Three receptor genes for plasminogen related growth factors in the genome of the puffer fish Fugu rubripes.
Plasminogen related growth factors (PRGFs) and their receptors play major roles in embryogenesis, tissue regeneration and neoplasia. In order to investigate the complexity and evolution of the PRGF receptor family we have cloned and sequenced three receptors for PRGFs in the teleost fish Fugu rubripes, a model vertebrate with a compact genome. One of the receptor genes isolated encodes the orthologue of mammalian MET, whilst the other two may represent Fugu rubripes orthologues of RON and SEA. This is the first time three PRGF receptors have been identified in a single species. (+info
Multiple binding sites in the growth factor receptor Xmrk mediate binding to p59fyn, GRB2 and Shc.
Melanoma formation in Xiphoporus is initiated by overexpression of the EGFR-related receptor tyrosine kinase Xmrk (Xiphoporus melanoma receptor kinase). This receptor is activated in fish melanoma as well as in a melanoma-derived cell line (PSM) resulting in constitutive Xmrk-mediated mitogenic signaling. In order to define the underlying signaling pathway(s), triggered by the activated Xmrk receptor, we attempted to identify its physiological substrates. Examination of the Xmrk carboxyterminus for putative tyrosine autophosphorylation sites revealed the presence of potential binding motifs for GRB2 as well as for Shc. Binding of these adaptor proteins to the Xmrk receptor was detected in vitro and in cells expressing the mrk kinase. The GRB2 and Shc interactions with the receptor could be disrupted individually by phosphotyrosine peptides containing putative Xmrk autophosphorylation sites, indicating direct binding of both proteins. Recruitment of GRB2 by the constitutively activated Xmrk receptor led to strong MAP kinase activation in Xiphoporus melanoma cells. We also identified a high-affinity binding site for src-kinases (pYEDL) in the Xmrk carboxyterminus. Competition experiments with phosphopeptides comprising this site confirmed that it is used for high-affinity binding of Xiphoporus fyn (Xfyn) to Xmrk in melanoma cells. Thus, Xmrk can initiate different signaling pathways by using multiple substrate-binding sites to trigger proliferation of pigment cells. (+info
Structure and expression of the highly repetitive histone H1-related sperm chromatin proteins from winter flounder.
In the late stages of spermatogenesis, winter flounder produce a family of high molecular mass (80-200 kDa) basic nuclear proteins (HMrBNPs) that combine with the normal complement of histones to produce condensed sperm chromatin with an increased nucleosomal repeat length. The HMrBNPs have a biased amino-acid composition in which Arg, Ser, Lys and Pro are abundant because of their presence in many simple peptide repeats. The organization of these repeats was deduced by cDNA cloning. The predominant repeating units are related 26- and 30-amino-acid sequences that in turn are linked by 6-amino-acid spacers to form 58- and 62-amino-acid repeats. Subsets of these repeats are also present, such as a dispersed 20-amino-acid repeat and a tandem array of nine heptapeptides at the C-terminus. The HMrBNPs appear to have evolved from an extreme H1 variant that has an N-terminal tail of HMrBNP-like sequence linked to an H1 globular region. Based on sequences of the most abundant HMrBNP cDNAs, and the lack of hybridization between HMrBNP mRNAs and a DNA probe for the H1 globular region, the latter domain appears to have been lost during expansion and amplification of the HMrBNP-like repeats. Transcripts of the HMrBNP and H1 variant genes are present in testis RNAs only during the mid-spermatid stage of spermatogenesis, at the same time that HMrBNPs in their highly phosphorylated form first appear in the nucleus. Judging by the lack of a lag between HMrBNP mRNA synthesis and translation, the mRNAs for these highly basic proteins are not stored for any length of time. Instead, the deposition of HMrBNPs onto DNA, which coincides with the major reorganization and silencing of the chromatin, may be controlled by dephosphorylation. (+info
Expression of the medaka (Oryzias latipes) Ol-Rx3 paired-like gene in two diencephalic derivatives, the eye and the hypothalamus.
Here we report the expression pattern of the homeobox Ol-Rx3 gene, a medaka gene homologous to the mouse, Xenopus, zebrafish and Drosophila Rx genes. Ol-Rx3 starts to be expressed, at late gastrula stages, in the presumptive territories of the anterior brain. Subsequently, transcripts are localised in an antero-ventral region of the prosencephalon and in the primordia of the optic vesicles. During organogenesis, distribution of Ol-Rx3 transcripts are gradually restricted to the floor of the diencephalon, the prospective territory of the hypothalamus and the neurohypophysis. During late development and in adult, Ol-Rx3 expression is maintained in hypothalamic nuclei bordering the third ventricle. In the optic vesicles, Ol-Rx3 expression is temporarily switched off when the eye cup morphogenesis is complete, but it is turned on again in the inner nuclear layer of the retina. Thus, the early expression pattern of Ol-Rx3 is in agreement with a conserved role in the specification of the ventral forebrain and eye field. Putative functions linked to late expression domains are discussed in light of the different hypothesis concerning the involvement of vertebrate Rx genes in the maintenance of particular cell fate. (+info
Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.
Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin. (+info
Changes in the levels of mRNAs for GH/prolactin/somatolactin family and Pit-1/GHF-1 in the pituitaries of pre-spawning chum salmon.
Changes in the levels of pituitary mRNAs encoding GH, prolactin (PRL) and somatolactin (SL) were determined in pre-spawning chum salmon (Oncorhynchus keta) caught at a few key points along their homing pathway in 1994 and 1995. Furthermore, we analyzed relationships between expression of pituitary-specific POU homeodomain transcription factor (Pit-1/GHF-1) and GH/PRL/SL family genes. In 1994, seawater (SW) fish and matured fresh-water (FW) fish were sequentially captured at two points along their homing pathway, the coast and the hatchery. In addition to these two points, maturing FW fish were captured at the intermediate of the two points in 1995. The levels of hormonal mRNAs were determined by a quantitative dot blot analysis using single-stranded sense DNA as the standard. Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis. In 1994, the levels of GH/PRL/SL family mRNAs except for PRL mRNA in the male FW fish were 1.8-4 times higher than those in the SW fish. In 1995, the level of PRL mRNA was somewhat sharply elevated in the maturing FW fish soon after entry into the FW environment, while that of SL mRNA was gradually increased during upstream migration from the coast to the hatchery. The levels of 3 kb Pit-1/GHF-1 mRNA in the FW fish were higher than those in the SW fish in both 1994 and 1995. The present results indicate that expression of genes for the GH/PRL/SL family and Pit-1/GHF-1 is coincidentally enhanced in homing chum salmon. Moreover, the present study suggests that expression of the SL gene is elevated with sexual maturation, whereas that of PRL gene is elevated with osmotic change during the final stages of spawning migration. (+info
Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element.
The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus. (+info
Tandem repeat structure of rhamnose-binding lectin from catfish (Silurus asotus) eggs.
The primary structure of catfish (Silurus asotus) egg lectin (SAL) was determined. SAL cDNA contained 1448-bp nucleotides and 308 amino acid residues, deduced from open reading frame. The SAL mature protein composed of 285-amino acid residues was followed by a predicted signal sequence having 23 residues. The mRNA of SAL was found to be expressed in eggs, but not in liver. SAL is composed of three tandem repeat domain structures divided into exactly 95 amino acid residues each, and all cysteine positions of each domain were completely conserved. Sequence homologies between the three domains, termed D1 (1-95), D2 (96-190) and D3 (191-285), were as follows; D1-D2, 28%; D2-D3, 33%; D1-D3, 43%. Two conserved peptide motifs, -(AN)YGR(TD)S(T)XCS(TGR)P- and -DPCX(G)T(Y)KY(L)-, appear to exist at the N- and C-terminal regions of each domain, respectively. The kinetic parameters of SAL obtained by measuring surface plasmon resonance were as follows: K(a) (M(-1)) for neohesperidosyl-BSA, 7. 1 x 10(6); for melibiosyl-BSA, 4.9 x 10(6); and for lactosyl-BSA, 5. 2 x 10(5). These results show that RBLs including SAL comprise a family of alpha-galactosyl binding lectins having characteristic tandem repeat domain structures. (+info