ARGUMENTS FOR SEQUENCING THE GENOME OF THE SEA
URCHIN STRONGYLOCENTROTUS PURPURATUS

Eric H. Davidson (California Institute of Technology)
R. Andrew Cameron (California Institute of Technology)
FOR THE SEA URCHIN GENOME ADVISORY GROUP
:
Robert C. Angerer (University of Rochester)
Lynne Angerer (University of Rochester)
Roy J. Britten (California Institute of Technology)
James A. Coffman (Stowers Institute for Medical Research)
William H. Klein (M. D. Anderson Cancer Center)
Donal Manahan (University of Southern California)
David R. McClay (Duke University)
Jonathan P. Rast (California Institute of Technology)
Victor D. Vacquier (Scripps Institute of Oceanography)
FOR BCM, HUMAN GENOME SEQUENCING CENTER:
Richard A. Gibbs, Director
George Weinstock, Co-Director
RATIONALE

1. Introduction

There are reasons to sequence the genomes of many different animals: every animal
genome holds secrets that when unlocked will yield invaluable mechanistic information that will
in some measure illuminate not only our own biology but that of the rest of the world as well.
However, in the case of most possible candidate genomes, it will be impossible to cash in on the
potential value of the sequence without greatly augmenting scientific efforts on the species
sequenced. Experimental systems that do not now exist or only barely exist in a few labs will
have to be developed; comparative explorations of interspecific differences across phylogenetic
distances that are often unclear will be required; and so will accumulation of repertoires of new
molecular and genomic data. In contrast, to cash in on the value of a sea urchin genome
sequence will require only the availability of the sequence: it will immediately be utilized by a
large assemblage of active laboratories. The wise decision made to sequence the Drosophila and
C. elegans genomes was based on the intense research use of these organisms and the large store
of knowledge already available. Of all remaining invertebrate genomes the same is most
pointedly the case for a sea urchin genome. En passant, one might note that by comparison there
is but a tiny handful of labs focusing on ascidians, although not one but two ascidian genomes
are now being sequenced. The sea urchin research community is possibly 20-40 times as large
as the ascidian community.

As detailed below, we have 75 letters from scientists in the US and elsewhere in support
of an effort to sequence the genome of the sea urchin Strongylocentrotus purpuratus, so the
opinions summarized in this document are not merely ours alone. S. purpuratus is the most
widely used of the several sea urchin species in use the world around, particularly for the kinds
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of molecular biology research that will be most immediately impacted by availability of genomic
sequence. There are reciprocal ways to look at the effect of an S. purpuratus genomic
sequencing project, and both are true: the large amount of funds and effort expended on sea
urchin research will be leveraged enormously by the availability of genomic sequence; and
equally, the value of the sequencing effort will be leveraged enormously by the already extant
commitment of research effort to this model system.
An important aspect of the sea urchin model system is the phylogenetic position of these
animals relative to ourselves. Figure 1 shows the relative positions of echinoderms, chordates,
C. elegans and Drosophila in animal phylogeny; i.e., of echinoderms, relative to the four genera
of animals from which genomes are so far sequenced.
These are all either chordates or ecdysozoans. The echinoderms and the hemichordates (a little
known, though very interesting group of marine worms) are sister groups. Echinoderms and
hemichordates are the only other living animal forms besides the chordates in the deuterostome
subgroup of the Animal Kingdom. In other words the chordates (including us) share a common
ancestor with echinoderms (including sea urchins). Therefore sea urchins are more closely
related to all other deuterostomes (including us) than is any deuterostome to any other animal
(e.g., flies or worms). So from the standpoint of phylogenetic position the sea urchin genome
would provide an invaluable outgroup for assessment of what is ancient in the regulatory
architecture and functionality of our own genome, what is chordate-specific, and the origin of
that which has been modified in evolution. Furthermore there remain some deep functional
mysteries about genomes that can only be solved by comparative examination across great
distances. For example, we noticed molecular linkages of a number of genes in the
S. purpuratus genome that are also linked in the mammalian MHC complex on a megabase
scale, even though sea urchins lack MHC genes: there must be a functional meaning to the
preservation of this system of linked genes over this huge evolutionary distance, but what is it?
Among the nonchordate deuterostomes the sea urchins are obviously the primary target for
investment in genomics since they are the only nonchordate deuterostomes that serve as major
current research models.

The detailed reasons why the S. purpuratus genome should be sequenced are ultimately
the same reasons that people work on this and other closely-related sea urchin species. In the
following we summarize the size and activity of the sea urchin research community, and the
major uses of this model system in central areas of biology, viz gene regulation molecular
biology; the cell biology and biochemistry of eggs, embryos, and the fertilization process; and
evolutionary biology. We also briefly review its medical relevance, and then propose a genomic
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sequencing strategy, in collaboration with our partners in this enterprise, Richard Gibbs and
George Weinstock of the Human Genome Sequencing Center, Baylor School of Medicine.

Gentaur Urchin Genome
2.
The Sea Urchin Research Community
A minimum criterion of accountability in respect to the expenditure of effort and public
resources needed to sequence an animal genome might be that there is a sufficient research
community to properly and immediately utilize the sequence.
Table 1 provides an overview of the dimensions of the sea urchin research community. There
are scores of sea urchin labs in the US, and sea urchins are also the research models in use in a
number of long-standing labs in Italy, Japan, as well as other countries. Hundreds of papers are
published each year describing results of research on sea urchins, and the US spends millions of
dollars supporting this research each year. A small sample of the papers published in prominent
scientific journals over the last two years is included at the end of this document as a
bibliography: these publications display the role of this model system in many areas of biology.
The sea urchin model system is most intensely used for research in gene regulatory molecular
biology, molecular embryology, fertilization biology, cell biology, and evolutionary biology, but
it also used for many other purposes such as marine population genetics, toxicology, nonadaptive
immune system biology, and so forth with the Gentaur Antibodies anti-Phospholipase C delta isoform Antibody
Table 1. Parameters of the Sea Urchin Research Community
Average number of attendees at the sea urchin meeting: 150 (steady for the last several years).
The number of laboratories worldwide that use sea urchins as a primary research organism: 143.
From the ISI database searched on the term "sea urchin", papers retrieved for 2000, 2001, 2002: 827.
Total grant dollars for sea urchin-based research at the NIH for the last fiscal year: $15M
The sea urchin research community is united and enthusiastic about the need for
S. purpuratus genomic sequence. As direct evidence for this statement, in Appendix 1 we have
excerpted some of the letters in our files from scientists whose main research model is the sea
urchin. Their names and affiliations of the authors of the 75 letters of support are also listed in
Appendix 1. It is obvious to one and all that in every area of mechanistic bioscience genomic
sequence is an invaluable resource, whether it be for study of gene function, or regulation
thereof.
The sea urchin research community has held a major meeting focussed on sea urchin
developmental cell and molecular biology, that lasts for about five days every year and a half
since 1981. At present this meeting is attended by about 150 people. The issue of sea urchin
genomics has been a prominent topic of discussion at the recent Sea Urchin Meetings, and the
Sea Urchin Genome Advisory Group was set up in consequence. The following proposal takes
into account the major needs of the community. These needs are focused directly on the
requirement for genomic sequence, since there are already extensive sea urchin EST data, BAC
libraries for a number of sea urchin species, arrayed cDNA libraries and other preparatory
genomics researches, detailed below.
Uses of Sea Urchin Embryos for Gene Regulation Molecular Biology
The area in which sea urchin research has had its major impact on the state of knowledge
in gene expression and gene regulation in development. This has been true for a long time:
maternal mRNA was discovered in sea urchin eggs; the first measurements that established the
complexity and prevalence distribution of mRNAs in any embryo were carried out on sea urchin
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embryos; the first measurements of transcription rates and average and specific mRNA turnover
rates as well as of protein synthesis rates in embryos were carried out on sea urchin embryos.
This was all between about 1965 and the early 1980's. These foundations of sea urchin embryo
molecular biology were in turn built on the century-long earlier history of experimental work on
sea urchin embryos. For example, pronuclear fusion at fertilization was first recognized in sea
urchin eggs in 1879 (by Fol), and the first realization that continuing gene expression is required
for embryogenesis to occur followed from experiments of Boveri carried out in 1904.
Since the mid-1980's there has been a great focus of attention in the world of sea urchin
developmental biology on the regulatory molecules that drive embryonic development:
transcription factors and signaling components. Because the sea urchin embryo can literally be
experimentally disassembled and reassembled, because the early cell lineage is invariant and
embryonic cell interactions and cell fates have been so well worked out, the molecular biology of
the embryo is uncommonly well integrated with knowledge of the process of embryogenesis. In
consequence, our level of understanding of early development in this embryo has achieved a
paradigmatic state.
There are four technological advances that place the sea urchin embryo at the forefront of
developmental regulatory genomics.
•A relatively very high throughput gene transfer system. Thousands of eggs can be
injected with expression constructs in a few hours, and the transcriptional readout obtained in
spatial and/or quantitative terms within a day or two.
•Technology for obtaining stable nuclear extract. Enormous numbers of sea urchin
embryos are available (literally trillions of embryo nuclei are extracted each year). These
extracts are used for purification and microsequencing of transcription factors, given only a
known genomic DNA target site.
•Use of morpholino substituted antisense oligonucleotides, and of mRNA encoding
Engrailed domain fusions. These perturbation reagents permit shut down of any specific
transcriptional regulatory process at will.
•Powerful and sensitive methods for whole mount in situ hybridization and
immunocytology. By these means normal or perturbed patterns of gene expression can be
visualized at single-cell resolution in sea urchin embryos.
As a result, we know a great deal about the signaling and transcription control processes
leading to cell specification in this embryo. The best characterized of all developmentally active
transcriptional control systems is a sea urchin cis-regulatory element (the endo16 cis-regulatory
system). The most comprehensive image of how maternal spatial cues initiate differential
transcription along the animal-vegetal axis has been constructed for sea urchin embryos. The
first large-scale gene regulatory network for a major process of development has been worked
out for sea urchin embryos (references included in bibliography). These are all genome-based
studies, and the rate of advance in this essential area, which lies at the heart of functional
genomics, will accelerate immediately as genomic sequence become available.
3.
Uses of Sea Urchins for the Cell Biology and Biochemistry of Eggs, Embryos, and
the Fertilization Process
There are many other areas in which sea urchin research has a very high connectivity
with respect to the general state of knowledge. This has long been so: for example cyclins were
first observed in sea urchin eggs; the role of cell adhesion in embryogenesis was first analyzed in
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sea urchins; cytonemes were discovered in sea urchin embryos. Among the areas of general
interest in which sea urchin research at present provides leading contributions are:
•Ca+2-mediated mechanisms of metabolic activation on fertilization.
•Mechanisms of sperm activation.
•Biochemistry underlying sperm flagellar motility.
•Biochemical basis of sperm-egg recognition.
•Structure/function analyses of cytoskeletal components, including microtubules,
microfilaments, and membrane substructures.
•Role of cytonemes in morphogenetic processes.
•Structure and function of centrioles.
•Response to metals and other toxic agents.
•Function of extracellular matrix in development.
•Biomineralization processes,
•Cellular mechanisms of morphogenesis.
Each of these areas resolves (more or less directly) into studies of the function of given
gene products. As for most areas of cell biology, progress will be greatly advanced when the
perturbations and structure-function assays afforded by experimental control of the underlying
molecular biology can be applied. The power of such approaches will in turn depend ultimately
on availability of genomic sequence.
4.
Uses of Sea Urchins for Evolutionary Biology
There are three areas in which sea urchins are very important in evolutionary biology, all
directly relevant to their genomics. These are, first, the amazing retention of syntenic relations
with mammals already noted above, in connection with Fig. 1; second, marine population
genetics, gene flow, and speciation; and third, comparative regulatory molecular biology.
With respect to the first of these, assembly of a genomic sea urchin sequence would
de facto produce a whole new field of "distant syntenics." The result could be completely novel
insights regarding the structure, evolution, and function of deuterostome genomes.
With respect to speciation and population structure, sea urchins have features that make
them very different from any animals whose genomes have yet been sequenced. They develop
indirectly by way of long-lived, feeding pelagic larvae.
Therefore there is continuous
intermixing of their gene pool: for example in S. purpuratus, which extends from Vancouver to
Mexico, there is no greater difference between genomes from individuals collected at the
extremes of their range than there is between the two haploid genomes of any given individual:
probably typical of many invertebrate marine organisms, the S. purpuratus population consists of
a huge, panmyctic gene pool. But it also has been a huge gene pool for a very long time, with
the result that the S. purpuratus genome is about 10-20X more polymorphic than are the
genomes of mammals. In other words, there are things to be learned from these animals here
that will illuminate large aspects of the biology of oceans and of organismal divergence and
speciation therein. Among the current fields of study are:
•Microsatellite genotyping in the context of wild populations and laboratory inbred
populations.
•Evolutionary rates of divergence, e.g., in populations isolated by the rise of the Isthmus
of Panama.
•Mechanisms of gene pool isolation by evolution of sperm/egg recognition barriers.
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In the area of comparative regulatory evolution, as in the area of "distant synteny," there
is a direct and relevant link between understanding how a sea urchin genome works and how our
own genomes work. It is likely to be possible to translate experimental evidence of the
regulatory network features for given developmental processes in sea urchin to our genomes:
once one knows what to look for, it is much easier to find it by comparative means alone. For
this it may be useful to use a "stepping stone" approach, i.e, from sea urchins to ascidians to
vertebrates. There is also an unlimited variety of fascinating comparative regulatory evolution
problems with respect to other forms. Three that are being worked on now are comparisons of
developmental regulatory processes between S. purpuratus and directly-developing sea urchins;
comparison to a distant echinoderm, the starfish; and comparison to hemichordates (see Fig. 1).
These kinds of studies will illuminate the ways in which developmental gene networks form and
reform, the basic process of evolution.
Uses of Sea Urchins for Studies Relevant to Human Disease
Ongoing medically relevant basic research is based on use of sea urchin gametes and
cells as model systems. For example, the membrane-bound receptor guanylate cyclase
implicated in the important human disease, heat-stable enterotoxin dysentery, was first isolated
from sea urchin sperm. The ubiquitous C+2 releasing second messenger, cyclic ADP ribose, was
discovered in sea urchin eggs and subsequently found to be important in calcium release in the
mammalian pancreas. Recently, a connection has been shown between β-catenin, a crucial
molecule in early embryonic cell specification, and the differentiation of metastatic cancer cells.
Human polycystic kidney disease leading to end-stage renal failure is the most frequent human
genetic disease among whites of European extraction. The disease is caused by mutations in
human polycystin, whose role in human physiology is unknown. The sea urchin sperm cell
receptor for egg jelly (REJ) is the only known protein in GenBank with homology to human
polycystin. It controls ion channel activity and by analogy polycystin may be an ion channel
regulatory protein whose mis-regulation could be the basis for this human disease. In another
area of cell biology, the sea urchin egg is the best model system for the fundamental cellular
processes of exocytosis and endocytosis, processes that lie at the heart of synaptic function,
insulin release in diabetes, rennin release in hypertension and immunoglobulin release in
immune system function. Furthermore envelope viruses such as influenza, hepatitis C and HIV
use these same cellular pathways to infect cells. In the sea urchin embryos these processes can
be studied in isolation.
PROPOSAL
The S. purpuratus Genome and Current Status of its "Pre-genomics"
The genome of S. purpuratus is 800 mb in size. It is 39% GC, and consists of about 25%
repetitive sequences, consisting of a complex set of diverse elements which occur at frequencies
ranging up to tens of thousands per genome. The repetitive sequences have been unusually well
studied, and all but the lowest frequency classes can be masked computationally by reference to
a library of repeat sequences. The genome has a typical short period interspersed sequence
organization, i.e., the single copy domains are punctuated by short repeat elements (a few
hundred base pairs in length) every couple of kb or so. There are also some clustered long repeat
domains, which include roughly a third of all the repeat sequence length: thus the vast majority
of repeat sequence elements are short. There are also on average several insertions per 100 kb of
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transposable element genes, usually the remains of reverse transcriptase genes. The genome
contains an ordinary frequency of microsatellites as well.
In addition to these general characteristics a large amount of effort has been put into
genomics in preparation for ultimate genome sequencing. The following has been achieved,
largely as a result of a two-year, $4 million Sea Urchin Genome Project funded by the Stowers
Institute for Medical Research, that ended in 2001:
•BAC libraries have been prepared and arrayed from S. purpuratus (17.5X coverage,
140 kb average length), and from three other sea urchin species at various evolutionary distances
from S. purpuratus (Paracentrotus lividus, Lytechinus variegatus, and Eucidaris tribuloides).
Further BAC libraries are in process. These libraries will enable interspecific sequence
comparisons around any given gene at any desired distance that is likely to be useful: the range
of divergence from S. purpuratus is from 50 my for L. variegatus (and less for P. lividus) to
250 my for Eucidaris.
•An S. purpuratus BAC-end sequencing project was carried out by Lee Hood and
colleagues which has resulted in about 8x104 sequence tags, including about 5% of the genome.
•Over 30 BACs have been fully sequenced, resulting in about 4.6 mb of assembled
genomic sequence.
•A custom-built sea urchin orthologie annotation program has been built, SUGAR (Sea Urchin
Genome Annotation Resource). From analysis of the genomic sequence in hand, an estimate of
about 22,000 genes is obtained with an average intergenic distance of 30 kb.
•About 16,000 S. purpuratus EST sequences exist (though they are not yet all organized
in appropriate data bases). There may be an additional EST data set several times this large soon
available from the Max-Planck Genome Institute in Berlin.
•Large, arrayed cDNA libraries have been built for every embryonic stage, for
unfertilized eggs, and for a number of adult tissues. These libraries are stored at Caltech, and
with the support of the NIH National Center for Research Resources, stamped high density filters
bearing these libraries are sent on request to any laboratory wishing access to them. Data
obtained from screening these libraries are accumulated on a central web site maintained at
Caltech.
•Software for interspecies genomic sequence comparison for the purpose of identifying
cis-regulatory sequence elements; for automated quantitation and comparison of arrayed filter
screens; and for construction of regulatory networks, has been built and tested, and is being
heavily used.
Whole Genome Sequencing Strategy
The methodology below is based on the approach being taken at the BCM-HGSC for the
rat genome project. However it also incorporates a new strategy, clone array pooled shotgun
sequencing (CAPSS) to introduce efficiencies and reduce costs. The overall approach is only a
suggestion, but likely represents a description that is close to the actual method that could be
used.
The sea urchin genome is about 800MB. There currently exists a BAC library of about
100,000 clones (average insert size 140 KB; 17.5x clone coverage). BAC end sequences (BES)
have been generated for 38,000 of these but no fingerprints. These existing resources will be
used to generate a 6x coverage draft sequence.
About 25000 of the clones (about 4x clone coverage), which have high confidence for
correct BES pairing, will be lightly sequenced (0.75x coverage) and the BES will be used with
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the resulting contigs to build a tiling path. In order to avoid having to make 25000 separate BAC
DNA preparations and libraries, the CAPSS strategy will be used. Cell cultures for the clones
will be distributed into a 158x158 array and the clones in rows and columns will be grouped into
316 pools. DNA preparations and shotgun libraries will be prepared from these pools and
sequenced to an average of between 0.75x coverage per clone (a total of 3x average sequence
coverage for the genome). The sequences from each row will be mixed with the sequences from
each column and co-assembled. The assemblies will be analyzed to identify contigs containing
both row and column reads, indicating the contigs corresponds to sequences from the BAC at the
intersection of the row and column that were mixed, thus deconvoluting the array. This will save
having to produce over 24000 BAC DNA preparations and shotgun libraries. It is possible that a
more conservative pooling scheme will be used, such as using 250 arrays of 10x10 clones. This
would save having to produce 20000 (25000 – 20x250) BAC DNA preparations and shotgun
libraries. Initial results on 10x10 arrays from the rat genome project show that this deconvolution
technique is successful. The current uncertainty lies with the technical issues in dealing with
larger arrays.
Once sequence information is available for the 25000 BACs, the overlap analysis will
follow the methodology currently being used at the BCM-HGSC for clone picking in the rat
genome project. The contigs will be anchored to the ends of the BACs (by identifying read pairs
where one read is in the vector and the other in a sequence contig) as well as linking contigs
together into scaffolds based on read pair information. Each BES will be compared to the
sequence contigs to find BACs that overlap and the size of the overlap region will be estimated
based on where the BES matches the ordered, oriented, and anchored contigs in the scaffolds.
This will be confirmed by comparing the sequence scaffolds in overlapping BACs. From this
information a tiling path with minimal overlaps will be generated. The informatics pipeline for
this method is currently in place at the BCM-HGSC and is being used to identify BACs to
sequence for the rat genome project.
Sequences from the BACs that are not in the tiling path will be added to the tiling path
and reassembled with the tiling path BACs. The tiling path that is produce will contain some
gaps and these will be filled by two methods. First the remaining BES will be compared to the
tiling path sequence to place the remaining BACs on the map. Candidates for gap filling will be
lightly sequenced and this information used to determine if the gap is completely filled. Any
gaps remaining after this process will be filled by screening the BAC library for gap fillers by
hybridization, using probes based on sequences at the end of contigs flanking gaps.
The sequence will be brought up to 6x by doing whole genome shotgun sequencing to 3x
coverage. The WGS reads will be binned to the proper BAC and assembled using the ATLAS
whole genome assembler, the method developed at the BCM-HGSC for assembling the rat
genome sequence from a mixed BAC-WGS approach. ATLAS initially finds overlaps between
WGS reads, then assigns these groups of reads to BACs based on sequence comparison, and
finally assembles the reads in each BAC. The software for clustering reads is also available on
the BCM-HGSC web site, where it is called the BAC fisher. This tool allows any investigator
who has some sequence information on a region of interest to pull out all reads of relevance
before the whole project is over.
Overall this approach would require about 9.6 million successful reads for a 6x sequence
coverage (500 bases per read), half in WGS and half in BAC sequencing. This is approximately
6 months sequencing at the BCM-HGSC if all capacity were directed at this project. In addition
the project will require from a few hundred to about 5000 BAC DNA preparations and shotgun
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libraries. This would take from a few weeks to few months if all capacity at the BCM-HGSC
were focused on this project. The overall cost is estimated at about $30 million.
How the Sequence Will Be Used.
The sequences determined by the sequencing center and the preliminary assembly of
them will be managed by the sequencing center. It is our aim to make further assembled and
annotated sequences immediately accessible, in both practical and intellectual terms, to the
community of experimentalists who will make use of them. These further assemblies and
annotations will be posted on the web site connected with the Sea Urchin Genome Project. The
sequence coverage for our proposed strategy will likely provide sufficient sequence for each
BAC to make an ordered and oriented assembly. The assembled sequences can then be
accessioned into our sequence database and cross-referenced to the macro-array location for the
original BAC clone.
In order to make the process of analyzing sequences convenient we have installed a web
based set of programs, the Cartwheel Project which has a loosely-coupled architecture built on
open source code. This system, designed by Titus Brown at Caltech, is in essence a
bioinformatics infrastructure which allows the user to have complete control over the analytical
process. They are currently supported at Caltech with funding from the NIH National Center for
from Gentaur Tubuline Research Resources. The analyses produced by Cartwheel are then viewable by programs such
as SUGAR, the Sea Urchin Genome Annotation Resource, a viewer designed to concurrently
represent a variety of genomic features on the sequence, including cDNA matches, repeat
sequence matches and genes predicted by several different prediction programs. The analyses
stored in Cartwheel can also be viewed with FamilyRelations, a graphical interface that shows
large sequence comparisons focusing on conserved elements between two genomes. Because
Cartwheel will adhere to a number of open protocols, most notably the Distributed Annotation
System (DAS) and Distributed Authoring and Versioning (WebDAV), it is both extremely
extensible and compatible with the many distinct formats and protocols used in bioinformatics
today. In particular, DAS will allow collaborative field-wide annotation of genome projects
based on data generated from and served by individual labs, a heretofore unprecedented ability.
We will immediately erect a community structure to conduct annotation and analysis
procedures on the sea urchin genome based on the computational arrangements described above.
We will apply for funds to expand the infrastructure to incorporate additional analysis systems as
needed so that we are able to annotate the sequence and post the results as they are obtained.
The Genome Advisory Group will carry oversight responsibility and install quality control
standards for the annotation process. Furthermore, the members of the sea urchin community
and other interested parties will be invited to join the annotation effort under the services
provided by DAS and DAV.
APPENDIX 1
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Names of People for Whom We Have Letters on File, Affiliation and Selected Excerpts.
Christiane Bierman, Harvard University
" The evolution of sperm-egg recognition is just one example of the many questions in evolutionary biology
that could be addressed much more effectively if comprehensive sequence information was available."
Roy J. Britten, California Institute of Technology
Charles Brokaw, California Institute of Technology
Bruce P. Brandhorst, Simon Fraser University, Canada
" I strongly support the proposal to complete the sequencing of the genome of the sea urchin S. purpuratus
and provide resources for some other echinoderms. I have been engaged in research on the development of
embryos of this species for over 30 years and am convinced that there are many important opportunities for
important advances in knowledge of development that will accrue from investing in the proposed genome
program."
Robert D. Burke, University of Victoria, Canada
"A complete sequence of Strongylocentrotus purpuratus genome will have to be done to understand the
evolutionary history of the vertebrates. The fact that echinoderms are the only major model organism that
shares a common ancestor with the chordates makes it critical that this project be given a high
priority........Sequencing the genome of a sea urchin will provide more useful comparative data than
sequencing another mammal."
Ron Burton, Scripps Institution of Oceanography
"As you know, I conduct research on the population genetics of sea urchins, including the purple sea urchin,
Strongylocentrotus purpuratus. I am most excited by the prospect of having the full genome of this species
sequenced."
Eugenio Carpizo-Ituarte, Universidad Autónoma de Baja California
David Carroll, Florida Institute of Technology
Douglas E. Chandler, Arizona State University
Gary N. Cherr, University of California, Davis
"The sea urchin embryo is a required model system in the U.S. regulatory control over the release of
pollutants to our nations waters, and has now been adopted in Europe and Asia. A better mechanistic
understanding of how pollutants impact gene expression during development is critical in developing more
accurate environmental assessment tools.....Having the entire genome available would greatly accelerate
these studies."
Kazuyoshi Chiba, Ochanomizu University, Japan
James A. Coffman, Stowers Institute for Medical Research
"The sea urchin is perhaps the best model system currently available for rapid experimental characterization
of gene cis-regulatory systems, and much of the time and labor-intensive preliminary work that is now
required to map and sequence genes in order to get at their cis-regulatory regions will be made unnecessary
by the availability of sequence from a whole genome project."
Alberto Darszon, Universidad Nacional Autonoma de Mexico
Maria Di Bernardi, Istituto di Biologia dello Sviluppo, Palermo, Italy
Marta Di Carlo, University of Palermo, Italy
Richard Emlet, Oregon Institute of Marine Biology
David Epel, Stanford University Hopkins Marine Station
"I strongly support the sea urchin genome project and can see many previously unimaginable projects that
can be initiated once this data is available and the answering of previously unapproachable questions."
Susan Ernst, Tufts University
"Remembering that in 1974 Larry Kedes and his co-workers cloned the first eukaryotic genes (the early
histone genes) from the sea urchin, it seems that this is a project that should have been started already. For
over 125 years research on sea urchin germ cells and embryos has been pivotal in establishing many
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fundamental concepts in cell, developmental and molecular biology. The community of investigastors using
sea urchins as a model system is vibrant and interactive.....This will benefit us all and, because of the
importance of the sea urchin as a cell, developmental and evolutionary system will also be a benefit to the
greater scientific community."
Charles A. Ettensohn, Carnegie Mellon University
"I am writing to indicate my enthusiastic support for a sea urchin whole-genome sequencing project. From
both developmental and evolutionary perspectives, such an effort will provide a wealth of
information....Beyond its importance in gene discovery, the sequence will greatly facilitate analysis of
transcriptional pathways during development....Finally, some of the most exciting applications of the
sequencing information lie at the interface between developmental and evolutionary biology. The compete
genomic sequence of a non-chordate deuterostome would provide important information concerning
developmental "inventions" of the chordates, and would shed light on genomic and developmental features
shared by the common ancestor of deuterostomes.
Carla Falugi, University of Milan, Italy
Jesus García-Soto, Universidad de Guanajuato
Giovanni Giudice, Università degli studi di Palermo, Italy
"It is of great importance to have the whole genome sequence of sea urchin because this animal still
represents one of the main model systems for early development."
Meredith Gould, Universidad Autonoma de Baja Calfiornia
Yukihisa Hamaguchi, Tokyo Institute of Technology
Jeff Hardin, University of Wisconsin
John Harding, Div. Comparative Medicine, NCRR, NIH
"The NCRR is highly supportive of genome sequencing efforts that will enhance the use of model organisms
such as the sea urchin. We share your enthusiasm for the further elucidation of sea urchin genome structure
and function, as exemplified by sequencing the sea urchin genome. Detailed knowledge of the sea urchin
genome sequence would clearly benefit the research efforts of users of the Sea Urchin Resource........."
Philippe Huitorel, Laboratoire de Biologie du Developpement, France
Kazuo Inaba, Tohoku University, Japan
Laurinda Jaffe, University of Connecticut Health Center
Hideki Katow, University of Tohoku, Japan
Takeo Kishimoto, Tokyo Institute of Technology
Ken Kitajima, Nagoya University Bioscience Center, Japan
"For the glycobiological studies, we have chosen sea urchins as model animals, because a relatively large
amount of gametic cells is available at once. Considering that this animal has been, long and exclusively,
used for understanding of the mechanistic foundations of various cellular processes, sea urchins could be
ideal model animals in the glycobiological field of study, in that we can easily link the presence of certain
glycan chains to the known biological processes."
Tetsuya Kominami, Ehime University, Japan
Hon Cheung Lee, University of Minnesota
"The point I would like to emphasize is that sea urchin is not just a marine invertebrate, but an extremely
versatile model system that can provide a wealth of information about a wide range of cellular functions with
practical and health relevance. Sequencing of the sea urchin genome should greatly facilitate the translation
of basic research to practical applications."
William J. Lennarz, State University of New York, Stony Brook
"I most enthusiastically support this initiative to get the complete genome of S. purpuratus. Having over half
of my lab working on projects on yeast, I have full well grown to appreciate the value of having the genome
completely sequenced. Since there has been so much work done on the development of the sea urchin,
complete knowledge of the genome will be invaluable."
Brian T. Livingston, University of South Florida
"My lab would benefit greatly from a sequenced genome, as this would facilitate dissection of gene
regulatory networks and the identification of new genes that play role in cell fate determination in sea urchin.
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We are also interested in investigating the evolution of the sea urchin genome relative to that of the
chordates."
Issei Mabuchi, The University of Tokyo
Valeria Matranga, C.N.R. – Istituto di Biologia dello Sviluppo, Italy
David McClay, Duke University
"We have 10 molecules that change during the 20 min it takes for an epithelial cell to convert into a
mesenchymal cell ..... we have obtained additional molecules that appear to be involved in controlling the
switch between an epithelial and a mesenchymal cell. Because of the importance of this transition in all of
biology and pathology, especially carcinogenic transformation, it is imperative to have efficient access to
new genes and their regulation in this process. Again, the availability of the genome and its accelerated
opportunity for gene discovery makes our study ever more efficient."
Hideo Mohri, Okazaki National Research Institutes, Japan
Giovanna Montana, University of Palermo, Italy
Masaki Morisawa, Misaki Marine Biological Station, The University of Tokyo
David Nishioka, Georgetown University
"One area of my own research on which sequencing the sea urchin genome will impact is the rational design
of new therapeutic drugs. In collaboration with the Cancer Therapy and Research center and the Institute for
Drug Development in San Antonio, I am using the early developing sea urchin as a system for testing newly
designed anticancer drugs."
Robert E. Palazzo, The University of Kansas
"Given the vast quantities of embryos that can be obtained, and their synchrony in development, the sea
urchin offers one of the few systems where large-scale purification of centrosomes is possible. This offers
the opportunity to isolate sufficient quantities of centrosomes for direct analysis. To this day we know very
little of centrosome composition."
Stephen R. Palumbi, Harvard University
"Last, the purple urchin will be one of the few species completely sequenced that has natural populations in
large abundance along with a huge store of intraspecific genetic diversity."
John S. Pearse, University of California, Santa Cruz
"A final problem I might mention is that of senility and aging. All the animal model systems with known or
soon-to-be-known genomic sequences are bilaterians with definitive live spans, and large efforts are being
made now to understand the genetic basis of senility by studying them. It seems to me that it could be
critical to know what genes are present or absent in species that don't undergo senility, such as sea urchins, to
fully understand the phenomenon of aging."
André Picard, CNRS UMR, France
" I answer here on behalf of the French community of labs working on sea urchin and starfish: probably
Christian Gache will have answered for himself, but he agreed to join us also......We all are strongly
interested by the whole-genome sequencing project on the sea urchin Strongylocentrotus purpuratus: the
main problem for cell biology, developmental biology and molecular biology on alternative (non-vertebrate)
models is to get the tools (cDNAs, recombinant proteins, antibodies)."
Dominic Poccia, Amherst College
"Our work I believe provides the most comprehensive study combining in vivo and in vitro experimentation
on sperm nucleus activation at fertilization, studies which would have been difficult or impossible with any
other organism. This work has contributed novel ideas on the roles of domains of male-specific nuclear
proteins in chromatin organization and on mechanisms of nuclear envelope disassembly and reassembly."
Rudolf A. Raff, Indiana University
Daniele Romancino, University of Palermo, Italy
Gerald Schatten, University of Pittsburgh School of Medicine
Sheldon S. Shen, Iowa State University
" I most emphatically support a sea urchin whole genome sequencing project, which is long overdue. As you
have noted in your letter, the sea urchin has played and continues to play an important role in our
understanding of development. Such a sequencing project has many potential benefits for the scientific
community and would greatly assist the progress of my own research. Even for an ion physiologist like
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myself, the need for specific gene sequence is necessary for fully understanding the roles of specific proteins
during development."
Greenfield Sluder, University of Massachusetts Medical Center
"For our work the sea urchin zygote is the very best model system out there.....Thus, for my research effort
the sea urchin genome project holds great benefit in that it will allow the generation of molecular reagents for
my planned research."
Andrew Smith, The Natural History Museum, London
L. Courtney Smith, The George Washington University
"Understanding the workings of the innate immune system in the sea urchin will illuminate the workings of
the more complex immune system, including the innate response, of mammals."
Michael J. Smith, Simon Fraser University, Canada
"I am writing in unequivocal support for the sea urchin whole genome-sequencing project. I have been
working on echinoderm developmental biology and molecular evolution, including sea urchins, for over 30
years. This project will materially contribute to our understanding of the evolution of genomes in
deuterostomes and will provide invaluable insights into functional correlates of genome sequence and
structure."
Giovanni Spinelli, Università de Palermo, Italy
Stephen A. Stricker, University of New Mexico
Norio Suzuki, Hokkaido University, Japan
Richard Tasca, NIHCD, NIH
"We recognize that our knowledge of sea urchin embryo development over the last quarter century has
provided novel insights into developmental mechanisms. Important advances have been made in
developmental gene regulation, cell specification, and other aspects of development using this model.
Genome sequencing of the most heavily used sea urchin species will further strengthen this model and that
will, in turn, greatly enhance the research efforts that we support on the very earliest stages of development
of many other organisms. In my opinion, sequencing the S. purpuratus genome should be completed as soon
as possible."
Victor Vacquier, Marine Biology Research Division, Scripps Institution of Oceanography
"Genomics will dominate experimental biology in the coming decade. The mechanism of gene network
control of development is one of the greatest and most complicated problems in all of biology. Organisms
that are proven models of great value, whose genomes are not sequenced, will be lost from future study. This
will result in a scientifically unhealthy intellectual narrowing of our general knowledge of biology."
Judith M. Venuti, Louisiana State University Health Sciences Center
Steven S. Vogel, Medical College of Georgia
Gary M. Wessel, Brown University
"While strong progress has been made studying fertilization in mice, research in two echinoderms, sea
urchins and starfish, have truly been instrumental in the driving research of the field fo the past hundred
years! These echinoderms will continue to lead progress in the future simply because of the practical
biological advantages of the system that you discuss in your proposal."
Michael Whitaker, University of Newcastle, United Kingdom
"I am writing to offer my strong support for the proposal to sequence one or two sea urchin genomes. Sea
urchin embryos offer advantages for biological and medical research in their cell and developmental biology.
Indeed, yours and Eric Davidson's work indicates that they may be the first organism in which the genetic
programme of regulative early development is described and understood. They are also unsurpassed as an
embryo in which to study the cell biology and cell physiology of early development and cell cycle
regulations."
Athula H. Wikramanayake, University of Hawaii at Manoa
"I am writing to strongly support the effort to have the Strongylocentrotus purpuratus genome sequenced and
to develop further resources for researchers working on sea urchins. The major research organism used in
my laboratory is the sea urchin and I expect it will continue to be the organism of choice for our research for
many years to come. My laboratory and many other laboratories working on sea urchin developmental
studies have already greatly benefited from the genomic resources for sea urchin developmental
studies...........I would expect that the whole genome sequence would further enhance our research and
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potentially allow us to make contributions that would have a significant impact on the elucidation of the
common molecular pathways involved in embryonic development."
Fred H. Wilt, University of California, Berkeley
Gregory A. Wray, Duke University
"I am writing to express my strong and enthusiastic support for your initiative to fully sequence the genome
of Strongylocentrotus purpuratus.....There is absolutely no question, however, that the lack of a complete
genomic sequence remains a major limitation for research on sea urchins....Complete sequencing of the S.
purpuratus genome would provide several immediate practical benefits, given the existing arrayed genomic
and cDNA libraries....These practical benefits would impact virtually every laboratory working on sea
urchins."

La Ode Muhammad Yasir Haya

Over population of marine sea urchins (Diadema setosum) may disrupt the growth of Crustose coralline causing the reduction of coral reefs growth. Consumption of Diadema setosum gonads as the alternative food may assists to preserve the balance of coral reefs ecosystem. The Objective of this study was to measure and evaluate the nutrient content of the gonad of Diadema setosum. Mass spectrophotometer was used to measure vitamin and albumin contents, Kjeldahl methods for protein content, and Atomization method for trace elements (Fe, Mg, and Zn) content. The presence of active compounds such as steroids, amino acids and antioxidants were identified by thin-layer chromatograph (TLC). Protein, albumin, vitamin A, vitamin E and trace elements (Fe, Mg, and Zn) were found in the gonad of Diadema setosum. Vitamin E (23.47 mg) was the highest nutrient content compared to other nutrient elements. The extracts of the gonad of Diadema setosum were found to have steroid, amino acids and antioxidant compounds. Overall, nutrient contents and active compounds in the gonad of Diadema setosum are essential components needed for immune system, therefore besides its potency as alternative food source, gonad of Diadema setosum has potency to become the source of immune-nutrient.

Joshua Zimmerberg, Chief, Laboratory of Cellular and Molecular Biophysics and Director,
NASA/NIH Tissue Culture Center
"Unfortunately, we cannot identify the essential proteins, although we already have them in hand, because
the technique appropriate to the amount of material isolated and purified is completely dependent upon valid
and complete sequence information at the genomic and proteomic level. We simply cannot answer our
reviewer's questions, based upon sequence data available to them for their organisms (yeast, human, mouse),
because we do not have comparable data for the sea urchin system."
APPENDIX 2
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Recent Citations Illustrating the Variety of Research Studies that Utilize Sea Urchins
Ameye L, De Becker G, Killian C, Wilt F, Kemps R, Kuypers S,
Dubois P. Proteins and saccharides of the sea urchin organic
matrix of mineralization: Characterization and localization in the
spine skeleton. J. Struct. Biol. 2001 Apr;134(1):56-66.
Angerer LM, Oleksyn DW, Levine AM, Li X, Klein WH, Angerer
RC. Sea urchin goosecoid function links fate specification along
the animal-vegetal and oral-aboral embryonic axes.
Development. 2001 Nov;128(22):4393-404.
Arenas-Mena C, Cameron AR, Davidson EH. Spatial expression of
Hox cluster genes in the ontogeny of a sea urchin. Development.
2000 Nov;127(21):4631-43.
Blank PS, Vogel SS, Malley JD, Zimmerberg J. A kinetic analysis
of calcium-triggered exocytosis. J. Gen. Physiol. 2001
Aug;118:145-56.
Cameron RA, Mahairas G, Rast JP, Martinez P, Biondi TR,
Swartzell S, Wallace JC, Poustka AJ, Livi

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