Copper-regulated transcription of metallothionein genes in the yeast Candida glabrata

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Title Copper-regulated transcription of metallothionein genes in the yeast Candida glabrata
Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Thorvaldsen, Joanne Louise
Date 1995-06
Description Copper is an essential metal ion for most organisms, yet the presence of excess copper ions is deleterious. Organisms have evolved systems to regulate the intracellular copper ion content tightly. The regulated synthesis of metal-binding metallothionein (MT) serves this purpose. This thesis presents a study on Cu-regulated MT synthesis in the yeast Candida glabrata. C. glabrata has three distinct MT genes (MTI, MTIIa and MTIIb) whose transcription is regulated by copper ions via the transcription factor AMT1. The first focus of this thesis is the in vivo analyses of the function and expression of the three MT genes. The second focus addresses the Cu-dependent function and structure of AMT1. To analyze the MTs in vivo it was necessary to develop tools to manipulate C. glabrata genetically. This included isolation of auxotrophic strains and identification of C. glabrata sequences which could confer autonomous replication in C. glabrata. The significance of MTII to copper tolerance was determined by disruption of MTIIa and MTIIb. The MT promoters were characterized using reporter constructs in C. glabrata. The Cu-dependent properties of AMT1 were studied using a heterologous expression system and purifying and analyzing the AMT1 DNA-binding domain. AMT1 was studied in Saccharomyces cerevisiae by measuring its ability to induce Cu-regulated expression from C. glabrata MT promoter sequences. The activity of AMT1 was also compared to ACE1, the analogous transcription factor in S. cerevisiae. A synthetic gene encoding the AMT1 DNA-binding domain was constructed enabling bacterial expression and purification of this domain. Various spectroscopic techniques were used to analyze how metal ions induce the AMT1 structure that interacts with DNA. In conclusion, my thesis describes cloning systems developed for C. glabrata, characterizes MT function and copper regulated MT promoter sequences in C. glabrata, demonstrates that AMT1 induces Cu-activated transcription of the three C. glabrata MT genes and that AMT1 is functionally homologous to ACE1 in S. cerevisiae, and presents evidence that a single Zn(II) and four clustered Cu(I) ions are responsible for inducing the AMT1 structure that interacts with DNA.
Type Text
Publisher University of Utah
Subject Genetics
Subject MESH Candida; Metallothionein; Copper
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of " Copper-regulated transcription of metallothionein genes in the yeast Candida glabrata". Spencer S. Eccles Health Sciences Library.
Rights Management © Joanne Louise Thorvaldsen.
Format application/pdf
Format Medium application/pdf
Format Extent 4,437,492 bytes
Identifier undthes,3825
Source Original University of Utah Spencer S. Eccles Health Sciences Library (no longer available)
Funding/Fellowship Predoctoral Fellowship T32 CA 09602 from the National Cancer Institute and by grant ES 03817 from the National Institutes of Health.
Master File Extent 4,437,525 bytes
ARK ark:/87278/s6542qd8
Setname ir_etd
ID 191341
Reference URL https://collections.lib.utah.edu/ark:/87278/s6542qd8
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