Doctoral Dissertations

Date of Award

12-1989

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

W. Stuart Riggsby

Committee Members

J. M. Becker, David A. Brian, D. W. Larimer

Abstract

Yeasts whose telomrphs (perfect stage) have not been isolated, as well as imperfect forms of yeasts whose telomorphs have been found are grouped in the genus Candida. Therefore the artificial genus Candida is comprised of members which are phylogenetically diverse. The main objective of the project reported here was to determine phylogenetic relatedness among members of the artificial genus Candida. Our approach involved comparison of nucleotide and deduced amino acid sequences of the Candida albicans and Candida tropicalis actin genes. Actin is a conserved geneic element present in all eucaryotes and is therefore an appropriate candidate to study systematics and evolutionary relatedness among distantly related species.

A 2.0 kb Sa/1 fragment which was reported to contain the actin gene of Candida albicans H317 was sequenced. This fragment was found to lack the first exon (minor exon) and part of the intron. Therefore a lambda genomic library of C. albicans H317 was generated and a clone containing the missing 5' region identified and sequenced. The actin gene of C. albicans encodes a protien containing 376 amino acids. It contains a single intron 644 bp in length located ten nucleotides downstream of the +1 translational start site. The mature actin mRNA transcript is 1.4 kbp long. Primer extension results suggested that the transcription start site is about 300 bp upstream of a 5' Sal I site. Comparison of the C. albicans actin gene sequence to that of S. cerevisiae. reveals 5% amino acid replacement substitution and 87.5 % nucleotide sequence similarity. In C. albicans, the region containing amino acid residues 189-205, is unusually rich in replacement substitutions.

Intron sequences, which evolve at a higher rate than coding sequences are expected to be divergent between two species, unless they are closely related. A subclone of the C. albicans actin gene, containing all of the intron except for the first 119 bp was found to not only hybridize to fragments of Eco Rl, Taq I and Alu I genomic digests of Type I Candida stellatoidea , but also to gave similar restriction endonuclease patterns for the Eco Rl and Taq I digests. Such similarity suggests that the two organisms must have diverged recently.

A 3.5 kbp Eco Rl genomic fragment containing the entire actin gene of Candida tropicalis was also cloned and sequenced. It encodes a protien 376 amino acids long and has an intron 689 bp in length located at the same position as the intron of the C. albicans and Saccharomyces cerevisiae actin genes. The nucleotide sequence of the C. tropicalis actin gene is 93.5 % similar to the C. albicans actin gene and 88 % similar to the S. cerevisiae actin gene.

Pairwise comparison of amino acid sequences of fungal actins show that and C. tropicalis actin is 98 % similar C. albicans actin and 96 % similar to theS. cerevisiae. actin. The analysis show that the two Candida species and S. cerevisiae are closely related. S. pombe and A. nidulans are distantly related to each other and distantly related to the two Candida species and to S. cerevisiae. We propose evolutionary relationships among these five fungi on the basis of amino acid sequence similarity of their actin genes.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS