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.

Isolation of Tyrosine Kinase Sequences by a Rapid Screening Procedure

Alexander Kamb, Harold Varmus and Cynthia Kenyon

Figure 1

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]

Figure 1