Candida Community News

The National Reserarch Council of Canada has been mandated by Merck Sharp & Dohme Corp. for the distribution of its Candida albicans GRACE (gene replacement and conditional expression) library.

First reported by Roemer et al. in 2003 (Molecular Microbiology 50(1), 167–181), the GRACE method allows for the transcriptional repression of tetracycline promoter-regulated genes. This collection has been replicated by members of the Malcolm Whiteway laboratory from individual tubes of the original library of 4,348 strains. Since this library was produced while the C. albicans annotation was still incomplete, several genes were inactivated in multiple independently derived mutants. We thus produced a non-redundant library containing a total of 2,357 different mutants (one mutant of each gene).

The mutants are supplied in 96 well plates of frozen culture stocks in YPD supplemented 200 ug/ml nourseothricin and 20% glycerol. In addition, we will provide you with the following information:

• the template for the 25 plate collection
• the master information file provided by Merck (with a sequencing-based update scheduled for early 2013)
• a list of non-growers on YPD, or empty wells

Distribution of the library is now conducted by the Leah Cowen laboratory at the University of Toronto.

The use of this library requires the execution of a fully executed MTA with Merck Sharp & Dohme Corp. Consequently, interested laboratories should contact Scott Walker at and provide full contact information of the requesting PI, full contact information of the PI’s institution that handles MTAs, and a brief statement of the proposed use of the strain collection(s) requested by the PI. Subsequently, a representative from Merck will contact Leah Cowen at to arrange distribution.

The cost of the library is $2350 CDN (+ tax and shipping if appropriate).

The National Reserarch Council of Canada has been mandated by Merck Sharp & Dohme Corp. for the distribution of its library of double barcoded Candida albicans mutants carrying heterozygous deletions.

This library is most commonly used in the Genome-Wide Fitness Test as first reported by Xu et al. in 2007 (PLoS Pathogens 3 e92). This powerful method has been shown to be very effective in identifying the mode of action of various antifungal compounds. The collection has been replicated by members of the NRC Genomics Team from individual tubes of the original library of 5,470 strains. Since it was was produced while the C. albicans annotation was still incomplete, some genes were inactivated in multiple independently derived mutants. We currently estimate the library to contain haploid mutations in 5,157 distinct genes.

The mutants are supplied in 60 µl of YPD with 25% glycerol, organized in 59 96-well plates. In addition, we will provide you with several documents including:

• An Excel file containing the strain coordinates and annotations.
• Sequences and structure of the insertions as determined by Sanger-type sequencing.
• Standard protocol for the microarray version of the Merck Fitness Test. Once the strain reannotation is complete, we hope to develop a version of the Fitness Test adapted for Next Generation Sequencing.

In order to validate the inactivated genes and barcode sequences, we isolated genomic DNA from pools of DBC strains produced by Merck in 2003 and 2007. We then used PCR to individually amplify the upstream and downstream barcodes along with genomic DNA from the insertion sites. These were subsequently sequenced in an Ion Torrent PGM. As of July 2013, we have validated 40.5% of the strains and this process will continue until the end of the year.

Distribution of the library is now conducted by the Leah Cowen laboratory at the University of Toronto.

The use of this library requires the execution of a fully executed MTA with Merck Sharp & Dohme Corp. Consequently, interested laboratories should contact Scott Walker at and provide full contact information of the requesting PI, full contact information of the PI’s institution that handles MTAs, and a brief statement of the proposed use of the strain collection(s) requested by the PI. Subsequently, a representative from Merck will contact Leah Cowen at to arrange distribution.

The cost of the library is $4500 CDN (+ tax and shipping if appropriate).

A memorial gathering in memory of Fred Sherman will be held at 10:00 am on Friday, December 6, 2013. The gathering will be held in the Ryan Case Methods Room (Rm #1-9576) of the University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester NY 14642. This event will be a celebration of the life and science of Fred, comprised of reminiscences about Fred by some who knew him well, followed by an opportunity for any guest to say a few words about Fred. For more information, contact Mark Dumont (), Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642 (Phone 585-275-2466). View his obituary.

An announcement from Oliver Homann:

Hello, I'd like to alert the community to the availability of a new platform-independent Java genome browser and motif analysis software called MochiView. Hopefully some of you may find it useful. The software was originally designed for visualization and analysis of C. albicans ChIP-Chip data, but has also been utilized for pure motif analysis as well as with ChIP-Seq/RNA-Seq data and with multiple additional genomes, including S. cerevisiae and humans. It takes ~10 minutes to download the software and install the necessary genome sequence and gene information (using the GFF files stored on SGD, CGD, or AspGD). A manuscript describing the software has just been published in BMC Biology. Visit the MochiView website to view:

• Demo videos
• List of features
• Software download
• Genome import instructions
• Motif library downloads

Contributions to the MochiView motif libraries would be appreciated! Contact us with suggestions, or to join the MochiView mailing list.

The A. P. Mitchell laboratory provided CGD with a list of genes in which multiple transformations using the published UAU1 method yielded no disruptants. In each case, at least 12 independent transformants were tested, indicating that the gene may be essential. The inability to disrupt these genes using the UAU1 construct does not constitute definitive proof that they are essential and investigators may want to use another method to attempt disruption of these genes. The file, UAU1_nondisruptable.txt, is available for download.

Carol Munro, University of Aberdeen, and Christophe d'Enfert, Institut Pasteur, have recently received funding from the Wellcome Trust, UK, to create three new resources for the Candida community. The three main goals of the project will be to create (1) a C. albicans ORFeome library by cloning every predicted ORF into a Gateway® vector in E. coli, (2) a library of bar-coded C. albicans over-expression vectors by placing each ORF under the control of a reverse Tetracycline promoter in a C. albicans integrative plasmid based on CIp10 and (3) a library of C. albicans strains each over-expressing a single gene by integrating the plasmids generated in (2) into the RPS1 chromosomal locus. Each ORF will be verified by sequencing both ends. The funding ends in September 2012 and at this point the 3 collections will be made available to the community and distributed for a small fee that will cover maintenance and distribution costs.

Contacts: Carol Munro, c.a.munro@abdn.ac.uk
Christophe d'Enfert, christophe.denfert@pasteur.fr

An announcement from David Soll:

The Developmental Studies Hybridoma Bank (DSHB) is a national resource established under the auspices of the National Institutes of Health to maintain and distribute at cost hybridomas and their monoclonal antibodies to the general scientific community. For example, monoclonals cost $25 per ml of supernatant, not $200 or $700 per ml. The DSHB now banks close to 1,000 hybridomas and filled close to 9,000 orders for multiple antibodies last year. It will rapidly grow in the near future due to its selection by the National Cancer Institute as the official bank and distributor of the 20,000 hybridomas, now being generated by the NCI Proteomics Initiative, and the antibodies they secrete against 5,000 human genes involved directly or indirectly in cancer. This represents antibodies against proteins encoded by one-fourth of the entire human genome.

The DSHB has now embarked on a new mission, to generate a bank, the DSHB-Microbe, that will collect and distribute hybridomas and their antibodies against microbial antigens - select viruses, bacteria, fungi, and parasites.

What will the DSHB do for you? It will provide you with relevant monoclonals at one-tenth to one-fifteenth the commercial price. It will relieve those of you who have made hybridomas from the burden of distribution. Most importantly, it will facilitate microbial research. It will maintain, reclone and characterize the hybridomas and secreted monoclonals, and help customers use them through a phone hot line.

What can you do? Send us your hybridomas for distribution. Remember, we distribute them, but you still own and can commercialize them. Alternatively, if you know of hybridomas that you would like made available at low cost and high quality, let us know their name, the scientist who generated them, and other details. We will contact the scientist and try to secure the hybridoma for the collection.

The DSHB has served the community of animal cell researchers for 20 years. Help me build the DSHB-Microbe so that it can do the same for microbiologists. [See our web page: dshb.biology.uiowa.edu].

Email: dshb@uiowa.edu

An announcement from Victoria Brown Kennerly:

We have generated DNA oligo arrays representing the Candida albicans genome for use in microarray experiments. They are now available to the community at cost: $100/array (3 genomes spotted per array).

Please see the following website for a complete description, or to order arrays: http://genome.wustl.edu/activity/ma/calbicans/.

One of the true pioneers of our field, Myra Kurtz, died at home on November 1, 2005, after a long struggle with cancer. Her loving family was by her side. She was 60 years old.

Myra's proudest professional accomplishment was her team's development of the hugely successful antifungal, caspofungin, at Merck. Her seminars were highlighted by "before" and "after" photographs documenting caspofungin's efficacy in treating esophageal candidiasis. We also remember Myra professionally as the first to develop C. albicans transformation methods, along with strains and vectors. Never satisfied with her success, Myra continued to improve upon her initial transformation strategies, and maintained an interest in new antifungal targets long after caspofungin proved to be a winner.

Myra's collegiality set the standard for our field. She was enthusiastic about publication of her group's results as a mechanism to share her information. She was a regular participant at meetings that focused on basic science, offering encouragement and advice. It was common knowledge that a request for meeting support sent to Myra would make its way quickly to the appropriate desk, and would not be unanswered.

Those of us lucky enough to know Myra personally remember her incredible breadth of interests and talents, fueled by endless energy. Whether the topic was poetry, beading, or cross-country skiing, Myra offered informed opinions based on considerable thought and experience. Most of all, Myra was fun! Her love of life was truly infectious.

Myra set the bar high. We are fortunate to have had such a gifted individual as our friend and colleague.

Aaron P. Mitchell

An announcement from Aaron Mitchell:

We have a new resource that will facilitate disruption of large numbers of genes at little cost to the investigator, using UAU1 methodology [Enloe, B., Diamond, A., and Mitchell, A. P. (2000) J Bacteriol 182, 5730; Davis, D. A., Bruno, V. M., Loza, L., Filler, S. G., and Mitchell, A. P. (2002) Genetics 162, 1573].

We have created Tn7-UAU1 transposon insertions in 4170 (or 65.5%) of the 6362 unique ORFs defined by the NRCC annotation http://candida.bri.nrc.ca/candida/index.cfm. The insertions are carried in a genomic library constructed from strain CAI4 DNA. Typical clones in the library are over 10 Kbp and are flanked by NotI restriction sites, permitting excision of the insertion-bearing C. albicans DNA for transformation. We have selected insertions that lie at least 300 bp from the nearest end of the clone, permitting efficient targeting into the C. albicans genome. The project is described at http://www.tigr.org/tdb/e2k1/caa1/. In our pilot studies, we have been able to construct multiple C. albicans insertion homozygotes in parallel.

We invite you to request up to 20 insertion-bearing clones that we will provide free of charge, and as quickly as possible. Requests must come from the lab head, and should be emailed to Aaron Mitchell apm4@columbia.edu.

To place a request, please follow this procedure.

1. Search the TIGR database for insertions in genes of interest. You may use your browser's search function, entering ORF19 designations, at http://www.tigr.org/tigr-scripts/e2k1/qzhao/complete_list.pl. The precise location of each insertion may be determined by following the "Seq_ID" link (2nd column from the left) to find the flanking sequence read with Tn7 primer "S" from one end of the transposon. (I can send the Excel file if you have trouble accessing the site.)

2. Note the "Clone_Name" (of the form "CAGxxxx") for the insertion of interest (listed in the 3rd column from the left).

3. Send the list of CAGxxxx clone numbers and shipping information to Aaron Mitchell apm4@columbia.edu. If you have a Fedex account, please send that information too.

4. You will receive an email acknowledging your request.

5. You will receive DNA samples spotted on sterile filter paper. We ask for your patience, because we are uncertain of demand right now, and there is some labor associated with filling each request. You should transform into E. coli, selecting AMP (and, if you wish, KAN) resistance.

6. In publications that benefit from these materials, please acknowledge: Qi Zhao and William C. Nierman (TIGR), Frank J. Smith and Aaron P. Mitchell (Columbia University), and NIH grant 1R01AI057804.

The annotation is now available for queries. Please take a look to see if your favorite genes (especially those already published) are present and properly annotated. This page allows you to search by gene name, orf19 number, contig, CandidaDB/GenBank number (activate the DBxref field) or description. If you have only an orf6 number, you can use our gene name translator. If unsuccessful, you can try submitting DNA or a.a. sequences on the right window of our Blast server. Once you find your gene(s), just press the button near the left end to see the complete annotation. Click twice on the sub-window to close it. If you wish to submit a few corrections, just e-mail me and we'll gladly update the information. If you wish to tackle more than a few genes (a gene family for ex.) please register and send Andre Nantel an e-mail. We'll provide you with more extensive access.

Andre Nantel, M.Sc., Ph.D.
Research Officer and Adjunct Professor
Biotechnology Research Institute
National Research Council of Canada
Montreal, PQ, Canada
andre.nantel@nrc-cnrc.gc.ca