Report of the Ciliate Genomics and Proteomics Workshop

International FASEB Conference on Ciliate Molecular Biology

Saxtons River -- August 9, 1999

Prepared by Eduardo Orias

 

Overview

 

The Ciliate Genomics and Proteomics Workshop was scheduled as a regular evening session of the 1999 FASEB Conference on Ciliate Molecular Biology, and was held on Monday August 9, 1999, 7:30-10 PM. This international workshop was co-organized by Peter Bruns (Cornell University, USA), Jean Cohen (Centre National de la Recherche Scientifique, France) and Eduardo Orias (University of California - Santa Barbara) and was attended by about 150 participants from North America, Europe and Japan. There were eleven oral presentations of current work relevant to ciliate genomics, mainly Tetrahymena. The presentations (5-15 min. long) focused on the highlights and summaries of the results, and each presentation was supplemented by a poster giving a more detailed description of the work. The presentations were divided into three groups: 1) useful biological and experimental features of selected organisms; 2) advances in Ciliate genomics and proteomics; 3) advances in the development of methods to fully exploit a genome sequence. An open ended period of general discussion followed the oral presentation segment.

 

The talks of the Genomics Workshop were followed by a period of lively general discussion. Regarding genome sequencing, a strong consensus was reached on two issues:

1) Among the Ciliates, the Tetrahymena genome should be given highest priority.

2) The macronucleus (the expressed, somatic nucleus) should be given higher priority than the non-expressed (germ line)micronucleus.

 

A group of about 30 investigators, strongly committed to work toward a Tetrahymena genome sequencing project, met again from 1:30-4:00 PM the next afternoon. The following actions were taken:

1. A subset of the participants committed themselves to fully explore options for shotgun cloning and sequencing the MAC genome.

2. A subset of the participants committed themselves to write up the compelling justifications for a genome sequence project based on insights about the fundamental questions addressed by their work.

3. A subset of the participants agreed to meet again in October 1999 in Chicago to formulate a final proposal to be used to seek extramural funding and sequencing facility commitment.

 

A group of Paramecium investigators also met in a follow-up meeting and decided to pursue a limited collaborative STS sequencing project by distributing the work among different laboratories.

 

The Genomics Workshop, and the entire conference as well, were permeated by a high level of excitement, enthusiasm and optimism driven by the quality of the experimental work reported, by the explosion of knowledge made possible by the (often collaborative) application of the latest genetic and molecular tools, and by the prospect that a genome sequencing project will lead to an even greater acceleration of the fundamental experimental contributions possible with the Ciliates in general, and Tetrahymena in particular.

 

Highlights of the workshop presentations and of additional relevant work presented during other sessions, as well as a more detailed report of the actions taken at the workshop, are listed below.

 

Salient features of the workshop presentations.

 

a) Features of Tetrahymena relevant to a genome sequencing project. Marty Gorovsky shared with the audience his February 1999 presentation on Tetrahymena to the NonMammalian Models Workshop convened by the Director of NIH, including a request for funds to support mainly a pilot Tetrahymena genome sequencing project.

 

b) Mapping progress. The Bruns group demonstrated the feasibility of constructing a deletion map of every chromosome, which will facilitate the efficient mapping and cloning of mutant genes with interesting phenotypes. The Orias group described recent progress in genetically mapping both the MIC and MAC genomes in concert. MAC chromosome pieces are the physical basis of coassortment groups. Macronuclear coassortment groups map to continuous segments of the MIC map, suggesting that gross DNA rearrangements do not accompany chromosome fragmentation and justifying the view that the MAC genome is in effect a natural restriction-like digest of the MIC genome that can be exploited for physical mapping and genome sequencing. A collaborative project between the Bruns and Orias groups has demonstrated the feasibility of their now ongoing project to make a linking library of inserts containing the Chromosome Fragmentation sequence (Cbs) to link MIC-adjacent MAC pieces into a complete physical map of every MIC chromosome. The approach will generate abundant links between the physical map and both the genetic maps and the genome sequence. The Karrer group (Marquette University, USA) provided an analysis of all the Tetrahymena DNA sequences listed in public databases. Their analysis demonstrates the very sharp separation in base composition between protein coding segments and the remaining sequences. This feature should greatly facilitate the identification of coding sequences and the annotation of the genomic sequence.

 

c) Sequencing strategies. Cliff Brunk (UCLA, USA) presented a strategy for increasing the efficiency of sequencing BAC clones.

 

d) Mutant-to-cloned-gene step. The Turkewitz group (University of Chicago, USA) has developed a method based on antisense repression that allows ready mutant isolation (with defective regulated secretion, in their case) and direct cloning of the affected gene. This illustrates the power and robustness of antisense technology in Tetrahymena.

 

e) Inducible promoters. The Miceli group (University of Camerino, Italy) reported work in progress for the isolation of genes involved in stress reactions, including the metallothionein gene from Tetrahymena and the hsp70 protein from the Hypotrich ciliate Euplotes. This work will further the use of ciliates as environmental biosensors. Inducible promoters will play a very important role in experimental analysis in Tetrahymena.

 

f) Proteomics. The Tiedtke group (University of Muenster, Germany) reported initial success in his project on the proteomics of the phagosome. This work exploits the speed with which Tetrahymena forms phagosomes (one per minute) at a single cellular location (the oral apparatus) and the ability to purify phagosomes in amounts higher by orders of magnitude than is practical from mammalian macrophages.

 

g) Paramecium Genomics. The Cohen group reported the development of an indexed macronuclear genomic library of 6-12 kb inserts that has been used successfully for gene cloning by complementation. The same group, in collaboration with the Pearlman group (York University, Canada) reported sequencing 400-500 nucleotides at each end of 41 of these clones (randomly selected). Eighteen high score BLAST matches to genes in one or more databases were found among the 82 sequence runs. Half of these coding sequences matched mammalian genes but failed to match yeast genes. The high frequency of matches is consistent with an earlier suggestion that coding sequences represent up to 50% of the Paramecium MAC genome. The Cohen group also presented a poster showing preliminary evidence of success in transforming the Paramecium micronucleus.

 

Additional genomics-relevant work presented at other sessions of the FASEB meeting.

 

- Gaertig's group (University of Georgia, USA) reported the discovery of kinesin 2 as the anterograde motor required for assembly and growth of the ciliary axoneme, which occurs at the tip. The gene knock-out has stubby cilia and cannot swim or carry out phagocytosis. Additional experiments showed that observed defects in cytokinesis and the lethality of the knock-out were secondary consequences of ciliary deficiency. This work illustrates the usefulness for Tetrahymena as an experimental system complementary to yeast for investigating fundamental problems, and the promise that the Tetrahymena genome sequence can be utilized effectively.

- Joint work of the Clark (Cornell University, USA), Dickerson (University of Georgia, USA) and Gaertig groups has accomplished the expression of Ichthyophthirius surface antigen on the surface of the Tetrahymena cell envelope. That ciliate is the causative agent of the fish disease known as "Ick" that plagues fisheries and pet stores. The work demonstrates the ability to express and correctly target foreign genes in Tetrahymena.

- The Bruns group showed successful examples of "chromosome painting" by FISH using DNA from purified MICs of unisomic strains, i.e. strains having copies of a single chromosome in their MIC. The group has elucidated the chromosome alignment in the "crescent", a very elongated form of the micronucleus that forms during prophase of meiosis I; they have showed that the centromeres of all 5 chromosomes are at one end, and all the telomeres are at the other. Chromosome painting greatly advances chromosome cytology in Tetrahymena and is a boon to genome mapping.

- David Asai and James Forney (Purdue University), editors of the volume of Methods of Cell Biology dedicated to Tetrahymena, announced that the volume will appear in September 1999 and circulated a spiral bound sample copy. This book will greatly facilitate the work of scientists who wish to switch to Tetrahymena in pursuit of the most favorable organism in which to investigate important problems of interest to them.

 

Major decisions reached at the workshop and their underlying rationales

 

1) Among the Ciliates, the Tetrahymena genome should be sequenced with highest priority for the following reasons:

- Genetic mapping of the MIC and MAC genomes is quite advanced in Tetrahymena but is essentially non-existent in any other ciliate;

- Physical mapping of the genome and correlation with the genetic maps are already underway in Tetrahymena.

- The highly site-specific fragmentation of the genome in Tetrahymena provides large, natural subunits for sequencing.

- Fortuitous biological advantages of Tetrahymena, e.g., cell culturing, genetic and physical mapping and ready maintenance of essential gene knockouts, greatly facilitate experimental analysis. The importance of these advantages is expected to increase after the genome is sequenced.

- Useful strains can be maintained frozen.

- A larger community currently works with the molecular biology of Tetrahymena than with any other ciliate.

 

2) The macronucleus (the expressed, somatic nucleus) should be given higher priority for sequencing than the non-expressed micronucleus:

- the MAC genome is 10-20% smaller

- in particular, the MAC genome contains far fewer repeated sequences, an important advantage for sequence assembly under shotgun sequencing.

- MAC DNA is about 20 times more abundant.

 

Actions taken at the follow-up Tetrahymena session.

 

Action 1. A subset of the participants agreed to fully explore alternative strategies for shotgun cloning and sequencing the MAC genome. Some advantages and disadvantages of the following options were stated:

a) Sequencing the entire 200 Mb genome as a unit.

b) Breaking up the MAC genome into 5-10 size-selected subsets of the macronuclear chromosome pieces and sequencing one subset at a time.

c) Sequencing individual, purified MAC pieces, one piece at a time.

d) Making first a MAC BAC library and sequencing BAC inserts.

e) Sequencing a cDNA library in parallel with the genome sequencing effort.

 

Action 2. A subset of the participants agreed to write up the compelling justifications for a genome sequence project from the vantage point of the individual focus of their work. Major foci include:

- the investigation of biological mechanisms of fundamental significance and of importance to human disease that cannot be approached in yeast because the biology is missing from that organism (e.g., regulated secretion, phagocytosis, ciliary motility, host factors for bacterial pathogenesis).

- Tetrahymena's unique evolutionary position as a free-living (thus having a genome coding for the required diversity of biological mechanisms) representative of the Alveolata, a major evolutionary group of "crown" eukaryotes that includes the Ciliates, Dinoflagellates and the (parasitic) Apicomplexa (e.g., Plasmodium, Toxoplasma).

- Tetrahymena's potential for biotechnology (e.g. as a system for the inexpensive and high level expression of foreign proteins).

 

Action 3. A subset of the participants agreed to meet again in October 1999 in Chicago to evaluate and collate the gathered information and to formulate a final proposal to be used to seek extramural funding sources and sequencing facility commitment.