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.
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