Worm Breeder's Gazette 14(1): 87 (October 1, 1995)

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.

How many PTKs to organise a worm?

Andrew Oates, Andrew Wilks

Growth Regulation Laboratory Ludwig Institute for Cancer Research PO Royal Melbourne Hospital, 3050 Australia

        Protein tyrosine kinases (PTKs) play important roles in
coordinating metazoan cell behavior, although only one such example,
let-23, is understood clearly in the nematode worm. Therefore we have
undertaken a  screen for PTK-related sequences from the C. elegans
genome using PCR and PTK specific primers. In order not to bias our
screen towards a specific developmental stage, genomic DNA rather than
cDNA was used as the template for amplification.   Degenerate
oligonucleotide primers complimentary to evolutionarily conserved
regions of the PTK catalytic domain were designed after Wilks (PNAS USA
86:1603-7, 1989),  with the modification that inosine was introduced at
locations where all four bases were possible (fig.1).

fig.1 Degenerate oligonucleotide PCR primers. (i=inosine)

HRDLAARN  (Hanks motif VIb)
5' CCGAATTCCA(C/T)(C/A)GIGA(C/T)(C/T)TIGCIGCI(C/A)GIAA 3'

DVWS(F/Y)GV  (partial Hanks motif IX)
5' CCGAATTCIACICC(A/G)(A/T)AI(G/C)(A/T)CCAIAC(G/A)TC 3'




        A complex library of kinase-related sequences has been
constructed by subcloning two distinct sizes of product of approximately
220bp and 300bp into pBluescript. Sequencing of 42 randomly chosen
clones from the library has revealed 14 distinct PTK genes, of which 10
have not been previously described. CeHD-9,-13,-17, and-15, were
sequenced as part of the genome project and can be found on contigs
b0523, M142, c01G6, and M79 respectively. Predicted introns occur in 8
of the 14 genes, varying in length fom 50 to 80 bp, but only
interrupting the coding sequence at four discrete locations. Shared
intron position was highly predictive of sequence similarity in the case
of CeHD-5 and -7 and in the case of CeHD-9,-13, and -22. The library is
currently being exhaustively screened, and the genes are being mapped to
the Cambridge YAC polytene grids (many thanks to Alan Coulson). These
data will be presented at a later date.


Predicted translation of kinase gene fragments.

clone
number
9    CLYGDG-K--VKISDFGLTR----RGTIYQLH---PETKSPIRWLAVETIRTMIRLLS-KT
22   CLYTDG-K--VKISDFGLTR----NGTVYEIK---PNTKSPIRWLAIETIKTMICS-E-NT
13   CLYGDG-K--VKIXDFGLTR----DGTIYQIK---PNTKAPIRWLAVEADEIHVYS-Q-KS
3    CLITKELN--VKISDFGLSV----NESETKMK---SLKKAPIRWLSPETFSKGLFN-E-KT
8    CLIS--FDGIVKIADFGLSKTLEKDQKAFKEALKEA----PPAWLAPECIQRE-SEFSTKT
17   CLVG-DTRT-IKIADFGLMRT-SYGSDYYKML---HRSWMPVRWMSKEAIEQGRFS-E-AA
20   ILLARDERT-VKICDFGLMRALKENEQMYTMA---PQKKVPFTWCPPEALRHRKFSSH-AS
28   ILVCSP--QCVKLADFGLSRALD-----YDAVYTARSGKLPIKWLAPE-VNYR-Q-FSMAS
41   ILVFSK--DKVKISDFGLSRALGVGKDYYKTNFNV-NLKLPIAWCAPECINYL-R-FTNAS
4    ILINNSLSV--KIADFGLARIL-MKENEYEA--RTGGARFPIKWTGPEAANYN-R-FTTKS
39   VLVGDKISGVPKVADFGLARKL-MEEDIYEA--RTG-AKFPIKWTAPEAATCG-N-FTVKS
15   CLVSEH--NIVKIADFGLAR-F-MKEDTYTA--HAG-AKFPIKWTAPEA--FN-T-FSSKS
5    VFVKR--NKMIRIGDFGLARHHS-KKSYYRMQCNPDTP-LPIFWLAPECFNES--KF-TES
7    VLIKR--NGVIRIADFGLARRHE-NKDYYRTR-SVGTA-IPLNWLAPECFEGSINKFDSKS