Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli.

Hemolysin genes of the reference strains of Actinobacillus pleuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia coli by using polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of the corresponding appA gene from A. pleuropneumoniae serotype 5. The three genes from serotypes 1, 2, and 5 have identical restriction maps and appear to encode a hemolysin which was previously identified in serotype 2 and designated HlyII. Gene appA is different from hlyIA encoding the major hemolysin type I (HlyI) which was identified earlier in serotype 1. Polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of hlyIA of serotype 1 showed that the gene encoding HlyI is present in serotype 1 but not in serotype 2, in contrast to the gene encoding HlyII that was present in both serotypes. This was confirmed by Western blot (immunoblot) experiments using monoclonal antibodies specific for either recombinant HlyI or recombinant HlyII, which showed that A. pleuropneumoniae serotype 1 strain 4074 produces both HlyI and HlyII, whereas serotype 2 strain S1536 produces only HlyII. The expression of both hemolysins was investigated in all serotypes by the use of monoclonal antibodies. HlyI was shown to be expressed by the reference strains of serotypes 1, 5a, 5b, 9, 10, and 11, whereas HlyII was shown to be expressed by the reference strains of all 12 serotypes tested except serotype 10. A. pleuropneumoniae serotype 1 strain 4074 is the first bacterium which has been shown to contain two different actively expressed RTX toxin genes. Comparison of our data with those from other groups shows that the originally described strongly hemolytic hemolysin type I (HlyI) corresponds to cytolysin I (ClyI) which was recently described by others, while the weakly hemolytic hemolysin type II (HlyII) seems to be identical to ClyII and AppA.     

Identification of a mutation in the Clock1 gene affecting zebrafish circadian rhythms

As part of an ongoing program to identify genes involved in maintaining circadian rhythms of zebrafish, 6,500 mutagenized genomes were screened for dominant mutants affecting circadian locomotor activity. Molecular analysis of one of these mutant lines, Clk1(dg3), revealed an I254N mutation in the PAS domain of the Clock1 protein. This isoleucine is tightly conserved in the Clock genes of several different species, and the I254N was not seen in any of the wild-type zebrafish population tested. Analysis of circadian activity rhythms as well as melatonin rhythms in homozygotes revealed the biological clock runs with a shortened period. The effect of this Clock1 mutation was characterized in vitro using a cell culture system where it appears to enhance the transactivation ability of the I254N Clock1 protein compared with that of the normal gene product.     

Identification and expression analysis of a novel splice variant of human Sprouty1 gene

Sprouty (SPRY) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor (FGF) and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. During large scale DNA sequencing of the human fetal brain cDNA library, we cloned a novel splice variant of human Sprouty1 gene, and termed it human Sprouty1b (SPRY1b). It has the same deduced protein as Sprouty1a, which has been reported. And like other members of Sprouty family, SPRY1b deduced protein also has a C-terminal cysteine-rich region. According to the search against human genome database, SPRY1b was mapped to 4q25-28. Expression analysis of SPRY1a and SPRY1b shows that they are hardly expressed in adult human, but have different expression patterns in fetus, which confirmed that SPRY1 is an important gene during fetal development.     

A gene expression screen in zebrafish embryogenesis.

A screen for developmentally regulated genes was conducted in the zebrafish, a system offering substantial advantages for the study of the molecular genetics of vertebrate embryogenesis. Clones from a normalized cDNA library from early somitogenesis stages were picked randomly and tested by high-throughput in situ hybridization for restricted expression in at least one of four stages of development. Among 2765 clones that were screened, a total of 347 genes with patterns judged to be restricted were selected. These clones were subjected to partial sequence analysis, allowing recognition of functional motifs in 163 among them. In addition, a portion of the clones were mapped with the aid of the LN54 radiation hybrid panel. The usefulness of the in situ hybridization screening approach is illustrated by describing several new markers for the characteristic structure in the fish embryo named the yolk syncytial layer, and for different regions of the developing brain.     

Embryonic retinal gene expression in sonic-you mutant zebrafish.

Hedgehog (Hh) signaling is required for proper eye development in vertebrates; known roles for Hh in the zebrafish include regulation of eye morphogenesis, ganglion cell neurogenesis, and photoreceptor differentiation. To gain insight into the mechanisms by which Hh signaling influences these developmental events, we have examined proliferation, cell death, and expression patterns of several retinal genes in the eyes of embryonic zebrafish lacking the sonic hedgehog gene. We find that features of the eye phenotype of the sonic-you (syu) mutant are consistent with multiple roles for the Hh signal during retinal development. Most interestingly, half of the mutant retinas failed to initiate cell differentiation and, instead, retained a neuroepithelial appearance. In the other half of the mutants, retinal cell differentiation was initiated, but not fully propagated. We also find that Hh signaling is important for retinal cell proliferation and retinal cell survival; together, these functions provide an explanation for progressive microphthalmia in the syu-/- mutant.     

Identification of a gene required for de novo DNA methylation of the zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for tail and notochord development. We have shown previously that ntl is de novo methylated during early embryogenesis. To find the gene that de novo methylates ntl and understand the meaning of this methylation, we cloned seven genes that encode the conserved catalytic domain of methyltransferases. We found that injection of antisense morpholino oligonucleotides against one of them, termed dnmt7, into eggs significantly reduced the level of ntl methylation, although no apparent phenotype was induced by the injection. Inhibition of Dnmt7 activity did not change the level of genome-wide methylation nor did it affect de novo methylation of injected plasmid DNA, indicating that Dnmt7 specifically methylates ntl in the genome.     

Identification of dynorphin a from zebrafish: a comparative study with mammalian dynorphin A

We report the cloning and molecular characterization of the zfPDYN. The complete open reading frame for this propeptide is comprised in two exons that are localized on chromosome 23. zfPDYN cDNA codes for a polypeptide of 252 amino acids that contains the consensus sequences for four opioid peptides: an Ile-enkephalin, the neo-endorphins, dynorphin A and dynorphin B. Upon comparison between zebrafish (zfDYN A) and mammalian dynorphin A (mDYN A) it has been stated that these two peptides only differ in two amino acids: the Leu(5) is replaced by Met(5) and the Lys(13) by Arg(13). Taking into consideration that mDYN A is able to bind to the three mammalian opioid receptors, we have compared the pharmacological profile of zfDYN A and mDYN A on the zebrafish opioid receptors. By means of radioligand binding techniques, we have established that these two dynorphins bind and activate all of the cloned opioid receptors from zebrafish (delta-, mu- and kappa-like), although with different affinities. zfDYN A and mDYN A displace [(3)H]-diprenorphine binding with K(i) values on the nanomolar range, showing greater affinity for zebrafish opioid receptor (ZFOR) 3 (kappa) receptor. ZFOR1 (delta) and ZFOR4 (delta) present higher affinity for zfDYN A than for mDYN A, while the opposing behavior is observed in ZFOR2 (mu). Functional [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) stimulation experiments indicate that these two peptides fully activate the zebrafish opioid receptors, although the mean effective dose (EC(50)) values obtained for ZFOR2 and ZFOR3 receptors are lower than those seen for ZFOR1 and ZFOR4. A comparative study indicates that mammalian and zebrafish opioid receptors might bind their corresponding dynorphin A in a similar fashion, hence suggesting an important role of the opioid system through the vertebrate evolution.     

Isolation and expression analysis of three zebrafish angiopoietin genes.

The Tie1 and Tie2 receptor tyrosine kinases and the Tie2 ligands, the angiopoietins, play critical roles in vertebrate vascular embryogenesis, helping to mediate the interaction between endothelial cells and the pericytes or vascular smooth muscle cells that envelop and support them. We have obtained full-length cDNA sequences for zebrafish orthologs of angiopoietin-1 (ang1), angiopoietin-2 (ang2), and angiopoietin-like-3 (angptl3). Ang1 is expressed in head ventral mesenchyme, in the ventromedial region of somites, in mesenchyme surrounding trunk axial vessels, and in the hypochord, a transient embryonic structure of endodermal origin that has been implicated in dorsal aorta assembly in both zebrafish and Xenopus. Ang2 is expressed in head and anterior trunk ventral mesenchyme and the developing pronephric glomeruli. Angptl3 is expressed in the yolk syncytial layer.     

Identification of the activation-induced cytidine deaminase gene from zebrafish: an evolutionary analysis

In the present study, we report the identification of the activation-induced cytidine deaminase (AID) encoding gene in frog, dog and chimpanzee, where both somatic hypermutation and class switch recombination (CSR) occurs and in zebrafish and fugu, species lacking CSR. The cDNA sequence of the zebrafish AID reported here suggests both N and C ends of the previously predicted protein sequence are incorrect. A comparison of AID sequences among mammals, birds, amphibians and fish revealed conserved aa residues which may be essential for AID activity, although the cytidine deaminase active motif in the latter is nine amino acids longer. Furthermore, an aa deletion, and extensive substitutions in the C terminal end of AID from bony fish indicate that the molecule may not yet have developed a capacity to recruit the specific cofactor(s) needed to initiate CSR.     

Identification and characterization of a second polygalacturonase gene of Aspergillus niger

Summary The filamentous fungus Aspergillus niger produces several endopolygalacturonases that are involved in the degradation of pectin. PGI, the enzyme representing the second most abundant activity in a commmercial enzyme preparation, was further characterized and the corresponding gene was isolated. The nucleotide sequence of the pgaI gene was determined and the protein coding region was found to be interrupted by two short introns, one of which has a unusual donor splice site. The deduced 368 amino acids long protein with a putative prepropeptide of 31 amino acids shows 60% sequence identity to PGII in the mature protein. PGI overproducting A. niger strains were obtained by contransformation with the cloned gene.     

Proteomic and gene expression analysis of zebrafish brain undergoing continuous light/dark stress

Several organisms irrespective of their complexity in structure and function have an inbuilt circadian rhythm. Zebrafish could be used as an alternate model animal in sleep research as it exhibits similar sleep–wake dynamics as mammals and Drosophila. In this study, we have analysed the adult zebrafish brain for its differential proteome and gene expression during perturbed light/dark cycle. A total of 53 and 25 proteins including sncb, peroxiredoxins and TCR alpha were identified based on two‐dimensional gel electrophoresis Fourier transform mass spectrometer/ion trap tandem mass spectrometer and differential in‐gel electrophoresis MALDI TOF MS/MS analysis, respectively, with at least 1.5‐fold changes between the control and experimental brains. Real time‐polymerase chain reaction revealed that many circadian pathway‐associated genes, such as per1b, bmal1b, cry1b, bmal2 and nr1d2, were differentially regulated during continuous light/dark exposures. It is hypothesized that the differential regulation of these genes might lead to the discovery of potential diagnostic markers for gaining insight into the light/dark‐associated stress in humans.     

Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates

Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or over-expression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation.