The primary objective is the establishment of a consortium of four groups to provide coordinated cDNA-based resources and services to EC workers. These resources will facilitate the development of physical maps and will provide ready access to candidate genes in a way that is much more effective and efficient than any so far available. The achievements of the project will be directly measurable in terms of the numbers of cDNAs accumulated and the demand from users for the resources.
It is also the objective to facilitate the exploitation of cDNA resources, developed under the contract for the Centralized Facility, in ways consistent with the scientific interests of the group in question and likely to further those interests and the general objectives of the Human Genome Analysis Programme such as the pervasion of new technologies, the improvement of the physical map, and the understanding of the genetic basis of biological functions. Access to the cDNA resources will not be restricted, however a small number of European groups will receive direct support through subcontracts.
A chromosomal deoxyribonucleic acid (cDNA) Resource has been created from which users can gain rapid access to any cDNA of interest. Using technology for normalizing cDNA libraries 21 000 normalized cDNA clones were added to the collection from average 15 week menstrual age brain tissue, liver tissue, kidney tissue and adrenal tissue. A relational database has been constructed which acts as an electronic reference library for cDNAs. A further 1200 sequences have been found by producing expressed sequence tags. Also, high density cDNA grids and normalized cDNA libraries are being developed.
With respect to circular ribonucleic acid (RNA) molecules containing human sequences a method has been developed for isolating rare sequences from the pool of human sequences expressed in brain or, indeed, any other tissue.
Also, more than 1000 cDNA clones from cDNA libraries from the left ventricle of human heart and from atrium were analysed by partial DNA sequence analysis. Similarity searches against the nucleic acid and protein databases resulted in the identification of more than 500 new and hitherto unknown partial messenger ribonucleic acid (mRNA) sequences transcribed in the human heart.
Sequence analysis of a cDNA library representing the expressed genes of the cell line W138 (derived from embryonic lung tissue) showed little similarity to sequences of the EMBL database. The fibroblast cell line is not regarded to be highly specialized but contained a high number of new genes.
Also, a cDNA library from gastric mucosa tissue was constructed and sequenced. 24 clones represented pepsinogen mRNA, and another 38 clones represented an insertion not identified so far. This is consistent with the distinct bands in a denaturing gel electrophoresis of the gastric mucosa mRNA.
The Genexpress programme team has implemented efficient, low cost, protocols to obtain finished sequences from cDNA inserts using a combination of primer walking and nested deletion methods, to build a fine resolution gene map by integration into the cytogenetic, genetic and physical maps of the human genome using polymerase chain reaction (PCR) based and hybridization methods.
Funding SchemeCSC - Cost-sharing contracts