Only three retrotransposons of the DIRS1 group have previously been described: DIRS1 from the slime mold Dictyostelium discoideum, PAT from the nematode Panagrellus redivivus, and Prt1 from the zygomycetous fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these elements suggest that they are related to the long terminal repeat (LTR) retroelements, such as the Ty3/gypsy retrotransposons and the vertebrate retroviruses. The DIRS1-group elements, however, have several unusual structural features which distinguish them from typical LTR elements: (1) they lack the capacity to encode DDE-type integrases or aspartic proteases; (2) they have open reading frames (ORFs) of unknown function; (3) they integrate without creating duplications of their target sites; and (4) although they are bordered by terminal repeats, these sequences differ from typical LTRs in that they are either inverted repeats or "split" direct repeats. Because of the small number of DIRS1-like elements described, and the unusual structures of these elements, little is known about their evolution, distribution, and replication mechanisms. Here, we report the identification of several new DIRS1-like retrotransposons, including elements from nematodes, sea urchins, fish, and amphibia. We also present evidence for the existence of DIRS1-like sequences in the human genome. In addition, we show that the lack of DDE-type integrase genes from elements of the DIRS1 group is explained by the finding that the previously uncharacterized ORFs of these elements encode proteins related to the site-specific recombinase of bacteriophage lambda. The presence of lambda-recombinase-like genes in DIRS1 elements also accounts for the lack of target-site duplications for these elements and may be related to the unusual structures of their terminal repeats. (+info)
One INK4 gene and no ARF at the Fugu equivalent of the human INK4A/ARF/INK4B tumour suppressor locus.
The INK4A/ARF/INK4B locus, conserved in mammals, encodes three polypeptides that regulate cell proliferation via the pRb and p53 tumour suppressor pathways. The locus is mutated in many cancers. The related, tandemly-linked INK4A and INK4B genes encode the p16(INK4A) and p15(INK4B) members of the INK4 family of cyclin-dependent kinase inhibitors which block phosphorylation of pRb, whereas the third product, ARF, derived from an alternative reading frame of INK4A, regulates p53 activity. We assessed the status of this unusual locus in the puffer fish, Fugu rubripes, and identified two INK4 genes using degenerate PCR and hybridization analyses. Sequence conservation and conservation of synteny between human and Fugu predict one gene to be an INK4A or INK4B homologue and the other an INK4D homologue. Analysis of the Fugu INK4A/B gene and the surrounding 40-kb of genomic DNA did not reveal the presence of any ARF-encoding potential or another related INK4 gene. We conclude that the gene duplication event that generated adjacent INK4A and INK4B genes and the association of ARF with the ancestral INK4A gene occurred after the divergence of the lineage leading to mammals from fish. Thus, unlike mammals, the fish p53 and pRb tumour suppressor pathways are not regulated by a single locus. (+info)
Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.
Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish. (+info)
Contrasting, species-dependent modulation of copper-mediated neurotoxicity by the Alzheimer's disease amyloid precursor protein.
The amyloid precursor protein (APP) of Alzheimer's disease (AD) has a copper binding domain (CuBD) located in the N-terminal cysteine-rich region that can strongly bind copper(II) and reduce it to Cu(I) in vitro. The CuBD sequence is similar among the APP family paralogs [amyloid precursor-like proteins (APLP1 and APLP2)] and its orthologs (including Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans), suggesting an overall conservation in its function or activity. The APP CuBD is involved in modulating Cu homeostasis and amyloid beta peptide production. In this paper, we demonstrate for the first time that Cu-metallated full-length APP ectodomain induces neuronal cell death in vitro. APP Cu neurotoxicity can be induced directly or potentiated through Cu(I)-mediated oxidation of low-density lipoprotein, a finding that may have important implications for the role of lipoproteins and membrane cholesterol composition in AD. Cu toxicity induced by human APP, Xenopus APP, and APLP2 CuBDs is dependent on conservation of histidine residues at positions corresponding to 147 and 151 of human APP. Intriguingly, APP orthologs with different amino acid residues at these positions had dramatically altered Cu phenotypes. The corresponding C. elegans APL-1 CuBD, which has tyrosine and lysine residues at positions 147 and 151, respectively, strongly protected against Cu-mediated lipid peroxidation and neurotoxicity in vitro. Replacement of histidines 147 and 151 with tyrosine and lysine residues conferred this neuroprotective Cu phenotype to human APP, APLP2, and Xenopus APP CuBD peptides. Moreover, we show that the toxic and protective CuBD phenotypes are associated with differences in Cu binding and reduction. These studies identify a significant evolutionary change in the function of the CuBD in modulating Cu metabolism. Our findings also suggest that targeting of inhibitors to histidine residues at positions 147 and 151 of APP could significantly alter the oxidative potential of APP. (+info)
V-SINEs: a new superfamily of vertebrate SINEs that are widespread in vertebrate genomes and retain a strongly conserved segment within each repetitive unit.
We have identified a new superfamily of vertebrate short interspersed repetitive elements (SINEs), designated V-SINEs, that are widespread in fishes and frogs. Each V-SINE includes a central conserved domain preceded by a 5'-end tRNA-related region and followed by a potentially recombinogenic (TG)(n) tract, with a 3' tail derived from the 3' untranslated region (UTR) of the corresponding partner long interspersed repetitive element (LINE) that encodes a functional reverse transcriptase. The central domain is strongly conserved and is even found in SINEs in the lamprey genome, suggesting that V-SINEs might be approximately 550 Myr old or older in view of the timing of divergence of the lamprey lineage from the bony fish lineage. The central conserved domain might have been subject to some form of positive selection. Although the contemporary 3' tails of V-SINEs differ from one another, it is possible that the original 3' tail might have been replaced, via recombination, by the 3' tails of more active partner LINEs, thereby retaining retropositional activity and the ability to survive for long periods on the evolutionary time scale. It seems plausible that V-SINEs may have some function(s) that have been maintained by the coevolution of SINEs and LINEs during the evolution of vertebrates. (+info)
New 3' elements control Pax6 expression in the developing pretectum, neural retina and olfactory region.
Pax6 is a key transcriptional regulator in eye, olfactory system, forebrain, pituitary cerebellum, spinal cord and pancreas development. Alternative splicing, promoter usage and multiple enhancers regulate the complex Pax6 spatio-temporal expression pattern. Chromosomal rearrangements which abolish PAX6 gene expression have been characterised downstream of the coding region. Through evolutionary sequence comparison and transgenic reporter studies, we have identified a new Pax6 3' cis-regulatory region. This region, C1170 Box 123, contains three distinct modules of human-mouse sequence conservation, while only Box 1 is conserved to Fugu. Both the human and the orthologous Fugu sequence direct similar reporter gene expression in the developing pretectum, neural retina and olfactory region, indicating evolutionary conservation of Pax6 regulatory mechanisms despite the low level of overall sequence conservation. (+info)
Xena, a full-length basal retroelement from tetraodontid fish.
Mobile genetic elements are ubiquitous throughout the eukaryote superkingdom. We have sequenced a highly unusual full-length retroelement from the Fugu fish, Takifugu rubripes. This element, which we have named Xena, is similar in structure and sequence to the Penelope retroelement from Drosophila virilis and consists of a single long open reading frame containing a reverse transcriptase domain flanked by identical direct long terminal repeat (LTR) sequences. These LTRs show an organization similar to the terminal repeats already described in the Penelope retrotransposon of Drosophila but are structurally and functionally distinct from the LTRs carried by LTR-retrotransposons. In view of their distinctness, we refer to these repeats as PLTRs (Penelope-LTRs). Whereas the element contains a reverse transcriptase, no other domains or motifs commonly associated with retroelements are present. In the full-length Fugu element, the 5' direct PLTR is preceded by an inverted PLTR fragment. Additional elements, many showing various degrees of deletion, are described from the Fugu genome and from that of the freshwater pufferfish Tetraodon nigroviridis. Many of these additional elements are also preceded by inverted PLTR sequences. Xena-like elements are also described from the genomes of several other organisms. The Penelope-Xena lineage is apparently a basal group within the retrotransposons and therefore represents an evolutionarily important class of retroelement. (+info)
Conserved regulation of the lymphocyte-specific expression of lck in the Fugu and mammals.
The lck gene encodes a lymphocyte-specific protein-tyrosine kinase that is implicated in T cell maturation and signaling. In mammals, the transcription of the lck gene is regulated by two independent promoters, the proximal promoter, which is active in thymocytes, and the distal promoter, which dominates in mature T cells. In the human and mouse lck gene loci, the two promoter elements are separated by at least 40 kb and 10 kb, respectively. In this study, we have cloned and sequenced 60 kb from the pufferfish (Fugu rubripes) lck locus. The promoter region of the Fugu lck spans only 4.2 kb and contains a proximal and a distal promoter in the 2.3-kb region adjacent to the coding sequence. By generating transgenic mice, we have demonstrated that the compact promoter of the Fugu lck contains regulatory elements that direct expression to lymphoid organs of mice. We were able to localize the regulatory elements to a short region of 830 bp without losing specificity to cultured human T cell line. These results show that the basic mechanisms that mediate lymphocyte-specific expression are conserved between teleosts and mammals. The short promoter of the Fugu lck isolated by us offers a powerful tool for labeling T cells, targeting expression, and manipulating T cell activity in fishes as well as in mammals. (+info)