Worm Breeder's Gazette 10(3): 60
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Studies on the genetic control of development have revealed several families of genes; for example, genes that encode transcription factors with motifs such as homeodomains, zinc fingers, and leucine zippers. Other gene families, for instance tyrosine kinases, may also play an important role in development. Tyrosine kinases comprise a large family in vertebrates and in Drosophila (1). The epidermal growth factor receptor and the src protein are tyrosine kinases that appear to participate in the regulation of cell proliferation. Recently, sevenless, a gene that determines a specific choice of cell fate in the Drosophila eye, has been shown to encode a tyrosine kinase. We have identified five tyrosine kinase sequences from C. elegans using an adaptation of the polymerase chain reaction (PCR). The method is simple and extremely rapid: short segments of conserved amino acid sequence within a protein family are used to design degenerate oligonucleotide primers. Two such regions are required for PCR. In the case of tyrosine kinases, we selected primers based on two sequences conserved among members of the src and abl subfamily of tyrosine kinases. These primers were twenty bases long with a degeneracy of 192 (tyr-kin-1) and 256 (tyr-kin-2). The primers were chosen to hybridize to opposite strands of genomic DNA such that the newly synthesized DNA strands overlapped. Repeated cycles of synthesis, denaturation, and primer annealing resulted in exponential amplification of the DNA between the primer ends. The amplified DNA fragments (86 bp in length) were cloned. Twenty individual clones from a single PCR experiment were analyzed and yielded five different sequences shown below. One sequence, tyr-kin (I), is identical to a region within a C. elegans gene previously isolated by Goddard et al. and believed to be an abl homolog (2). The other four sequences possess certain residues common to most tyrosine kinases (see figure). The generality of the method has been demonstrated by isolating a sequence from C. elegans similar to the Drosophila segment polarity gene, wingless, and two sequences from C. elegans that are similar to the mab-5 homeobox (see article in last WBG by Costa et al.). The amplified fragments for two of these three sequences were larger than expected and proved to have short introns. C. elegans is ideally suited to this approach because genes are generally simple and introns are generally small. Nevertheless, using this method we have also identified six new potassium channel sequences in humans. We currently are characterizing further the genes that correspond to these different C. elegans sequences. We intend to analyze the distribution of their transcripts by in situ hybridization (with M. Weir) and to generate and study null mutations in the different genes. [See Figure 1]