UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR)

WORKPLAN of the PARASITE GENOME COMMITTEE April 1996

Rationale

The Parasite Genome Committee was created for coordination of research on the analysis and mapping of genomes of the TDR target parasites. The sequenced genome map of the parasite will provide a powerful tool for identification and cloning of target genes for vaccines, diagnostics and drug development. Understanding the mechanisms of drug resistance and various functions of the parasite is enormously facilitated with known genome sequence. Development of physical maps is the first step and a one-time investment to serve the scientific community. It is understood, that TDR can provide only a fraction of the funds required for such high technology research, therefore, a close collaboration with other institutions would be necessary.

Objectives

The activities of the Parasite Genome Committee are carried out by five networks corresponding to the target parasites: Filaria, Leishmania, Trypanosoma brucei, T. cruzi and Schistosoma. Malaria and leprosy are not included because there are independent networks for the Plasmodium and M. leprae genomes which are being supported outside TDR. Each network has a workplan that has been agreed upon by the Committee. In all the networks, much emphasis will be put on the development of a common database system, and on the training and participation of Disease Endemic Country (DEC) scientists, to produce low resolution maps of the genome of the five parasites and promote gene manipulation techniques.

The Filaria genome network:- will focus research on Brugia malayi. High quality cDNA libraries have been constructed and evaluated for all developmental stages of the parasite. 2400 cDNA clones have been sequenced and over 1000 new Brugia genes have already been identified (March 1996). The known map of C. elegans will be used extensively to rapidly identify numerous genes, thereby facilitating efforts of the network. Coordinator: Dr Steven A. Williams.

The Leishmania genome network:- will focus on a single L. major strain (Friedlin). An international consortium has been established to sequence the entire genome from cosmids of the Friedlin strain within the next 5 years. The cosmid library, and/or high density filters prepared from it, can be obtained by all interested centres from a central resource laboratory. The resource also includes EST sequence information on many new genes, clones for which can be obtained by any parties interested in their development as drug targets or vaccines. Coordinator: Professor Jenefer M. Blackwell.

The Trypanosoma brucei genome network:- will not focus on any particular cloned isolate because a substantial number of markers are available which can be used for physical mapping and have been developed using different isolates. However, genomic libraries are needed and will be constructed by a selected laboratory. This will act as a trypanosomatid reference centre to maintain large DNA libraries for distribution. Coordinator: Professor John Donelson.

The Trypanosoma cruzi genome network:- has chosen a single well-developed CL-Brener strain from Brazil. The main activities include the construction of genomic libraries from different stages of the parasite. Small sections will be mapped initially with the development of the entire map in view, using contig libraries. Coordinator: Professor Alberto Carlos C. Frasch.

The Schistosoma genome network:- has selected S. mansoni and S. japonicum. Priority has been given to four objectives towards mapping of the schistosome genome: 1) Resource development; stage-specific cDNA libraries are available, however, development of large fragment genomic libraries is needed; 2) Gene discovery, including identification of stage-specific genes; 3) low resolution physical mapping; and 4) development of genome databank. Coordinator: Professor Mette Strand.

Progress and Expected Outcomes

During the first year of Parasite Genome activity, five networks have been set up and consolidated. Planning meetings have been held to select parasite strains for research and to draw up guidelines towards attaining the Programme's objectives. All networks successfully fulfilled an important task to construct parasite stage specific cDNA and large fragment genome libraries (YAC, BAC, Cosmids). Thousands of EST and chromosome markers have been obtained and the process of chromosome mapping was initiated. Thousands of clones of cDNA have been fingerprinted for a Leishmania's strain representing 80-90% of the entire genome. More than 100 contigs are now available for sequencing. Significant expansion of these activities is expected during the next biennium, in the hope that with the introduction of high precision robotics the genomes of the representative parasites will be sequenced within the next 5 years.

Possibilities for Collaboration in Workplan Activities

Research proposals with budgets up to US$50 000 addressing one of the objectives mentioned above are invited. Projects are funded for three years after which investigators should reapply, either for a continuation or for a new line of research. At this point, competitive proposals are encouraged to assure continuation of particularly interesting research leads but the Committee will not fund parallel work at different institutions. Interested investigators should consult with the Committee manager before applying.

How to apply

Researchers interested in collaborating the above mentioned activities should request application forms from the Communications department of TDR. [Email: TDRNEWS@WHO.CH]

Next proposal deadline: 23 July 1996 ------------

All specific correspondence related to research covered by the Parasite Genome Committee should be sent to:

Dr B. Dobrokhotov Manager of the Parasite Genome Committee UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) World Health Organization, 20 Avenue Appia, CH-1211 Geneva 27, Switzerland

Tel: (41.22) 791.3816/3724 Fax: (41.22) 791.4854 Internet: Dobrokhotovb@WHO.CH

-----------WorkPlan Tables for 1996, in list form follows----------

TRYPANOSOMA CRUZI GENOME PROJECT WORKPLAN FOR 1996-1997 Coordinator: Dr Alberto Carlos C. Frasch

OBJECTIVES PLANNED ACTIVITIES ---STATUS 1. Characterization of T. cruzi CL-Brener reference clone a) Characterization Growth and differentiation Susceptibility to chemicals Identification of Molecular markers ---Significant Progress achieved b) Karyotype analysis Identification of chromosomes Identification of markers for each chromosome ---42 chromosomes and 8 linkage groups identified 2. Library, contig and map construction of CL-Brener clone a) Contig construction Contig construction with cosmid libraries ---cDNA, YAC, BAC and cosmid libraries constructed b) Map construction Physical map ---To be initiated in 1996 3. Gene identification in CL-Brener clone a) Library cDNA libraries from the different forms of the parasite b) Sequencing Sequencing of random clones ---To be initiated in 1996 4. Genomic sequencing a) Sequencing Assignment of contigs 5. Integrated database a) Reference center Entering data using AceDB software ---Created

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FILARIAL GENOME NETWORK WORKPLAN FOR 1996-1997 Coordinator: Dr Steven A. Williams

OBJECTIVES PLANNED ACTIVITIES ---STATUS 1. Gene Discovery

a) cDNA libraries Prepare and evaluate cDNA libraries from B. malayi in transition between the life cycle stages ---7 cDNA libraries constructed

b) EST analysis Preliminary EST analysis on 200 clones from each of the cDNA libraries ---2400 EST sequenced 1000 new genes identified

c) Subtraction and EST Analysis Subtract the most common clones from all of the libraries and EST analysis on 800 additional clones from each cDNA library ---Ongoing d) Network Database Establish an interactive DNA database and communication network - will integrate DNA sequence and mapping data as well as bibliographic and other information ---The database is in place and development is ongoing 2. Genome Mapping

a) Cosmid Library Construct a B. malayi genomic cosmid library for mapping ---To be initiated in 1996

b) YAC Library Construct a B. malayi YAC genomic library for mapping ----To be initiated in 1996

c) Mapping Construct a low to medium resolution physical map ---To be initiated in 1996

3. Individual Gene Analysis and Vaccine Study

a) Library Screening Screen cDNA libraries with infected and immune sera ---Ongoing

b) Gene Study Detailed DNA sequence analysis and mapping of genes with potential as vaccine candidates and drug targets ---Ongoing

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THE SCHISTOSOME GENOME PROJECT WORKPLAN FOR 1996-1997 Coordinator: Dr Mette Strand

OBJECTIVES PLANNED ACTIVITIES ---STATUS

1. Resource Development

a) Development of stage-specific cDNA libraries 1. Cercariae 2. Schistosomula 3. Juvenile worms 4. Female adult worms 5. Male adult worms 6. Egg Stage-specific libraries are available, however only libraries #2, 4 and 6 are in phagemids. Criteria for new libraries: Directional library in phagemids; cDNA inserts >500bp average cDNA inserts>1000bp 106 or greater recombinants 95% of recombinants should contain specific insert <1% of recombinants should contain insert of host origin, RDNA or mitochondia transcripts ---The majority of sequencing has been carried with adult worm libraries

b) Development of large fragment genomic libraries 1) YACs ---Available 2) Cosmids ---To be initiated

2. Gene Discovery a) Expressed sequence tags (ESTs)

Identify genes from different developmental stages by use of Subtractive libraries and Differential Display techniques ---1400 EST sequenced, 640 new genes identified

Identification of rare mRNAs ---To be initiated

3. Chromosome Mapping Generate low resolution physical map ---To be initiated

a) YAC hybridization Map cosmids and individual genes ---Preliminary stage, screening

b) Cosmid hybridization Map genes to cosmids, Generate linkage maps ---To be initiated in 1996

c) Molecular characterization of chromosome structure Map YACs to chromosomes to develop contigs for each chromosome (FISH, CISS, PRINS)

4. Development of Genome Databank Standardize information and create genomic database available via Internet ---To be created in 1996

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AFRICAN TRYPANOSOME GENOME PROJECT WORKPLAN FOR 1996-97 Coordinator: Dr J.E. Donelson

OBJECTIVES PLANNED ACTIVITIES ---STATUS

1. Gene Discovery

a) EST analyses Create small scale cDNA plasmid preparations on at least 1500 cDNAs and conduct 5" single pass sequencing on them ---1000 EST sequenced

b) Differential gene expression Use the technique of Differential Display to identify differentially expressed RNAs in long slender & stumpy forms of the bloodstream stage

c) Network database Establish an interactive DNA database and communication network for both the EST data and genomic mapping data ---Created in 1995

2. Genomic DNA Contig Mapping

a) Library characterization Screen the P1 library with several hundred ESTs & known genes to map genes to DNA inserts

b) Chromosome characterization Map several hundred ESTs and known genes to T.b. brucei chromosomes resolved on PFG gels

c) Contig mapping Construct overlapping contig maps of P1 clones across several chromosomes

d) Database conversion Adapt the C. elegans AceDB database for use in storing and analysing trypanosome genomic DNA contig data

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THE LEISHMANIA GENOME PROJECT WORKPLAN FOR 1996-97 Coordinator: Dr J. Blackwell

The Committee agreed that a chromosome by chromosome approach on multiple species/isolates/clones of Leishmania was inefficient and not likely to lead to a complete physical map for the genome within an acceptable time period. All future WHO/TDR effort should be put into contiging a single clone of L. major using a global approach.

1. Complete PFGE karyotype mapping of physical linkage groups for LEM1317. Complete comparative PFGE maps for L. major LV39 and Friedlin strains to confirm that linkage groups are maintained across species, and hence reassure the network that one clone of Leishmania was suitable for mapping/sequencing programmes.

2. Continued sequencing ESTs from L. major developmental libraries. Goal 1000 new sequences. Objectives: to provide markers for mapping/contig construction; gene identification.

3. Cosmid contiging of L. major Friedlin, global strategy using overlapping EST content, end rescue and fingerprinting techniques.

4. Based on availability of cosmid contigs across ~80-90% of the L. major Friedlin genome, an international consortium has now been established in the network, with the aim of completing genomic sequencing for L. major Friedlin within 5 years.

5. Routine entry of all sequencing data onto the public databases (EMBL and GenBank), and entry of all genome information (including sequence data) onto LeishDB. Dissemination via disc or internet of LeishDB to all members of the network, and other interested laboratories.

(par_geno.txt)vbb.2apr96 email version of par_geno.wpd/11mar96 .