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D Hamer

Publications and source records attributed to D Hamer.

33 records · Page 2Linked to original sources
PubMed1990

Cloned yeast and mammalian transcription factor TFIID gene products support basal but not activated metallothionein gene transcription.

Transcription factor IID (TFIID), the "TATA binding factor," is thought to play a key role in the regulation of eukaryotic transcriptional initiation. We have studied the role of TFIID in the transcription of the yeast metallothionein gene, which is regulated by the copper-dependent activator protein ACE1. Both basal and induced transcription of the metallothionein gene require TFIID and a functional TATA binding site. Crude human and mouse TFIID fractions, prepared from mammalian cells, respond to stimulation by ACE1. In contrast, human and yeast TFIID proteins expressed from the cloned genes do not respond to ACE1, except in the presence of wheat germ or yeast total cell extracts. These results indicate that the cloned TFIID gene products lack a component(s) or modification(s) that is required for regulated as compared to basal transcription.

Animals↗
PubMed1990

A gene that encodes a protein consisting solely of zinc finger domains is preferentially expressed in transformed mouse cells.

We describe the cloning and characterization of the mouse MOK-2 gene, a new member of the Krüppel family of zinc finger proteins. Sequencing of both cDNA and genomic clones showed that the predicted MOK-2 protein consists of seven zinc finger domains with only five additional amino acids. The finger domains of MOK-2 are highly homologous to one another but not to those of other zinc finger proteins. MOK-2 is preferentially expressed in transformed cell lines, brain tissue, and testis tissue. Its possible role in cellular transformation is discussed.

Amino Acid Sequence↗
PubMed1990

The DNA and Cu binding functions of ACE1 are interdigitated within a single domain.

We present genetic and biochemical evidence that the amino-terminal region of ACE1, the activator of yeast Cu-metallothionein gene transcription, is composed of a single domain in which the DNA- and Cu-binding residues are interdigitated. Analysis of truncation mutants showed that both the DNA and Cu interactions functions of ACE1 are contained within an amino-terminal 101 amino acid peptide that can fold into a protease-resistant domain structure. Studies of point mutants revealed that two basic residues within this domain are required for efficient DNA binding although not for productive interaction with Cu. Mutations at these sites alter the specificity of ACE1 for two binding sites in the upstream activation region, both of which are shown to be necessary for efficient transcription in vivo. Systematic mutagenesis of the 12 cysteine residues in ACE1 showed that all 11 cysteines within the minimal DNA-binding domain are required for ACE1 to undergo a Cu-induced conformational switch into an active DNA-binding protein. A twelfth cysteine, located outside the DNA-binding domain, is not required for proper folding. The critical basic and cysteine residues of ACE1 are interdigitated, thereby providing an unusual example of overlapping small molecule and DNA binding functions within a directly regulated transcription factor. In contrast, the carboxyl-terminal region of ACE1 is shown to contain a constitutive trans-activation domain that is spatially distinct and functionally dissociable from the DNA- and Cu-binding domain.

Amino Acid Sequence↗
PubMed1989

Cooperative activation of a eukaryotic transcription factor: interaction between Cu(I) and yeast ACE1 protein.

Cu ions activate yeast metallothionein gene transcription by altering the conformation and DNA-binding activity of the ACE1 transcription factor. We show that this conformational switch occurs in an all-or-none highly cooperative fashion (Hill coefficient = 4). Analysis of the subunit composition of ACE1 bound to DNA indicates that cooperativity results from the binding of multiple Cu(I) ions to the cysteine-rich DNA-binding domain. Surprisingly, DNA has little effect on the interaction between Cu(I) and ACE1 as assayed by partial proteolysis; this suggests that the effect of the metal on DNA binding is primarily kinetic rather than thermodynamic. Although Ag(I) also activates ACE1, it acts less cooperatively than the smaller Cu(I) ion and the resulting metalloprotein has a reduced affinity for DNA. The cooperative interaction between Cu and ACE1 allows the cell to respond to a small change in metal concentration by a large change in gene expression.

Copper↗
PubMed1989

Copper and the ACE1 regulatory protein reversibly induce yeast metallothionein gene transcription in a mouse extract.

We describe a cell-free system in which the transcription of the yeast metallothionein gene is inducible by the addition of metal ions plus a specific regulatory protein. Efficient transcription requires the complete yeast ACE1 metalloregulatory protein, including both its DNA-binding and transactivation domains; a mouse nuclear extract providing RNA polymerase and general transcription factors; a template containing the ACE1 binding site; and Cu(I). Because the binding of ACE1 to DNA is dependent on Cu, it is possible to inhibit transcription by the use of Cu-complexing agents such as CN-. We have used this specific inhibition to show that the ACE1 regulatory protein is required for the maintenance as well as the formation of a functional preinitiation complex. The ability to reversibly induce yeast metallothionein gene transcription in vitro provides a powerful system for determining the molecular mechanism of a simple eukaryotic regulatory circuit.

Animals↗
PubMed1989

Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

Animals↗
PubMed1988

Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein.

Copper homeostasis in yeast involves a copper binding protein, metallothionein, and a trans-acting regulatory protein that activates transcription of the metallothionein gene in response to copper ions. We show that the regulatory protein specifically binds to the metallothionein gene control sequences in the presence, but not in the absence, of copper. Both the DNA binding and metalloregulatory functions of the transacting factor are contained within its aminoterminal domain, and partial proteolysis experiments show that copper activates this domain by causing a major switch in its conformation. Silver also activates the DNA binding domain in vitro and induces metallothionein gene transcription in vivo. We propose a novel copper cluster model for the DNA binding domain based on its surprising structural similarities to metallothionein itself.

Base Sequence↗
PubMed1988

Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants.

Cu-metallothionein was purified from Saccharomyces cerevisiae harboring plasmids containing mutated CUP1 metallothionein genes resulting in deletions at the carboxy-terminal end of the polypeptide. The truncated polypeptides are recovered as polypeptides of 35 and 48 residues in length. The Cu-S cluster in the wild-type metallothionein and the two truncates were characterized. The truncated proteins, designated T35 and T48, contain 4 and 2 fewer cysteinyl residues, respectively, compared to the 12 cysteines in wild-type metallothionein; yet the mutant molecules bind Cu(I) ions in a stoichiometry comparable to the wild-type protein, i.e. 7-8 mol eq. The Cu(I) ions bound to T48 are as tenaciously bound as those bound to the wild-type molecule. The electronic transitions in the ultraviolet are similar for Cu-T48 and the wild-type protein. Both mutants and wild-type Cu-protein exhibit luminescence. The corrected emission maxima occurs at 609 nm with a corrected excitation peak near 277 nm. The luminescence quantum yield and lifetime of fluorescence decay of Cu-T48 and wild-type Cu-metallothionein are similar. The absolute quantum yield of the wild-type Cu-protein luminescence is 0.0058 and has a 440-ns lifetime. The similar fluorescence rate constant in the two molecules suggests they possess a similar chromophore. The Cu-T35 protein is more labile than Cu-T48 or the wild-type protein in the association of Cu(I) ions and the air sensitivity of the electronic transitions and luminescence. Although T48 lacks 2 of the 12 cysteines in the wild-type protein, we are unable to detect any differences in the properties of the native metal clusters in the two molecules; T35 lacking 4 cysteinyl residues forms a Cu(I) cluster with properties significantly different from the wild-type molecule. Properties of the Cu-thiolate cluster were also studied in Cu(I)-reconstituted samples. The cluster in wild-type metallothionein forms in all-or-nothing fashion. This conclusion is based on copper binding stoichiometry and luminescence studies. The relative quantum yield of samples with intermediate Cu(I) levels was constant, consistent with all-or-none cluster formation.

Amino Acids↗
PubMed1984

Copper metallothionein of yeast, structure of the gene, and regulation of expression.

Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copper-binding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.

Amino Acid Sequence↗
PubMed1984

A mouse histone H4 gene carried by an SV40 vector is accurately expressed in infected monkey cells.

We present evidence that a cloned mouse histone H4 gene contains all the information required for the generation of functional H4 mRNA. The cloned mouse gene, flanked by spacer sequences extending 228 bp at the 5' end and 1100 bp at the 3' end, was introduced into the late region of the SV40 genome and the recombinant virus was used to infect cultured monkey kidney cells. RNA mapping studies demonstrated that the H4 transcripts from the infected cells could be initiated at either the mouse or viral promoter and that the majority of the RNA had the same 3' end as authentic mouse H4 RNA. The mouse RNA was incorporated into polysomes and there was a specific increase in H4 protein synthesis in cells infected with the recombinant virus. The distribution of the H4 transcripts between the polysomal and postpolysomal fractions suggests that RNA initiated at the mouse promoter is more efficiently bound to polysomes than is the hybrid RNA initiated at the SV40 promoter.

Animals↗
PubMed1984

Efficient production of hepatitis B surface antigen using a bovine papilloma virus-metallothionein vector.

We have developed a highly efficient system for producing hepatitis B virus surface antigen in cultured mammalian cells. This system utilizes a recombinant bovine papilloma virus in which the hepatitis surface antigen coding sequences are inserted into the 5' untranslated region of the mouse metallothionein-I gene. Mouse fibroblasts stably transformed with this molecule produce surface antigen at levels as high as 10 mg/L/24 h and can be maintained in continuous culture for up to 85 days.

Animals↗