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PMID: 11438645 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

Molecular and cellular biology ·Vol. 21 ·No. 15 ·2001-08-00 ·Pages 4875-88

Stoyan T, Gloeckner G, Diekmann S, Carbon J

Abstract

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

MeSH Terms
Amino Acid Sequence Anti-Bacterial Agents/pharmacology Basic Helix-Loop-Helix Leucine Zipper Transcription Factors Binding Sites Candida/genetics,metabolism Centromere/metabolism Chromatin/metabolism Chromosome Segregation Chromosomes/metabolism,physiology,ultrastructure Cloning, Molecular DNA/metabolism DNA-Binding Proteins/physiology Dose-Response Relationship, Drug Doxycycline/pharmacology Epitopes Fluorescent Dyes/pharmacology Fungal Proteins/physiology Gene Deletion Genetic Complementation Test Indoles/pharmacology Kinetics Methionine/metabolism Models, Genetic Molecular Sequence Data Mutagenesis Mutation Phenotype Plasmids/metabolism Precipitin Tests Promoter Regions, Genetic Protein Binding Protein Structure, Tertiary Recombinant Proteins/metabolism Saccharomyces cerevisiae/genetics Saccharomyces cerevisiae Proteins Sequence Analysis, DNA Sequence Homology, Amino Acid Structure-Activity Relationship Time Factors
Chemicals
Anti-Bacterial Agents Basic Helix-Loop-Helix Leucine Zipper Transcription Factors CBF1 protein, S cerevisiae Chromatin DNA-Binding Proteins Epitopes Fluorescent Dyes Fungal Proteins Indoles Recombinant Proteins Saccharomyces cerevisiae Proteins DAPI DNA Methionine Doxycycline
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Stoyan T
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106, USA. stoyan@lifesci.ucsb.edu
Gloeckner G
Diekmann S
Carbon J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.21.15.4875-4888.2001
Email
stoyan@lifesci.ucsb.edu
Published
2001-08-00
Pages
4875-88
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC87199
Subset
IM
Grants
NCI NIH HHS · R01 CA011034 · United States
NCI NIH HHS · CA-11034 · United States
Databases
GENBANK
AF233343
External Links
PubMed source DOI: 10.1128/MCB.21.15.4875-4888.2001 PMC Full Text View journal details
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