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A Emery-Goodman

Publications and source records attributed to A Emery-Goodman.

6 recordsLinked to original sources
PubMed1993

The putative iron-responsive element in the human erythroid 5-aminolevulinate synthase mRNA mediates translational control.

The 52-nucleotide 5'-untranslated region of the human erythroid 5-aminolevulinate synthase mRNA contains a 28-nucleotide iron-responsive element-like stem-loop motif. We fused the 5'-untranslated region upstream to the coding sequence of the human growth hormone cDNA. A chimeric construct containing a mutated variant of the presumptive iron-responsive element was similarly synthesized. Translation of the wild type chimeric transcript was markedly repressed (approximately 95%) in rabbit reticulocyte lysates as opposed to the mutant. Both transcripts translated with comparable efficiency in wheat germ extracts. Purified placental iron regulatory factor selectively and markedly inhibited translation of the wild type chimeric transcript (> 90%) when tested in wheat germ extracts. By contrast, translations of either the mutant chimeric transcript or other control mRNA species were unaffected. The proximal position of the iron-responsive element relative to the cap site was shown to be important for translational control, in vitro. Our studies suggest that interaction of the iron regulatory factor with the iron-responsive element sterically hinders formation of the preinitiation complex, resulting in translational repression. Thus inactivation of the repressor protein by critical levels of iron or heme would trigger translation of this mRNA in erythroid cells. Consequently, protoporphyrin and heme synthesis would be subtly coordinated with iron supply.

5-Aminolevulinate Synthetase↗
PubMed1993

Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase.

Iron regulatory factor (IRF) is a cytoplasmic mRNA-binding protein that coordinates post-transcriptionally the expression of several important proteins in iron metabolism. Binding of IRF to iron-responsive elements (IRE) in the 5' untranslated region (UTR) of ferritin and erythroid 5-aminolevulinic acid-synthase mRNAs inhibits their translation, whereas binding to IREs in the 3' UTR of transferrin receptor (TfR) mRNA prevents the degradation of this mRNA. IRF binds RNA strongly after iron deprivation, but is inactive, yet present, under conditions of high cellular iron supply. Recently, IRF was also shown to have aconitase activity indicating the existence of an Fe-S cluster in the protein. In the current study we expressed human IRF in insect cells from recombinant baculovirus and analysed IRE-binding and aconitase activities under various culture conditions. Newly made apoprotein, synthesized in the absence of iron, was fully active in IRE-binding, but showed no aconitase activity. In contrast, IRF made by cells grown in high iron medium bound RNA poorly, but exhibited high aconitase activity with a Km of 9.2 microM for cis-aconitate. Apo-IRF was converted in vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost its RNA-binding capacity. These results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster.

Aconitate Hydratase↗
PubMed1992

Epstein-Barr virus burden in Hodgkin's disease is related to latent membrane protein gene expression but not to active viral replication.

The Epstein-Barr virus (EBV) has been increasingly detected in Hodgkin's disease (HD), but its role in pathogenesis remains uncertain. We analyzed 20 specimens of HD known to contain EBV DNA by a sensitive reverse transcriptase polymerase chain reaction (RT-PCR). The cases were assessed for the presence of RNA transcripts of the BNLF1 gene (coding for the viral latent membrane protein [LMP]) and the late replicative gene BLLF1 (coding for the principle envelope glycoprotein [gp220/350]). LMP RNA transcripts were found in 9 of 20 (45%) cases, mostly those containing many copies of viral DNA and of mixed cellularity (MC) histological subtype. Only one LMP RNA-positive case was also positive for RNA transcripts of the active replication gene BLLF1. Our results show that viral burden in HD is not primarily related to active viral replication, but is associated with LMP gene expression.

Adolescent↗
PubMed1992

Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation.

Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene.

Amino Acid Sequence↗