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CILIATEMOLBIO-L  September 2006

CILIATEMOLBIO-L September 2006

Subject:

Cloning by Complementation proposal

From:

Robert Coyne <[log in to unmask]>

Date:

Tue, 5 Sep 2006 15:54:31 -0400

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (108 lines)

Dear colleagues,
I am writing to ask for your help regarding a proposal that I (as PI) and Ed Orias are planning to
submit to the NIH. The objective is to develop an efficient means to clone genes of interest from
Tetrahymena thermophila by functional complementation of mutations (often referred to as
“forward genetics”).  This will allow us to harness in Tetrahymena “the awesome power of
genetics” in unbiased mutant phenotype searches, which is so advantageously exploited in other
model organisms. A more detailed description of the specific aims, the proposed resource, and
how it would be used by the community is attached at the end of this message. We also intend to
propose, as part of the project, to hold training workshops for interested researchers -- at any
career level and working with any organism -- on the methods and resources (genetic, molecular,
informatic, etc.) relevant to Tetrahymena forward genetics, so that any researcher interested in
using this resource can be brought up to speed in a week or two.
This proposal will be submitted in response to NIGMS Program Announcement 04-135, “Tools for
genetic and genomic studies in emerging model organisms” (http://grants2.nih.gov/grants/
guide/pa-files/PA-04-135.html).  The aim of this NIH program is to facilitate the use of genome
sequence to “exploit the full potential of novel or developing model systems for comparative and
functional genomic studies”. Although the project would most directly and immediately benefit the
work of Tetrahymena researchers, we expect indirect benefits to the work of other researchers as
well. It is important that the application document every type of benefit to convince reviewers of
the resource’s value.
Now to the immediate point:  WE NEED YOUR HELP TO MAKE THIS HAPPEN!  If you see benefits of
this resource to your work, please communicate this in a letter of support for this project,
addressed to me (fax or email preferred) at the address below.  In order to assemble the vital
supporting data for the application, it would be very helpful if your letter could address any or all
of the following topics: a) a concise paragraph or two explaining how this resource would facilitate
your current or future research, b) how it could enable possible novel avenues of research in your
project, and c) very importantly, what the potential significance of this research is for
fundamental, biomedical or biotech research. It would also be very helpful to know if you already
have collections of mutants whose phenotypes have been analyzed but the genes determining the
phenotypes have not yet been identified, or if the availability of this resource would induce you to
undertake new genetic screens. If you can also envision the use of this resource in undergraduate
research, which is relevant to the training of future biomedical researchers, please address this
potential use in the letter. For non-Tetrahymena researchers, if you can visualize how this
resource can be used to facilitate your work, e.g. by identifying genes of interest in a close relative
and by raising the profile of ciliates as model organisms, please include this information in your
letter.
The deadline for this proposal submission is October 1, 2006.  I apologize for asking you to take
time out of your busy schedules on such short deadline, but please send your letters to me as
soon as you can before September 23.  And if you have any ideas for novel uses of these
resources that could strengthen the application and are not addressed in this request letter,
please feel welcome to let me know directly.
If you intend to submit a letter, please let me know your intent right away, so I can quickly assess
the extent of support. Please contact me or Ed if you have any questions. Thank you very much for
your help.

Regards,
Bob

Bob Coyne
The Institute for Genomic Research
9712 Medical Center Dr.
Rockville, MD  20850

Fax: 301-294-3142
email: [log in to unmask]


Specific aims, the proposed resource and how it could be used by the community

Specific aims
1) To finish relating the MAC genome sequence scaffolds to their MIC chromosomal locations. This
would involve a very light (0.25-fold genomic coverage) WGS MAC sequencing of inbred strain C3,
identifying SNP and VNTR polymorphisms by aligning with the existing strain B sequence
scaffolds, and mapping these DNA polymorphisms (and associated scaffolds) to chromosome
arms, using C3 matings to existing B nullisomic strains. With this information, the number of
clones needed to ensure rescue of any mutation mapped to a particular chromosome arm (e.g.
Specific Aim #4) will be reduced approximately 10-fold.
2) To make an arrayed library of 15-20-kb inserts with 6 to 10-fold insert coverage of the MAC
genome in a novel linear E. coli vector engineered for Tetrahymena transformation.
3) To end-sequence and map each insert to the MAC genome sequence assemblies and to genetic
maps of the MIC and the MAC. Using these maps, to create a rearrayed overlapping tiled set of
clones to cover as much as possible of the genome with as few clones as possible (approximately
10,000).
4) To demonstrate the power of this forward genetics system by identifying the mutant gene in a
number of mutants of interest already available in the research community.

A brief description of the proposed resource: Lucigen Corp. has recently succeeded in generating
a library of Tetrahymena macronuclear genomic DNA with long inserts - up to 20 kb - in a novel
linear E. coli vector.  Test sequencing of a sample of this library has shown a high proportion of
stable inserts, as evidenced by aligning end sequences with the Tetrahymena MAC assemblies. We
propose to modify the vector so that part of it can replicate as a MAC minichromosome, i.e., by
adding a Tetrahymena origin of replication, a selectable marker, and flanking chromosome
breakage sites (Cbs) to obtain the release of the autonomously replicating linear episome and
telomere addition. The modified vector will be tested for transformation efficiency and mutant
rescue capability with a well-characterized gene.  We anticipate completing this test in time for the
review of the grant application. When funds from the project become available, we would make a
size-selected 15-20 kb library using the modified vector.  Inserts would be end-sequenced and
mapped to the MAC genome assembly to generate a tiled collection of clones that ideally cover the
entire genome (somewhere around 10,000 clones, or about twenty six 384-well plates).
Meanwhile, and in coordination with the Orias lab’s NCRR-funded and on-going efforts to use
HAPPY mapping to complete the assemblies of the MAC and MIC genomes, we would finish the
genetic maps necessary to associate each genomic scaffold (and therefore each 20-kb insert) with
its MIC chromosomal arm location.  Copies of the entire library would be made available without
restriction to those in the research community who need it to advance their work.

How we envision the resource being used by the community to identify the gene responsible for a
mutant phenotype of interest:  Anyone with a germline mutation and a gene to be identified would
transform mating mutant cells by electroporation with DNA made from all or part of the library.  If
a selectable phenotype is associated with the wild type allele, using the whole library may be
sufficient to identify the transforming insert.  If not, you would first genetically map the mutation
to a chromosome arm -- simply by doing one set of parallel matings to a panel of seven
nullisomic strains and examining the phenotypes of the progeny. This would reduce the number
of clones necessary to use in transformation, and increase the number of potential wild type
transformants, by about 10-fold.  After rescue, the transforming DNA insert would be identified
(e.g. the ends could be amplified by inverse PCR from the known vector sequence) and mapped to
the genome sequence.  Once the transforming insert is identified, its individual predicted genes
(about four on average) would be subcloned into the same or another transformation vector and
used to transform mutant cells to pinpoint the gene of interest.

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