Showing papers on "Psychological repression published in 1991"

Transcriptional regulation of a pair-rule stripe in Drosophila.

Abstract: The periodic, seven-stripe pattern of the primary pair-rule gene even-skipped (eve) is initiated by crude, overlapping gradients of maternal and gap gene proteins in the early Drosophila embryo. Previous genetic studies suggest that one of the stripes, stripe 2, is initiated by the maternal morphogen bicoid (bcd) and the gap protein hunchback (hb), while the borders of the stripe are formed by selective repression, involving the gap protein giant (gt) in anterior regions and the Kruppel (Kr) protein in posterior regions. Here, we present several lines of evidence that are consistent with this model for stripe 2 expression, including in vitro DNA-binding experiments and transient cotransfection assays in cultured cells. These experiments suggest that repression involves a competition or short-range quenching mechanism, whereby the binding of gt and Kr interferes with the binding or activity of bcd and hb activators at overlapping or neighboring sites within the eve stripe 2 promoter element. Such short-range repression could reflect a general property of promoters composed of multiple, but autonomous regulatory elements.

Active repression of transcription by the engrailed homeodomain protein.

Abstract: The Drosophila engrailed gene product (En) is a homeodomain-containing protein that contributes to segmental patterning. In transfection assays it acts as a transcriptional repressor. We show that En is an active repressor, blocking activation by mammalian and yeast activators that bind to sites some distance away from those bound by En. Active repression is distinct from the effects of passive homeodomain-containing proteins, which repress when competing with activators for binding sites and activate when competing with En. Active repression activity maps outside the En homeodomain, and this activity can be transferred to a heterologous DNA binding domain.

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII.

Abstract: Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.

Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression

Abstract: Glucose (catabolite) repression is mediated by multiple mechanisms that combine to regulate transcription of the GAL genes over at least a thousandfold range. We have determined that this is due predominantly to modest glucose repression (4- to 7-fold) of expression of GAL4, the gene encoding the transcriptional activator of the GAL genes. GAL4 regulation is affected by mutations in several genes previously implicated in the glucose repression pathway; it is not dependent on GAL4 or GAL80 protein function. GAL4 promoter sequences that mediate glucose repression were found to lie downstream of positively acting elements that may be "TATA boxes." Two nearly identical sequences (10/12 base pairs) in this region that may be binding sites for the MIG1 protein were identified as functional glucose-control elements. A 4-base-pair insertion in one of these sites causes constitutive GAL4 synthesis and leads to substantial relief (50-fold) of glucose repression of GAL1 expression. Furthermore, promoter deletions that modestly reduce GAL4 expression, and therefore presumably the amount of GAL4 protein synthesized, cause much greater reductions in GAL1 expression. These results suggest that GAL4 works synergistically to activate GAL1 expression. Thus, glucose repression of GAL1 expression is due largely to a relatively small reduction of GAL4 protein levels caused by reduced GAL4 transcription. This illustrates how modest regulation of a weakly expressed regulatory gene can act as a sensitive genetic switch to produce greatly amplified responses to environmental changes.

RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes.

Abstract: We show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is dependent on RPD1 (and RPD3 [M. Vidal and R. F. Gaber, Mol. Cell. Biol. 11:6317-6327, 1991]). Genes regulated by stimuli as diverse as external signals (PHO5), cell differentiation processes (SPO11 and SPO13), cell type (RME1, FUS1, HO, TY2, STE6, STE3, and BAR1), and genes whose regulatory signals remain unknown (TRK2) depend on RPD1 to achieve maximal states of transcriptional regulation. RPD1 enhances both positive and negative regulation of these genes: in rpd1 delta mutants, higher levels of expression are observed under repression conditions and lower levels are observed under activation conditions. We show that several independent genetic screens, designed to identify yeast transcriptional regulators, have detected the RPD1 locus (also known as SIN3, SD11, and UME4). The inferred RPD1 protein contains four regions predicted to take on helix-loop-helix-like secondary structures and three regions (acidic, glutamine rich, and proline rich) reminiscent of the activating domains of transcriptional activators.

GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats.

Abstract: Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.

Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway.

Abstract: Transcription of the fbpl gene, encoding fructose-l,6-bisphosphatase, of Schizosaccharomyces pombe is subject to glucose repression. Previous work has demonstrated that several genes {g/t genes} are required for this repression. In this report we demonstrate that one of these genes, git2, is the same as the cyrl gene, which encodes adenylate cyclase, and that loss-of-function mutations in git2 cause constitutive fbpl transcription. Addition of cAMP to the growth medium suppresses the transcriptional defect in git2 mutants as well as in strains that carry mutations in any of six additional g/t genes. Similarly, exogenous cAMP represses [bpl transcription in wild-type cells grown on a derepressing carbon source. Different levels of adenylate cyclase activity in different git2 mutants, coupled with the result that some git2 mutants display intragenic complementation, strongly suggest that adenylate cyclase acts as a multimer and that different git2 mutations alter distinct activities of adenylate cyclase, including catalytic activity and response to glucose. Additional experiments demonstrate that this cAMP signaling pathway is independent of the S. pombe rasl gene and works by activation of cAMP-dependent protein kinase.

Human cytomegalovirus ie2 negatively regulates alpha gene expression via a short target sequence near the transcription start site.

Abstract: Repression of human cytomegalovirus alpha (immediate-early) gene expression is under the control of the viral ie2 gene. Here we show that ie2 negatively regulates gene expression directed by the strong cytomegalovirus enhancer via a specific 15-bp target sequence (which we term cis repression signal [crs]). This crs is located between -14 and +1 relative to the transcription start site and will function in an orientation-independent fashion, consistent with repression occurring at the transcriptional level. Repression is dominant over transactivation by ie1 gene products. The crs (5'-CGTTTAGTGAACCGT-3') does not contain previously recognized binding sites for cellular transcription factors, and a precise copy is not found elsewhere in the human cytomegalovirus genome. The position of the signal near the transcription start site appears to be important in function; addition of the crs near the transcription start site of a heterologous promoter, from the thymidine kinase gene of herpes simplex virus type 1, conferred cytomegalovirus ie2-dependent repression upon this promoter. Thus, we propose that an ie2 gene product or an induced cellular protein mediates repression by binding to crs. Negative regulation of alpha gene expression may be important during viral replication or latency.

Concentration-dependent transcriptional activation or repression by Krüppel from a single binding site

Abstract: One of the gap class of segmentation genes, Kruppel (Kr), is required for normal thorax and abdominal development of the Drosophila embryo. Its gene product, a zinc-finger type protein, forms a bell-shape concentration gradient in a central position of the blastoderm. Genetic and molecular studies suggested that the Kr protein (KR) may act both as a positive and as a negative regulator of transcription on several other genes of the zygotic segmentation hierarchy. We have examined the regulatory potential of Kr by a series of cotransfection experiments in the Drosophila Schneider cell line system. Different doses of Kr expression plasmid were tested for their ability to drive reporter gene expression mediated by a single 11-base pair KR in vitro binding site common to several putative Kr target genes. Our results show that low amounts of Kr expression plasmid lead to transcriptional activation, whereas high amounts result in repression. Distinct portions of KR other than the DNA-binding domain are required for gene activation and repression, suggesting that KR itself can act as a concentration-dependent positive and negative regulator of transcription.

Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO.

Abstract: Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the 11-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc- mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc- mutants but exhibited wild-type inducible expression.

Functional analysis of E2-mediated repression of the HPV18 P105 promoter.

Abstract: Transcription of the transforming genes E6 and E7 of the human papillomavirus type 18 (HPV18) can be repressed by the product of the E2 open reading frame. Mutations were introduced in the cis-responsive elements upstream from the E6 and E7 transcriptional promoter, P105. The effect of these mutations in the absence or presence of E2 was examined in the human cervical carcinoma cell line C33, transfected with expression plasmids. In the presence of HPV18 E2, repression was relieved only when both of the two E2 binding motifs, immediately proximal to the P105 TATA box, were mutated. Mutation of a third E2 binding site, 110 nucleotides upstream of the TATA box, led to a slight but consistent activation in the presence of the full-length E2. Therefore, three E2 binding sites appear to be involved in the negative regulation of the P105 promoter by HPV18 E2. In the presence of the BPV1 E2 protein, only the E2 binding site most proximal to the promoter TATA box was involved in the P105 repression. The comparative analysis of the in vitro and in vivo properties of the human and bovine E2 proteins on the HPV18 P105 promoter provides new insights into the mechanism of transcriptional repression, which appears to involve steric hindrance at the site of transcriptional initiation.

Repression of the interferon signal transduction pathway by the adenovirus E1A oncogene

Abstract: The signal transduction pathway initiated by type I interferon (alpha and beta interferons) is inhibited by expression of the adenovirus type 5 E1A oncogene. Cotransfection analyses with the E1A oncogene and an interferon-stimulated reporter gene show that mutations within an amino-terminal domain of the E1A oncoprotein are defective in transcriptional repression. Cotransfection experiments also revealed that the transcriptional repression is mediated through the interferon-stimulated response element (ISRE) found within the promoter of interferon-stimulated genes. Since interferon treatment activates a latent cytoplasmic DNA-binding factor that can recognize the ISRE and subsequently stimulate transcription, the appearance of this factor was analyzed in a cell line that constitutively expresses the E1A oncogene. The DNA binding activity of this transcriptional activator was found to be inhibited in the E1A-expressing cell line. In vitro cytoplasmic mixing experiments with extracts from control and E1A-expressing cells identified a specific component of this multimeric transcription factor to be defective.

A general topoisomerase I-dependent transcriptional repression in the stationary phase in yeast.

Abstract: This paper shows that in the yeast Saccharomyces cerevisiae the levels of most mRNAs decrease, in a temporally orchestrated manner, as cells approach and enter the stationary phase. The decreased level of mRNAs is primarily due to transcriptional repression because the overall rate of in vivo transcription by RNA polymerase II is similarly reduced in the stationary phase. The reduction in mRNA levels and the general transcriptional repression are both dependent on topoisomerase I (encoded by TOP1). Specifically, these two processes are much slower in top1 mutants, as their mRNA levels and transcriptional rate remain unchanged for a longer period of time in the stationary phase before they start to decrease. In contrast, the mRNA levels in the stationary phase are not affected by perturbation of topoisomerase II activity. TOP1-dependent repression operates even on HSP26 and SSA3, which have been shown previously to be transcriptionally induced in early stationary phase. Thus, their mRNA levels are high upon the entry of the cells into the stationary phase but gradually decrease, by a TOP1-dependent mechanism, later in the stationary phase. A minor population of mRNAs is not subjected to the TOP1-dependent regulation, as their levels do not change in stationary phase. The possible role of topoisomerase I in the general transcriptional repression is discussed.

Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae.

Abstract: Genes encoding enzymes in the threonine/methionine biosynthetic pathway were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.

Activation and repression of transcription by the gap proteins hunchback and Krüppel in cultured Drosophila cells.

Abstract: We have studied the ability of the Drosophila gap proteins Kriippel and hunchback to function as transcriptional regulators in cultured cells. Both proteins bind to specific sites in a 100-bp DNA fragment located upstream of the segment polarity gene engrailed, which also contains functional binding sites for a number of homeo box proteins. The hunchback protein is a strikingly concentration-dependent activator of transcription, capable of functioning both by itself and also synergistically with the pair-rule proteins fushi tarazu and paired. In contrast, Kriippel is a transcriptional repressor that can block transcription induced either by hunchback or by several different homeo box proteins. While repression of the homeo box protein activators requires a Kriippel-hinding site on the DNA, repression of hunchback can occur efficiently in the absence of a KruppeZ-binding site. We discuss the possible molecular mechanisms underlying these activities, as well as the potential significance of these results with respect to segmentation in Drosophila.

AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae.

Abstract: We have cloned a DNA fragment complementing the aar1 mutation defective in the a1-alpha 2 repression of the alpha 1 cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR1 gene is identical with TUP1, which is allelic to the SFL2, FLK1, CYC9, UMR7, AMM1, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the beta subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR1 gene in alpha and a/alpha cells conferred the nonmating phenotype, and the a/alpha diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for a1-alpha 2 repression of RME1 and for alpha 2 repression of a-specific genes.

Repression of the Human Glycoprotein Hormone α-Subunit Gene by Glucocorticoids: Evidence for Receptor Interactions with Limiting Transcriptional Activators

Abstract: Expression of the glycoprotein hormone alpha gene is regulated divergently by glucocorticoids in different cell types. Coexpression of the glucocorticoid receptor (GR) with an alpha-CAT reporter gene caused activation of alpha promoter activity in fibroblasts, but repression in JEG-3 choriocarcinoma cells, indicating that cell-specific factors dictate positive vs. negative regulation of this promoter by GR. Cell-specific sequences and other enhancer elements in the the alpha gene have been relatively well characterized in JEG-3 cells, and this model was used to further examine the mechanism of transcriptional repression by glucocorticoids. Promoter mutagenesis indicated that the degree of GR-mediated repression was impaired by a variety of deletional and site-directed mutations between -171 and -111 bp, a region that includes both cell-specific and cAMP response elements (CREs). In an attempt to further localize a negative glucocorticoid response element (GRE) sequence, binding studies were used to assess GR interactions with alpha promoter DNA sequences. Using avidin-biotin complex DNA binding assays, a series of overlapping alpha promoter DNA sequences between -170 to 29 basepairs were tested, but each failed to bind GR, whereas a control GRE avidly bound receptor. Similarly, in competition assays in transfected CV-1 cells, the alpha gene 5'-flanking sequence did not compete for GR stimulation of a glucocorticoid responsive reporter gene, whereas a sequence that contains known GR-binding sites (murine mammary tumor virus) effectively inhibited GR-mediated expression. The absence of high affinity GR-binding sites in the alpha promoter suggested that mutations that affected GR inhibition may have eliminated recognition sites for transactivators, which are themselves targets for the GR, rather than altering specific negative GRE sites in the DNA sequence. To examine this possibility, GR repression was studied using chimeric transcription factors. The transcription-activating domains of several different proteins (CREB, thyroid hormone receptor, or VP16) were linked to the DNA-binding domain of Gal-4, and transcription was driven by the Gal-4 recognition site (UAS). GR markedly repressed transactivation by Gal-4-CREB and, to a lesser degree, the Gal-4-thyroid hormone receptor and Gal-4-VP16 chimeric proteins. Repression occurred when UAS was linked to either the alpha promoter or to the E1B promoter. Thus, inhibition occurs in the absence of either the CRE or the proximal alpha promoter. These results support a mechanism in which GR-mediated repression in JEG-3 cells occurs by receptor interference with the transactivating potential of enhancer-binding proteins or associated transcription factors.

P regulatory products repress in vivo the P promoter activity in P-lacZ fusion genes.

Abstract: The transposition of P elements in Drosophila melanogaster is regulated by products encoded by the P elements themselves. The molecular mechanisms of this regulation are complex and still unclear. We have assayed in vivo the effects of P regulatory products on the P promoter itself by using P-lacZ fusion genes. We have found that all the P-lacZ insertions are repressed in a P background. This repression occurs in all the tissues observed and at all the developmental stages. The amount of transcripts specific for P-lacZ is substantially reduced in a P background. These results suggest that P trans-acting products can exert a direct repression on the P promoter transcription.

Regulation of the adhE gene, which encodes ethanol dehydrogenase in Escherichia coli.

Abstract: The respiratory control of adhE, which encodes ethanol (alcohol) dehydrogenase in Escherichia coli, was examined at the transcriptional level by using various phi (adhE9-lacZ) adhE+ merodiploid strains. Expression of the adhE9-lacZ operon fusion was increased about eightfold by anaerobic growth. Under anaerobic growth conditions, provision of nitrate lowered the level of expression. Nitrate repression was more severe than aerobic repression. From analyses of various mutants with mutations related to nitrate reduction, nitrate repression appears to result from two effects. (i) When nitrate was present, NarL, the positive regulator of the nar operon, exerted a direct repression on adhE expression, which was demonstrable even aerobically. (ii) The chemical reduction of nitrate exerted an indirect effect by altering the cellular redox potential.

Sum1-1: A Suppressor of Silencing Defects in Saccharomyces Cerevisiae

Abstract: The repression of transcription of the silent mating-type locus HMRa in the yeast Saccharomyces cerevisiae requires the four SIR proteins, histone H4 and a flanking site designated HMR-E. The SUM1-1 mutation alleviated the need for many of these components in transcriptional repression. In the absence of each of the SIR proteins, SUM1-1 restored repression in MAT alpha strains; thus, SUM1-1 appeared to bypass the need for the SIR genes in repression of HMRa. Repression was not specific to the genes normally present at HMR, since the TRP1 gene placed at HMR was repressed by SUM1-1 in a sir3 strain. Therefore, like the mechanisms of silencing normally used at HMR, silencing by SUM1-1 was gene-nonspecific. SUM1-1 suppressed point mutations in histone H4, but failed to suppress strongly a deletion mutation in histone H4. Similarly, SUM1-1 suppressed mutations in the three known elements of HMR-E, but was unable to suppress a deletion of HMR-E. These epistasis analyses implied that the functions required for repression at HMR can be ordered, with the SIR genes and silencer elements acting upstream of SUM1-1. SUM1-1 itself may function at the level of chromatin in the assembly of inactive DNA at the silent mating-type loci.

Characterization of the cis elements involved in basal and E2-transactivated expression of the bovine papillomavirus P2443 promoter.

Abstract: Transcriptional transactivation and repression by the viral E2 proteins are important regulatory mechanisms for the papillomaviruses. In the bovine papillomavirus type 1 (BPV-1), several viral promoters can be transactivated by E2 through E2-dependent enhancer elements located in the viral long control region (LCR), including promoters involved in E2 expression itself. This report demonstrates that the BPV-1 P2443 promoter is transactivated by E2-responsive elements in the LCR and that this promoter is responsible for a major part of the expression of the E2 and E5 gene products. Characterization of the cis elements involved in P2443 regulation indicated that the single E2-binding site directly upstream of P2443 is not required for either the E2 transactivation or for any E2 repression of the basal or transactivated activity of this promoter. Therefore, cooperative interactions between E2 bound at the LCR and E2 bound near P2443 do not have any role in the regulation of this promoter. Further definition of the cis regulatory elements of this promoter indicated that a binding site for the transcriptional factor Sp1 exists directly upstream of the P2443 TATA box and is critical for the basal level of transcription from this promoter. Disruption of this Sp1 site eliminated P2443 promoter activity in transient expression assays for E2 and E5 and resulted in a loss of transforming activity when introduced into the full viral genome.

Proopiomelanocortin Gene Promoter Elements Required for Constitutive and Glucocorticoid-Repressed Transcription

Abstract: The POMC gene is expressed predominantly in the anterior pituitary. The high level of POMC transcription in this tissue is modulated by peptide hormones and repressed by glucocorticoids. In this present study we have investigated promoter elements required for the high basal transcription and glucocorticoid repression using transient transfection and in vitro transcription assays. We first determined that the region between −77 to −51 of the promoter, which has previously been shown to harbor a glucocorticoid receptorbinding site, is required for high basal expression both in vivo and in vitro. This promoter domain is also required for glucocorticoid repression of transcription in vivo. Two sitedirected mutants within this area both decreased basal transcription, but were fully repressed by glucocorticoids, implying that the −77 to −51 region is a complex regulatory region harboring separable basal and glucocorticoidrepressible elements. Electrophoretic mobility shift and exonuclease III footprinting anal...

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

Abstract: ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.