The price of repression.
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Binding, Competitive↗
Biomedical subjects
A D Johnson
Publications and source records attributed to A D Johnson.
Angioscopic versus angiographic detection of thrombus during coronary interventional procedures.
The purpose of this study was to compare angiography and angioscopy for the detection of thrombus during coronary interventional procedures. The diagnosis of coronary thrombus has important clinical implications. Angioscopy can directly visualize the coronary luminal surface and may be more accurate than angiography in the diagnosis of thrombus. Angiography and angioscopy were sequentially performed in 75 patients undergoing a variety of interventional cardiology procedures during 117 distinct procedural time points. The angiographic presence of thrombus was defined as a noncalcified filling defect outlined on at least 3 sides by contrast media. The angioscopic presence of thrombus was defined as red material protruding into the lumen (intraluminal thrombus) or adherent to the luminal wall (mural thrombus) that persisted despite flushing. Thrombus was detected on 14 occasions (12.0%) by angiography compared with 48 (41.0%) by angioscopy (p < 0.05). In 4 of the 14 episodes (28.6%) of angiographic filling defects, angioscopy found no evidence of thrombus and provided an alternative explanation for the angiographic filling defect. When angioscopy was used as a reference standard, the sensitivity of thrombus detection by angiography was 20.8%, with a specificity of 94.2% and a predictive value of 71.4%. The sensitivity of angiography for the detection of intraluminal (protruding into the lumen) thrombus was 100% compared with only 10% for mural (adherent to the luminal wall) thrombus (p < 0.05). Angioscopy was significantly more accurate than angiography for detecting coronary thrombus and may be considered an improved reference standard for this diagnosis.
Aged↗
DNA bending by the a1 and alpha 2 homeodomain proteins from yeast.
Structural and biochemical studies of monomer homeodomain-DNA complexes have not so far revealed any cases of pronounced DNA distortion. In this paper we show that multimeric complexes of the yeast homeodomain proteins a1 and alpha 2 induced significant bends in their operators upon binding. Based on a series of circular permutation experiments, we found that a dimer of alpha 2 bound to operator DNA produced a mild bend in the DNA, whereas the alpha 2-MCM1-DNA and the a1-alpha 2-DNA complexes exhibited much sharper bends. As these latter two complexes represent the in vivo form of DNA-bound a1 and alpha 2, we conclude that, in the cell, these homeodomain proteins are associated with pronounced bends in DNA. We discuss possible roles for these bends in transcriptional repression.
Base Sequence↗
IgG but not other classes of anti-[(H2A-H2B)-DNA] is an early sign of procainamide-induced lupus.
A longitudinal study was undertaken to characterize the autoantibodies induced during the course of procainamide treatment and to relate this information to the appearance of symptomatic drug-induced lupus. IgG, IgA, and IgM Abs to histones, native and denatured DNA, chromatin, and (H2A-H2B)-DNA were determined by ELISA in serial serum samples obtained over the course of an average of 2.1 yr on 22 patients undergoing treatment with procainamide and on an additional 9 patients after discontinuation of procainamide because of drug-induced lupus. Ten patients in the prospective group developed lupus-like symptoms after an average of 1.8 +/- 2.1 yr of procainamide treatment. Of the total of 19 patients with drug-induced lupus, 16 had IgG Abs to the (H2A-H2B)-DNA complex at the time of diagnosis; this autoantibody was first detected 0.9 +/- 1.3 yr before diagnosis in 7 patients. In contrast, the 9 patients who remained asymptomatic during treatment with procainamide for an average of 4.3 +/- 2.2 yr had negligible levels of IgG anti-[(H2A-H2B)-DNA], although IgA and IgM Abs of this specificity were not uncommon. Abs to denatured DNA and histones were elicited coordinately, but these specificities did not discriminate symptomatic from asymptomatic procainamide-treated patients. We conclude that chronic exposure to procainamide commonly elicited autoantibodies with specificities for denatured epitopes on DNA and histones and for native regions on the (H2A-H2B)-DNA subunit of chromatin. However, rapid switch to the IgG class of anti-[(H2A-H2B)-DNA] occurred only in patients who went on to develop symptomatic disease.
Antibodies, Antinuclear↗
A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression.
A series of mutations in the homeo domain of the yeast alpha 2 protein were constructed to test, both in vivo and in vitro, predictions based on the alpha 2-DNA cocrystal structure described by Wolberger et al. (1991). The effects of the mutations were observed in three different contexts using authentic target DNA sequences: alpha 2 binding alone to specific DNA, alpha 2 binding cooperatively with MCM1 to specific DNA, and alpha 2 binding cooperatively with a1 to specific DNA. As expected, changes in the amino acid residues that contact DNA in the X-ray structure severely compromised the ability of alpha 2 to bind DNA alone and to bind DNA cooperatively with MCM1. In contrast, many of these same mutations, including a triple change that altered all the "recognition" residues of helix 3, had little or no effect on the cooperative binding of alpha 2 and a1 to specific DNA, as determined both in vivo and in vitro. These results show that the ability of a homeo domain protein to correctly select and repress target genes does not necessarily depend on the residues commonly implicated in sequence-specific DNA binding.
Amino Acid Sequence↗
A Xenopus laevis gene encoding EF-1 alpha S, the somatic form of elongation factor 1 alpha: sequence, structure, and identification of regulatory elements required for embryonic transcription.
Transcription of the Xenopus laevis EF-1 alpha S gene commences at the mid-blastula stage of embryonic development and then continues constitutively in all somatic tissues. The EF-1 alpha S promoter is extremely active in the early Xenopus embryo where EF-1 alpha S transcripts account for as much as 40% of all new polyadenylated transcripts. We have isolated the Xenopus EF-1 alpha S gene and used microinjection techniques to identify promoter elements responsible for embryonic transcription. These in vivo expression studies have identified an enhancer fragment, located approximately 4.4 kb upstream of the transcription start site, that is required for maximum expression from the EF-1 alpha S promoter. The enhancer fragment contains both an octamer and a G/C box sequence, but mutation studies indicate that the octamer plays no significant role in regulation of EF-1 alpha S expression in the embryo. The presence of a G/C element in the enhancer and of multiple G/C boxes in the proximal promoter region suggests that the G/C box binding protein, Sp1, plays a major role in the developmental regulation of EF-1 alpha S promoter activity.
Amino Acid Sequence↗
Crystallization and preliminary X-ray diffraction studies of an a1/alpha 2/DNA ternary complex.
Crystals have been obtained of a ternary complex containing the yeast a1/alpha 2 homeodomain heterodimer bound to a 21-base pair DNA site containing two 5' overhanging bases at each end. The crystals are grown from cobaltic hexamine and form in space group P6(1) or P6(5) with a = b = 133 A, c = 45.4 A. Crystals that are flash-frozen at -179 degrees C diffract to 2.7 A along the c-axis and to 2.4 A in perpendicular directions. The crystals contain one protein-DNA complex in the crystallographic asymmetric unit.
Base Sequence↗
Molecular mechanisms of cell-type determination in budding yeast.
Studies of cell-type determination in the yeast Saccharomyces cerevisiae have revealed a regulatory network of proteins that are highly conserved in evolutionary terms. In the past few years, genetic, biochemical, and structural approaches have shown what many of these components do, how they fit together, and how they cooperate to regulate the expression of many different target genes.
Biological Evolution↗
Identification of genes required for alpha 2 repression in Saccharomyces cerevisiae.
Transcriptional repression of the a-specific genes in Saccharomyces cerevisiae alpha cells involves the concerted action of several proteins. The homeodomian protein alpha 2, together with MCM1, recruits two general transcriptional repressors, SSN6 and TUP1, to the promoters of a-specific genes. SSN6 and TUP1 then mediate repression of the a-specific genes. SIN4, another general negative regulator, is required for this repression, but unlike tup1 or ssn6 deletions, sin4 deletions cause only partial loss of repression. We have screened for other genes required for a-specific gene repression in alpha cells. In addition to recovering multiple alleles of previously identified genes required for this process (referred to as alpha 2 repression), we have identified four other genes, designated ARE1, ARE2, ARE3, and ARE4 (for alpha 2 repression). Recessive mutations in the ARE genes cause partial loss of a-specific gene repression and cause pleiotropic phenotypes similar to those resulting from mutations in SSN6, TUP1, or SIN4, suggesting that the ARE genes are general negative regulators. Based on our initial analysis, we propose that two distinct classes of general negative regulators cooperate to bring about full levels of alpha 2 repression. The sequence of ARE1 revealed that it encodes a CDC28-related protein kinase, identical to UME5, and thus suggests that protein phosphorylation plays a role in alpha 2 repression.
DNA-Binding Proteins↗
Differential distribution of 70-kD heat shock protein in atherosclerosis. Its potential role in arterial SMC survival.
Smooth muscle cell death may contribute to necrotic plaque rupture and subsequent thromboembolus. Stress-induced synthesis of heat-shock proteins (HSPs) normally protects cells from death, but vascular HSPs may become insufficient as cytotoxicity increases in advanced plaques. To determine whether vascular HSP content is altered near necrosis, we compared 70-kD HSP (HSP70) distribution between fibrotic and necrotic plagues in immunostained carotid endarterectomy specimens. Average levels of HSP70 immunoreactivity were compared by video densitometry between fibrotic and necrotic plaques or between their underlying media. Both necrotic plaques and their underlying media contained significantly more HSP70 staining than did fibrotic tissues. To test whether cellular HSP70 correlated with resistance to toxicity in vitro, aortic smooth muscle cells (aSMCs) were heat shocked to induce endogenous HSPs or given 2 to 50 micrograms/mL purified HSP70. Cells were then serum deprived or exposed to 12 to 96 mumol/L cholestanetriol (C3ol) or 25-hydroxycholesterol, and survival was determined. Cellular HSP70 content was assayed by immunoblotting, and protein synthesis was monitored by 35S radiolabeling. Serum deprivation inhibited general protein synthesis but induced HSP70; C3ol exposure inhibited both overall protein and HSP70 synthesis, including post-heat shock. Induction of endogenous HSPs or 10 micrograms/mL exogenous HSP70 improved viability of serum-deprived cells (P < .05 and P < .01, respectively), while only exogenous HSP70 protected against C3ol (P < .002). The results suggest that insufficient HSP70 accumulates in aSMCs residing near necrosis to protect against plaque toxicity; aSMC death might then occur, allowing resident macrophages to degrade and destabilize the matrix, leading to rupture.
Animals↗
The involvement of slaframine and swainsonine in slobbers syndrome: a review.
The history of "slobbers syndrome," a mycotoxicosis associated with Rhizoctonia leguminicola infestation of pastures and stored forages, is discussed. The chemistry and physiological effects of the two known biologically active alkaloids of R. leguminicola, slaframine and swainsonine, are described. Slaframine administration is generally associated with increased exocrine function, especially salivation. Ingestion of swainsonine may be linked to serious and potentially lethal central nervous system defects similar to that described for locoism. However, the singular effects of these alkaloids do not completely account for the total clinical picture noted in the field during the occurrence of slobbers syndrome. It is possible that this phenomenon is the result of an interaction between both known and unidentified biologically active metabolites of R. leguminicola.
Alkaloids↗
The WD repeats of Tup1 interact with the homeo domain protein alpha 2.
Tup1 and Ssn6 transcriptionally repress a wide variety of genes in yeast but do not appear to bind DNA. We provide genetic and biochemical evidence that the DNA-binding protein alpha 2, a regulator of cell-type-specific genes, recruits the Tup1/Ssn6 repressor by directly interacting with Tup1. This interaction is mediated by a region of Tup1 containing seven copies of the WD repeat, a 40 amino acid motif of unknown function found in many other proteins. We have found that a single WD repeat will interact with alpha 2, indicating that the WD repeat is a protein-protein interaction domain. Furthermore, a fragment of Tup1 containing primarily WD repeats provides at least partial repression in the absence of Ssn6, suggesting that the repeats also mediate interaction between Tup1 and other components of the repression machinery.
Amino Acid Sequence↗
The predominant form of fibroblast growth factor receptor expressed by proliferating human arterial smooth muscle cells in culture is type I.
Fibroblast growth factors (FGF) and their specific receptors (FGFR) have diverse roles, including induction of proliferation in smooth muscle cells which contributes to restenosis after coronary artery balloon angioplasty. The relative levels of expression of the four major types of FGFR were studied in 13 different human arterial smooth muscle cell isolates. Cell lines were established by the explant technique from intima/media tissue samples obtained from patients undergoing either coronary artery bypass surgery or cardiac transplantation procedures. Expression of FGFR isoforms was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using primers for the conserved tyrosine kinase (TK) domain followed by Southern blotting with TK insert probes unique to each isoform. The data indicate that FGFR I is the major form of FGF receptor mRNA expressed by proliferating human arterial smooth muscle cells. This strongly suggests that it is this type of FGFR that mediates the signal transduction cascade associated with mitogenesis in proliferating human smooth muscle cells.
Arteries↗
pXeX, a vector for efficient expression of cloned sequences in Xenopus embryos.
We describe the construction of pXeX, a plasmid vector that efficiently expresses cloned sequences in Xenopus embryos. This plasmid contains the transcription regulatory regions from the Xenopus laevis elongation factor-1 alpha-encoding gene (EF-1 alpha). Expression of cloned sequences commences in blastula-stage embryos, coincident with transcriptional activation of the embryonic genome, and transcripts may persist until the tadpole stage of development.
Animals↗
Interaction between two homeodomain proteins is specified by a short C-terminal tail.
Two yeast homeodomain proteins, a1 and alpha 2, interact and cooperatively bind the haploid-specific gene (hsg) operator, resulting in the repression of a set of genes involved in the determination of cell type. The cooperative binding of a1 and alpha 2 to DNA can be reconstituted in vitro using purified fragments of a1 and alpha 2. Only the homeodomain is needed for a1, but for alpha 2 a C-terminal 22-amino-acid tail is required as well. As most of the specificity of DNA binding appears to derive from a1, we proposed that alpha 2 functions in the a1/alpha 2 heterodimer to contact a1 with its tail. By construction and analysis of several chimaeric proteins, we investigate how two DNA-binding proteins, one with low intrinsic specificity (alpha 2) and one with no apparent intrinsic DNA-binding ability (a1), can together create a highly specific DNA-binding activity. We show that the 22-amino-acid region of alpha 2 immediately C-terminal to the homeodomain, when grafted onto the a1 homeodomain, converts a1 to a strong DNA-binding protein. This alpha 2 tail can also be attached to the Drosophila engrailed homeodomain, and the chimaeric protein now binds cooperatively to DNA with a1, showing how a simple change can create a new homeodomain combination that specifically recognizes a new DNA operator.
Animals↗
Heterodimerization of the yeast homeodomain transcriptional regulators alpha 2 and a1 induces an interfacial helix in alpha 2.
The homeodomain proteins a1 and alpha 2 act cooperatively to regulate cell type specific genes in yeast. The basis of the cooperativity is a weak interaction between the two proteins which forms heterodimers that bind DNA tightly and specifically. In this paper, we examine the mechanism of heterodimerization. We show that two relatively small fragments of a1 and alpha 2 are capable of heterodimerization and tight DNA binding. The alpha 2 fragment contains the homeodomain followed by the natural 22 C-terminal amino acids of the protein; these 22 amino acids are unstructured in the alpha 2 fragment. The a1 fragment contains only the homeodomain, indicating that the a1 homeodomain mediates both DNA binding and protein-protein interactions with alpha 2. We used isotope-edited NMR spectroscopy to study the interaction in solution of these two fragments. Samples in which only the alpha 2 fragment was uniformly labeled with 15N allowed us to visualize changes in the NMR spectra of the alpha 2 fragment produced by heterodimerization. We found that the a1 homeodomain perturbs the resonances of only the C-terminal tail of alpha 2; moreover, contact with a1 converts a portion of this tail (residues 193-203) from its unstructured state to an alpha-helix, as determined by J coupling and NOE measurements. Thus the heterodimerization of two homeodomain proteins involves the specific interaction between a tail of one protein and the homeodomain of the other. This interaction is accompanied by the acquisition of secondary structure in the tail.
Amino Acid Sequence↗
Transcriptional repression directed by the yeast alpha 2 protein in vitro.
The alpha 2 protein, a homeodomain protein involved in specifying cell type in the budding yeast Saccharomyces cerevisiae, is a transcriptional repressor. alpha 2 binds cooperatively with Mcm1, a serum response factor-related protein, to the a-specific gene operator. The alpha 2-Mcm1 complex in turn recruits Ssn6 and Tup1 to the operator, and we believe that these latter two proteins are responsible for the transcriptional repression. Placement of the a-specific gene operator in any of a variety of positions upstream of a test promoter leads to repression of that promoter in vivo. In this respect, the a-specific gene operator resembles a negatively acting enhancer. Here we describe the in vitro reconstitution of this example of negative control from a distance. We observe repression in vitro in the absence of exogenously added activator protein and on templates that lack binding sites for known activator proteins, and we infer that alpha 2-directed repression acts on the general transcription machinery.
DNA-Binding Proteins↗