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Physarum Genome Project Update by Jonatha Gott; December, 2004
Background:
On August 4th, 2004, the NIH announced that Physarum polycephalum was one of 18
new model organisms whose genomes will be sequenced as part of the Comparative
Genome Evolution project of the National Human Genome Research Institute (NHGRI).
The anticipated end-product of this whole genome shotgun sequencing project is an
assembled genome sequence with ~6x coverage. The press release can be viewed at
http://www.genome.gov/12511858. NIH has designated Jonatha Gott (Case Western
Reserve University, Cleveland, OH USA) as its contact person within the Physarum
community.
An initial working group comprised of many of the people that participated in gathering
information for the NHGRI panel met in Innsbruck, Austria on July 12th, 2004 to discuss
which strain should be used for DNA preparation, how the data should be handled, and
how additional funding needed for mining the genome could be acquired. Participants
were Sandie Baldauf (University of York, UK), Jonatha Gott (Case Western Reserve
University, Cleveland, USA), Eggehard Holler (University of Regensburg, Germany),
Wolfgang Marwan (University of Hertfordshire, UK), Gérard Pierron (Institut André
Lwoff, Villjuif, France), Pauline Schaap (University of Dundee, UK), and Georg
Golderer, Stefan Leitner, Ernst R. Werner, Gabriele Werner-Felmayer (University of
Innsbruck, Austria). Since that time the email discussion list has been expanded
significantly; please contact Jonatha Gott (see the membership list) if you would like to
be added to the email list.
Strain to be used as the source of DNA for genomic sequencing:
The working group initially decided that DNA should be prepared from diploid nuclei of
LU897 x LU898 plasmodia or from haploid nuclei of LU897 amoebae, with flow
cytometry testing of the strains in two different laboratories (Villejuif and Innsbruck) to
assess the uniformity of each DNA preparation. The initial choice of LU897 x LU898
was made, in part, because this strain was used for the construction of a normalized
cDNA library that is being used to identify ESTs, which would make the gene annotation
easier. However, upon further reflection and subsequent discussion via email, a
consensus emerged from the working group that the axenic haploid amoebal strain
LU352 should be used. Use of a haploid strain will simplify genome assembly, while
use of an axenic strain will avoid problems caused by contaminating bacterial DNA.
Gérard Pierron is currently growing LU352 obtained from Roger Anderson and will be
carrying out flow cytometry on that strain soon. If the DNA content appears consistent,
Gérard will isolate DNA from this strain for sequencing.
Information from Roger Anderson regarding LU352: LU352 was derived as a progeny
of the cross CLd-AXE x LU213 and is largely isogenic with all of the Colonia-inbred LU
strains. It is haploid and contains mutations in at least 2 genes that allow it to grow in
liquid culture. The CLd-AXE parent was the result of prolonged axenic culture, so
LU352 may contain additional mutations. LU352 has the genotype matA2 gadAh
npfC5; the gad mutation promotes apogamic development, but the npfC5 mutation
prevents it.
Physarum database:
In order to maximise the benefits of the Physarum genome project, the Physarum
research community needs a fully functional database that is accessible to all. The
construction and maintenance of such a database is a major undertaking, requiring a
significant amount of effort and funding, and Pauline Schaap suggested partnering with
the Dictyostelium group. Discussions with Rex Chisholm, one of the lead developers of
the Dictyostelium database (http://dictybase.org), were extremely helpful and led to this
suggested course of events:
1. Partner with the Dicty database, using their servers/hardware and "database
schema". Rex suggests making cosmetic changes to the "look" of our site to clearly
delineate the Physarum section of the database, but this is apparently trivial to do if we
give them an idea of what we want.
2. Once the assembled sequence is available, have the sequencing center run an
automated annotation to localize likely genes and send to the Dictybase group. Rex
estimates that it would take them about one month to go from an empty database to an
annotated genome coupled to known Genbank and PubMed entries and any available
EST sequences!!! There will also be automatic links set up with Genbank and PubMed
to link relevant new material to the Physarum site. This they could do largely without
input or funding from us. At this point the site is fully functional, but it would be more
accurate and useful if active curating kicks in, so
3. Hire a curator to compile data from available databases and the literature regarding
ESTs, in situ hybridizations, phenotypes, available mutants, microarray data, etc. and
information from the Physarum community for manual annotation of each gene. The
curator is responsible for quality control and will be the contact person for the entire
Physarum community. All data to be added to the site will be sent to the curator to
provide uniformity and continuity. The Dictybase group have offered to train the curator
we hire, most likely a post-doc level person with computer savvy and
knowledge/understanding of the Physarum literature. We will have to come up with
funding to pay our curator and part of a computer developer's salary (see below).
This isn't entirely altruistic on the part of the Dictybase group, as they would get some
support for personnel and hardware updates, plus they become even more
indispensable in terms of their own grant renewal. In return, we get a functional
database and a means disseminating the genome data, two key elements to getting
downstream funding for Physarum genome mining. This appears to be a win-win
situation.
Funding:
As the NIH contact person, Jonatha Gott would likely write a grant (with heavy input and
letters of support from the Dictybase group and the Physarum community) asking NIH
and/or NSF for roughly $100,000/year for salaries, fringe benefits, hardware updates,
etc. to fund the Physarum database. Rex thought that we would have a very good shot
at getting funding since the costs are VERY minimal relative to starting from scratch (i.e.
buying the hardware, hiring the computer people to develop the software, etc.)
The initial working group also agreed that a world-wide and concerted effort to raise
additional funding for various projects will be necessary in order to provide the
background information for annotating the genes (such as sequencing EST libraries) as
well as for retrieving functional information once the assembled sequence is available.
Those of the Physarum community who are interested in participating in one of these
“mining” projects should contact Jonatha Gott, Center for RNA Molecular Biology, Case
Western Reserve University, School of Medicine, Cleveland, Ohio, 44106 USA; email:[email protected] , who has agreed to co-ordinate these joint research efforts.
Thoughts on resource sharing:
We are requesting that sequence data from the Physarum genome project be released
to the public as soon as it is generated and hope that everyone in the Physarum
community will freely share all of the resources that have been generated in their
respective laboratories. Although a number of valuable resources exist in the Physarum
community, including cDNA and genomic libraries, strain collections, plasmids,
antibodies, etc., there is currently no single place that anyone interested in accessing
these resources can go to find out what is available. The Physarum genome project
would seem to provide the perfect context for organizing such a database. Would
anyone be willing to undertake a survey of members of the Physarum community to find
out what is available?