Tetrahymena Group



Tetrahymena thermophila is a microbial eukaryote and a model system of proven utility. Each cell contains two genomes. The transcriptionally active macronuclear genome consists of ~250 macronuclear chromosomes, generated by site-specific, genetically-determined fragmentation of the 5 germline chromosomes of the transcriptionally inert micronucleus. These fragments represent a natural, complete, high copy number YAC-like library of the expressed genome. Efforts are already underway to RAPD map and to order the macronuclear pieces into a complete physical map exploiting the unique 15-base pair sequence which is necessary and sufficient to cleave the micronuclear chromosomes into macronuclear pieces. We believe that the macronuclear chromosomes offer unique advantages for sequencing the entire genome by direct shotgun sequencing and request funds for a pilot project. Funds are requested to assess the feasibility of this approach and to develop important complementary technologies for functional analyses, as well as for database and community training resources.

Because ORFs are readily delineated - 25 % GC average genomic content - and introns are relatively small and rare, we think that direct genome sequencing could make it unnecessary to obtain a set of fully sequenced cDNAs. The pilot sequencing project will tell us if our belief is correct. So at the present time we are not requesting cDNA sequencing.

Prioritized list of resources needed:

1. To explore direct shotgun sequencing of the entire genome by sequencing macronuclear chromosomes (genome size = 200 Mb, total number of macronuclear chromosomes ~ 250, average size ~ 700 Kb, range 300 Kb - 3.3 Mb). A pilot project will sequence 10-15% of the genome. The initial work will focus on those macronuclear chromosomes known to contain genes for proteins which are likely to be tightly co- regulated (and thus possibly on the same macronuclear chromosome) with other proteins (e.g. genes for regulated secretion of stored protein products, genes for ciliary dyneins, etc.) ORFs would be identified and knocked out to determine the fraction of the expressed genome and the fraction of essential genes. The genes chosen are a subset of those found in Tetrahymena that are involved in processes of significance for human biology and disease that cannot be studied in yeast. The work will use a combination of available technologies for separating macronuclear chromosomes, shotgun cloning and sequencing and can be started now.

Estimated duration: one-two years. Amount requested: $3 million total.


2. Improve genomic technology in Tetrahymena. This would include:

  • construction of higher resolution maps of macronuclear and germline chromosomes.
  • cloning by complementation
  • insertional mutagenesis
  • development of highly engineered strains:
  • an inducible-repressible system
  • efficient maintenance of free plasmids

Estimated duration, 3 years. Amount requested: $2 million total.

3. Database resources to make sequences and results readily available not only to the Tetrahymena community but also the entire scientific community. For the time being it would seem most cost effective to affiliate with an existing genome database, adopt their format and augment their resources to support this effort.

Duration: on-going. Amount requested: $200,000 per year.

4. Yearly course to train interested biologists on Tetrahymena-specific genetic and molecular methods.

Duration: on-going for a 5 year trial period. Amount requested: $30,000 per year.

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