Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans

Science

Volumn 285, Issue 5431, 1999, Pages 1271-1275

  Hull, Christina M ^a   Johnson, Alexander D ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BINDING PROTEIN; PHOSPHATIDYLINOSITOL KINASE; POLYNUCLEOTIDE ADENYLYLTRANSFERASE; TRANSCRIPTION FACTOR;

ARTICLE; CANDIDA ALBICANS; GENE LOCUS; GENETIC ANALYSIS; LIFE CYCLE; MATING; NONHUMAN; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE; SEQUENCE ANALYSIS; TRANSCRIPTION REGULATION;

1-PHOSPHATIDYLINOSITOL 4-KINASE; AMINO ACID SEQUENCE; CANDIDA ALBICANS; GENE DELETION; GENE EXPRESSION REGULATION, FUNGAL; GENES, FUNGAL; GENES, MATING TYPE, FUNGAL; HOMEODOMAIN PROTEINS; INTRONS; MOLECULAR SEQUENCE DATA; OPEN READING FRAMES; OPERATOR REGIONS (GENETICS); POLYNUCLEOTIDE ADENYLYLTRANSFERASE; RECEPTORS, STEROID; RECOMBINATION, GENETIC; REPRESSOR PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TRANSCRIPTION, GENETIC; TRANSFORMATION, GENETIC;

EID: 0033588031     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5431.1271     Document Type: Article

References (30)

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4. 32P in random prime reactions with the Megaprime DNA Labeling Kit (Amersham). Probe hybridizations, plaque lifts, phage purifications, and plasmid excisions were carried out according to B. Braun's Plaque lift/Phage screening protocol (www.sacs.ucsf. edu/home/JohnsonLab/). For each screening, ∼10,000 plaques were screened and estimated to represent 2.5 to 5 genome equivalents. Genomic inserts were sequenced in full at the University of California, San Francisco, Biomolecular Resource Center.
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10. Disruption constructs for MTLa1 and MTLα2 were constructed with the URA blaster construct pMB-7 (27). The MTLa1 deletion construct primers 5′-cagcacatgcatgcgagctcctttcagatcaagaaacagtttca-3′ and 5′-cagcacatgtcgacggtacccgtcctcagtatagatgc-3′ yielded a PCR fragment with Asp 718 and Sac I ends, and the primers 5′-cagcacatgtcgacggtgccacaataactccactg-3′ and 5′-cagcacatgcatgcgagtgcccgtggtaatacaaa-3′ yielded a PCR fragment with Sal I and Sph I ends. The fragments were cut and ligated sequentially into pMB-7. The Asp 718 to Sac I digest was performed as a partial digest on the PCR product because of a Sac I site in the fragment. The MTLa1 homeodomain deletion construct primers 5′-cagcacatgcatgcgagctcctttcagatcaagaaacagtttca-3′ and 5′-cagcacatgtcgacggtacccgtcctcagtatagatgc-3′ yielded a PCR fragment with Asp 718 and Sac I ends, and the primers 5′-cagcacatgcatgcgagctcaaagtgtagagaaactagttc-3′ and 5′-cagcacatgtcgacggtacctaactaattattttatttcctcccctttta-3′ yielded a PCR fragment with Sal I and Sph I ends. The fragments were cut and ligated sequentially into pMB-7. The MTLα2 deletion construct primers 5′-gaagatctgagctcagtctatcttgatttaggg-3′ and 5′-ggaagatctgtcttgttattgatgtgag-3′ yielded a PCR fragment with Bgl II and Sac I ends, and the primers 5′-aactgcagcttcgtataggtgtgcacttt-3′ and 5′-aactgcagaagcttgactctttggtcatgccttcc-3′ yielded a PCR fragment with Hind III and Pst I ends. The fragments were ligated sequentially into pMB-7.
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12. Supplemental information on isolation of C. albicans genomic DNA and PCR conditions is available on Science Online at www.sciencemag.org/feature/data/ 1041394.shl. Primer sequences used in PCR are as follows: A, 5′-gttacaccacaatcaacacc-3′; B, 5′-ttacatgttggtgaacctaaag-3′; C, 5′-cagcacatgcatgcgagctcaaagtgtagagaaactagttc-3′; D, 5′-cagcacatgtcgacggtacctaactaattattttatttcctcccctttta-3′; E, 5′-cgggatccgaaacgacaaagactagac-3′; F, 5′-cgggatccctaggttgaatttgaacttg; hisG1, 5′-gcgcgtggcgatgcacatggtcag-3′; hisG2, 5′-gcgcggcggttgagtagctct-3′.
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17. The starting plasmid for GFP repression reporters was pYGFP3 (gift from B. Cormack) (28). pYGFP3 (pAJ699) was modified by the addition of the C. albicans URA3 gene into the Sal I site of the vector with a PCR fragment with Sal I restriction sites on the ends by using primers 5′-tcgcctcgagtcgacgggcccagtactaataggaattg-3′ and 5′-tctcggagctcgagtcgacgggcccaggaccacctttgattgt-3′. The resulting fragment was digested with Sal I ligated into the Sal I site of pYGFP3 to create pAJ717. A URA3 3′-untranslated region was inserted immediately downstream of the GFP gene. The UTR was generated as a PCR fragment with Pst I restriction sites on the ends with primers 5′-cgggatccgcgcgctgcagtagttagttaaatgtgaagggggag-3′ and 5′-cgggatccttaattaactgcagaggaccacctttgattgt-3′. The resulting fragment was digested with Pst I and ligated into the Pst I site of pAJ717 to create pAJ724. The C albicans ADH1 promoter was inserted immediately upstream of the GFP gene. ADH1-Bgl II was created in a chimeric PCR reaction to generate an ADH1 promoter with Hind III restriction sites on the ends and a Bgl II restriction site located between the predicted upstream activating sequences and TATA box. Two starting fragments with overlapping ends were generated with primers 5′-cgggatccaagctttaacaaatgaa-3′, 5′-ataagagatctcttgcttgcatgacg-3′, and 5′-cgggatccaagcttaattgtttttgtatttg-3′, 5′-gcaagagatctcttattcagaattttcag-3′. These PCR products were mixed in a final PCR reaction to generate the full-length ADHI-Bgl II promoter with primers 5′-cgggatccaagctttaacaaatgaa-3′ and 5′-cgggatccaagcttaattgtttttgtatttg-3′. The resulting ADH1-Bgl II promoter fragment was digested with Hind III and cloned into pAJ724 to generate CaADH1-GFP (pAj868). Reporter plasmids with three tandem represser binding sites were constructed by cloning double-stranded oligonucleotides into the Bgl II site of the CaADH1-GFP plasmid. In each case two oligonucleotides containing three binding sites were phosphorylated and annealed to one another to generate a double-stranded duplex with single-stranded Bgl II-compatible overhangs. Single binding sites for each are highlighted in bold type. (i) pAJ888, S. cerevisiae haploid-specific gene site 5′-atctgatgtaattaattacatgaattgatgtaattaattacatgaattgatgtaattaattacatga- 3′ annealed to 5′-gatctcatgtaattaattacatcaattcatgtaattaattacatcaattcatgtaattaattacatca- 3′. (ii) pAJ898, S. cerevisiae haploid-specific gene site with point mutations that abolish a1/α2 binding in S. cerevisiae (18). 5′-gatctgctgtaattaattccatgaattgctgtaattaattccatgaattgctgtaattaattccatga- 3′ annealed to 5′-gatctcatggaattaattacagcaattcatggaattaattacagcaattcatggaattaattacagca- 3′. (iii) pAJ1081, a1/α2 binding sequence from the C. albicans CAG1 promoter 5′-gatctgatgtgatttttaacatggattgatgtgatttttaacatggattgatgtgatttttaacatgg- 3′ annealed to 5′-gatcccatgttaaaaatcacatcaatccatgttaaaaatcacatcaatccatgttaaaaatcacatca- 3′. (iv) pAJ933, a1/α2 binding sequence from the C albicans CAG1 promoter with point mutations analogous to the mutations in the S. cerevisiae binding site that abolish a1/α2 binding 5′-gatctgctgtgatttttaccatggattgctgtgatttttaccatggattgctgtgatttttaccatgg- 3′ annealed to 5′-gatcccatggtaaaaatcacagcaatccatggtaaaaatcacagcaatccatggtaaaaatcacagca- 3′. Reporter constructs were linearized through a Bsp El site and integrated into the ADHI promoter. The pattern of fluorescence was confirmed for five transformants for each reporter.
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30. We thank J. Rine, J. Thorner, I. Herskowitz, D. Ganem, and R. Brazas for helpful discussions; I. Herskowitz, R. Smith, D. Inglis, A. Uhl, D. Kadosh, A. Tsong, B. Braun, and R. Brazas for comments on the manuscript; and B. Cormack for use of unpublished reagents. Special thanks to B. Braun for use of his genomic library and for technical assistance throughout, to R. Taylor at the UCSF BRC Facility for her sequencing expertise, and to members of the Johnson lab for their support. This work was supported by NIH grant GM37049 to A.D.J.