Worm Breeder's Gazette 9(1): 20
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.
We have previously described the isolation of C. elegans genomic clones that hybridize to cDNA sequences from two different Drosophila myosin light chain (MLC) genes (1985 CSH meeting abstract). The Drosophila genes used as probes (kindly supplied by S. Falkenthal & N. Davidson) represent the single 'regulatory' and 'essential' MLC genes in Drosophila. We designate our regulatory and essential clones as Class I and Class II, respectively. Five separate chromosomal sites are represented in our current collection of overlapping clones, including 3 Class I sites and 2 Class II sites. Class I and Class II clones do not cross-hybridize even under conditions of reduced stringency. J. Sulston and A. Coulson have provided us with overlapping cosmid clones for 3 of the 5 chromosomal sites. These will be used in localizing the MLC genes on the genetic map. The DNA sequences of 2 Class I genes (designated MLC-1 and MLC-2) confirm that they encode MLC proteins. The two genes are clustered at one chromosomal site and are divergently transcribed. Evidence that MLC-1 and MLC-2 are, in fact, expressed and not pseudogenes is discussed below. The complete sequence of the coding region of MLC-2 has been determined, together with several hundred base pairs of flanking sequence. A protein sequence of 170 amino acids has been inferred from the DNA sequence. A region corresponding to approximately 20 C-terminal amino acids of MLC-1 remains to be sequenced. There is a single Ile/Val substitution when MLC-1 is compared to MLC-2, and the MLC-1 sequence contains 5 extra amino acids at its amino terminal end. Each gene contains 3 small introns ranging in size from 47 to 54 bp. The entire MLC-2 protein sequence has been compared to those of the Drosophila and chicken skeletal L2 regulatory MLC proteins. There is 39% identity between the nematode and chicken MLCs and 34% identity between the nematode and Drosophila MLCs (as compared to 28% identity between the Drosophila and chicken proteins). Certain regions of the regulatory MLC-1 and MLC-2 amino acid sequences also show significant homology to essential MLCs from other organisms (eg. Drosophila, chicken, rabbit) as well as to a variety of other calcium binding proteins, including calmodulin, troponin C, and parvalbumin. The regulatory MLCs are no more closely related to essential MLCs than they are to other proteins in the super-family of calcium binding proteins. Northern blots indicate that a Class I probe, including a portion of both MLC-1 and MLC-2, hybridizes to two relatively abundant mRNAs. These mRNAs are small (~0.7-1.2 kb) as expected for MLC mRNAs. We believe that these mRNAs are transcripts of the MLC-1 and MLC-2 genes, because on Southern blots MLC-1 and MLC-2 do not cross-hybridize to any genomic DNA sequences except each other. A Class II probe also hybridizes to two relatively abundant small mRNAs distinct from those detected with the Class I probe. A Class I probe derived from a second chromosomal site detects 3 RNAs on a Northern blot (of approx. 2.2, 1.9, and 1.1 kb). Preliminary results suggest that one of the larger RNAS may encode a troponin T-like protein as judged by homology of sequenced fragments to troponin T from rabbit skeletal muscle.