1. Microarrays: Goal
to make technique accessible to as broad a group as possible:
one reader per 20 active-user laboratories; for laboratories that are
making their own arrays, one per 20 active-user laboratories.
We endorse the academic use
as well as development of the technique. This can best be accomplished
at present by the method of polynucleotide arrays (Pat Brown/Synteni).
There was considerable interest
by some people for purchasing ready-made arrays. The manner in which
the NIH would subsidize this is unclear. The cost per array should ideally
be no more than $50-100 per array.
We recommend that the NIH
support, perhaps via instrumentation grants, the purchase of arrayer
(robot for spotting) and reader. We appreciate that these pieces of
equipment are under development but feel that the yeast community has
immediate use of this technique and that their use will lead to improvements
in the technique.
Approximate cost for arrayer:
Approximate cost for reader:
Cost per array: at present,
primer pairs for amplifying all yeast ORFs cost approximately $30,000
and will produce ca. 1000 arrays, for a cost per array of $30.
Arrays need a set of standard
controls to allow comparison from experiment to experiment and from
laboratory to laboratory.
2. Mass spectrometric
identification of components of protein complexes.
We anticipate that the demand
for mass spectrometric analysis will increase as genome sequence information
increases. We therefore recommend the establishment of a small number
of national centers to which samples from any number of organisms are
sent for analysis.
Approximate cost for instrumentation:
Personnel cost (operator/technician):
3. Deconvolution microscope:
approximately 1 per 3-4 user laboratories.
is particularly critical for analysis of small cells such as
yeast and Chlamydomonas. It is preferable to confocal microscopy
because it can take thin optical sections and tilt the sample to assess
co-localization. In addition, the software for image analysis is able
to remove out-of-focus information and produce sharper images.
Approximate cost: $150,000
for 3-I; $300,000 for DeltaVision.
Purchase of such microscopes
is not expected to be funded through RO1 grants.
1. Heterologous cDNA libraries
for regulated expression in yeast: care needs to be given to design
of the vector and to produce full-length cDNA. Libraries should be made
from human and mouse, as well as Xenopus, and other model organisms.
Approximate cost: $75,000-$100,000
for one-two years.
Construction of such libraries
might be coordinated with ongoing efforts to construct full-length cDNA
libraries for CGAP and mouse GAP projects.
2. Unigene set of plasmids
carrying each ORF. This set can be used for: (a)
cloning by complementation, (b)
facile construction of derivative sets allowing inducible expression,
overexpression, epitope-tagged, etc. in a variety of vectors (integrating,
high copy, etc.), and (c)
amplification of ORFs for array construction.
The initial unigene set of
plasmids can in principle be converted to the variant sets using, for
the example, the method of Elledge and colleagues.
Approximate cost: $50,000
for construction of unigene set.
3. Chemical libraries.
Such libraries could be used for: (a)
determining the response pattern of cells to drugs, (b)
determining sensitivity of given strains to drugs, and (c)
carrying out medium-scale (nonrobotic) screening of mutants with the
These libraries would be
used in the laboratories of the investigators and supplied in 96-well
General comment about
distribution of genomic resources:
A mechanism for distributing
genomic resources such as the unigene set of plasmids and heterologous
cDNA libraries at nominal cost needs to be developed. The methods for
distributing such material to the yeast community will be a preview
of what will be required for distribution of comparable materials to
researchers working on other model organisms. Distribution of genomic
resources through commercial channels is currently prohibitively expensive
for allowing full access to many important reagents, for example, those
necessary to produce DNA microarrays.
1. Importance: The
Saccharomyces Genome Database was recognized as being enormously
successful and essential not only for the yeast community but valuable
for others as well. The successful functioning of SGD leads to additional
suggestions for ways in which its usefulness can be expanded. SGD is
in many respects a model for genome databases.
a) The SGD should be a
repository for unpublished information on mutant phenotypes.
b) The SGD should have
more effective links to other organism databases.
c) The SGD should consider
curating fission yeast.
d) The SGD should curate
pathways such as metabolic, signal transduction, etc.
e) SGD should consider
curating and cross-referencing expression array data and developing
methods for sorting existing array data. We recognize this as a major
challenge, but it is an extraordinarily important undertaking.
f) The SGD should consider
developing a phenotype-based search engine.
g) The SGD should provide
image data for protein localization or at least link to the Yale Genome
h) Attempts should be made
to give different organism databases the same look and feel.
OTHER MODEL ORGANISMS
A common theme for essentially
all the organisms was the need for (a) EST sequencing, (b) increased genomic
curation, (c) microarray capability, and (d) ultimately genomic sequence.
unique feature exceptional opportunity to study structure of
flagella (ca. 250 proteins): wish list physical map and EST.
pombe: has many functional and structural differences from S.
cerevisiae but has many of the same advantageous technical features
(which can be enhanced). Top priority request: capability of microarrays;
next: enhanced database with annotation. Fission yeast should be included
in various yeast initiatives.
rationale: the filamentous fungi have a dramatically different
life style from budding yeast; this is reflected currently in its EST
sequences, many of which are novel.
A variety of filamentous
fungi were discussed in addition to Neurospora, including Aspergillus
nidulans (a pathogen of immunocompromised individuals) and Ashbya
gossypii (a cotton pathogen whose genome appears related to budding
yeast prior to duplication).
One criterion for choosing
a filamentous fungus for conferring expanded genomic resources is that
it have homologous recombination to facilitate genetic analysis.
4. Model organisms for
infectious disease: several infectious agents exist that have great
potential as model organisms because of their relevance to important
infectious diseases but are experimentally manipulable. These organisms
include Toxoplasma, Candida albicans, Cryptococcus
neoformans, Ustilago maydis (corn smut), and Histoplasma
capsulatum. As noted above for Neurospora and other filamentous
fungi, an important criterion for choosing model infectious disease
organisms for expanded genomic resources is that it have homologous
recombination to facilitate genetic analysis.