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Transcript profiling of Candida albicans amino acid starvation response

 

 

Introduction                                                                                                                

The response to amino acid starvation (General Amino Acid Control) is a very well characterized mechanism in the yeast Saccharomyces cerevisiae. It involves the transcription factor Gcn4, which activates target genes via the responsive element GCRE in response to amino acid starvation. The regulation of GCN4 activation is dependent upon four small open reading frames upstream of start codon in the GCN4 mRNA, which regulate the efficiency and kinetics of GCN4 translation. Translational Induction of GCN4 involves the phosphorylation of the initiation factor eIF2a subunit by the Gcn2 protein kinase. The Candida albicans GCN4 homologue was shown to mediate a similar response under inducible conditions, and also to be partly involved in the morphogenetic switch from yeast to pseudohyphal growth (Tripathi et al. (2002) EMBO Journal 21, 5448-5456). The C. albicans Gcn2 homologue shares 37% overall identity with ScGcn2, and 47% identity within the protein kinase domain. Although CaGcn2 is an eIF2a kinase, which phosphorylates this factor in response to amino acid starvation, strains lacking functional Gcn2 are resistant to the histidine analogue 3-Aminotriazole (3AT) and moreover are still capable of initiating the general amino acid response. 

In these experiments, transcript profiling was performed to examine the CaGcn4 and CaGcn2 regulons.  Each experimental condition was pair-hybridized with a control mRNA population which was the resulting pool of three independent wild type cell cultures (CAF2-1) growing in synthetic complete medium. Triplicate hybridizations were performed for each strain using three independent cultures. Total mRNA was extracted from three independent cultures for the wild type and gcn4 strains, and from three independent knockouts in the gcn2 null background. Hybridizations were performed using glass microarrays with probes for 5907 C. albicans open reading frames. For each strain, cells were grown to mid-exponential phase (O.D600nm 0.5) before being transferred to fresh SC or SC lacking histidine and containing 40 mM 3AT for 3h.

These data are provided by Hélène Tournu, Abigail Mavor, Gwyneth Bertram, and Alistair Brown.  For additional information, please contact Alistair Brown (al.brown@abdn.ac.uk). This work was supported by the EC (QLK2CT-2000-00795), the Welcome trust (063204),  and the BBSRC (G11780, P10256, P17124).

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Details of Procedures

Species

Candida albicans

Strains

Wild type (wt): CAF2-1 (Fonzi & Irwin, 1993)

Dgcn4 (D4): GTC43, gcn4::hisG-URA3-hisG/gcn4::hisG (Tripathi et al., 2002)

Dgcn2 (D2): HTC43, HTC47 and HTC51, gcn2::hisG-URA3-hisG/gcn2::hisG (Tournu et al., submitted)

Experimental populations

Cultures in complete glucose medium (SC) grown at 30°C for 3h from exponentially growing cultures (Tournu et al., submitted).

Cultures in 3-Aminotriazole (3AT) grown in SC lacking histidine and containing 40 mM 3AT at 30°C from exponentially growing cultures (Tournu et al., submitted)

RNA isolation

By dropping cell samples into liquid N2 and trizol extraction, as described  (Murad et al., 2001)

labeling

Direct labelling with Cy3/C5-dCTP as described here

Targets

Cy3/Cy5-labelled cDNA

Microchip features

C. albicans glass microarrays with probes for 5907 genes as described here

Microchip manufacturer

Eurogentec SA

Hybridization method

As described here

Imaging

ScanArray light scanner (Packard Bioscience, version 2.11)

Data capture

QuantArray software (Packard Bioscience, version 2.0). Each images were scanned manually and data points were flagged if necessary (dust, local dye background, etc..). These flagged data points were automatically excluded for data normalisation in GeneSpring.

Low quality analysis

GeneSpring v. 5.0

Normalisation method

Intensity-dependent method (Lowess) applied to the print-tip (GeneSpring v 5.0)

High quality analysis

Significance Analysis of Microarrays, SAM (Tusher et al., 2001)

 

 

 

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Normalized Data

The normalized intensities of the 18 hybridisations (triplicate hybridizations for each strain in each growth condition) are available in normalized data.xls. Transcript profiling analysis was carried out comparing the effect of amino acid starvation upon wild type, gcn4 null and gcn2 null backgrounds in C. albicans. Genes significantly regulated in response to 3AT were identified for each strain using Significance Analysis of Microarrays or SAM. The false positive threshold was set at one for each pair-comparison (wt-SAM.xls, gcn4-SAM.xls, and gcn2-SAM.xls). Interestingly, around 50% of the GCN4-activated target genes are found significantly up regulated in a gcn2 null (see Venn diagrams). The Venn diagram gene lists can be found in gene-lists.xls.


Raw Data                                                                                                                

Comparison*

Cy3 labelling

Cy5 labelling

Date of hybridization

Slide

Batch

Name of .txt file

Download all .txt file

cwtSC/wtSC

cwt-SC

wt-SC

28/01/03

1

J020B

Export J020B1- CAF2+HIS_1

cwtSC/wtSC

cwt-SC

wt-SC

28/01/03

2

J020B

Export J020B2-CAF2+HIS_2

cwtSC/wtSC

cwt-SC

wt-SC

28/01/03

3

J020B

Export J020B3-CAF2+HIS_3

cwtSC/wt3AT

cwt-SC

wt-3AT

30/01/03

13

J020B

Export J020B13-CAF2+3AT_1

cwtSC/wt3AT

cwt-SC

wt-3AT

30/01/03

14

J020B

Export J020B14-CAF2+3AT_2

cwtSC/wt3AT

cwt-SC

wt-3AT

30/01/03

15

J020B

Export J020B15-CAF2+3AT_3

cwtSC/D4SC

cwt-SC

D4-SC

03/02/03

4

J020B

Export J020B4-gcn4+HIS_1

cwtSC/D4SC

cwt-SC

D4-SC

03/02/03

5

J020B

Export J020B5-gcn4+HIS_2

cwtSC/D4SC

cwt-SC

D4-SC

03/02/03

6

J020B

Export J020B6-gcn4+HIS_3

cwtSC/D43AT

cwt-SC

D4-3AT

13/02/03

17

J020B

Export J020B17-gcn4+3AT_1

cwtSC/D43AT

cwt-SC

D4-3AT

13/02/03

18

J020B

Export J020B18-gcn4+3AT_2

cwtSC/D43AT

cwt-SC

D4-3AT

13/02/03

19

J020B

Export J020B19-gcn4+3AT_3

cwtSC/D2SC

cwt-SC

D2-SC

23/06/03

8

E120c

Export E120c8-gcn2+HIS_1

cwtSC/D2SC

cwt-SC

D2-SC

26/03/03

15

E120c

Export E120c15-gcn2+HIS_2

cwtSC/D2SC

cwt-SC

D2-SC

01/07/03

26

E120c

Export E120c26-gcn2+HIS_3

cwtSC/D23AT

cwt-SC

D2-3AT

23/06/03

9

E120c

Export E120c9-gcn2+3AT_1

cwtSC/D23AT

cwt-SC

D2-3AT

26/03/03

16

E120c

Export E120c16-gcn2+3AT_2

cwtSC/D23AT

cwt-SC

D2-3AT

01/07/03

27

E120c

Export E120c27-gcn2+3AT_3

*cwt-SC, control RNA from wild type CAF2-1 (pool of three mRNAs populations obtained from three independent cell cultures); wt-SC, each

individual mRNA population from CAF2-1 culture grown in SC; wt-3AT, individual CAF2-1 culture grown in SC-HIS+40 mM 3AT for 3h; D4-SC,

gcn4 null (GTC43) grown in SC; D4-3AT, gcn4 null grown in 3AT conditions; D2-SC, each individual gcn2 null (three independent knockouts,

HTC43, HTC47 and HTC51) grown in SC; D2-3AT, gcn2 nulls grown in 3AT conditions.

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