyze and determine hNPM function. We also observed that hNPMc is expressed at far lower levels than its wild-type counterpart and is almost undetectable at 36hpf while hNPMwt continues to be expressed. Unlike mammals, two endogenous zebrafish NPM1 proteins were identified and named, zNpm1a and zNpm1b. Both zNpm1a and zNpm1b proteins are ubiquitously expressed in the embryo and demonstrated nucleolar localization. Expression of hNPMwt resulted in its colocalization with endogenous zNpm while hNPMc was able to bring about the export of both zebrafish proteins to the cytoplasm through heterotypic interactions. Co-immunoprecipitation experiments confirmed the interaction between human and zebrafish NPM1 proteins and zNpm1a and zNpm1b were both able to bind and co-immunoprecipitate with hNPM1 and hNPMc. These experiments suggest that transient hNPMc expression during zebrafish hematopoiesis may mimic its function in human leukemic blasts and provide clues to its functional role in AML. Comparable protein levels of either hNPMwt or hNPMc were expressed in embryos, confirmed by western blot at 22–24 hpf, and analyzed by whole mount in situ hybridization (WISH) using antisense RNA markers of specific hematopoietic lineages. Expression of hNPMc caused an increase in cMYB expression at 36 hpf, indicating an increase in the hematopoietic stem cell compartment. Furthermore, myeloid precursors (PU.1 at 22 hpf) also showed an increase upon hNPMc expression; however, mature myeloid cell (MPO and L-plastin at 26 hpf) levels were not increased relative to those in control hNPMwt injected embryos. Interestingly, the expression of hNPMc in p53 mutant embryos resulted in elevated levels of both PU.1 and MPO expressing cells, suggesting that hNPMc in zebrafish can activate p53 dependent cell cycle arrest, senescence or cell death in PU.1 cells prior to differentiation. These in vivo studies of hNPMc function during zebrafish hematopoietic differentiation suggest that hNPMc expression may increase the stem cell/ myeloid precursor compartment and can activate a p53 dependent cell death response in myeloid cells. Taking advantage of the zebrafish system in these continuing studies will further address how hNPMc expression may contribute to leukemogenesis.

Phosphoglycerate kinase 1 expression responds to freezing, anoxia, and dehydration stresses in the freeze tolerant wood frog, Rana sylvatica

Natural freezing survival by wood frogs (Rana sylvatica) involves multiple organ-specific changes in gene expression. Screening of a cDNA library made from brain of frozen frogs revealed freeze-responsive up-regulation of the glycolytic enzyme, phosphoglycerate kinase 1 (PGK1). Northern blots showed an approximately two-fold increase in pgk1 transcripts in brain of frozen frogs whereas PGK1 protein levels rose by three- to five-fold within 4-8 hr of freezing. Freezing also elevated pgk1 transcripts in liver but not in skin. Both transcript and protein levels also rose in response to two of the component stresses of freezing (anoxia and dehydration) with a particularly pronounced (11-fold) increase in PGK1 protein in brain in response to anoxia. Amino acid sequence analysis showed 92.5% identity between wood frog and Xenopus laevis PGK1 and 86-88% identity with the zebrafish, chicken, and human protein. Four unique amino acid substitutions in the wood frog protein could be important in maintaining the functional conformation of the wood frog protein at low body temperatures. Elevated amounts of PGK1, one of the ATP-generating reactions of glycolysis, in wood frog brain during freezing would enhance the glycolytic capacity of the organ and support the maintenance of cellular energetics under the ischemic conditions of the frozen state.

Knockdown of a galectin-1-like protein in zebrafish (Danio rerio) causes defects in skeletal muscle development

We previously identified and characterized four galectin-1-like proteins in zebrafish, Drgal1-L1, Drgal1-L2, Drgal1-L3, and one splice variant of Drgal1-L2, of distinct ontogenic expression. Drgal1-L1 is maternal; Drgal1-L2 is zygotic and strongly expressed in the notochord, while Drgal1-L3 is both maternal and zygotic. Knockdown experiments in zebrafish embryos using a morpholino-modified antisense oligo targeted to the 5'-UTR sequence of Drgal1-L2 resulted in a phenotype with a bent tail and disorganized muscle fibers. This effect was dose-dependent as follows: 62-66% at 17 ng, 29-35% at 5.7 ng, 21-28% at 1.9 ng, and 14-17% at 0.6 ng. However, no (or a negligible number of) Drgal1-L1 knockdown embryos showed similar morphological defects, indicating that the observed effects are sequence-specific, and not due to the toxicity of the morpholino-modified oligos. Further, ectopic expression of native Drgal1-L2 specifically rescued the phenotype, as co-injection of the full-length sense Drgal1-L2 mRNA with Drgal1-L2-MO yielded 60-62% normal embryos. As the notochord serves as the primary source of signaling molecules required for proper patterning of adjacent tissues, such as neural tube, somites, and heart, these results suggest that galectins produced by the notochord play a key role in somitic cell differentiation and development.

Structures of the wild-type and activated catalytic domains ofBrachydanio rerioPolo-like kinase 1 (Plk1): changes in the active-site conformation and interactions with ligands

Polo-like kinase 1 (Plk1) is a member of a family of serine/threonine kinases involved in the regulation of cell-cycle progression and cytokinesis and is an attractive target for the development of anticancer therapeutics. A zebrafish homolog of the human Plk1 (hPlk1) kinase domain (KD) was identified that can be expressed in large quantities in bacteria and crystallizes readily, whether in a wild-type form or as a variant containing the activating Thr196-->Asp substitution, in one space group and under similar conditions both in the absence and presence of active-site compounds. This construct was validated by testing a panel of hPlk1 inhibitors against human and zebrafish proteins and it was shown that the selected small molecules inhibited the homologs with a high degree of correlation. Crystal structures of ligand-free wild-type and activated zebrafish Plk1 (zPlk1) KDs revealed the organization of the secondary structural elements around the active site and demonstrated that the activation segment was disordered in the activated form of the domain but possessed a well defined secondary structure in the wild-type enzyme. The cocrystal structure of wild-type zPlk1 KD with ADP documented the hydrolysis of ATP and revealed the phosphorylation site. The cocrystal structure of the activated KD with wortmannin, a covalent inhibitor of Plk1 and PI3 kinases, showed the binding mode of the small molecule to the enzyme and may facilitate the design of more potent Plk1 inhibitors. The work presented in this study establishes the zPlk1 KD as a useful tool for rapid low- and high-throughput structure-based screening and drug discovery of compounds specific for this mitotic target.

Genes required for functional glycosylation of dystroglycan are conserved in zebrafish

Mutations in human genes encoding proteins involved in α-dystroglycan glycosylation result in dystroglycanopathies: severe congenital muscular dystrophy phenotypes often accompanied by CNS abnormalities and ocular defects. We have identified the zebrafish orthologues of the seven known genes in this pathway and examined their expression during embryonic development. Zebrafish Large, POMT1, POMT2, POMGnT1, Fukutin, and FKRP show in situ hybridization patterns similar to those of dystroglycan, with broad expression throughout early development. By 30 h postfertilization (hpf), transcripts of all these genes are most prominent in the CNS, eye, and muscle, tissues that are predominantly affected in the dystroglycanopathies. In contrast, Large2 expression is more restricted and by 30 hpf is confined to the lens, cerebellum, and pronephric duct. We show that the monoclonal antibody IIH6, which recognizes a glycoform of dystroglycan, also detects the zebrafish protein. Injection of morpholino oligonucleotides against zebrafish Large2 resulted in loss of IIH6 immunostaining. These data indicate that the dystroglycan glycosylation pathway is conserved in zebrafish and suggest this organism is likely to be a useful model system for functional studies.

Goodpasture Antigen-binding Protein and Its Spliced Variant, Ceramide Transfer Protein, Have Different Functions in the Modulation of Apoptosis during Zebrafish Development

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the alpha3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPDelta26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpDelta26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpDelta26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.

The Zebrafish Secretome

The secretome is a functionally rich proteome subset, including cellular membrane and extracellular proteins processed through the secretory pathway. In this study, Danio rerio and Homo sapiens RefSeq proteins were analyzed with SignalP, TargetP, Phobius, and pTarget algorithms. About 16.5% of the zebrafish proteome and 17.0% of the human proteome possessed predicted N-terminal signal sequences. Nearly half of these proteins were subsequently classified as soluble, as they lacked predicted transmembrane domains. The soluble proteins were further subclassified, predicting 1345 (3.8%) zebrafish and 1207 (3.2%) human proteins as extracellular. Comparison of the zebrafish and human soluble secretome proteins identified 372 as orthologs, on the basis of reciprocal BLAST best hits. The computational characterization of the zebrafish proteins found many more members of the secretome than annotated in the SwissProt database. Only 180 of the 2078 zebrafish SwissProt protein entries, and 995 of the 19,294 human SwissProt protein entries were annotated with secreted protein locales. A specific investigation of the fibroblast growth factor and matrix metalloproteinase (MMP) protein families confirmed the prediction data and generated annotation of three additional putative MMP zebrafish proteins. This study presents the first known published description of the zebrafish secretome since the completion of the zebrafish genome sequencing project.

Molecular Identification and Functional Characterization of the Kisspeptin/Kisspeptin Receptor System in Lower Vertebrates1

The KISS1 gene encodes the kisspeptin neuropeptide, which activates the KISS1 receptor (KISS1R; G protein-coupled receptor 54; GPR54) and participates in neuroendocrine regulation of GnRH secretion. To study the physiological function(s) and evolutionary conservation of KISS1, we cloned opossum, Xenopus, and zebrafish kiss1 cDNAs. Processing zebrafish, Xenopus, or opossum KISS proteins would liberate a carboxy-terminal amidated peptide with 52, 54, or 53 amino acid residues, respectively. Phylogenetic analysis of all known vertebrate KISS1 peptides showed clear clustering of the sequences according to canonical vertebrate classes. The zebrafish kiss1 gene consists of two exons and one intron. Real-time PCR analysis of two kiss1R cloned from zebrafish brain found expression of kiss1, kiss1ra, and kiss1rb, with kiss1ra-more similar to other piscine Kiss1 receptors-highly expressed in the gonads and kiss1rb in other nonbrain tissues. In females kiss1 mRNA levels gradually increased during the first few weeks of life to peak in fish with ovaries containing mature oocytes, while in males kiss1 mRNA levels peaked after 6 wk postfertilization when the testes exhibited initial stages of spermatogenesis and decreased after puberty. Zebrafish kiss1ra and kiss1rb were expressed differentially with similar patterns in both genders. These results indicate that the Kiss1/Kiss1r system may participate in puberty initiation in fish as well. Like human KISS1R, Kiss1ra transduces its activity via the PKC pathway, whereas Kiss1rb does so via both PKC and PKA pathways. The human KISS1R was highly activated by both huKISS10amide and zfKISS10amide, whereas both zebrafish Kiss1 receptor types were less sensitive to amidation.

Brd4 associates with mitotic chromosomes throughout early zebrafish embryogenesis

Brd4 is a member of the BET (bromodomains and extraterminal) subfamily of bromodomain proteins that includes chromatin-modifying proteins and transcriptional regulators. Brd4 has a role in cell cycle progression, making it indispensable in mouse embryos and cultured cells. The N-terminal domain of Brd4 participates in a fusion oncogene. Brd4 associates with acetylated histones in chromatin, and this association persists during mitosis implicating Brd4 in epigenetic memory. Brd4 sequence, particularly the bromodomains and ET domain, is conserved in the zebrafish and Xenopus laevis proteins reported here. Brd4 is expressed and localized on mitotic chromosomes in early zebrafish embryos before and after the midblastula transition (MBT), indicating that the Brd4-chromosome association is a conserved property that is maintained even before zygotic transcription. The association of Brd4 with acetylated histones may also be conserved in early embryos as we found that histones H3 and H4 are already acetylated during pre-MBT stages.

Spectral alignment for top-down MS | BiSSCat | Zebrafish protein database | Automatic GUI | Assessing the quality of protein-protein interactions | TagFinder

Spectral alignment for top-down MS | BiSSCat | Zebrafish protein database | Automatic GUI | Assessing the quality of protein-protein interactions | TagFinder

BIM and other BCL-2 family proteins exhibit cross-species conservation of function between zebrafish and mammals

Here we investigate the function of zebrafish Bcl-2 family proteins and demonstrate important conservation of function across zebrafish and mammalian systems. We have isolated a zebrafish ortholog of mammalian BIM and show that it is the most toxic of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian BIM gene. The zebrafish bad gene shows a complete lack of embryonic lethality, but like mammalian BAD, its pro-apoptotic activity is regulated through phosphorylation of critical serines. We also found that the pattern of mitochondrial dysfunction observed by zebrafish BH3 domain peptides in a mammalian cytochrome c release assay recapitulates the pattern of embryonic lethality induced by the respective mRNA injections in vivo. In contrast to zebrafish Bim, Bid exhibited only weak binding to zebrafish Bcl-2 and moderate-to-weak overall lethality in zebrafish embryos and isolated mitochondria. Given that zebrafish Bcl-2 binds strongly to mammalian BID and BIM peptides and proteins, the protein identified as the zebrafish Bid ortholog has different properties than mammalian BID. Overall, our results demonstrate the high degree of functional conservation between zebrafish and mammalian Bcl-2 family proteins, thus validating the zebrafish as a model system to further dissect the molecular mechanisms that regulate apoptosis in future forward genetic and chemical modifier screens.

Transcriptional studies of a novel family of “short C1q” domain proteins in zebrafish

Transcriptional studies of a novel family of “short C1q” domain proteins in zebrafish

Salmon louse (Lepeophtheirus salmonis) transcriptomes during post molting maturation and egg production, revealed using EST-sequencing and microarray analysis

BackgroundLepeophtheirus salmonis is an ectoparasitic copepod feeding on skin, mucus and blood from salmonid hosts. Initial analysis of EST sequences from pre adult and adult stages of L. salmonis revealed a large proportion of novel transcripts. In order to link unknown transcripts to biological functions we have combined EST sequencing and microarray analysis to characterize female salmon louse transcriptomes during post molting maturation and egg production.ResultsEST sequence analysis shows that 43% of the ESTs have no significant hits in GenBank. Sequenced ESTs assembled into 556 contigs and 1614 singletons and whenever homologous genes were identified no clear correlation with homologous genes from any specific animal group was evident. Sequence comparison of 27 L. salmonis proteins with homologous proteins in humans, zebrafish, insects and crustaceans revealed an almost identical sequence identity with all species.Microarray analysis of maturing female adult salmon lice revealed two major transcription patterns; up-regulation during the final molting followed by down regulation and female specific up regulation during post molting growth and egg production. For a third minor group of ESTs transcription decreased during molting from pre-adult II to immature adults. Genes regulated during molting typically gave hits with cuticula proteins whilst transcripts up regulated during post molting growth were female specific, including two vitellogenins.ConclusionThe copepod L.salmonis contains high a level of novel genes. Among analyzed L.salmonis proteins, sequence identities with homologous proteins in crustaceans are no higher than to homologous proteins in humans. Three distinct processes, molting, post molting growth and egg production correlate with transcriptional regulation of three groups of transcripts; two including genes related to growth, one including genes related to egg production. The function of the regulated transcripts is discussed in relation to post molting morphological changes in adult female salmon louse. There is clear evidence that transcription of the major yolk proteins is not induced before some of the post molting growth of abdomen and the genital segment has occurred. A hallmark for the observed growth is transcription of many putative cuticula proteins prior to the size increase.

A novel mutation in JARID1C/SMCX in a patient with autism spectrum disorder (ASD)

We describe a nondysmorphic patient with developmental delay and autism spectrum disorder who has a missense mutation in the Jumonji AT-rich interactive domain 1C (JARID1C) gene. This child first presented at 30 months of age with stereotyped and repetitive behaviors, impairment in social reciprocity and in the use of multiple nonverbal behaviors, and developmental delay primarily in the language domain. A diagnosis of autism was made and subsequently confirmed at the current age of 47 months. Cytogenetic and fragile X studies were normal. Mutational analysis revealed a novel missense mutation in exon 16 of the JARID1C gene that results in an arginine to tryptophan substitution at amino acid 766 (R766W). Sequence alignment analysis with multiple available eukaryotic sequences including the homologous proteins of mouse and zebrafish demonstrated that the affected amino acid is conserved. JARID1C has not previously been implicated in autism susceptibility. Recent novel molecular evidence suggests that it is a histone demethylase specific for di- and trimethylated histone 3 lysine 4 (H3K4) and functions as a transcriptional repressor by fostering REST-mediated neuronal gene regulation. The JARID1C-regulated genes SCN2A, CACNA1H, BDNF, and SLC18A1 have previously been associated with autism and cognitive dysfunction. This patient brings the total number of reported JARID1C mutations to 14. This presentation both extends the range of neurocognitive phenotypes attributable to mutations in this gene and illustrates the importance of molecular studies and DNA sequence analysis for accurate diagnosis of monogenic causes of autism.

Zn(ii) ions bind very efficiently to tandem repeat region of “prion related protein” (PrP-rel-2) of zebra-fish. MS and potentiometric evidence

Multi-histidine peptide fragments of zebra-fish prion protein are effective ligands for Zn(II) ions. Moreover the formation of a dinuclear complex species with a longer peptide can suggest the existence of the cooperative effect in the metal ion binding.

Molecular cloning and biochemical characterization of sialidases from zebrafish (Danio rerio)

Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT-PCR (reverse transcription-PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.

Cytoplasmic Domain of Zebrafish Myelin Protein Zero: Adhesive Role Depends on β-Conformation

Solution spectroscopy studies on the cytoplasmic domain of human myelin protein zero (P0) (hP0-cyt) suggest that H-bonding between β-strands from apposed molecules is likely responsible for the tight cytoplasmic apposition in compact myelin. As a follow-up to these findings, in the current study we used circular dichroism and x-ray diffraction to analyze the same type of model membranes previously used for hP0-cyt to investigate the molecular mechanism underlying the zebrafish cytoplasmic apposition. This space is significantly narrower in teleosts compared with that in higher vertebrates, and can be accounted for in part by the much shorter cytoplasmic domain in the zebrafish protein (zP0-cyt). Circular dichroism measurements on zP0-cyt showed similar structural characteristics to those of hP0-cyt, i.e., the protein underwent a β→ α structural transition at lipid/protein (L/P) molar ratios >50, and adopted a β-conformation at lower L/P molar ratios. X-ray diffraction was carried out on lipid vesicle solutions with zP0-cyt before and after dehydration to study the effect of protein on membrane lipid packing. Solution diffraction revealed the electron-density profile of a single membrane bilayer. Diffraction patterns of dried samples suggested a multilamellar structure with the β-folded P0-cyt located at the intermembrane space. Our findings support the idea that the adhesive role of P0 at the cytoplasmic apposition in compact myelin depends on the cytoplasmic domain of P0 being in the β-conformation.