Back to Galar Fungail home page
Introduction
Candida albicans DNA micro-arrays have been designed by the Galar Fungail Consortium on the basis of Assembly 6 of the C. albicans genome sequence provided by the Stanford Genome Technology Center.
In order to design these arrays, identification and annotation of genes in the C. albicans genome have been performed. This annotation is available in the CandidaDB C albicans genomics database developed by the Galar Fungail Consortium. Using this information, oligonucleotides were defined with the primer3 software in order to produce 300-400 nt probes preferentially located at the 3'-end of all C. albicans genes. Probes have been obtained by amplification of C. albicans genomic DNA and spotted on aldehyde coated glass slides. The arrays provide probes for 5907 C. albicans genes as well as various controls (C. albicans mitochondrial genes, bacterial an S. cerevisiae genes, intergenic regions). Arrays are available from Eurogentec S.A.
For more informations on the C. albicans genome annotation and CandidaDB, click here
For a Table summarizing the data in CandidaDB, click here
For more informations on the C. albicans micro-arrays, click here
To access files for use with the micro-arrays, click here
Identification of C. albicans genes and annotation
The following strategy has been followed to develop CandidaDB:
The identification of open reading frames was performed on Assembly 6 of the C. albicans genome sequence provided by the Stanford Genome Technology Center.
All open reading frames which encode proteins of more than 150 amino acids or have a homologue in public databases or have a significant coding probability according to GeneMark prediction were subjected to a manual annotation using GMP-Tool-box.
Duplicated protein coding sequences which occur in the C. albicans genome because of heterozygosity were subsequently identified. In this case, only one allele is present in CandidaDB but information about the location of the other allele in Assembly 6 of the C. albicans genome is provided.
Annotation of all remaining protein coding sequences was done using the following convention:
Gene names: The standard convention for naming C. albicans genes was used. Only genes that have already been characterized or can be postulated to encode a functional homologue of the more closely related Saccharomyces cerevisiae gene are named according to this convention. Genes that did not meet these criteria have been designated IPFxxxx where IPF stands for "Individual Protein File".
Gene names tags: Several tags have been added to gene names to take into account the occurence of frame-shifts, contig breaks and exon/intron structures that are present in the current version of the C. albicans genome:
".5" and ".3" indicate that the protein coding sequence only corresponds to the 5'- or 3'-end of a gene and that the corresponding 3'- or 5'-end has not been defined.
".5f" and ".3f" indicate that the protein coding sequence only corresponds to the 5'- or 3'-end of a gene and that the corresponding 3'- or 5'-end is found contiguously on the genome.
".5eoc" and ".3eoc" indicate that the protein coding sequence only corresponds to the 5'- or 3'-end of a gene and is located at the end of a Contig in Assembly 6 of the C. albicans genome available at the Stanford Genome Technology Center.
".exon1" and ".exon2" indicate exon regions of genes that have a probable exon/intron structure.
Functions: Functions were assigned on the basis of published data when available or homology to proteins of known function. In this case, the proposed function is followed by a "(by homology)" tag to emphasize that it has not been validated by experimental data and therefore should be used cautiously.
S. cerevisiae homologue: Informations are provided on the most closely related gene in S. cerevisiae.These include when available gene name, alternate gene names, function, e-value of blastp of the C. albicans protein vs the S. cerevisiae proteome, reciprocity of the blastp result.
In addition to these informations and to the different search and genome browsing tools, CandidaDB provides links to the sequence of the Assembly6 Contig in which a coding sequence is located and to the corresponding ORF and its annotation on the Stanford Genome Technology Center website.
WARNING: For the purpose of developing CandidaDB, we have generated a virtual genome sequence by linking Contig DNA sequences of Assembly 6 available at the Stanford Genome Technology Center. Contig sequences are separated by stretches of 500 undefined nucleotides (X). Linkage of the Contig sequences is independent of their actual order on the C. albicans genome. Therefore, the locations of the coding sequences on this virtual genome do not reflect their actual location on the C. albicans genome. Location of the coding sequences on the Contig they originate is also provided in CandidaDB.
If
you use CandidaDB in a publication, please quote the following:
"Nucleotide sequence data for Candida albicans were obtained from
the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida.
Sequencing of C. albicans was accomplished with the support of the NIDR
and the Burroughs Wellcome Fund.
Informations about coding sequences and proteins were obtained from CandidaDB available at http://www.pasteur.fr/Galar_Fungail/CandidaDB/ which has been developed by the Galar Fungail European Consortium (QLK2-2000-00795)."
Any
comments concerning CandidaDB are welcome and should be sent to denfert@pasteur.fr.
C. albicans micro-arrays
PCR primers of 18-22 bases in length were designed using primer3 software to amplify a 3'-region of 300-400 bp from each ORF. Primers were in order i) to be optimally located in the 3'-end of the corresponding gene, ii) to limit the simultaneous amplification of regions corresponding to distinct genes, and iii) to limit potential cross-hybridizations. For this purpose all pairs defined using primer3 and all the corresponding predicted PCR products were compared using blastn to the C. albicans genome sequence (Assembly 6). Only those PCR primer pairs that did not give more than ten significant hits (p < 0.05) to the genome, that were not susceptible to amplify another ORF and whose corresponding PCR products did not match another ORF over more than 40 contiguous bp or 100 discontinuous bp were retained.
In a previous study (Murad et al., 2001, see information here), we had defined primer pairs for 2016 C. albicans genes. Of these only 1331 fit the above criteria. Definition of oligonucleotides was performed for all remaining ORFs included in CandidaDB. When the above criteria were not met for a pair of primers, a new pair was defined by varying primer3 parameters for oligonucleotide definition and by extending the region for oligonucleotide definition to the whole ORF rather than the most 3' 600 nt. A total of 6038 primer pairs were defined that correspond to 6003 entries in CandidaDB. This is summarized in a Table availale here with information on 1) the sequence of the primer pairs, 2) their location in the ORF, 3) a link to the sequence of the PCR product, 4) a link to the result of a blastn comparison to all ORFs available in CandidaDB, and 5) an evaluation of the potential for cross-hybridization of each probe. Two criteria are provided: #cross-hyb with Sc/Self>0.3 ie the number of blastn matches with a score that is more than 0.3x the self-score of the PCR product; #cross-hyb with log(E-value)<-20 ie the number of blastn matches with an e-value below 1e-20. Using these criteria, 303 of the 6038 probes had a potential of cross-hybridization.
These oligonucleotides were synthesized with a 5'-tag (5'-oligonucleotide, 5'-CGACGCCCGCTGATA: 3'-oligonucleotide, 5'-GTCCGGGAGCCATC') to facilitate subsequent re-amplification of the PCR products. Using these oligonucleotides, the ORFs were PCR-amplified from the C. albicans SC5314 genome and subsequently reamplified using amine-modified oligonucleotides corresponding to the 5'- and 3'-tags. The purity and length of all PCR products were checked by agarose gel electrophoresis. A total of 5943 PCR products corresponding to 5907 genes, which satisfied our quality controls, were spotted in duplicate onto aldehyde coated glass slides. Candida albicans genomic DNA, E. coli ORFs and S. cerevisiae ORFs were included on the micro-arrays as controls.
DNA-arrays described above are available from Eurogentec SA. Further informations on the arrays produced by Eurogentec are available at http://www.eurogentec.com/code/en/geno_dnaa_prod_cand.htm.
Files to download
To download the zip archive with information on all primer pairs and corresponding probes, click here
Upon unzipping, an excel file and two folders will be obtained. They should be placed in the same directory in order that the html links available in the excel file are active.
Files for use with the different batches of arrays:
Each zip archive corresponds to the information provided by Eurogentec SA with a specific batch of C. albicans arrays. It contains:
1. A Read_Me_first file
2. A PowerPoint file with the array coordinates
3. A JPEG file and the corresponding .gr file with data of a QC experiment obtained after labelling of the array with terminal transferase
4. A albicans.gal and a GeneFunctionsalbicansImagene.txt file to be used with the scanning software;
5. A GeneFunctionsalbicans.xls with the localization of individual genes on the array ( block, column and row locations). When available, the potential gene function is provided.
6. A scorecard 4 pins vertical.gal corresponding to the DNA of the ScoreCard kit (Amersham) spotted on the array.
7. Detailed protocoles for micro-array hybridization as provided by Eurogentec
| Batch | Zip archive |
| March 2002 | download |
| CaB030C | download |
| CaB100C | download |
| CaG010Cs1 | download |
| CaG030Cs1 | download |
| CaL090B | download |
| CaL170B | download |
To download the zip archive with files for use with GeneSpring, click here
Place the zip archive in the data folder of GeneSring and unzipit. The new genome is called GeneSpringalbicans.
Credits
Annotation of the C. albicans genome was performed under the supervision of Christophe d'Enfert with the help of Lionel Frangeul (Institut Pasteur). It has involved Alistair Brown and Abigail Mavor (University of Aberdeen, UK), Claude Gaillardin and Djamila Onésime (INRA, Grignon, France), Joachim Ernst and Krishnamurthy Shankarling (University of Duesseldorf, Germany), Angel Dominguez, Maria-Carmen Lopez and Nuria Martin (University of Salamanca, Spain), Jose Perez Martin (CSIC, Madrid, Spain), Piet de Groot and Frans Klis (University of Amsterdam, the Netherlands), Luis Castillo and Rafael Santandreu (University of Valencia, Spain), Oliver Bader, Chantal Fradin, Donita Kunze and Bernhard Hube (Robert Koch Institute, Berlin, Germany) and Fredj Tekaia, Sylvie Rodriguez and Susana Garcia (Institut Pasteur, Paris, France).
Implementation of CandidaDB has been achieved by Christophe d'Enfert, Louis Jones, Ivan Moszer and Lionel Frangeul (Institut Pasteur). The original SubtiList database was realized by Ivan Moszer with the invaluable help from Claudine Médigue and Alain Viari.
The design of the DNA-arrays has been performed by Christophe d'Enfert with the help of Fredj Tekaia and Lionel Frangeul (Institut Pasteur)
This work was supported by grants the Ministère de la Recherche et de la Technologie (P.R.F.M.M.I.P. - Réseau Infections Fongiques) and the European Commission (Galar Fungail network).
Sequence data for Candida albicans was obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida. Sequencing of Candida albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund.
Primer3 was developed at Whitehead_Institute and Howard Hughes Medical Institute. The development of Primer3 and the Primer3 web site was funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute.