SAMPLING THE GENOMIC POOL OF PROTEIN-TYROSINE KINASE GENES USING THE POLYMERASE-CHAIN-REACTION WITH GENOMIC DNA

The polymerase chain reaction (PCR), with cDNA as template, has been w idely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expressio n. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as P CR template to identify members of protein families in an expression-i ndependent manner. This test involved amplification of DNA encoding pr otein tyrosine kinase (PTR) genes from the genomes of three animal spe cies that are well known developmental models; namely, the mouse Mus m usculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kina ses were 13 members of the PTR family that had not been reported previ ously. Selected PTKs from this screen were shown to be expressed durin g development, demonstrating that the amplified fragments did not aris e from pseudogenes. This approach will be useful for the identificatio n of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest , this method offers an opportunity to sample the genome for new membe rs irrespective of their expression pattern. (C) 1998 Academic Press.