Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin, oriP. This activation involves the direct interaction of EBNA1 dimers with multiple sites within the two noncontiguous functional elements of the origin, the family of repeats (FR) element and the dyad symmetry (DS) element. The efficient interaction of EBNA1 dimers bound to these two elements in oriP results in the formation of DNA loops in which the FR and DS elements are bound together through EBNA1. In order to elucidate the mechanism by which EBNA1 induces oriP DNA looping, we have investigated the DNA sequences and EBNA1 amino acids required for EBNAl-mediated DNA looping. Using a series of truncation mutants of EBNA1 produced in baculovirus and purified to apparent homogeneity, we have demonstrated that the EBNA1 DNA binding and dimerization domain is not sufficient to mediate oriP DNA looping and that an additional region(s) located between amino acids 346 and 450 is required. Single EBNAl-binding sites, separated by 930 bp of plasmid DNA, were also shown to support EBNAl-mediated looping, indicating that the formation of large EBNA1 complexes, such as those observed on oriP FR and DS elements, is not a requirement for looping.
The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.
The caudal neurosecretory system of the flounder (Platichthys flesus) has been examined by immunocytochemistry and in situ hybridization for the expression of parathyroid hormone-related protein (PTHrP) and calcium-sensing receptors (CaSR). The N-terminus nucleotide and deduced amino acid sequences of flounder PTHrP were determined and used to prepare oligonucleotide probes and homologous antiserum. The Dahlgren cells of the posterior spinal cord and their axons contained PTHrP protein which was also detected around the capillaries of the urophysis. PTHrP gene expression was abundant in the Dahlgren perikarya and axons in the spinal cord, but it was absent from nerve endings in the urophysis. Calcium-sensing receptor protein was present in the Dahlgren perikarya and axons, also with abundant gene expression, but there was neither protein nor mRNA in the urophysis.There were no apparent differences between freshwater-and seawater-adapted fish in either CaSR or PTHrP expression in the caudal neurosecretory system. These observations suggest that Dahlgren cells produce PTHrP which may be released from axons abutting capillaries in the urophysis. However, the sensing of ionic calcium appears to be confined to the perikarya of the Dahlgren cells in the spinal cord neuropil, suggesting that they are responsive to calcium in the central nervous system rather than the general circulation.
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