Ran binding protein RanBP1 in zebrafish embryonic development.

Ran, which functions in nucleocytoplasmic transport and mitosis, binds to and is regulated in part by RanBP1. We have identified a zebrafish RanBP1 cDNA and report that it encodes for a polypeptide of 233 amino acids with considerable similarity to human and Xenopus RanBP1, despite the fact that it is 10% longer due to an extension at its carboxy terminus. RanBP1 mRNA is present as a maternal transcript and is expressed ubiquitously throughout the developing embryo. At the protein level, RanBP1 is present at all embryonic stages. Surprisingly, the ectopic overexpression of the protein had no obvious effect on embryogenesis. Attempts were also made to down-regulate RanBP1 activity by RNA interference. Injecting double-stranded RNA augmented both the mortality rate and the frequency of induced defects. Specific defects accompanied by changes in RanBP1 expression were not seen, leading us to propose that RNAi is not a reliable method for deregulating the activity of constitutively expressed genes, like RanBP1, in zebrafish. Mol. Reprod. Dev. 59:235-248, 2001.

Differential distribution of the G protein gamma3 subunit in the developing zebrafish nervous system.

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein alpha subunit was the sole regulator of intracellular molecules. The G protein betagamma complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein gamma subunit. The 666 nucleotides of the zebrafish G protein gamma subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse gamma subunits. BLAST search analysis of GenBank revealed that the zebrafish gamma subunit is 93% identical and 97% similar to the mammalian gamma3 subunit. The gamma3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected gamma3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish gamma3 subunit to form a signaling heterodimeric complex with a beta subunit was tested using a human beta2 subunit. The gamma3 formed a heterodimer with beta2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the beta2gamma3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a betagamma heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.

The Translocon-Associated Protein beta (TRAPbeta) in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors.

The normal translocation of nascent polypeptides into the lumen of the endoplasmic reticulum (ER) is thought to be aided in part by a translocon-associated protein (TRAP) complex consisting of 4 protein subunits. The association of mature proteins with the ER and Golgi, or other intracellular locales, such as lysosomes, depends on the initial targeting of the nascent polypeptide to the ER membrane. A similar scenario must also exist for proteins destined for secretion. We have identified a member of the TRAP complex using a two hybrid screen to isolate proteins that bind to zebrafish (Danio) Ran binding protein 1. The polypeptide predicted from the largest open reading frame contains 183 amino acids with a 86 and 87% sequence identity to the TRAPbeta subunits in human and chicken, respectively. Sequence analysis identified a cleavable amino-terminal signal peptide in the zebrafish TRAPbeta subunit and a region of the protein spans the membrane of the endoplasmic reticulum. A reverse transcriptase-polymerase chain reaction assay showed that TRAPbeta mRNA is expressed in the developing zebrafish embryo. TRAPbeta mRNA is maternally supplied to the egg and is expressed constitutively throughout development and in the adult. This pattern of expression indicates that the message encoding part of the machinery targeting nascent polypeptides to the ER lumen is available at the onset of embryogenesis when the rate of translation increases exponentially over that occurring in the oocyte. In situ hybridization was used to test whether or not TRAPbeta transcripts might become localized and/or enriched in the developing embryo. Homogeneous staining is seen in the blastula and early gastrula stages. At mid-to-late gastrula stages, however, the message becomes enriched in the developing notochord and polster, or hatching gland rudiment. The TRAPbeta gene, mapped using the LN54 mouse-zebrafish radiation hybrid panel to linkage group 19, resides next to a gene (Z15451) which has sequence homology to notch2 and vascular endothelial growth factor. TRAPbeta, however, does not appear to belong to a group of genes which are syntenic with orthologues or paralogues on human chromosomes.

Induction and regulation of CYP2E1 in murine liver after acute and chronic acetone administration.

The effects of acute and chronic acetone administration on hepatic Cyp2e1 were investigated in mice. Acute treatment consisted of a single dose of acetone (5 ml/kg) given intragastrically, whereas the chronic regimen consisted of 1% acetone in drinking water for 8 days. We examined 1) relative induction of Cyp2e1 protein by immunoblotting, 2) relative induction of enzyme catalytic activity (p-nitrophenol hydroxylation), and 3) Cyp2e1 mRNA levels associated with acute and chronic treatment regimens. Western immunoblotting, using a monoclonal antibody (Mab 1-98-1) specific for rat ethanol-inducible P-450, detected a band of M(r) 51,000 in liver microsomes of both control and acetone-treated mice. Densitometric quantitation showed significant enhancement of the intensity of this band by 4.4- and 5.3-fold after acute and chronic acetone treatments, respectively. Hydroxylation of p-nitrophenol was increased 2.3-fold in microsomes from livers exposed acutely to acetone, as compared with an increase of 3.7-fold in microsomes from livers exposed chronically. The induction of Cyp2e1 protein, as well as of catalytic activity, by acetone was not accompanied by significant alterations in the levels of Cyp2e1 mRNA. These results demonstrate a difference in induced increases of Cyp2e1 between acute and chronic acetone treatments: significantly higher induction of both protein and catalytic activity is induced by treatment under chronic vs. acute conditions.