FGSC Aspergillus Homepage Archiv

ASPERFEST4:

The Fourth International Aspergillus Meeting will be at Asilomar, Sun-Tues, 3/18/07 -3/20/07, immediately preceeding the 24th Fungal Genetics Conference. Meeting Registration should be open in October. Watch our homepage(http://www.fgsc.net/Aspergillus/asperghome.html) for more info on Asperfest4... and to see minutes and photos from Asperfest3.

ASPERGILLUS GENE NAMING AT UNIPROT/SWISS PROT:

In an effort to make their comparative data as easy to interpret as possible, the UniProtKB/Swiss-Prot database (http://www.expasy.org/sprot/) is now propagating S.cerevisiae names to Aspergillus genes found based on orthology(atg8 for ATG8 rather than atgH). If a name is already assigned, UniProtKB/Swiss-Prot will add the S. cerevisiae name as a synonym.

Please note: This convention was adopted by UniProtKB/Swiss-Prot without consultation of the AGRPC. We are just passing this on for your information.

VEA IN KU DELETES AND PCR PROTOCOL FROM BERL OAKLEY:

Ana Calvo has sequenced the veA locus in several of the Ku strains (nkuA deletants) with the following results: TNO2, TNO2A3, TNA25, TNOA21 and TNO2A7 are veA1.

In addition, we have had several inquiries about the fusion PCR procedure we use. The procedure is robust and almost always produces fusion PCR products that are pure enough to be used successfully for transformation without band purification. An advantage over the procedure previously described (Yang et al., 2004, Eukaryot. Cell 3:1359-1362) by the Osmani lab (which has, itself, been used successfully many times) is that it requires two less primers. The procedure is described in detail here

The 3rd International Aspergillus meeting was held as a satellite meeting at the 8th European Conference on Fungal Genetics. The site at University of Natural Resources and Applied Life Sciences (BOKU), at the end of the excellent Vienna metro system was comfortable and convenient. There were 165 participants representing 24 countries. The scientific program featured 10 talks and 87 posters presented by scientists at all career stages.

The 4th International Aspergillus meeting will be held at the Asilomar Conference Center as a satellite meeting to the 24th Fungal Genetics Conference.

Dear Aspergillus Researcher,

In response to community input at last year's Aspergillus meeting, an A. nidulans action committee was appointed by the AGRPC to lay the groundwork for a proposal(s) to generate genome-wide community resources (see - http://www.fgsc.net/Aspergillus/Asper2minutes.doc -). We are therefore writing regarding potential grant applications and funding opportunities for genome based Aspergillus community resource development.

Much has been achieved over the last year with the publishing of three Aspergillus genome papers in Nature, the generation of strains and PCR techniques for improved gene targeting, the generation of microarrays and the recent improved annotation of the A. nidulans genome. Clearly we would be wise to capitalize on this momentum.

For any grant applications significant input from the community will be required. Below are potential areas for community resource development:

- improved genome annotation based upon cDNA sequence.
- bioinformatics and database development and maintenance.
- deletions and phenotypic characterization.
- promoter replacement for gene rundown and over expression.
- GFP or similar visual tagging.
- affinity tagging, protein purification and MS analysis of protein complexes.
- yeast two-hybrid screens.
- resources based upon technological advances, such as generating
synthetic lethal screen methodologies.
- development of automated robotics for gene manipulation.
- microarray experiments.
- random insertional mutagenesis.

We ask that you consider these areas for potential resource development and consider what your lab would find most useful. In addition, please indicate which other areas you think are important to develop.

The other major issue is how to garner funding for such projects. The NIH is currently running a funding mechanism called TOOLS FOR GENETIC AND GENOMIC STUDIES IN EMERGING MODEL ORGANISMS. The next grant submission date for these grants is June 1, 2006. We could possibly submit a grant(s) for this deadline depending on what other projects are being developed and the level of interest within the community for the different projects. This funding mechanism is probably only open for A. nidulans.

Do any of you know of other realistic funding mechanisms for generating community based resources? Are there any potential commercial or international sources of funding or people we could approach. Do you know of any resources that could be utilized?

Finally, it would be helpful to know of any ongoing projects so that we may coordinate our efforts.

Please address your input to all members of the action committee for community resource development so that we can discuss further at the European Aspergillus Meeting.

The A. nidulans Action Committee thanks you for your input.

Stephen A. Osmani <osmani.2@osu.edu>, Berl Oakley <oakley.2@osu.edu>, Michelle Momany <momany@plantbio.uga.edu>, Gerhard Braus <gbraus@gwdg.de>, Mark Caddick <caddick@liverpool.ac.uk>, Gustavo Goldman <ggoldman@usp.br>, Michael Hynes <mjhynes@unimelb.edu.au>, Nancy Keller <npk@plantpath.wisc.edu>

A. nidulans annotation release 4 has been posted at FGI (http://www.broad.mit.edu/annotation/fungi/aspergillus_nidulans/ see "What's new?" link for more info). Release 4 incorporates manual curation of questionable gene calls and maps of the 70mers used on the PFGRC oligo arrays. This improved annotation was made possible by funding from NIAID to the PFGRC (http://www.niaid.nih.gov/dmid/genomes/pfgrc/default.htm) and lots of cooperation between Jennifer Wortman’s group at TIGR and James Galagan’s group at Broad. The improved annotation has been deposited at GenBank, but might not yet be posted.

Recently, we have deleted the ku70 and ku80 homologues in Aspergillus fumigatus. As expected, these strains showed increased frequency of homologous recombination. These studies were published in the January issue of Eukaryotic Cell (Krappmann et al. 2006. Eukaryot Cell 5:212-5 and da Silva et al, 2006 Eukaryot Cell 5:207-11). In order to make these strains available to the community, we are depositing them in the FGSC. We hope that the community may organize genome wide gene targeting projects for A. fumigatus.

FGSC #	SPECIES	GENOTYPE	DEPOSITED BY	ORIGINAL NUMBER
A1145	A. nidulans	pyrG89; pyroA4;nkuA::argB; riboB2	Berl Oakley	TN02A7
A1146	A. nidulans	wA3; pyroA4; argB2; nku::argB	Berl Oakley	TN02
A1147	A. nidulans	pyrG89; argB2; pabaB22, nku::argB; riboB2	Berl Oakley	TN02A25
A1148	A. nidulans	pyrG89; nkuB::A. fumigatus riboB; pyroA4, nkuA::argB, riboB2	Berl Oakley	TN12
A1149	A. nidulans	pyrG89; pyroA4; nkuA::argB	Berl Oakley	TN02A3
A1150	A. nidulans	pyroA4; nkuA::argB; riboB2	Berl Oakley	TN02A21
1151	A. fumigatus	pyrG^{AF::Delta KU80} pyrG-	Gustavo Goldman	KU80 DELTA
1152	A. fumigatus	wt	Gustavo Goldman	NOT CEA17
1153	A. nidulans	yA1 pabaA1 pyroA4 argB2 nkuA::bar	Michael Hynes	MH11046
1154	A. nidulans	yA1 pabaA1 niiA4 nkuA::bar	Michael Hynes	MH11057
1155	A. nidulans	pyrG89 pyroA4 nkuA::bar	Michael Hynes	MH11068
1157	A. fumigatus	akuA::ptrA	Sven Krappmann	AfS28
1158	A. fumigatus	akuA::loxP-hygro^R/tk	Sven Krappmann	AfS34
1159	A. fumigatus	akuA::loxP	Sven Krappmann	AfS35

The paper describing the efficient gene targeting system involving the knockout of the A. nidulans Ku70 homolog (nkuA), and the development of heterologous selectable markers, has been accepted for publication in Genetics and is available on line, prior to publication. We (the Osmani, Hynes and Oakley labs) have used the system for GFP and mRFP fusions, for promoter replacements and for knocking out essential genes (gene replacement/heterokaryon rescue) and non-essential genes. Most experiments were done with linear fragments generated by fusion PCR although plasmids were used in some experiments. We have consistently obtained correct targeting frequencies of about 90% and integration of additional sequences at other places in the genome is very rare. The heterologous selectable markers are important because if one uses an A. nidulans gene as a selectable marker, one can get integration of the transforming sequence into the chromosomal copy of the gene (assuming the chromosomal copy has not been deleted). Here is the link for the paper.

http://www.genetics.org/cgi/content/abstract/genetics.105.052563v1?ck=nck

Those of you that have been using the strains should feel free to publish data based on the use of the strains. The Ku knockout strains and the heterologous selectable markers (pyroA and riboB from A. fumigatus as well as the bar gene under the control of an A. nidulans promoter) are available from the FGSC. My lab plans to deposit fusion PCR cassettes using these markers as we construct and test them.

The publication of the genome paper and the development of an efficient gene targeting system means that this is a good time to revisit the possibility of carrying out genome-wide gene targeting projects. There has also been significant reannotation of the A. nidulans sequence based on cDNA sequences, although this does not appear to have reached the databases yet. This should greatly facilitate gene targeting projects. I will send out an e-mail next week with some thoughts and questions about genome wide gene targeting projects.