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I Sadowski

Publications and source records attributed to I Sadowski.

At least 19 recordsLinked to original sources
PubMed2001

A two-hybrid system for transactivator bait proteins.

We describe a two-hybrid strategy for detection of interactions with transactivator proteins. This repressed transactivator (RTA) system employs the N-terminal repression domain of the yeast general repressor TUP1. TUP1-GAL80 fusion proteins, when coexpressed with GAL4, are shown to inhibit transcription of GAL4-dependent reporter genes. This effect requires the C-terminal 30 residues of GAL4, which are required for interaction with GAL80 in vitro. Furthermore, repression of GAL transcription by TUP1-GAL80 requires SRB10, demonstrating that the TUP1 repression domain, in the context of a two-hybrid interaction, functions by the same mechanism as endogenous TUP1. Using this strategy, we demonstrate interactions between the mammalian basic helix-loop-helix proteins MyoD and E12, and between c-Myc and Bin-1. We have also identified interacting clones from a TUP1-cDNA fusion expression library by using GAL4-VP16 as a bait fusion. These results demonstrate that RTA is generally applicable for identifying and characterizing interactions with transactivator proteins in vivo.

Animals↗
PubMed2000

Multiple signals regulate GAL transcription in yeast.

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.

Alleles↗
PubMed2000

Two regulators of Ste12p inhibit pheromone-responsive transcription by separate mechanisms.

The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. Ste12p is negatively regulated by two inhibitor proteins, Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216-688)] from a GAL promoter causes FUS1 induction in a strain expressing wild-type STE12, suggesting that this region can cause the activation of endogenous Ste12p. Residues 262 to 594 are sufficient to cause STE12-dependent FUS1 induction when overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p in yeast extracts. In contrast, recombinant glutathione S-transferase-Dig2p binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but not DIG1, from a GAL promoter inhibits transcriptional activation by an Ste12p DBD-VP16 fusion. Furthermore, disruption of dig1, but not dig2, causes elevated transcriptional activation by a LexA-Ste12p(216-688) fusion. Ste12p has multiple regions within the C terminus (flanking residue 474) that can promote multimerization in vitro, and we demonstrate that these interactions can contribute to the activation of endogenous Ste12p by overproduced C-terminal fragments. These results demonstrate that Dig1p and Dig2p do not function by redundant mechanisms but rather inhibit pheromone-responsive transcription through interactions with separate regions of Ste12p.

Fungal Proteins↗
PubMed1999

Streptococcal meningitis: effect of CSF filtration on inflammation and neuronal damage.

The effect of CSF filtration on inflammation and neuronal damage was studied in experimental Streptococcus pneumoniae meningitis. New Zealand white rabbits received either antibiotic therapy alone (ceftriaxone i.v., 20 mg/kg bolus, 10 mg/kg maintenance dose; n = 10) or ceftriaxone plus CSF filtration (n = 11) 12 h after intracisternal infection. Immediately after the onset of antibiotic therapy 300 microliters cisternal CSF was removed, passed through a miniaturized CSF-1 filter at a constant flow of 20 microliters/min, and then reinjected. This procedure was repeated six times at intervals of 20 min. Antibiosis plus CSF filtration caused a transient reduction in CSF bacterial titers and leukocyte counts compared with antibiosis alone (P = 0.04 and 0.02 5 h after initiation of therapy). CSF lipoteichoic acid concentrations were not reduced. The concentration of neuron-specific enolase in CSF and the density of apoptotic neurons in the dentate gyrus were almost equal 12 h after the onset of treatment. Adjuvant CSF filtration accelerated the elimination of viable bacteria from CSF in comparison to antibiotic treatment alone. Parameters of neuronal destruction, however, were not reduced.

Animals↗
PubMed1999

GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8.

Phosphorylation of the yeast transcription factor GAL4 at S699 is required for efficient galactose-inducible transcription. We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro. RNA Pol II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating GAL4 at S699. Mutations at GAL4 S699 and srb10 are epistatic for GAL induction, demonstrating that SRB10 regulates GAL4 activity through this phosphorylation in vivo. These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation.

Animals↗
PubMed1999

Quinupristin/dalfopristin attenuates the inflammatory response and reduces the concentration of neuron-specific enolase in the cerebrospinal fluid of rabbits with experimental Streptococcus pneumoniae meningitis.

The inflammatory response following initiation of antibiotic therapy and parameters of neuronal damage were compared during intravenous treatment with quinupristin/dalfopristin (100 mg/kg as either a short or a continuous infusion) and ceftriaxone (10 mg/kg/h) in a rabbit model of Streptococcus pneumoniae meningitis. With both modes of administration, quinupristin/dalfopristin was less bactericidal than ceftriaxone. However, the concentration of proinflammatory cell wall components (lipoteichoic acid (LTA) and teichoic acid (TA)) and the activity of tumour necrosis factor (TNF) in cerebrospinal fluid (CSF) were significantly lower in the two quinupristin/dalfopristin groups than in ceftriaxone-treated rabbits. The median LTA/TA concentrations (25th/75th percentiles) were as follows: (i) 14 h after infection: 133 (72/155) ng/mL for continuous infusion of quinupristin/dalfopristin and 193 (91/308) ng/mL for short duration infusion, compared with 455 (274/2042) ng/mL for ceftriaxone (P = 0.002 and 0.02 respectively); (ii) 17 h after infection: 116 (60/368) ng/mL for continuous infusion of quinupristin/dalfopristin and 117 (41/247) ng/mL for short duration infusion, compared with 694 (156/2173) ng/mL for ceftriaxone (P = 0.04 and 0.03 respectively). Fourteen hours after infection the median TNF activity (25th/75th percentiles) was 0.2 (0.1/1.9) U/mL for continuous infusion of quinupristin/dalfopristin and 0.1 (0.01/3.5) U/mL for short duration infusion, compared with 30 (4.6/180) U/mL for ceftriaxone (P = 0.02 for each comparison); 17 h after infection the TNF activity was 2.8 (0.2/11) U/mL (continuous infusion of quinupristin/dalfopristin) and 0.1 (0.04/6.1) U/mL (short duration infusion), compared with 48.6 (18/169) U/mL for ceftriaxone (P = 0.002 and 0.001). The concentration of neuron-specific enolase (NSE) 24 h after infection was significantly lower in animals treated with quinupristin/dalfopristin: 4.6 (3.3/5.7) microg/L (continuous infusion) and 3.6 (2.9/4.7) microg/L (short duration infusion) than in those treated with ceftriaxone (17.7 (8.8/78.2) microg/L) (P = 0.03 and 0.009 respectively). In conclusion, antibiotic treatment with quinupristin/dalfopristin attenuated the inflammatory response within the subarachnoid space after initiation of antibiotic therapy. The concentration of NSE in the CSF, taken as a measure of neuronal damage, was lower in quinupristin/dalfopristin-treated rabbits than in ceftriaxone-treated rabbits.

Animals↗
PubMed1998

Mapping a protein-binding site on straightened DNA by atomic force microscopy.

We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (> 5 kb) at nanometer resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface. The immobilized molecules were detected by AFM. The straightened DNA displayed a sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein-binding site. Using GAL4, a yeast transcription factor, we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site. The technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-molecule level.

Aluminum Silicates↗
PubMed1998

Naturally occurring human immunodeficiency virus type 1 long terminal repeats have a frequently observed duplication that binds RBF-2 and represses transcription.

Approximately 38% of human immunodeficiency virus type 1 (HIV-1)-infected patients within the Vancouver Lymphadenopathy-AIDS Study have proviruses bearing partial 15- to 34-nucleotide duplications upstream of the NF-kappaB binding sites within the 5' long terminal repeat (LTR). This most frequent naturally occurring length polymorphism (MFNLP) of the HIV-1 5' LTR encompasses potential binding sites for several candidate transcription factors, including TCF-1alpha/hLEF, c-Ets, AP-4, and Ras-responsive binding factor 2 (RBF-2) (M. C. Estable et al., J. Virol. 70:4053-4062, 1996). RBF-2 and an apparently related factor, RBF-1, bind to at least four cis elements within the LTR which are required for full transcriptional responsiveness to protein-tyrosine kinases and v-Ras (B. Bell and I. Sadowski, Oncogene 13:2687-2697, 1996). Here we demonstrate that representative MFNLPs from two patients specifically bind RBF-2. In both cases, deletion of the MFNLP caused elevated LTR-directed transcription in cells expressing RBF-2 but not in cells with undetectable RBF-2. RBF-1, but not RBF-2, appears to contain the Ets transcription factor family member GABPalpha/GABPbeta1. Taken together with the fact that every MFNLP from a comparative study of over 500 LTR sequences from 42 patients contains a predicted binding site for RBF-2, our data suggest that the MFNLP is selected in vivo because it provides a duplicated RBF-2 cis element, which may limit transcription in monocytes and activated T cells.

DNA Replication↗
PubMed1997

Characterization of the basal and pheromone-stimulated phosphorylation states of Ste12p.

The Saccharomyces cerevisiae transcription factor Ste12p is required for basal and activated expression of pheromone-responsive genes, and for invasive growth in haploid cells. In diploid yeast, Ste12p is implicated in pseudohyphal development. The ability of Ste12p to effect these various responses in three different cell types must require stringent regulation of its transcriptional activation function and interaction with additional transcription factors. We have examined the phosphorylation state of Ste12p in untreated and pheromone-treated haploid cells, and found eight constitutively phosphorylated peptides. Phosphorylation at the constitutive sites does not require the protein kinases of the pheromone-response pathway. Treatment of haploid yeast with mating pheromone causes the appearance of novel relatively minor phosphorylations on Ste12p. Brief [35S]methionine labeling reveals novel pheromone-dependent, electrophoretically slower migrating Ste12p species. Similarly, the sole difference we observe in tryptic phosphopeptides generated from Ste12p from pheromone-treated and untreated cells is the transient appearance of two novel minor hydrophobic phosphopeptides. The pheromone-dependent phosphorylation of Ste12p requires an intact pheromone-response pathway and localization of Ste12p to the nucleus, but does not require the Ste12p DNA-binding domain. We conclude from these experiments that the pheromone-response pathway induces the formation of specific hyperphosphorylation on Ste12p, which can only be detected as apparently minor modifications in vivo. We argue that, if Ste12p is regulated by direct pheromone-responsive phosphorylation, then that phosphorylation must be represented by the two novel phosphopeptides. However, we cannot exclude the possibility that pheromone-responsive transcription is controlled by direct phosphorylation of a target other than Ste12p.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Ras-responsiveness of the HIV-1 LTR requires RBF-1 and RBF-2 binding sites.

Lentiviruses characteristically form latent integrated proviruses whose transcription can be induced by cell regulatory signals. The HIV-1 LTR responds to multiple signals, including the Ras pathway. We report here that Ras-responsive HIV-1 transcription requires two previously undescribed factors, RBF-1 and RBF-2 in Jurkat T cells. RBF-1 binds to Ets-like motifs located between nucleotides -151 and -142, and within the NF-kappaB binding sites, but is distinct from Ets-1 or Elf-1. RBF-2 binds the HIV-1 LTR at nucleotides -131 and -121 and immediately 3' of the TATA box. Both RBF-1 and RBF-2 contain DNA binding subunits of relative molecular weight 100 kilodaltons. Mutation of the RBF-1 and RBF-2 binding elements (RBEs) prevents Ras stimulation of HIV-1 LTR-directed transcription. These data define a mechanism for Ras responsiveness of HIV-1 transcription that involves the previously uncharacterized factors RBF-1 and RBF-2.

Base Sequence↗
PubMed1996

Phosphorylation of Ga14p at a single C-terminal residue is necessary for galactose-inducible transcription.

Gal4p regulates expression of genes necessary for galactose catabolism in Saccharomyces cerevisiae. We have previously shown that phosphorylation of Gal4p requires both its DNA binding and transcriptional-activation functions and have suggested that phosphorylation occurs as a consequence of interaction with general transcription factors. In this study, we show that phosphorylation occurs rapidly on a limited fraction of overexpressed Gal4p present in a sodium dodecyl sulfate-extractable subcellular fraction while a significant fraction remains stably unphosphorylated. Taken together with our previous observations, we conclude that Gal4p is phosphorylated only if it becomes localized to the nucleus and is capable of both DNA binding and transcriptional activation. We demonstrate that Gal4p is multiply phosphorylated at both the C and N termini, and we identify the precise locations of three sites of phosphorylation at serines 691, 696, and 699. Of these sites, only serine 699 must be phosphorylated for galactose-inducible transcription to occur. Mutation of S-699 to alanine significantly impairs GAL induction by galactose in GAL80+ cells but does not affect transcriptional activation by Gal4p in gal80- cells. In gal80- cells, Gal4p phosphorylation, including that of serine 699, is stimulated by the presence of both galactose and glucose, indicating that phosphorylation at this site is not specifically activated by galactose. Serine 699 phosphorylation requires Gal4p's DNA binding function and is influenced by the function of the RNA polymerase II holoenzyme component Gal11p. These results suggest that a phosphorylation on Gal4p, likely resulting from interaction with the holoenzyme, modulates the induction process by regulating interaction between Gal4p and Gal80p.

Base Sequence↗
PubMed1993

Inactivation of a yeast transactivator by the fused HIV-1 proteinase: a simple assay for inhibitors of the viral enzyme activity.

The human immunodeficiency virus type 1 (HIV-1) proteinase (PR) and its flanking sequences have been fused in frame between the DNA-binding domain and the transcription-activation domain of the yeast protein, GAL4. As has been shown before with the 3C proteinase of Coxsackie virus B3 (CVB3) [Das Mahapatra et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4159-4162], the GAL4::PR fusion protein retains its GAL4 function, providing the PR is inactive. When PR is active, its autocatalytic activity in the hybrid protein is shown to inactivate the transactivation function of GAL4. This provides a simple assay to monitor PR activity. A dose-dependent effect of a potent PR-specific inhibitor is demonstrated in this system and illustrates the sensitivity of the assay. The assay is used for high throughput screening to identify novel inhibitors of the viral PR, and provides a method to generate and analyze mutants and revertants of the PR.

DNA-Binding Proteins↗
PubMed1993

Wild type GAL4 binds cooperatively to the GAL1-10 UASG in vitro.

Transcription of the genes required for utilization of galactose in Saccharomyces cerevisiae is controlled primarily by the transcriptional activator protein GAL4. The upstream activating sequences for galactose (UASG) of most GAL genes have multiple sites to which GAL4 can bind. In this report we compare the binding properties of wild type GAL4 and derivatives of GAL4 bearing the N-terminal DNA-binding domain to multiple DNA-binding sites in vitro. To produce wild type GAL4, we constructed a recombinant baculovirus for expression in insect cells. Recombinant wild type GAL4 was found to bind efficiently to an oligonucleotide containing a near-consensus 17-mer GAL4 DNA-binding site in electrophoretic mobility shift assays. Footprinting experiments revealed that wild type GAL4 binds cooperatively to the four GAL4 DNA-binding sites of the GAL1-10 UASG; however, in contrast an N-terminal fragment of GAL4 containing only the DNA-binding/dimerization domains binds to each of these sites with slightly different affinity. With increasing concentrations of GAL4(1-147), the four sites become filled in the following order: site II, site IV, site I, and site III. In experiments with wild type GAL4, these four sites become fully occupied at approximately the same concentration of protein. In footprints of wild type GAL4 on the USAG, enhancements and protections of DNase I-sensitive cleavages are detectable between sites III and IV, indicative of formation of a loop between these distantly spaced sites. Binding of wild type GAL4 to a strong near-consensus binding site assists binding to an adjacent mutant site in both electrophoretic mobility shift and footprinting assays. GAL4(1-147) and GAL4(1-147) fused to portions of GAL4's activating region II were incapable of cooperative DNA binding in our assays. We conclude from these observations that wild type GAL4 has a cooperative DNA-binding function that is distinct from the DNA binding and dimerization or transcriptional activation functions, and likely plays and important role in precise regulation of GAL gene transcription.

Animals↗
PubMed1993

GAL4 is regulated by a glucose-responsive functional domain.

The Saccharomyces cerevisiae transcriptional activator GAL4 is regulated by the presence of available carbon sources. Galactose induces activity by inhibiting the negative regulator GAL80, while glucose, the preferred carbon source, antagonizes GAL4 function by several mechanisms. In the present study we present evidence that one mechanisms for inhibition of GAL transcription by glucose involves direct inhibition of the GAL4 protein. We demonstrate that a large, previously uncharacterized, central region of GAL4 contains at least three 'inhibitory domains' and a 'glucose response domain' (GRD). Deletion of the entire central region eliminates direct inhibition of GAL4 by glucose, and furthermore, fusion of the central region to a heterologous transcriptional activator confers inhibition by glucose. The central region inhibitory domains constitutively inhibit transcriptional activation when the GRD is absent. Direct inhibition of GAL4 activity can be detected within 30 min following glucose addition and may represent an early mechanism promoting a switch from galactose to glucose utilization. A model for the regulatory role of the central region is presented, involving interaction with an additional protein that inhibits GAL4 activity when glucose is present.

Amino Acid Sequence↗
PubMed1992

GAL4 fusion vectors for expression in yeast or mammalian cells.

We describe two sets of vectors, one for yeast (pY1, pY2 and pY3) and one for mammalian cells (pM1, pM2, and pM3), that simplify the production of fusion proteins containing the DNA-binding domain of GAL4. This protein fragment, consisting of GAL4 amino acid (aa) residues 1-147, binds to a specific 17-bp nucleotide sequence, but is incapable of activating transcription unless fused to a protein that can contribute an activating function. Genetic strategies exploiting this property of GAL4 (aa 1-147) have been developed to characterize transcription factor functional domains, protein-protein interactions, and site-specific proteolysis. The vectors we describe allow fusion to the C terminus of GAL4 (aa 1-147) in any reading frame, and thus facilitate these experimental strategies.

Amino Acid Sequence↗
PubMed1992

A genetic system for studying the activity of a proteolytic enzyme.

We describe a genetic system for monitoring the activity of a specific proteolytic enzyme by taking advantage of the properties of the yeast transcriptional activator GAL4. The GAL4 protein contains two separable and functionally essential domains: the amino-terminal DNA binding domain and the carboxyl-terminal transcriptional activating domain. We constructed two hybrid proteins by inserting between the DNA binding domain and the activation domain of GAL4 either (i) a self-cleaving protease (3C protease of a picornavirus, coxsackievirus B3) or (ii) a mutant form of the protease that is unable to cleave. We show that, although the hybrid protein containing the mutant protease activates transcription of GAL1-lacZ reporter gene, the hybrid protein bearing the wild-type protease is proteolytically cleaved and fails to activate transcription. Our approach to monitor the proteolytic activity could be used to develop simple genetic systems to study other proteases.

Amino Acid Sequence↗