Research Projects in Genomics

The long-term goal of this proposal is to isolate, characterize, and define the functional roles of all genes essential, important, and ancillary to the water and ion stress response and tolerance phenotype of plants. Over the last decade, it has become clear that responses to water deficit and ion imbalance are governed by complex molecular and biochemical signal transduction processes, which coordinately act to determine tolerance or sensitivity at the whole-plant level. Within the last five years, however, advances in genomics, informatics, and functional genomics have made it technically feasible to gain a complete understanding of how many genes become integrated to effect abiotic stress tolerance. To tackle the genetic basis of abiotic stress tolerance in higher plants in the most efficient, comprehensive, integrative way possible, we have formed a consortium between Purdue University (R. Bressan, P. Hasegawa and J. Pardo), Oklahoma State University (R. Burnap, J. Cushman and R. Prade), and the University of Arizona (H. Bohnert, D. Galbraith and J-K. Zhu).Ý Each participant has documented, extensive experience in this research area with proven records of productivity and in many instances past or present collaborations.

We will employ four distinct, yet complementary approaches to isolate, characterize, and assess the function of the core-set of stress-related genes involved with the water and ion stress response and tolerance phenotype in plants. The first approach will encompass the identification of plant genes whose expression is altered in response to stress using EST sequencing and microarray analysis. We select Arabidopsis, Mesembryanthemum, and rice as small-genome models that represent the primary plant genetic models for glycophytes and halophytes, and a crop species. The second approach will entail the discovery of plant genes by complementation of stress-associated mutants in other model eukaryotes. This approach, which relies on fundamental homologies in cellular responses to stress exploits organisms for which the genome has been completely sequenced (yeast, Synechocystis), and for which powerful recombination-expression systems have recently been developed (Aspergillus). The third approach will involve the comparative study of gene expression in halophytes and glycophytes. Recent studies of halophytes have revealed the existence of mechanisms of stress tolerance that are not present or not appropriately expressed in glycophytes. Dunaliella salina and Mesembryanthemum crystallinum.will be analyzed by EST/Microarray analysis. The fourth objective will comprise a functional analysis of stress-related transcripts by monitoring in situ localization using promoter trapping approaches.

These approaches represent a logical extension of ongoing work in our individual groups. We will foster interaction and integration of Consortium activities through daily interactions, workshops/ meetings and extended work periods in member laboratories for our students and postdoctoral fellows to ensure a new generation of researchers trained in multi-faceted and interdisciplinary problem solving. The impact of abiotic stress on crop productivity is remarkable according to USDA statistics and amounts to two-thirds of all yield reductions in agriculture. This proposal is exceptionally timely, combines unique expertise, is hypothesis-driven and culminates in a clearly defined goal - understanding number, nature and networking of genes and physiological mechanisms that constitute plant abiotic stress tolerance.

Large-scale genomics has become a basic research and technology tool necessary for the competitive development of the biological sciences in general, biotechnology, and industrial microbiology. This seed-proposal is the result of an effort by the world wide Aspergillus community, and for the first time brings together academic and industrial collaborators in a consortium that will make publicly available all the generated DNA information. It is important for the success of the project, to be viewed as a collaborative and fair arrangement between the industries providing the financial support and the university researcher doing the sequencing. Therefore, the Result of this Project is the Sequence and will NOT constitute new discovery. Any NEW discovery that utilizes the Sequence (as a tool) is not part of the project.

What is required to obtain the complete chromosome IV DNA sequence. According to the Lander and Waterman [1, 2] analysis based on random selection of clones, the 2.9 megabase A. nidulans chromosome IV will require sequencing of 60,000, 1kb insert, clones and is predicted to result in a DNA sequence contained in 5 contigs (the gaps are mathematical). In a real experiment, the random sequencing approach of a 38.8 kb DNA segment of A. nidulans chromosome VIII required 854 sequencing reactions [3], indicating that the sequencing of 2.9 Mb will require approximately 62,000 reactions. If the random approach is followed throughout the project, 30 Mb of DNA information will be generated, that theoretically covers chromosome IV 10 times at a price of $4.1 million ($1.42 per base).

What can be done in a seed project. We plan to run 30,000 sequencing reactions. This represents approximately 15 Mb of DNA information, a 5X sequence redundancy and half of the reactions needed to obtain the complete DNA sequence using the industry standard random approach. The predicted cost for the final sequence is $510 thousand (35 cents per base - done at TAMU/OSU). To reduce the unnecessary redundancy of the currently utilized and wide-spread random approach we will test a non random approach to select clones for sequencing. A non random selection strategy, such as tiling [4], reduces significantly the number of clones without compromising the quality of the final DNA sequence. Thus, it is possible, but not guaranteed, that with 30,000 sequencing reactions we determine the complete DNA sequence of chromosome IV.

The goal of this proposal is to seed (prime) a broader and worldwide initiative that aims the 31 Mb genomic DNA sequence of the ascomycete Aspergillus nidulans. with the DNA sequence of chromosome IV. The specific aims for this project are:

a- determine the genomic DNA sequence of chromosome IV,
b- link to the Internet, with unrestricted access to the community, of a user friendly interfaced and fully integrated Aspergillus genome database,
c- reach the end of the project with at least one established Aspergillus genome sequencing center and one central automated data processing and
presentation resource.

Large-scale sequencing of chromosome IV. Production of 30,000 sequencing reactions is predicted and will require 468, 64 well, gels. Assuming 5 gels a week the sequencing component can be completed in 93 weeks. The predicted weekly minimal module is: 320 reactions, 160 templates.

Anchoring the chromosome IV DNA sequence to the physical and genetic map. Full integration of the genetic and physical map with the chromosome IV DNA sequence will be obtained by sequencing the ends of the cosmids that make up the minimal tiling (146 templates, 292 reactions).

DNA libraries and clone management. A One or more, 1.5 kb insert, sequencing libraries will be constructed for chromosome IV. The primary library will be partitioned into smaller overlapping sub-libraries based on information extracted from DNA/DNA hybridization experiments. Large-scale sequencing of individual sub-libraries will begin as soon as they become available, and we will gradually switch to a non random sequencing approach as we eliminate redundancy and successfully test a non random sampling strategy. The partitioning of the primary library into overlapping sub-libraries has several advantages: i) it insures that sequencing data are collected throughout the entire chromosome, ii) it allows extensive elimination of redundancy of individual sub-libraries, but retains sufficient redundancy to assure accurate base-pair readings, iii) it allows large-scale sequencing and redundancy elimination to be performed in parallel, and iv) it will reduce the cost of large-scale sequencing projects by several fold.

Genome database The assembled DNA sequence will be made publicly available immediately after annotation through submission to GenBank. For the Aspergillus database, the same information will be mirrored in a high-speed client dedicated server, and a fully featured database will be available over the Internet. The main goal is the construction, maintenance and regular updates of a Web site that would contain the molecular and genetic information based on what is publicly available. Web maintenance and update would be automated and include, basic HTML based interface, CGI-scripts that implement published algorithms for pattern identification, java based web pages and, a dedicated web-crawler. For the fungal community, access to an automated Web site will be a major advantage, because search and pattern recognition databases will allow user induced updates and identify the new information that is deposited into publicly available databases.

References. 1. Arratia, R., et al., Genomic mapping by anchoring random clones: A mathematical analysis. Genomics, 1991. 11: 806-827. 2. Lander, E.S. and M.S. Waterman, Genomic mapping by fingerprinting random clones: A mathematical analysis. Genomics, 1988. 2: 231-239. 3. Kupfer, D.M., et al., Multicellular ascomycetous fungal genomes contain more than 8000 genes. FGB 21: 364-372. 4. Prade, R.A., et al., In vitro reconstruction of the Aspergillus nidulans genome. submitted, 1996: .