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Summary:
Dictyostelium discoideum has been an
important experimental organism for the analysis of fundamental biological
processes, including cell migration (Parent
and Devreotes, 1999), cell – cell signaling and signal transduction (Parent and Devreotes, 1996; van Es and
Devreotes, 1999), phagocytosis (Titus,
2000), signaling during morphogenesis and cell fate choice (Meili et al., 2000) and recently for the growth and study of
intracellular microbial pathogens such as Legionella (Solomon et al., 2000). Because of
the unique experimental opportunities it provides, the NIH has included Dictyostelium
in its list of important model organisms.
One goal for the NIH support of model organisms is to provide
infrastructure that facilitates the use of the experimental system. A critical component of this infrastructure
is a well-organized and up-to-date on-line bioinformatics resource that
organizes and presents information about the experimental system. As the genome sequence is completed,
experimental results from functional genomics studies, proteomics, genetic
screens and from single gene functional studies represent an important resource
for the community using Dictyostelium in their studies. In addition, making this information, as
well as specific protocols for working with Dictyostelium readily available on
the internet will facilitate the use of the system by investigators wishing to
apply its unique experimental advantages to their studies. Our goal is to expand the current, volunteer
effort to provide a one-stop resource for Dictyostelium bioinformatics by
obtaining support for personnel and hardware.
This site would also serve as a front end for the stock center whose
establishment is described in a companion document. Together these services support the entire community of
investigators interested in the use of Dictyostelium as a model system.
Introduction:
D. discoideum was
has many experimental advantages for studying fundamental cellular processes
with powerful combination of molecular genetic, biochemical and cell biological
tools. These processes include
cytokinesis, motility, phagocytosis, chemotaxis, signal transduction, and
aspects of development such as cell sorting, pattern formation, and cell-type
determination. The combination of
biochemical, molecular genetic, and cell biological tools that can be applied
to Dictyostelium are unique among model organisms. Each experimental system has both strengths and weaknesses. The strengths of Dictyostelium include its
easy growth and synchronous development.
This makes possible the economical culture of large quantities of both
wildtype and mutant strains that facilitate biochemical analysis. The range of available cell biological
assays has made Dictyostelium the experimental system of choice for a variety
of fundamental studies on motility, signaling, cytokinesis and phagocytosis –
and especially important, these cell biological studies can be readily
correlated with biochemical analysis.
For example, Dictyostelium has been one of the most important systems
for analyzing the function of actin-associated proteins in cell locomotion,
regulation of cell shape and cell behavior.
Similarly the function of myosin outside of the context of muscle has
been more extensively studied in Dictyostelium than any other organism (Chen et al., 1994; Chen et al., 1995; De
Lozanne and Spudich, 1987; Knecht and Loomis, 1987; Moores et al., 1996; Ruppel
and Spudich, 1996; Zang et al., 1997). The
development of live cell imaging coupled to the ready manipulation of gene
expression has produced important new understanding about cytokinesis (Weber et al., 1999), phagoctyosis (Weber
et al., 1999), and chemotaxis (Parent
et al., 1998). The
transient recruitment of cellular components to the membrane during chemotaxis
was first demonstrated using Dictyostelium (Parent
et al., 1998). Molecular
genetic manipulation is now allowing careful dissection of the pathways leading
from binding of chemoattractant to its cell surface receptor to reorganization
of the cytoskeleton to produce directed cell migration (van Es and Devreotes, 1999).
The value of Dictyostelium as a model
organism is being greatly enhanced by the development of an expressed sequence
tag database, progress toward the completion of the genome sequence by an
international consortium, development of proteomics and DNA array technology,
and development of large screens to identify novel components of the signaling
and motility systems. These
developments, coupled with the unique experimental opportunities available
through the use of Dictyostelium as an experimental organism are drawing
investigators from other fields. For
example, the growth of intracellular parasites, such as Legionella (Solomon et al., 2000), in Dictyostelium has attracted investigators
interested in understanding the cellular requirements for intracellular
parasite growth. Dictyostelium can
provide them with a genetic system for identifying key host cell genes required
for this growth and may eventually lead the way to novel means of interfering
with this process. Similarly, Alexander
and his colleagues have identified Dictyostelium mutants resistant to the
cancer chemotheraputic agent cisplatin.
By identifying the mutant pathways in these strains, Dictyostelium may
contribute to an understanding of the mechanisms of drug resistance that
develop during chemotherapy. The use
of Dictyostelium for similar studies by investigators not familiar with the
system, as well as increased progress in areas where Dictyostelium has already
proven its value would be greatly facilitated by a comprehensive, well
organized on-line informatics resource.
For the last seven years we have been
developing an on-line bioinformatics resource for Dictyostelium – the Dictyostelium
Virtual Library (http://dicty.cmb.northwestern.edu/dicty/). This effort began with a listserv type
mailing list that allowed rapid communication between investigators using
Dictyostelium. An electronic newsletter
that provides abstracts of papers once they are accepted for publication has
facilitated sharing of information between investigators. The newsletter is also searchable using a
search engine on the front page of the web site. A compilation of the Dictyostelium literature, produced and maintained
by Dr. Jakob Franke (Columbia University) can be downloaded through the
Dictyostelium web site. The current web
site also has an on-line, user-modifiable database of investigators using
Dictyostelium. A database of
Dictyostelium genes using a modified version of AceDB, called DictyDB has been
developed by Dr. Doug Smith (UCSD) and is available through the central
Dictyostelium web site. In addition,
there are links to web sites for Dictyostelium laboratories with web
sites. The Dictyostelium Virtual
Library also includes links to all of the Dictyostelium genome sequencing
centers, a limited number of protocols and methods for using Dictyostelium, a
listing of Dictyostelium genes that have been disrupted by gene targeting and
their phenotypes. However useful these
volunteer efforts have been, their scope has surpassed our ability to provide
continuous updates and links for protocols, vectors, maps, gene function,
etc. Investigators using Dictyostelium
now require a central, dedicated bioinformatics resource.
Goal of
Dictyostelium on-line informatics resource.
The goal of establishing a comprehensive
Dictyostelium bioinformatics resource is to provide a single portal through
which all Dictyostelium bioinformatics can be accessed. A central goal is to establish, maintain and
annotate relational databases that allow investigators to utilize DNA sequence,
global expression data, mutant phenotypes, and physiological and biochemical
results in a concerted fashion. In
addition we will make available protocols for working with Dictyostelium. These goals are currently being pursued for
other model organisms for which the genome sequences have been completed,
including yeast (http://genome-www.stanford.edu)
, Drosophila (http://www.flybase.bio.Indiana.edu
and http://fruitfly.bdgp.berkeley.edu/)
, C. elegans (http://elegans.swmed.edu/)
and mouse (http://www.informatics.jax.org). Our goal for this resource is not to
reinvent the overall schemes being developed by these and similar efforts, but
rather to apply the best features of them to the data being obtained for
Dictyostelium. This allows us to focus
our efforts on annotation rather than database development. For example, the Gene Ontology consortium
represents an on-going effort to establish common vocabularies by which gene
function can be described (Ashburner
et al., 2000). We
anticipate affiliating the Dictyostelium informatics resource with this
effort. In a similar fashion, in a
collaborative effort with other organism-based bioinformatics resources,
whenever possible, the Dictyostelium resource would use existing relational
database structures so as to facilitate direct comparisons between
organisms.
The resource will also serve as the portal
for the stock center that is being proposed by Drs. Richard Kessin and Jakob
Franke. The databases developed by the
informatics resource will contain data about strain availability. We propose to develop a system that links
strain availability directly to DNA sequences, gene expression, disruption,
mutant phenotypes and the literature. A
single click on a button would generate a request to the stock center for the
strain.
We envision this effort starting out
modestly with two or three curators familiar with both the informatics side of
the effort and with Dictyostelium as an organism and a database engineer to
handle the data structures and web presentation. See the section below on implementation.
One of the most important questions that
will arise is: why support Dictyostelium informatics? Perhaps one of the best arguments comes from its unique place in
the biological kingdom. Dictyostelium
sits at the transition between free-living single cells and multicellular
organisms. During its lifecycle
Dictyostelium exists both as single cells (during vegetative growth) and as a
multicellular tissue capable of morphogenesis (during development). Because of this position, understanding the
complement of Dictyostelium genes may give insight into the biological
functions important for multicellularity.
As a free-ranging amoeba, Dictyostelium is a consummate phagocyte. Comparisons with cells such as yeast and
macrophages may give significant insight into this critical biological
process. Similarly, as an amoeba,
Dictyostelium exhibits extensive motility and chemotaxis. As such it will likely provide a valuable
insight into the collection of functions required for ameboid movement – a
function that is significant for important health related issues such as wound
healing, mounting of an immune response and susceptibility to parasitic
diseases.
When these unique biological features are
coupled with the ease and efficiency of molecular genetic manipulation, the
ability to validate biochemical pathways experimentally because of the ease of
growing adequate quantities of cells for biochemical analysis, and the strong
cell biology available in Dictyostelium, we believe Dictyostelium provides a
unique model system of particular advantage for studies on cell motility,
signal transduction, phagocytosis and the mechanisms important for
multicellularity.
Since there is a strong on-going
international genome sequencing effort, a large and growing expressed sequence
tag database, the genomic data is being generated and accumulating rapidly. For example the Japanese effort has already
identified over 3000 gene families in Dictyostelium. The resource will provide an ongoing mechanism to correlate this
data with the variety of other data available for Dictyostelium. Delaying the implementation of this resource
will decrease the immediacy and impact of much of this data.
Implementation of
the Resource.
The Dictyostelium Bioinformatics Resource will share a great deal of similarity in scope and concept with a number of other successful model organism sites such as the Drosophila Flybase sites at http://flybase.harvard.edu:7081/ and http://flybase.bio.indiana.edu/, the yeast Saccharomyces Genome Database at http://genome-www.stanford.edu/Saccharomyces/ and the C. elegans resource http://elegans.swmed.edu/. These resources all share a number of common elements – they have a shallow navigational structure to allow researchers to easily move between sections of the resource, they attempt to put a consistent interface and terminology on a number of formally independent data sources. For example, with Flybase, an investigator can easily flip between different ‘data views’ of available information for Drosophila, including genetic maps, genes, clones, alleles, gene products, and stocks. We would like to take these very good ideas and implement them in a way that makes sense for Dictyostelium and the community of investigators who use or who might wish to take advantage of the experimental opportunities this organism provides. We would also like to build in ‘web portal’ concepts, allowing researchers to build a profile of sequences, genes, phenotypes and/or strains they are interested in and receive automated notification whenever new entries are added to the system.
We propose using Oracle, an industry standard relational database, as the underlying data engine for the Dictyostelium Bioinformatics Resource. We believe that this will allow development of a more versatile site and will allow us to take better advantage of much of the research and optimization that has gone on in the past few years and will continue to go on in commercial sector database design. At Northwestern we have assembled a team with experience taking standard ‘E-commerce’ technologies and they have applied these technologies to bioinformatics problems in both the basic and clinical sciences. We propose to take advantage of the expertise and the experience of this team to build a modern and extensible Dictyostelium web site.
Architecture and technologies. In addition to using Oracle as the data engine for storage and retrieval of sequence, strain and other relevant data, we will couple Oracle to the web using standard SQL queries run through Allaire ColdFusion. We have extensive experience using ColdFusion, currently the best rapid prototyping and application development platform availabe for database to web development. We have been using ColdFusion in production applications since 1997. The current version works natively with EJB (Enterprise Java beans) and can translate data from a number of different sources into XML without additional coding. By relying on mainstream commercial packages we will be able concentrate on bioinformatics rather than technology development.
Implementation. The data repository for the Dictyostelium Bioinformatics Resource will be held in an Oracle 8i database running on Sun servers. This system is in use in many high-throughput, high transaction volume applications in industry and academia. This will give us a solid, high-throughput technology platform on which to build our analysis tools. The Oracle database will house genetic, sequence, strain, gene profile, bibliographic and phenotypic data. These data will be collated from multiple sources including the current genome projects, published single gene functional studies, and the published Dictyostelium literature. All queries, reports, and results will be dynamically generated from the database and be accessible to members of the Dictyostelium community via the internet using middleware built with Allaire ColdFusion. The existing Dictyostelium site uses this technology to present data from the investigator database (http://www.basic.nwu.edu/dicty/search.cfm).
Because the database and the middleware are ‘connected’ using standard SQL, once a particular report has been written the report can be easily stored and run at any future time. All our reports will be written using a combination of Allaire ColdFusion and standard SQL (Structured Query Language, a standard programming language for ‘querying’ databases), making all the results instantly web accessible anywhere in the world. Further, we have used these technologies to generate automatic messaging and email-based notification systems for both event-driven and date-driven notifications.
We are also anxious
to participate in collaborative interactions with other organism based
informatics resources to facilitate comparisons of data housed for different
organisms. We would propose the
establishment of a consortium of organism based informatics resources that
could standardize technologies and share applications.
The facility will be
directed by Rex Chisholm, Professor of Cell and Molecular Biology at
Northwestern University Medical School.
Dr. Chisholm has worked with Dictyostelium for over twenty years. He has been responsible for establishing and
maintaining the existing Dictyostelium internet resources at http://dicty.cmb.northwestern.edu/dicty/. In addition, the technical aspects of the
resource will be overseen by Dr. Warren Kibbe, an experienced bioinformaticist
who has substantial experience with interfacing relational database with
biologically based internet sites. An
experienced Oracle database engineer, Ms. Lorraine Padour is already part of
the Northwestern University Medical Informatics resource and a portion of her
effort will be available to this project.
We will also hire two curators familiar with Dictyostelium to actively
populate, maintain and annotate the databases.
A budget our estimated first year costs is attached.
Questions to be
addressed in a concept paper:
·
By what
process did the community obtain input and reach a consensus about the priority
for the proposed project? At the most recent International
Dictyostelium Conference, Dictyostelium 1999 held in Bar Harbor, Maine, a
community board was established. The
composition of the community board is international in nature (see
http://dicty.cmb.northwestern.edu/dicty/pnd/organizing_committee.htm). The first priority of the organizing
committee was to develop the interactive web site at http://dicty.cmb.northwestern.edu/dicty/pnd/
for the trans-NIH Coordinating Committee for Non-Mammalian Models (see http://www.nih.gov/science/models). The community board has selected
establishment of the Dictyostelium bioinformatics resource and the stock center
as its highest priorities together with the completion of the genome sequence. In addition, over 400 investigators who are
listed in the investigator database use the current configuration of the
Dictyostelium Virtual Library. The
community already recognizes the current site as the central Dictyostelium
portal. It averages over 1500 visits
each month indicating significant use for a resource that is significantly less
comprehensive than the proposed effort.
·
Other
Sources of Support: The
sequencing of the Dictyostelium genome is currently supported by the NIH
through a grant to Baylor College of Medicine from the National Institute for
Child Health and Development. In
addition the European Community and the German National Science Foundation have
also supported Dictyostelium genome sequencing. Each of these projects has an informatics component. However, the focus of these informatics
components is the analysis of genome sequence and cDNA sequences. The focus of this request is to consolidate
current and past gene by gene based research, genomic expression studies, mutant
phenotypes, and other similar data together with that being generated by the
genome sequencing and EST efforts into an easy to use on-line system.
·
Advantages
and Limitations: The primary advantage of establishing this
resource is that it will provide a single source for bioinformatics of
Dictyostelium. This facility will
maximize the investment made in Dictyostelium genome sequencing and the large
number of single gene, cell biological and biochemical studies by making them
all accessible online through a single web portal. This will benefit both investigators experienced with
Dictyostelium and those without experience using the experimental system, but
interested in taking advantage of some of its unique experimental
opportunities.
·
Do we need
the genomic resources now? Two main factors argue for implementing this
resource as soon as possible. First,
there is already a large amount of data to be cataloged. This includes both genomic and cDNA
sequences generated by the international genome sequencing consortium and the
Japanese cDNA project. In addition a
large number of gene disruption strains has already been produced, several
large genetic screens have been undertaken and extensive gene regulation
studies have been completed. We
currently estimate that approximately 95% of the Dictyostelium genes have
representative sequences available, that approximately 50% of the genes are
fully sequenced and that approximately 80% of the entire genome has been
sequenced. In addition, around 30% of
the developmentally regulated genes have already been characterized by
mutational analysis. Delay in
implementing the resource results in lost opportunities for investigators both
inside and outside the field. A second
factor is that costs for establishing and maintaining the resource will
unlikely decrease in the future.
Because of its unique position in the biological kingdom having an
active Dictyostelium bioinformatics resource is also likely to enhance the
value of similar resources for other organisms by facilitating organism to
organism comparisons of core proteome size as well as specialized
functions. By working closely with the
other organism based informatics resources a Dictyostelium resource would be
able to enhance development of database structures so as to encompass a wide
variety of organisms with different niches in the biological kingdom. Thus there
·
Are there
plans to develop the resources outside the US? No. The Dictyostelium Virtual library is
already serving as the central portal for Dictyostelium information and the Dictyostelium
resource would work with genomics and proteomics efforts elsewhere to ensure
that it meets their needs. Participants
from each major genomics activity would serve an the advisory board for the
Dictyostelium informatics resource.
·
What are the
unique advantages of this organism: The advantages of Dictyostelium are discussed above in the introduction
and the section entitled “What unique features does Dictyostelium offer that
justify this effort?” In addition, the
community has created a on-line tutorial
(http://dicty.cmb.northwestern.edu/dicty/pnd/) that highlights many of the
unique features of Dictyostelium.
·
What
scientific advances will be made possible? As noted above, the unique position of Dictyostelium in the biological
kingdom makes it a particularly useful organism that will help define the genes
important for multicellularity. In
addition, research in areas of fundamental biological inquiry where
Dictyostelium excels, such as cell motility, signal transduction and
phagocytosis are likely to benefit from the availability of this resource. For example, the ability to begin to
correlate mutations in signal transduction pathways with cytoskeletal mutants
are likely to have a great impact on understanding of chemotaxis. Similarly the recent interest by the
infectious disease community in using Dictyostelium as a host for intracellular
microorganisms will certainly benefit since this effort is being spearheaded by
investigators who are unfamiliar with Dictyostelium. For them the internet presence we propose to develop will serve
as a valuable resource for those new to the organism.
·
What is the
cost? Initially we request
funds to purchase a dedicated server and hire two full time
curators/informaticians to define database structure (primarily by
collaborating with similar efforts in other organisms) and to begin to populate
and annotate the data and technical support to guide construction of the
datastructures and development of the web interfaces. We expect the initial start up costs to be about $40,000 plus the
salary and fringe benefits and operating budget of about $200,000
annually. A detailed budget is
appended.
·
What is the
duration? A primary reason
for requesting support through this mechanism is the need for on-going support for
this activity. We initially request
five years of support initially. At the
end of the five year period the success of the resource and its value to the
Dictyotelium community and the biomedical research community at large should be
evaluated.
·
How will the
repository be supported after the completion of the project? It is
anticipated that the resource would require NIH or other extramural support as
long as it serves the needs of the community.
As bioinformatics develops, it may be that the organism based
information resources could be housed in a centralized operation similar to
Genebank, housed perhaps by the National Center for Biotechnology
Information. However, certainly during
the early stages it will be necessary that the curators have experience with
Dictyostelium as an experimental model to efficiently annotate and organize the
experimental data.
·
How will the
resources be made available to the research community? This will be an on-line resource available to
everyone on the internet.
·
What
resources currently exist? The
current on-line resource, the Dictyostelium Virtual Library, is operated on a
strictly volunteer basis. The rate at
which data about this organism has outstripped our ability to keep the
information up-to-date. Our goal is to
obtain resources that provides the resources necessary to have a focused and
dedicated effort to grow the existing resource into a first rate bioinformatics
effort for Dictyostelium.
·
What is the
size of the research community? The size of the community is roughly five hundred active scientists in
80 laboratories. Because of the cell
biological and genetic possibilities of the organism, the community is growing.
It is also widely used as a teaching tool. The community is organized and has a
board of directors that is international in scope and seeks to make research on
the organism as efficient and as cooperative as possible. The record of
cooperation in this field, like that in the C. elegans community, is
excellent.
·
Who will
benefit? We imagine that
anyone actively using Dictyostelium as an experimental organism would benefit
from this resource. In addition, the
resource will also be of great value to anyone who might be interested in using
the organism. Finally we believe the
entire field of functional genomics will benefit because Dictyostelium has a
unique position at the intersection between single celled and multicellular
organisms. Efforts to define core
proteomes and specialized proteomes important for fundamental biological functions
such as multicellularity, chemotaxis, and phagocytosis will benefit certainly
benefit from a concerted effort to make Dictyostelium bioinformatics readily
available to the biological research community.
·
What will be
the benefits? The primary benefit
will be an improvement in the accessibility, organization and usability of
bioinformatics as it relates to Dictyostelium.
The existing data represents a valuable resource much of which has been
funded by the NIH and NSF. The NIH is
already a substantial contributor to the genome sequencing effort. A concerted effort to produce and maintain a
Dictyostelium bioinformatics resource will ensure that the investments already
made by NIH in studies using Dictyostelium as well as the Dictyostelium genome
are maximized. This resource will
ensure that the valuable data produced from studies using Dictyostelium as a model organism will be
available to the entire research community in a format that facilitates
organism by organism comparisons and stimulates cross-talk between researchers
using different model organisms.
We believe that developing an integrated informatics infrastructure that is tightly coupled to the Dictyostelium research community will be an important factor in allowing Dictyostelium to fulfill its unique role as a model organism. The goal of the Bioinformatics Resource is to rapidly build modular, scalable, and cost effective tools for the biomedical research community. By careful consideration of scaling issues in the initial design we hope to concentrate on the biological information collection, analysis and distribution methods as we move forward rather than needing to “reinvent” the underlying technology infrastructure as new techniques or technologies emerge. We believe that funding of this resource will produce more efficient use of NIH resources currently committed to studies using Dictyostelium. In addition we believe that wide availability of this information will facilitate biomedical research in general by stimulating the use of Dictyostelium for studies that take advantage of its unique features, such as has recently occurred in the infectious disease community with the use of Dictyostelium as a host for intracellular bacterial parasites. Together with its position in the evolutionary “tree of life” at the junction between single celled and multicelled organisms, ready availability of informatics resources will facilitate understanding of both the core proteomes and the specific collections of genes important for multicellularity as well as the fundamental cell biological processes to which Dictyostelium has already become a proven and important contributor.
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Peter v. Sengbusch - b-online@botanik.uni-hamburg.de