The general aim of this project is to develop methods for identifying transcribed sequences for large chromosomal regions and to apply this methodology to produce integrated transcriptional, genetic and physical maps of human chromosome 21 and mouse chromosome 16. The significance of this work lies in understanding the structural and functional organization of mammalian chromosomes, in characterizing the differences in organization of syntonic regions of mouse and human genomes, in determining the nature of chromosome breakpoints in evolution, and in identifying genes of significance in Down syndrome. A method for screening large, arrayed genomic libraries with cDNA probes to detect transcribed sequences was developed. The cDNA probes were qualitatively depleted of highly repeated sequences by hybridization with genomic DNA immobilized on finely divided cellulose. The method detects genomic clones of genes expressed at greater than or equal to 0.01% of total mRNA. A large, arrayed library of genomic fragments specific for mouse chromosome 16 was constructed and screened with cDNA probes from adult liver, adult cortex, and developing cerebellum. 319 expressed sequences were detected. Two methods were developed for increasing the sensitivity of the cDNA screenings: size fractionation of the template mRNA prior to reverse transcription and pre-annealing of cDNA with cognate mRNA to enrich for low abundance sequences. Genomic libraries for human chromosome 21 are being prepared both from yeast artificial chromosome (YAC) clones from band 21q22 and from a somatic cell hybrid containing only human chromosome 21. These will be screened with cDNA probes for various human tissues and the expressed sequences placed on physical and genetic maps.
