SULLIVAN DC, CHAPMAN SW, JOSHNI M, CHAPMAN BA; Interscience Conference on Antimicrobial Agents and Chemotherapy.
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 1999 Sep 26-29; 39: 587 (abstract no. 2077).
Univ. Mississippi Med. Ctr., Jackson, MS.
BACKGROUND: Infection with Blastomyces dermatitidis is initiated by inhalation of mycelial phase conidia into the lungs where it converts to a yeast phase at 37[o]C. Genes involved in response to temperature shift are proposed to play a role in the pathogenesis.METHODS: dermatitidis was grown at 25[o]C on a shaking incubator. Aliquots of the culture were removed, the cells collected by centrifugation, and frozen at -80[o]C. The culture was shifted to 37[o]C and aliquots were collected at 24, 48 and 72 hr post temperature shift. The RNA arbitrarily primed (RAP)-PCR method was used for fingerprinting gene transcripts. RNA was isolated by the guanidinium isothiocyanate method and reverse transcribed with an 18 base arbitrary primer. Second strand synthesis used the same primer in one round of low stringency PCR followed by high stringency PCR (40 cycles). S32 labeled products were analyzed on polyacrylamide/urea gels and autoradiographed. PCR products selectively expressed at 25[o]C or 37[o]C were excised from the gel, reamplified by PCR, cloned and sequenced.RESULTS: Two clones were expressed at 25[o]C but absent or greatly reduced at 37[o] C: one showed 88% homology to the 40S ribosomal protein S23. Four clones were expressed at 37[o]C but absent or greatly reduced at 25[o]C. Three were homologous to reported amino acid sequences: cytoplasmic dynein heavy chain (76%; Neurospora crassa, an amphipathic helix SIN gene product) (73%; Saccromyces cerevisiae), and a yeast amino acid permease (58%; S. cerevisiae).CONCLUSIONS: Cytoplasmic dynein is required for nuclear migration in S. cerevisiae and nuclear distribution in Aspergillus nidulans and N. crassa. The SIN3 protein is a transcriptional regulator that can play a role in both activation and repression of transcription. Our findings suggest these genes may play a similar role in the transition from mycelial to yeast phase following temperature shift in B. dermatitidis.
Publication Types:
Keywords:
- Amino Acid Sequence
- Base Sequence
- Blastomyces
- DNA Primers
- DNA, Complementary
- Gene Expression Profiling
- Polymerase Chain Reaction
- RNA
- Saccharomyces cerevisiae
- Temperature
- genetics
Other ID:
UI: 102246832
From Meeting Abstracts