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A D Johnson

Publications and source records attributed to A D Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1994

Operator-constitutive mutations in a DNA sequence recognized by a yeast homeodomain.

Homeodomain proteins regulate transcription in organisms as diverse as yeasts, mammals and plants, often effecting key decisions in development. Although homeodomains can selectively recognize certain DNA sequences, a question has arisen as to how specific this interaction is and how much it contributes to the ability of these proteins to properly select target genes in the cell. This question is particularly an issue in cases where the homeodomain proteins recognize DNA cooperatively with other DNA-binding proteins. In this paper, we examine the issue of DNA binding specificity for the homeodomain of the yeast alpha 2 protein (which recognizes the a-specific gene operator cooperatively with the MCM1 protein) by examining both in vivo and in vitro the effects of point mutations in its recognition sequence. We found that most changes in the homeodomain recognition sequence produced only small effects on both homeodomain affinity as measured in vitro (with and without the helper protein MCM1) and operator function as determined in vivo. This tolerance for operator mutations illustrates in a systematic way the modest DNA-binding specificity of the alpha 2 homeodomain and contrasts with the behavior of many of the bacterial and phage repressors where single point mutations in the operator can have dramatic effects on affinity. This tolerance for different sequences may arise from the fact that most of the interactions made between the alpha 2 homeodomain and the DNA occur through long amino acid side chains; we suggest that these side chains can reconfigure in order to create surfaces complementary to many different DNA sequences. The relaxed DNA-binding specificity of homeodomain proteins such as alpha 2 may be an important feature that permits new regulatory circuits to evolve rapidly from existing components.

Base Sequence↗
PubMed1994

Recognition of a DNA operator by a dimer composed of two different homeodomain proteins.

The yeast homeodomain proteins a1 and alpha 2 interact to form a heterodimer that binds DNA with high specificity. The DNA recognition element consists of two similar half sites, arranged with dyad symmetry and separated by a fixed number of base pairs. We demonstrate that in the a1 alpha 2-DNA complex, one of these half-sites is bound by a1 while the other is bound by alpha 2. These assignments allow a comparison of the chemical and nuclease protection patterns produced by both proteins when bound together to the hsg operator. Contrary to simple expectations, we propose that the a1 and alpha 2 homeodomains are arranged on the DNA in tandem, despite the fact that the recognition sequence is dyad symmetric.

Base Sequence↗
PubMed1993

Yeast a1 and alpha 2 homeodomain proteins form a DNA-binding activity with properties distinct from those of either protein.

The yeast a1 and alpha 2 proteins are examples of homeodomain proteins that display cell-type-specific expression. They are co-expressed in only one type of cell, the a/alpha cell, where they repress the expression of a group of target genes. Using purified proteins, we demonstrate that a1 and alpha 2 form a highly specific DNA-binding activity, which recognizes an operator found upstream of each target gene. These proteins interact with DNA to form a ternary complex in which both a1 and alpha 2 contact the DNA through their respective homeodomains. An alpha 2 homodimer can recognize the same operator as the a1/alpha 2 heterodimer, but the affinity and specificity of the alpha 2 homodimer for DNA are much weaker than those of the a1/alpha 2 heterodimer. This difference results in part from the fact that the heterodimer is formed using a set of protein-protein contacts that is distinct from those that form the alpha 2/alpha 2 homodimer. Although a1 contacts DNA in the presence of alpha 2, it does not on its own bind DNA specifically, even at very high concentrations. These results suggest that the dimerization of heterologous partners can produce a DNA-binding activity that is not a simple hybrid of the known properties of each constituent.

Base Sequence↗
PubMed1993

Origin of a novel allele in a mammalian hybrid zone.

The occurrence of rare or novel alleles has been documented in at least 23 different hybrid zones spanning vertebrate and invertebrate taxa. As most novel alleles either occur at high frequencies in hybrid populations or are exclusively restricted to hybrids, it has seemed probable that hybridization has a role in their origin; however, the molecular nature of these novel alleles and the mechanisms responsible for their origin remain obscure. We examined the complete coding sequences of six alleles of alcohol dehydrogenase in a mammalian hybrid zone between two species of pocket gophers (Geomys). One of these sequences encodes a novel electromorph that had been identified in earlier allozyme studies; this novel allele differs from one of the parental alleles by a single base substitution. This substitution generates an amino acid replacement that affects the net charge of the translated protein. This resultant charge change is congruent with the observed allozyme mobility patterns. Our data thus provide evidence for simple DNA substitution as a mechanism for the origin of this novel hybrid-zone allele.

Alcohol Dehydrogenase↗
PubMed1993

A short, disordered protein region mediates interactions between the homeodomain of the yeast alpha 2 protein and the MCM1 protein.

Homeodomains are folded into a characteristic three-dimensional structure capable of recognizing DNA in a sequence-specific manner. We show that correct target site selection by the yeast alpha 2 protein requires, as well as its homeodomain, an adjacent short and apparently unstructured region of the protein. This flexible homeodomain extension is responsible for specifying an interaction with a second regulatory protein, MCM1, which permits the cooperative binding of the two proteins to an operator. Two additional experiments suggest that this extension-homeodomain arrangement is likely to have some generality. First, when the extension of alpha 2 is grafted onto the Drosophila engrailed homeodomain, it yields a protein with the DNA binding specificity of engrailed and the ability to bind cooperatively to DNA with MCM1. Second, the alpha 2 extension specifies interaction not only with the yeast MCM1 protein, but also with the related human protein SRF.

Amino Acid Sequence↗
PubMed1993

Atherosclerosis alters the localization of HSP70 in human and macaque aortas.

Evidence suggests an important role for heat shock proteins (HSPs) in the evolution of atherosclerotic necrotic cores. The present study compared normal-appearing and atherosclerotic aortas obtained from control and diet-induced atherosclerotic cynomolgus macaques and from human autopsies, with respect to the localization and content of 70-kDa HSPs (HSP70). The distribution pattern of HSP70 was determined by immunostaining tissue sections with anti-HSP70 monoclonal antibody against both constitutive and inducible isoforms. Changes in HSP70 staining with developing atherosclerosis were quantitated using video morphometry. Total aortic HSP70 content was evaluated by Western blotting tissue homogenates. In both macaque and human aortas, HSP70 staining was homogeneous in normal-appearing regions, but developed a heterogeneous pattern in the presence of atherosclerosis. Immunostaining for three other HSPs (90, 65, and 28 kDa) confirmed the change as HSP-specific. Video morphometry indicated a significant positive association between severity of atherosclerosis and altered patterns of HSP70 staining. However, Western blots detected no difference in total HSP70 content of either human or macaque aortas with plaque progression. The data suggest HSP70 localization changes in aortas during atherosclerosis evolution without affecting overall aortic HSP70 content. Such changes in HSP70 localization may reflect differences in the cellular response and resistance to cytotoxic conditions present within the plaque, which could influence the expansion of necrotic cores.

Animals↗
PubMed1993

Exogenous HSP70 becomes cell associated, but not internalized, by stressed arterial smooth muscle cells.

Cell death within atherosclerotic plaques leads to necrosis and rupture, resulting in vascular occlusion. We have previously demonstrated that addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSMCs or acts from outside cells to preserve viability, cultured rabbit aSMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or 125I-radiolabeled HSP70. Cell-associated HSP70 was analyzed using Western blotting, fluorescence spectroscopy, and gamma counting/autoradiography. Surface binding of HSP70 to aSMCs was differentiated from uptake by using trypsin treatment to degrade non-internalized HSP70. Specificity of HSP70 binding was tested by inhibiting uptake of 125I-HSP70 with excess unlabeled HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogenous HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels present in unstressed aSMCs. However, < 5% of the exogenous HSP70 was trypsin-insensitive, indicating that bound HSP70 was not internalized. Binding of 125I-HSP70 was inhibited by both unlabeled HSP70 and BSA, implying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSMCs, but it did not specifically inhibit synthesis of endogenous HSPs after heat shock. The results indicate that exogenous HSP70 protects viability of stressed aSMCs through interactions with the cell surface rather than via internalization.

Animals↗
PubMed1993

A conditional allele of the Saccharomyces cerevisiae HOP1 gene is suppressed by overexpression of two other meiosis-specific genes: RED1 and REC104.

The HOP1 gene of Saccharomyces cerevisiae is believed to encode a protein component of the synaptonemal complex, the structure formed when homologous chromosomes synapse during meiotic prophase. Five new mutant alleles (three conditional, two nonconditional) of HOP1 were identified by screening EMS-mutagenized cells for a failure to complement the spore viability defect of a hop1 null allele. Two high copy plasmids were found that partially suppress the temperature-sensitive spore inviability phenotype of one of these alleles, hop1-628. The suppression is allele-specific; no effect of the plasmids is observed in hop1 null diploids. Mutation of either of the two suppressor genes results in recessive spore lethality, indicating that these genes play important roles during meiosis. The DNA sequence of one high copy suppressor gene matched that of RED1, a previously identified meiosis-specific gene. Our data strongly support the idea that RED1 protein is also a component of the synaptonemal complex and further suggest that the RED1 and HOP1 gene products may interact. The second suppressor maps to the right arm of chromosome VIII distal to CDC12 and is REC104, a meiosis-specific gene believed to act early in meiosis.

Alleles↗
PubMed1993

The carboxy-terminal tail of the homeo domain protein alpha 2 is required for function with a second homeo domain protein.

The homeo domain protein alpha 2 from Saccharomyces cerevisiae has two roles in the a/alpha cell: With MCM1, alpha 2 turns off transcription of a-specific genes; with a1 (a second homeo domain protein), alpha 2 represses transcription of haploid-specific genes. From the carboxy-terminal side of the alpha 2 homeo domain extends an unstructured 22-amino-acid residue tail. In this paper we show that the carboxy-terminal tail of alpha 2 is required for formation of a stable a1/alpha 2-operator complex and is thus required for a1/alpha 2-mediated repression of transcription. In contrast, the tail is dispensable for alpha 2/MCM1-mediated repression. These results indicate that a short, unstructured tail mediates the interaction between two homeo domain proteins.

Base Sequence↗
PubMed1993

The yeast alpha 2 protein can repress transcription by RNA polymerases I and II but not III.

The alpha 2 protein of the yeast Saccharomyces cerevisiae normally represses a set of cell-type-specific genes (the a-specific genes) that are transcribed by RNA polymerase II. In this study, we determined whether alpha 2 can affect transcription by other RNA polymerases. We find that alpha 2 can repress transcription by RNA polymerase I but not by RNA polymerase III. Additional experiments indicate that alpha 2 represses RNA polymerase I transcription through the same pathway that it uses to repress RNA polymerase II transcription. These results implicate conserved components of the transcription machinery as mediators of alpha 2 repression and exclude several alternate models.

Base Sequence↗
PubMed1993

Pancreatic splenic lobe organ culture system: viability and amylase release.

An organ explant culture system for the intact chick pancreatic splenic lobe (SL) was characterized for exocrine function. Organ cultures were prepared using the pancreatic splenic lobe from 9- and 15-day-old male broiler chicks (Arbor Acres x Arbor Acres) to characterize amylase release as well as tissue integrity during 2, 4, 8, and 12 h of incubation. Light microscopy studies indicated necrosis of the exocrine pancreatic acini after 4 h of incubation. Changes in islets of Langerhans were noted 4 h after incubation, but islet structural integrity remained intact for up to 12 h of incubation. Lactate dehydrogenase (LD) levels measured in the culture medium did not increase significantly from 2 to 4 h of incubation. After 4 h of incubation, total LD levels increased (P < .05) for the 9-day-old SL cultures, and LD levels increased (P < .01) per unit weight of SL for the 15-day-old cultures. Medium amylase activity did not increase after 2 h of incubation. Large increases occurred for total amylase activity and amylase activity per unit weight of SL between 4 and 12 h of incubation for both 9- and 15-day-old cultures. Histological examination as well as increases in LD and amylase activities for total and per unit weight of SL in the incubation medium suggest that the viability of the 9- and 15-day-old SL organ incubation system decreases after 4 h.

Aging↗
PubMed1992

Ssn6-Tup1 is a general repressor of transcription in yeast.

The homeodomain protein alpha 2 and the SRF-like protein Mcm1 are required to establish cell type in the yeast Saccharomyces cerevisiae. Together, these regulatory proteins recognize a specific DNA operator, marking a set of genes for transcriptional repression. In this paper, we show that occupancy of the operator by alpha 2-Mcm1 is not sufficient to bring about repression. Rather, repression is effected only when Ssn6 (a TPR protein) and Tup1 (a beta-transducin repeat protein) are also present in the cell. We show that Ssn6 represses transcription when brought to a promoter by a bacterial DNA-binding domain and that Tup1 is required for this repression. Based on these and other results, we propose that Ssn6-Tup1 is a general repressor of transcription in yeast, recruited to target promoters by a variety of sequence-specific DNA-binding proteins.

Immunoblotting↗
PubMed1992

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer.

DNA recognition sequences for dimeric proteins typically contain two types of information. The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites. We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type. Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats. We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators. These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell.

Base Sequence↗
PubMed1992

Meiotic induction of the yeast HOP1 gene is controlled by positive and negative regulatory sites.

The process of meiosis and sporulation in the yeast Saccharomyces cerevisiae is a highly regulated developmental pathway dependent on genetic as well as nutritional signals. The HOP1 gene, which encodes a component of meiotic chromosomes, is not expressed in mitotically growing cells, but its transcription is induced shortly after yeast cells enter the meiotic pathway. Through a series of deletions and mutations in the HOP1 promoter, we located two regulatory sites that are essential for proper regulation of HOP1. One site, called URS1H, brings about repression of HOP1 in mitotic cells and functions as an activator sequence in cells undergoing meiosis. The second site, which we designated UASH, acts as an activator sequence in meiotic cells and has similarity to the binding site of the mammalian CCAAT/enhancer binding protein (C/EBP). Both sites are required for full meiotic induction of the HOP1 promoter. We conclude that in mitotic yeast cells, the URS1H site maintains the repressed state of the HOP1 promoter, masking the effect of the UASH site. Upon entry into meiosis, repression is lifted, allowing the URS1H and UASH sites to activate high-level transcription.

Base Sequence↗
PubMed1991

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.

Base Sequence↗
PubMed1991

Crystallization and preliminary X-ray diffraction studies of a MAT alpha 2-DNA complex.

Crystals have been obtained of the DNA-binding domain of the yeast MAT alpha 2 repressor bound to a 21 base-pair DNA site. The crystals are grown from polyethylene glycol and CaCl2 and form in space group P2(1) with a = 60.1 A, b = 39.4 A, c = 68.7 A and beta = 98 degrees. They diffract to 2.9 A resolution and contain one protein-DNA complex in the crystallographic asymmetric unit.

Base Sequence↗
PubMed1991

Secondary structure of the homeo domain of yeast alpha 2 repressor determined by NMR spectroscopy.

The yeast alpha 2 protein is a regulator of cell type in Saccharomyces cerevisiae. It represses transcription of a set of target genes by binding to an operator located upstream of each of these genes. The alpha 2 protein shares weak sequence similarity with members of the homeo domain family; the homeo domain is a 60-amino-acid segment found in many eukaryotic transcriptional regulators. In this paper we address the question of whether alpha 2 is structurally related to prototypical members of the homeo domain family. We used solution 1H and 15N nuclear magnetic resonance [NMR] spectroscopy to determine the secondary structure of an 83-amino-acid residue fragment of alpha 2 that contains the homeo domain homology. We have obtained resonance assignments for the backbone protons and nitrogens of the entire 60-residue region of the putative homeo domain and for most of the remainder of the alpha 2 fragment. The secondary structure was determined by using NOE connectivities between backbone protons, 3JHN-H alpha coupling constants, and dynamical information from the hydrogen exchange kinetics of the backbone amides. Three helical segments exist in the alpha 2 fragment consisting of residues 11-23, 32-42, and 46-60 (corresponding to residues 138-150, 159-169, and 173-187 of the intact protein). The positions of these three helices correspond extremely well to those of the Drosophila Antennapedia (Antp) and engrailed (en) homeo domains, whose three-dimensional structures have recently been determined by NMR spectroscopy and X-ray crystallography, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗