A series of plasmids was constructed to study the effect of two enhancers, the simian virus 40 72-base-pair repeat and the Harvey sarcoma virus 73-base-pair repeat, on the mouse ma'J-globin promoter. These plasmids contain the mouse Pm"-globin promoter linked to the Escherichia coli galK gene, thus allowing galactokinase enzyme activity to be used as a measure of promoter function. In CV-1 (primate) cells, it was found that an enhancer is required for optimal promoter activity and that the simian virus 40 (primate) enhancer increases galactokinase fourfold more than the Harvey sarcoma virus (mouse) enhancer. In L (mouse) cells, however, the Harvey sarcoma virus enhancer is 1.3-fold stronger than the simian virus 40 enhancer. These data support the hypothesis that enhancer activity can be species specific. Furthermore, when both enhancers are present on the same plasmid, their effect is additive on the ,-globin promoter whether the plasmid is in CV-1 cells or L cells.The mouse ,B-globin gene serves as a valuable model for the study of gene expression in mammals. We wished to develop expression vectors which would allow us to optimize the expression of cloned mouse 3-globin genes that are transferred into mouse tissue culture cells or into intact animals. Important elements that have been shown to stimulate the expression of certain mammalian genes have recently been discovered. These are cis DNA sequences called enhancers (1,2,(4)(5)(6)(7)(11)(12)(13)(14). As a first step toward obtaining regulated globin gene expression in mice, we examined the effect of two different enhancing sequences, the monkey simian virus 40 (SV40) 72-base-pair (bp) repeat and the mouse Harvey sarcoma virus (HaSV) 73-bp repeat, on the expression of a cloned mouse P'ai-globin promoter in both monkey (CV-1) cells and mouse (L) cells.Specifically, we asked several questions. (i) Studies have been performed with the SV40 enhancer with its natural promoter, the SV40 early promoter (2,4,6,11,13). Does the SV40 enhancer have a similar effect on the mouse pglobin promoter? (ii) The SV40 and the murine Moloney sarcoma enhancer act differently on the SV40 early promoter (11), and the SV40 and polyoma enhancers show cell-specific stimulation of the SV40 and rabbit P-globin promoters (4). Is this cell (species)-specific effect a general one? (iii) Do two different enhancers have an additive effect on promoter activity?To answer these questions, we constructed a series of plasmids containing the mouse jmajglobin promoter linked to the Escherichia coli galactokinase gene (galK). This allows us to measure galactokinase (Gal K) levels as a function of the activity of the P-globin promoter.Some constructs also contain a second assayable E. coli gene, gpt, which is driven by the SV40 early promoter, giving an internal control for transformation frequency. These constructions are based on the system described by Schumperli et al. (16). The SV40 72-bp repeat and the HaSV 73-bp repeat were added singly and in combination to these constructs. The Gal K and gpt...

Mechanisms of post-transcriptional regulation of gene expression include control of initiation of translation and regulation of mRNA degradation. Among the best-studied models for these processes is regulation of proteins involved in iron homeostasis. These control mechanisms involve functional iron-responsive elements (IREs) in the 5¢-UTRs or 3¢-UTRs of mRNAs that interact with iron regulatory proteins (IRPs), depending upon the amount of iron present in the cell. Two IRPs have been identified: IRP-1, which contains a 4Fe)4S iron-sulfur cluster [1], and IRP-2, which does not [2,3]. IRP-1 has 30% amino acid identity to mitochondrial aconitase [4], a 4Fe)4S enzyme involved in the tricarboxylic acid cycle. IRP-1 is generally believed to interconvert between an enzymatically inactive IRE-binding state and a nonbinding form with aconitase activity, the latter requiring an intact 4Fe-4S cluster. Thus, the simple model for iron sensing by IRP-1 involves direct association of iron with the ironsulfur center to form a complete 4Fe)4S cluster.A linkage between cellular iron levels and energy metabolism is suggested by the influence of agents that Iron regulatory protein-1 binding to the iron-responsive element of mRNA is sensitive to iron, oxidative stress, NO, and hypoxia. Each of these agents changes the level of intracellular ATP, suggesting a link between iron levels and cellular energy metabolism. Furthermore, restoration of iron regulatory protein-1 aconitase activity after NO removal has been shown to require mitochondrial ATP. We demonstrate here that the iron-responsive element-binding activity of iron regulatory protein is ATP-dependent in HepG2 cells. Iron cannot decrease iron regulatory protein binding activity in cell extracts if they are simultaneously treated with an uncoupler of oxidative phosphorylation. Physiologic concentrations of ATP inhibit ironresponsive element ⁄ iron regulatory protein binding in cell extracts and binding of iron-responsive element to recombinant iron regulatory protein-1. ADP has the same effect, in contrast to the nonhydrolyzable analog adenosine 5¢-(b,c-imido)triphosphate, indicating that in order to inhibit iron regulatory protein-1 binding activity, ATP must be hydrolyzed. Abbreviations AMP-PNP, adenosine 5¢-(b,c-imido)triphosphate; ATP-cS, adenosine 5¢-O-(3-thiotriphosphate); CCCP, carbonyl cyanide m-chlorophenylhydrazone; EMSA, electrophoretic mobility shift assay; IRE, iron-responsive element; IRP, iron regulatory protein.

Iron may populate distinct hepatocellular iron pools that differentially regulate expression of proteins such as ferritin and transferrin receptor (TfR) through iron-regulatory mRNA-binding proteins (IRPs), and may additionally regulate uptake and accumulation of non-transferrin-bound iron (NTBI). We examined iron-regulatory protein (IRP) binding activity and ferritin/TfR expression in human hepatoma (HepG2) cells exposed to iron at different levels for different periods. Several iron-dependent RNA-binding activities were identified, but only IRP increased with beta-mercaptoethanol. With exposures between 0 and 20 microg/ml iron, decreases in IRP binding accompanied large changes in TfR and ferritin expression, while chelation of residual iron with deferoxamine (DFO) caused a large increase in IRP binding with little additional effect on TfR or ferritin expression. Cellular iron content increased beyond 4 days of exposure to iron at 20 microg/ml, when IRP binding, TfR, and ferritin had all reached stable levels. However, iron content of the cells plateaued by 7 days, or decreased with 24 h exposure to very high concentrations (>50 microg/ml) of iron. These results indicate that iron-replete HepG2 cells exhibit a narrow range of maximal responsiveness of the IRP-regulatory mechanism, whose functional response is blunted both by excessive iron exposure and by removal of iron from a chelatable pool. HepG2 cells are able to limit iron accumulation upon higher or prolonged exposure to NTBI, apparently independent of the IRP mechanism.

The interaction of enhancers with different promoters was studied by measuring the influence of two enhancers (from simian virus 40 and from Harvey sarcoma virus) on the activity of expression vectors that are identical except for their promoter region. The promoters examined were from the simian virus 40 early region, with or without its own 72-base-pair repeat, and the mouse IInaJor-globin gene. It is clear that the promoter acted upon strongly influences the level of activity of an enhancer.It is known that DNA sequences called enhancers can activate certain cloned genes (1, 2, 7, 9, 15-17, 21, 26). A number of enhancers have been identified, both of viral and cellular origin (1,7,12,15,21,22,(24)(25)(26). Enhancers act in cis in an orientation-independent manner (10,26,30). Factors known to influence enhancer activity include the cell line the enhancer is in and, in some cases, its position relative to the promoter being tested (1,4,6,10,20,26 that galactokinase activity could be used to measure promoter function.The basic plasmid used for these constructions was pSVK100, described in detail elsewhere (27). It contains the SV40 early promoter driving the E. coli galK gene (Fig. 1). The 72-bp repeat (enhancer) was cleaved from pSVK100 by digestion with PvuII and SphI, which deletes 142 bp, leaving the terminal 21 bp of one enhancer (26). This deletion is sufficient to eliminate detectable enhancer activity (11,14,26). The resultant DNA was ligated to give pZP1 (Fig. 1). The exact deletion size was confirmed by DNA sequencing (data not shown). pZP1, then, contains the SV40 early promoter with no enhancer (SV40d). The HaSV enhancer was added to give pZP2, whereas the SV40 enhancer was added to pZP1 at the BamHI site, ca. 2 kilobase pairs from its location in pSVK100, giving pZP1-gpt. The final plasmid, pZP2-gpt, contains both enhancers and was constructed by inserting the BamHI fragment just described into pZP2, which already has the HaSV enhancer. A schematic of these plasmids is shown in Fig. 2A. Figure 2B shows the parallel constructions with the mouse maJor-globin promoter. The following plasmids are identical except for their promoter: pZP1 and pPB12; pZP2 and pPB12H; pZP1-gpt and pPB22; pZP2-gpt and pPB22H. Details of the construction of pPB12, pPB12H, pPB22, and pPB22H have been reported (4). (Tables 1 and 2). Transfections and galactokinase assays were as described previously (3,4). A comparison of the ratios of the galactokinase observed in the SV40-enhanced plasmid to that in the unenhanced plasmid in CV-1 cells for the two promoters indicated a large difference in enhancement, i.e., the SV40 enhancer activates the ,Bglobin promoter much more than the SV40d promoter (a ratio of 13 for the ,B-globin promoter and 1.3 for the SV40d promoter). The SV40 enhancer was 60% stronger with the Pglobin promoter than with the SV40d promoter in L cells, whereas in R1610 cells, the SV40 enhancer was fivefold stronger with the ,B-globin promoter. The situation was similar for the HaSV enhancer; in all cases it was m...

Iron regulatory protein 1 (IRP-1) is a bifunctional protein involved in iron homeostasis and metabolism. In one state, it binds to specific sequences in the mRNA's of several proteins involved in iron and energy metabolism, thereby influencing their expression post-transcriptionally. In another state it contains a [4Fe-4S] iron-sulfur cofactor and displays aconitase activity in the cytosol. We have shown that this protein binds and hydrolyzes ATP, with kinetic and thermodynamic equilibrium constants that predict saturation with ATP, favouring a non-RNA-binding form at normal cellular ATP levels, and thus pointing to additional function(s) of the protein. Here we show for the first time that the RNA-binding and aconitase forms of IRP-1 can undergo interconversion dependent on the density of cells growing in culture. Thus, in high density confluent cultures, compared with low density, actively proliferating cultures, cytosolic aconitase activity is increased whereas RNA binding activity is diminished. This is accompanied by a decrease in transferrin receptor expression in confluent cells, possibly due to loss of the transcript-stabilizing activity of bound IRP-1. In high density HepG2 cultures, cytosolic glutamate and the ratio of reduced-to-oxidized glutathione were increased. We propose that increased cytosolic aconitase activity in confluent cultures may divert cytosolic citrate away from the fatty acid/membrane synthetic pathways required by dividing cells, into a glutamate-dependent maintenance of cellular macromolecular synthesis. In addition, this may confer additional protection from oxidative stress due to down-regulation of iron acquisition from transferrin and increased glutamate for glutathione synthesis.