Open Access Research article

Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

Jorge Rodriguez-Caban1, Waleska Gonzalez-Velazquez1, Lizaida Perez-Sanchez1, Ricardo Gonzalez-Mendez2 and Nuri Rodriguez-del Valle1*

Author Affiliations

1 Department of Microbiology and Medical Zoology, Medical Sciences Campus, University of Puerto Rico, PO Box 365067, San Juan, PR 00936-5067, USA

2 Department of Radiological Sciences, Medical Sciences Campus, University of Puerto Rico, PO Box 365067, San Juan, PR 00936-5067, USA

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BMC Microbiology 2011, 11:162  doi:10.1186/1471-2180-11-162

Published: 11 July 2011

Additional files

Additional file 1:

DNA and Amino acid sequence SSDCL-1. The partial DNA and derived amino acid sequence of the ssdcl-1 gene. Non-coding regions are given in lower case letters, coding regions and amino acids are given in upper case letters. The helicase domain is shadowed in yellow, the dsRNA binding domain is shadowed in blue green and the RNAse 3 domain is shadowed in gray. The putative intron is given in lower case red letters.

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Additional file 2:

Amino acid sequence alignments of SSDCL-1 to other fungal DCL-1 homologues. The predicted amino acid sequence of S. schenckii SSDCL-1 and DCL-1 homologues from other fungi were aligned using M-Coffee. In the alignment, black shading with white letters indicates 100% identity, gray shading with white letters indicates 75-99% identity, gray shading with black letters indicates 50-74% identity. Important domains are highlighted in colored boxes. The helicase domain, dsRNA binding domain and the RNAse III domains are highlighted in green, red and blue boxes, respectively.

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Additional File 3:

pSD2G, sscmk1 inserts and colony PCR. This file shows pSD2G (pSD2G) from the Fungal Genetic Stock Center. It has a geneticin resistance cassette and two trpC promoters flanking the multiple cloning site (MCS). File 3A and 3B show the nucleotide sequences of the sscmk1 gene inserted into pSD2G: a 405 bp insert from the 3' region and a 432 bp insert from the 5' region of the gene. These inserts were amplified by PCR from cDNA containing the coding sequence of the sscmk1 gene, cloned in pCR®2.1-TOPO, excised by digestion with restriction enzymes and cloned in the MCS of pSD2G to produce pSD2G-RNAi1 and pSD2G-RNAi2, respectively. File 3C Shows the results of the colony PCR of various S. schenckii transformants. Cell suspensions of S. schenckii transformants were used as templates for PCR using the G418 (for)/G418 (rev) primer pair as described in Methods. Lane 4 shows the 123 bp DNA ladder. Lanes 1, 2, 3, 5 and 6 shows the bands obtained when the cells transformed with pSD2G-RNAi1 from colonies 14, 15, 18, 19 and 21 were used as template, respectively. In lanes 7 and 8, suspensions of non-transformed cells were used as templates for PCR. A band of the expected size, 622 bp, due to the presence of the geneticin resistance cassette was observed in transformed yeast cells.

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Additional File 4:

cDNA and derived amino acid sequence of the S. schenckii HSP90 homologue isolated using yeast two-hybrid assay. The cDNA and derived amino acid sequence of the SSHSP90 identified in the yeast two-hybrid assay as interacting with SSCMK1 is shown. Non-coding regions are given in lower case letters, coding regions and amino acids are given in upper case letters. The HATPase domain is shaded in yellow and the sequence isolated in the yeast two-hybrid assay is shaded in gray. Red letters mark the conserved MEEVD domain in the C terminal domain of HSP90, necessary for the interaction with tetratricopeptide repeat containing proteins.

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Additional File 5:

Amino acid sequence alignment of SSHSP90 to other fungal HSP90 homologues. The predicted amino acid sequence of S. schenckii SSHSP90 and HSP90 homologues from other fungi were aligned using M-Coffee. In the alignment, black shading with white letters indicates 100% identity, gray shading with white letters indicates 75-99% identity, gray shading with black letters indicates 50-74% identity. Important domains, the HATPase domain and theHSP 90 domain, are highlighted in blue and red boxes, respectively. The C terminal domain is indicated with a blue line.

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