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A D Johnson

Publications and source records attributed to A D Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans.

Candida albicans, the most prevalent fungal pathogen in humans, is thought to lack a sexual cycle. A set of C. albicans genes has been identified that corresponds to the master sexual cycle regulators a1, alpha1, and alpha2 of the Saccharomyces cerevisiae mating-type (MAT) locus. The C. albicans genes are arranged in a way that suggests that these genes are part of a mating type-like locus that is similar to the mating-type loci of other fungi. In addition to the transcriptional regulators a1, alpha1, and alpha2, the C. albicans mating type-like locus contains several genes not seen in other fungal MAT loci, including those encoding proteins similar to poly(A) polymerases, oxysterol binding proteins, and phosphatidylinositol kinases.

1-Phosphatidylinositol 4-Kinase↗
PubMed1999

Phenotypic switching in Candida albicans is controlled by a SIR2 gene.

We report the cloning of a gene from the human fungal pathogen Candida albicans with sequence and functional similarity to the Saccharomyces cerevisiae SIR2 gene. Deletion of the gene in C. albicans produces a dramatic phenotype: variant colony morphologies arise at frequencies as high as 1 in 10. The morphologies resemble those described previously as part of a phenotypic switching system proposed to contribute to pathogenesis. Deletion of SIR2 also produces a high frequency of karyotypic changes. These and other results are consistent with a model whereby Sir2 controls phenotypic switching and chromosome stability in C.albicans by organizing chromatin structure.

Amino Acid Sequence↗
PubMed1999

Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity.

The Sir2 protein mediates gene silencing and repression of recombination at the rDNA repeats in budding yeast. Here we show that Sir2 executes these functions as a component of a nucleolar complex designated RENT (regulator of nucleolar silencing and telophase exit). Net1, a core subunit of this complex, preferentially cross-links to the rDNA repeats, but not to silent DNA regions near telomeres or to active genes, and tethers the RENT complex to rDNA. Net1 is furthermore required for rDNA silencing and nucleolar integrity. During interphase, Net1 and Sir2 colocalize to a subdomain within the nucleous, but at the end of mitosis a fraction of Sir2 leaves the nucleolus and disperses as foci throughout the nucleus, suggesting that the structure of rDNA silent chromatin changes during the cell cycle. Our findings suggest that a protein complex shown to regulate exit from mitosis is also involved in gene silencing.

Base Sequence↗
PubMed1999

A trans-acting peptide activates the yeast a1 repressor by raising its DNA-binding affinity.

The cooperative binding of gene regulatory proteins to DNA is a common feature of transcriptional control in both prokaryotes and eukaryotes. It is generally viewed as a simple energy coupling, through protein-protein interactions, of two or more DNA-binding proteins. In this paper, we show that the simple view does not account for the cooperative DNA binding of a1 and alpha2, two homeodomain proteins from budding yeast. Rather, we show through the use of chimeric proteins and synthetic peptides that, upon heterodimerization, alpha2 instructs a1 to bind DNA. This change is induced by contact with a peptide contributed by alpha2, and this contact converts a1 from a weak to a strong DNA-binding protein. This explains, in part, how high DNA-binding specificity is achieved only when the two gene regulatory proteins conjoin. We also provide evidence that features of the a1-alpha2 interaction can serve as a model for other examples of protein-protein interactions, including that between the herpes virus transcriptional activator VP16 and the mammalian homeodomain-containing protein Oct-l.

Amino Acid Sequence↗
PubMed1999

Differential activation of the SMalphaA promoter in smooth vs. skeletal muscle cells by bHLH factors.

E-box/basic helix-loop-helix (bHLH)-dependent regulation of promoters for skeletal muscle-specific genes is well established, but similar regulation of smooth muscle-selective promoters has not been reported. Using transient transfection assays of smooth muscle alpha-actin (SMalphaA) promoter-chloramphenicol acetyltransferase (CAT) reporter constructs in rat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, we identified two activator elements, smE1 and smE2, with sequences corresponding to E-box (5'-CAnnTG-3') motifs. In L6 myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completely abolished promoter activity. In contrast, mutation of smE1 and smE2 was required to reduce promoter activity in SMCs. Supershift analyses identified a myogenin-containing complex as the predominant smE1 and smE2 binding activity in skeletal muscle, and myogenin overexpression transactivated the promoter. Supershift analyses with SMC extracts demonstrated that the bHLH protein upstream stimulatory factor (USF) bound smE1, and USF overexpression transactivated the promoter in an smE1-dependent manner. In summary, our results provide novel evidence implicating E-box elements in directing expression of the SMalphaA promoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.

Actins↗
PubMed1999

Characterization of an E-box-dependent cis element in the smooth muscle alpha-actin promoter.

Identification of the regulators of smooth muscle specific gene expression is critical for understanding smooth muscle cell (SMC) differentiation and the alterations in SMC phenotype seen in vascular diseases. Previous studies have identified that a 2-bp mutation in a conserved cis-acting element (TGTTTATC) in the promoter of the chicken smooth muscle (SM) alpha-actin gene abolished nuclear factor binding and decreased transcriptional activity of a 271-bp SM alpha-actin promoter fragment when transfected into rat aortic SMC. However, the promoter region containing this conserved sequence has negative cis regulatory activity when studied in homologous systems. The goal of the present studies was to further characterize the transcriptional activity of the rat SM alpha-actin promoter region between -224 and -236 that is conserved across mammals. DNAse I analysis and electrophoretic mobility shift assays demonstrated that SMC nuclear proteins bound an extended sequence (TGTTTATCCCCATAA). Transient transfection experiments of wild-type and mutant rat SM alpha-actin promoter-luciferase constructs into rat aortic SMC revealed that promoter activity was enhanced by mutations of specific nucleotides in the TGTTTATCCCCA region. Interestingly, the TGTTTATCCCCA element in the rat SM alpha-actin promoter is centered between 2 canonical E-boxes. Mutations of the flanking E-boxes abolished the enhancement in promoter activity seen with mutation of the TGTTTATCCCCA element alone. Thus studies provide evidence for a regulatory cassette in the rat SM alpha-actin promoter that regulates gene expression via combinatorial interactions between 2 E-boxes and a newly described TGTTTATCCCCA element.

Actins↗
PubMed1998

Mutations in chromatin components suppress a defect of Gcn5 protein in Saccharomyces cerevisiae.

The yeast GCN5 gene encodes the catalytic subunit of a nuclear histone acetyltransferase and is part of a high-molecular-weight complex involved in transcriptional regulation. In this paper we show that full activation of the HO promoter in vivo requires the Gcn5 protein and that defects in this protein can be suppressed by deletion of the RPD3 gene, which encodes a histone deacetylase. These results suggest an interplay between acetylation and deacetylation of histones in the regulation of the HO gene. We also show that mutations in either the H4 or the H3 histone gene, as well as mutations in the SIN1 gene, which encodes an HMG1-like protein, strongly suppress the defects produced by the gcn5 mutant. These results suggest a hierarchy of action in the process of chromatin remodeling.

Acetyltransferases↗
PubMed1998

The C-terminal domain of Sin1 interacts with the SWI-SNF complex in yeast.

In the yeast Saccharomyces cerevisiae, the SWI-SNF complex has been proposed to antagonize the repressive effects of chromatin by disrupting nucleosomes. The SIN genes were identified as suppressors of defects in the SWI-SNF complex, and the SIN1 gene encodes an HMG1-like protein that has been proposed to be a component of chromatin. Specific mutations (sin mutations) in both histone H3 and H4 genes produce the same phenotypic effects as do mutations in the SIN1 gene. In this study, we demonstrate that Sin1 and the H3 and H4 histones interact genetically and that the C terminus of Sin1 physically associates with components of the SWI-SNF complex. In addition, we demonstrate that this interaction is blocked in the full-length Sin1 protein by the N-terminal half of the protein. Based on these and additional results, we propose that Sin1 acts as a regulatable bridge between the SWI-SNF complex and the nucleosome.

Binding Sites↗
PubMed1998

Two MCAT elements of the SM alpha-actin promoter function differentially in SM vs. non-SM cells.

Transcriptional activity of the smooth muscle (SM) alpha-actin gene is differentially regulated in SM vs. non-SM cells. Contained within the rat SM alpha-actin promoter are two MCAT motifs, binding sites for transcription enhancer factor 1 (TEF-1) transcriptional factors implicated in the regulation of many muscle-specific genes. Transfections of SM alpha-actin promoter-CAT constructs containing wild-type or mutagenized MCAT elements were performed to evaluate their functional significance. Mutation of the MCAT elements resulted in increased transcriptional activity in SM cells, whereas these mutations either had no effect or decreased activity in L6 myotubes or endothelial cells. High-resolution gel shift assays resolved several complexes of different mobilities that were formed between MCAT oligonucleotides and nuclear extracts from the different cell types, although no single band was unique to SM. Western blot analysis of nuclear extracts with polyclonal antibodies to conserved domains of the TEF-1 gene family revealed multiple reactive bands, some that were similar and others that differed between SM and non-SM. Supershift assays with a polyclonal antibody to the TEF-related protein family demonstrated that TEF-1 or TEF-1-related proteins were contained in the shifted complexes. Results suggest that the MCAT elements may contribute to cell type-specific regulation of the SM alpha-actin gene. However, it remains to be determined whether the differential transcriptional activity of MCAT elements in SM vs. non-SM is due to differences in expression of TEF-1 or TEF-1-related proteins or to unique (cell type specific) combinatorial interactions of the MCAT elements with other cis-elements and trans-factors.

Actins↗
PubMed1997

Control of filament formation in Candida albicans by the transcriptional repressor TUP1.

The pathogenic yeast Candida albicans regulates its cellular morphology in response to environmental conditions. Ellipsoidal, single cells (blastospores) predominate in rich media, whereas filaments composed of elongated cells that are attached end-to-end form in response to starvation, serum, and other conditions. The TUP1 gene, which encodes a general transcriptional repressor in Saccharomyces cerevisiae, was isolated from C. albicans and disrupted. The resulting tup1 mutant strain of C. albicans grew exclusively as filaments under all conditions tested. TUP1 was epistatic to the transcriptional activator CPH1, previously found to promote filamentous growth. The results suggest a model where TUP1 represses genes responsible for initiating filamentous growth and this repression is lifted under inducing environmental conditions.

Amino Acid Sequence↗
PubMed1997

A complex composed of tup1 and ssn6 represses transcription in vitro.

The Saccharomyces cerevisiae Tup1 protein is a member of a family of WD repeat containing proteins that are involved in repression of transcription. Tup1, along with the Ssn6 protein, represses a wide variety of genes in yeast including cell type-specific and glucose-repressed genes. Tup1 and Ssn6 are recruited to these specific gene sets by interaction with sequence-specific DNA binding proteins. In this work, a protein complex containing Ssn6 and Tup1 was purified to determine its composition. The size of the complex is estimated to be 440 kDa. Tup1 and Ssn6, which are both phosphoproteins, are the only proteins present in stoichiometric amounts in the complex. We also demonstrate that this purified complex represses transcription in an in vitro assay.

Cell-Free System↗
PubMed1997

Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3.

The SIR2, SIR3, and SIR4 silent information regulator proteins are involved in the assembly of silent chromatin domains in the budding yeast Saccharomyces cerevisiae. Using a series of biochemical experiments, we have studied protein-protein interactions involving these proteins. We found that yeast extracts contained a SIR2/SIR4 complex that was associated with little or no SIR3. However, truncations of the N-terminal two-thirds of the SIR4 protein allowed it to efficiently associate with SIR3, suggesting that the N-terminal domain of SIR4 inhibited its interaction with SIR3. We propose that the SIR3 and SIR4 proteins interact only during the assembly of the SIR protein complex at the silencer and that an early step in assembly unmasks the SIR4 protein to allow its association with SIR3. To test whether the interactions observed in yeast extracts were direct, we tested these SIR-SIR interactions using bacterially expressed SIR proteins. We observed direct interactions between SIR4 and SIR2, SIR4 and SIR3, SIR2 and SIR3, SIR2 and SIR2, and SIR4 and SIR4, indicating that the associations observed in yeast extracts were direct.

Binding Sites↗
PubMed1997

Residues in the WD repeats of Tup1 required for interaction with alpha2.

The yeast transcriptional repressor Tup1 contains seven WD repeats which interact with the DNA-binding protein alpha2. We have identified mutations in Tup1 that disrupt this interaction. The positions of the amino acids changed by these mutations are consistent with Tup1 being folded into a seven-bladed propeller like that formed by another WD repeat-containing protein, the beta subunit of the heterotrimeric G protein used in signal transduction. Our results also indicate that the interaction between Tup1 and alpha2 resembles the interaction between Gbeta and G alpha, suggesting that a similar structural interface is formed by WD repeat proteins that are used in both transcriptional regulation and signal transduction.

Amino Acid Sequence↗
PubMed1996

Accessibility of alpha 2-repressed promoters to the activator Gal4.

It has been proposed that eukaryotic repressors of transcription can act by organizing chromatin, thereby preventing the accessibility of nearby DNA to activator proteins required for transcription initiation. In this study, we test this idea for the yeast alpha 2 repressor using a simple, artificial promoter that contains a single binding site for the activator protein Gal4 and a single binding site for the repressor alpha 2. When both the repressor and the activator are expressed in the same cell, the artificial promoter is efficiently repressed. In vivo footprinting experiments demonstrate that Gal4 can occupy its binding site even when the promoter is repressed. This result indicates that alpha 2-directed repression must result from interference with some stage in transcription initiation other than activator binding to DNA.

Base Sequence↗
PubMed1996

Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells. Association with the activated phenotype.

Proliferation and phenotypic modulation of smooth muscle cells (SMCs) are major components of the vessel's response to injury in experimental models of restenosis. Some of the growth factors involved in restenosis have been identified, but to date little is known about the transcription factors that ultimately regulate this process. We examined the expression of the four members of the myocyte enhancer binding factor-2 (MEF2) family of transcription factors in cultured rat aortic SMCs (RASMCs) and a rat model of restenosis because of their known importance in regulating the differentiated phenotype of skeletal and cardiac muscle. In skeletal and cardiac muscle, the MEF2s are believed to be important for activating the expression of contractile protein and other muscle-specific genes. Therefore, we anticipated that the MEF2s would be expressed at high levels in medial SMCs that are producing contractile proteins and that they would be downregulated along with the contractile protein genes in neointimal SMCs. On the contrary, we observe that MEF2A, MEF2B, and MEF2D mRNAs are upregulated in the neointima, with the highest levels in the layer of cells nearest to the lumen, whereas MEF2C mRNA levels do not appreciably increase. Moreover, few cells in the media are making MEF2 proteins detectable by immunohistochemistry, whereas large numbers of neointimal cells are positive for all four MEF2s. These data suggest that the MEF2s are involved in the activated smooth muscle phenotype and not in the maintenance of contractile protein gene expression.

Animals↗
PubMed1995

The tetratricopeptide repeats of Ssn6 interact with the homeo domain of alpha 2.

The tetratricopeptide repeat (TPR) is a 34-amino-acid degenerate sequence motif that is found in a large variety of proteins, both prokaryotic and eukaryotic. TPRs are usually found in tandem arrays of up to 16 copies. In this paper we identify a direct interaction between the TPRs of Ssn6, a general transcriptional repressor, and alpha 2, a cell-type regulator in Saccharomyces cerevisiae. Five of the Ssn6 TPRs were tested individually, and all were found to interact specifically with alpha 2. These results suggest a model for TPR-protein interactions and for the role that a tandem array of TPRs may have in mediating transcriptional repression.

Amino Acid Sequence↗
PubMed1995

Crystal structure of the MATa1/MAT alpha 2 homeodomain heterodimer bound to DNA.

The Saccharomyces cerevisiae MATa1 and MAT alpha 2 homeodomain proteins, which play a role in determining yeast cell type, form a heterodimer that binds DNA and represses transcription in a cell type-specific manner. Whereas the alpha 2 and a1 proteins on their own have only modest affinity for DNA, the a1/alpha 2 heterodimer binds DNA with high specificity and affinity. The three-dimensional crystal structure of the a1/alpha 2 homeodomain heterodimer bound to DNA was determined at a resolution of 2.5 A. The a1 and alpha 2 homeodomains bind in a head-to-tail orientation, with heterodimer contacts mediated by a 16-residue tail located carboxyl-terminal to the alpha 2 homeodomain. This tail becomes ordered in the presence of a1, part of it forming a short amphipathic helix that packs against the a1 homeodomain between helices 1 and 2. A pronounced 60 degree bend is induced in the DNA, which makes possible protein-protein and protein-DNA contacts that could not take place in a straight DNA fragment. Complex formation mediated by flexible protein-recognition peptides attached to stably folded DNA binding domains may prove to be a general feature of the architecture of other classes of eukaryotic transcriptional regulators.

Amino Acid Sequence↗