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Transcript Profiling of Repressors Involved in Morphogenesis in Candida albicans

This experiment has examined the Nrg1, Tup1 and Ssn6 regulons in C. albicans On the basis of their roles in S. cerevisiae, Tup1 and Ssn6 were predicted to form a complex that represses the transcription of target genes. C. albicans Nrg1 is known to be a zinc finger DNA binding protein that binds to the NRE to mediate transcriptional repression (Murad et al., 2001), probably by bringing the Tup1-Ssn6 co-repressor complex to the target promoters. Therefore, significant overlap was expected between the Tup1 and Ssn6 regulons, and the Nrg1 regulon was expected to be a subset of the Tup1 and Ssn6 regulons.

Transcript profiling has been used previously to determine the roles of Nrg1 and Tup1 in C. albicans using membrane arrays covering 2002 ORFs. (Murad et al., 2001). Since then, the Stanford Genome Technology Center released Assembly 6 of the C. albicans genome, with 10X coverage. The European consortium Galar Fungail annotated the ORFs highlighted by the sequencing and the resulting database, CandidaDB, has been used to design glass slide arrays with 5896 ORFs representing (almost) the complete C. albicans genome. These have been used to analyse the transcriptome of nrg1, tup1, smooth ssn6 , wrinkly ssn6 , and CAI4 cells growing in YPD at 30¡C. cDNA from three independent cultures of each strain (labelled with Cy5) was co-hybridised with a common RNA control (labelled with Cy3).

These data were provided by Alistair Brown and coll., Aberdeen Fungal Group, University of Aberdeen, UK and will appear in Garcia-Sanchez, S., Mavor, A., Russel, C. L., Argimon, S., Dennison, P., Enjalbert, B. and Brown, A.J.P. (2005) Global roles of Ssn6 in Tup1- and Nrg1-dependent gene regulation in the fungal pathogen, Candida albicans. Mol. Biol. Cell, in press

Experimental Data

Species	Candida albicans

Strains	wt: CAI4 (Fonzi & Irwin, 1993)
	nrg1: MMC4 (Murad et al., 2001)
	tup1: BCa2-10 (Braun & Johnson, 1997)
	ssn6: SGC124 - wrinkly and smooth forms; Garcia-Sanchez (submitted)
Growth Conditions	All strains (all Ura^-) were grown in YPD (+Ade, +Uri) at 30¡C to mid-exponential phase (equivalent to OD₆₀₀ 0.6-0.8).
RNA extraction	Harvested cells were frozen by dropping them into liquid N₂. RNA was extracted by the Trizol protocol as described in SOPs.
Labelling	Direct labelling with Cy3/Cy5-dUTP as described in SOPs.
Control RNA (cRNA)	Control (Cy3) on each array was from a pool of four CAF2-1 (Fonzi & Irwin, 1993) RNA samples grown in YPD (+Ade, +Uri) at 30¡C to mid-exponential phase.
Microarray	Glass microarrays with probes for 5907 C. albicans ORFs as described.
Microarray Manufacturer	Eurogentec SA
Hybrisation Method	As described in SOPs.
Imaging & Data Capture	ScanArray Lite (Perkin Elmer). Software: ScanArray and QuantArray
Normalisation Method	Intensity dependent (Lowess) normalisation by GeneSpring 7.0 (Silicon Genetics)
Quality analysis	In QuantArray, spots containing dust or dye smears that would give artificially high signals were flagged manually; GeneSpring did not use these data in further analysis. Genes that were significantly regulated in each mutant strain compared with CAI4 were identified using SAM software (Tusher et al., 2001) with parameters that minimised the number of possible false positives (FDR<10%).

Microarray Data

Sample	Cy3 Labelling	Cy5 Labelling	Date of Hybridisation	Microarray Batch No.	Microarray Slide No.	QuantArray Text file	GeneSpring Data in Excel
CAI4 #1	cRNA	CAI4	10-Mar-03	B030C	61	Export B030C-61 CAI4#1	Venn diagrams Nrg1-Tup1-smooth Ssn6-wrinkly ssn6 lists
CAI4 #2	cRNA	CAI4	07-Apr-03	B030C	28	Export B030C-28 CAI4#2
CAI4 #3	cRNA	CAI4	05-May-03	B030C	30	Export B030C-30 CAI4#3
nrg1 #1	cRNA	MMC4	12-Feb-03	J020B	11	Export J020B-11 nrg1#1
nrg1 #2	cRNA	MMC4	06-Feb-03	J020B	12	Export J020B-12 nrg1#2
nrg1 #3	cRNA	MMC4	06-Feb-03	J020B	16	Export J020B-16 nrg1#3
tup1 #1	cRNA	BCa-9	19-Mar-03	J020B	29	Export J020B-29 tup1#1
tup1 #2	cRNA	BCa-9	19-Mar-03	B030C	26	Export B030C-26 tup1#2
tup1 #3	cRNA	BCa-9	19-Mar-03	B030C	27	Export B030C-27 tup1#3
smooth ssn6 #1	cRNA	SGC124	02-May-03	J020B	30	Export J020B-30 ssn6#1
smooth ssn6 #2	cRNA	SGC124	02-May-03	B030C	36	Export B030C-36 ssn6#2
smooth ssn6 #3	cRNA	SGC124	05-May-03	B030C	37	Export B030C-37 ssn6#3
wrinkly ssn6 #1	cRNA	SGC124	24-Aug-04	A150D	36	Export A150D-36 wkssn6#1
wrinkly ssn6 #2	cRNA	SGC124	24-Aug-04	A150D	37	Export A150D-37 wkssn6#2
wrinkly ssn6 #3	cRNA	SGC124	24-Aug-04	A150D	38	Export A150D-38 wkssn6#3
						Download all text files	Download all data

Additional control experiments: transcript profiling of CAI8 vs CAI4

Sample	Cy3 Labelling	Cy5 Labelling	Date of Hybridisation	Microarray Batch No.	Microarray Slide No.	QuantArray Text file	GeneSpring Data in Excel
CAI8 vs CAI4	CAI4	CAI8	10-March-05	J260D	9	Export J260D-9 8vs4#1	Interpretation
CAI8 vs CAI4	CAI8	CAI4	10-March-05	J260D	10	Export J260D-10 8vs4#2
CAI8 vs CAI4	CAI4	CAI8	10-March-05	J260D	11	Export J260D-11 8vs4#3
CAI8 vs CAI4	CAI8	CAI4	10-March-05	J260D	12	Export J260D-12 8vs4#4
						Download all text files	Download all data

GF2 laboratories home pages

Useful links

Christophe d'Enfert
Fungal Biology and Pathogenicity
Institut Pasteur
25, rue du Docteur Roux
75015 Paris, France
gf2@pasteur.fr

	Galar Fungail 2
Interaction of fungal pathogens with host cells: a post-genomic approach

Microarray Data

Additional control experiments: transcript profiling of CAI8 vs CAI4

GF2 laboratories home pages

Useful links

Christophe d'Enfert Fungal Biology and Pathogenicity Institut Pasteur 25, rue du Docteur Roux 75015 Paris, France gf2@pasteur.fr

Christophe d'Enfert
Fungal Biology and Pathogenicity
Institut Pasteur
25, rue du Docteur Roux
75015 Paris, France
gf2@pasteur.fr