Worm Breeder's Gazette 14(1): 38 (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.
Department of Neurology, Baylor College of Medicine, Houston, TX.
We are interested in determining the possible use of C. elegans as an animal model to study the protein(s) indicated in the human disease Myotonic Dystrophy, (DM). We therefore performed a computer search of the dbEST database to locate cDNAs that were similar in sequence to the DM protein kinase gene, (DMPK). We found four different sequences with protein sequence similarities ranging from 50 to 95% over the short regions that had been sequenced. One cDNA (Accession # T01329) in the databases was originally found by Anthony Kerlavage at the Institute for Genomic Research in Gaithersburg, MD. The clone, CEESP52, came from a C. elegans embryonic cDNA library. One open-reading frame of the clone contained a protein sequence that matches at a score of 71% identity and 95% similarity to part of the catalytic domain of the human DM kinase gene. We have hybridized a probe made from CEESP52 to the Yac contig library and determined that the gene containing the coding sequences is found on Yac Y38B5. Yac Y38B5 is covered by known cosmids except for an approximately 20kb gap in the cosmid contigs. A Southern indicated that the region sequenced did not hybridize to any of the cosmids, therefore we believe that at least part, if not all, of the gene is located in the gap region. We have run pulse field gels and isolated Yac Y38B5 DNA and will be screening subclones of the Yac for the corresponding gene. We will then perform rescue experiments of known mutations or construct our own mutant. As another approach to locating DMPK homologs we have used is employing a monoclonal and polyclonal antibodies made to the human DMPK protein on Western blots containing C. elegans homogenates. The monoclonal antibody identified an approximately 80kDa band on the Western, whereas the polyclonal detected 5 different bands. We are in the process of isolating these genes from a lgt11 embryonic library. Below is a translated protein sequence match of the 4 closest homologs to DMPK found in the dbEST database over a overlapping 100 amino acid region. Two other cDNA sequences in the dbEST database have good similarity scores to DMPK equivalent to the C. elegans Ndr peptide score, but are not in the same region. Both have been mapped and are found on chromosome I (1,2). The partial sequence of the Ndr protein has been recently published and has homologs in both Drosophila and humans (3). Upper Sequence : Human DMPK (all matches to the Human DMPK) 2nd Sequence : Accession # T01329 peptide Match Percentage : 71% 3rd Sequence : C. elegans Ndr peptide Match Percentage : 45% 4th Sequence : Accession # D37290 peptide Match Percentage : 35% . : . : . : . : . : 1 DFEILKVIGRGAFSEVAVVKMKQTGQVYAMKIMNKWDMLKRGEVSCFREE |||:|||||:|||:|||||:|: |::|||||:|||:|:||:| :||||| 1 DFEVLKVIGKGAFGEVAVVRMRGVGEIYAMKILNKWEMVKRAETACFREE :| |||||||||:|| :|: :||::|||||: | :|: : : : | | 1 HFKSLKVIGRGAFGEVRLVQKHDTGHIYAMKILRKSEMVEKEQTAHVRAE :| :|:|:|:||: :| :|: | :||||:: | :| : : | 1 NFALLRVLGKGAYGKVFLVRKDHNTIYAMKVLRKTRVLTKQKXLEHTMAE . : . : . : . : . 51 RDVLVNGDRRWITQLHFAFQDENYLYLVMEYYVGGDLLTLLSKFGERIP ||||| |||||||:||:|||||: ||:||:||:|||:||||||| ::|| 51 RDVLVYGDRRWITNLHYAFQDEKNLYFVMDYYIGGDMLTLLSKFVDHIP ||:| ::| |: :: ::||| : ||||||: |||::||| | : 51 RDILSEADCDWVVKMYYSFQDYSNLYLVMEFLPGGDMMTLLIKKATLTE | || :: | :||| : |::|||| ||:| | | | 51 RQVLERLRTPFLVNLXYAFQTDXKLHIVMEYVRGGELFTHLCSRGH 1) Gilchrist, E. and Baillie, D., (1995) A C. elegans Myotonic Dystrophy gene homologue. Abstracts of the 10th International C. Elegans meeting. University of Wisconsin, Madison. P233. 2) Wissmann, A., McGhee, J., Mains, P., (1995) Let-502 is a homolog of human Myotonic Dystrophy kinase. Abstractions of the 10th International C. elegans meeting. University of Wisconsin, Madison. P548. 3) Millward, T., Cron, P., Hemmings, B., (1995) Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase. Proc. Natl .Acad. Sci. USA 92:5022-6.