Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.     

Helix 1/8 interactions influence the activity of nuclear receptor ligand-binding domains

The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)gamma and nerve growth factor inducible (NGFIB)beta and identify residues important for stabilization of the intramolecular interactions induced by PPARgamma ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPARgamma as a dynamic structure.     

Human egasyn binds beta-glucuronidase but neither the esterase active site of egasyn nor the C terminus of beta-glucuronidase is involved in their interaction

Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.     

Structural and functional organization of a porcine gene coding for nuclear factor I

This paper describes the structure of a 70-kb porcine gene for nuclear factor I, including its promoter region, comprising a total of 11 exons. Different mRNAs that we have isolated as cDNAs from both porcine liver and human HeLa cells presumably are generated from this gene by differential splicing events. One cDNA species from porcine liver that lacks exon 9 carries coding information for a protein of 439 amino acids. The in vitro translated protein displays all the properties of an NFI-like protein with high affinity toward the sequence element TGG(N)6GCCAA, as shown by gel shift analysis, and no or little affinity toward CCAAT box containing sequences. Cotranslation experiments with full-length and truncated variants of the protein demonstrate that it binds as a dimer to its cognate DNA recognition sequence. Its DNA-binding domain which is retained in all cDNA clones was mapped by deletion analysis to the 250 N-terminal amino acids of the protein. No structural homologies are observed between this protein and other known DNA-binding proteins; instead, the protein contains a novel alpha-helical sequence motif consisting of several lysine residues spaced at intervals of seven amino acids which we have termed the "lysine helix". The C-terminal portion of the protein derived from full-length cDNAs encodes a short amino acid sequence which is identical with the heptapeptide repeat CT7 observed in the C-terminal domain of the largest subunits of yeast and mouse RNA polymerase II. This region is removed by differential splicing in some of the NFI/CTF cDNAs and thus may be of functional significance.