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 widely 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 expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, 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 kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification 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 members irrespective of their expression pattern.
Oates
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AC
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Victoria, Australia.
Wollberg
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P
Achen
M G
MG
Wilks
A F
AF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D019143:Evolution, Molecular; D018507:Gene Expression Regulation, Developmental; D017238:Genes, Helminth; D017344:Genes, Insect; D016678:Genome; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011544:Pseudogenes; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D004247:DNA; D011505:Protein-Tyrosine Kinases