Two independent approaches to the study of muscle genes have converged to demonstrate that the
sup-10 gene encodes a regulatory myosin light chain (MLC) protein. Sup-10, described by I. Greenwald and R. Horvitz (1986, Genetics 113: 63-72), is a member of a group of interacting genes involved in muscle structure and function. These genes include
unc-93,
sup-10, 500) and
sup-10(
n983) are rare alleles that confer a characteristic uncoordinated ('rubber band') muscle defective phenotype. Null alleles of
unc-93,
sup-10 and
sup-18 are phenotypically wild-type and act as recessive suppressors of
unc-93(
e1500) and
sup-10(
n983). The wild-type null phenotype for four of the five genes and the patterns of cross-suppression suggest that these genes are members of a multi- gene family. We have been using transposon tagging to clone four of these genes. Alleles of
sup-10,
sup-9 and
sup-18 were isolated from strains with
unc-93(
e1500) or
sup-10(
n983) in a
mut-2(
r459) background (see Levin and Horvitz, 1987 C. elegans meeting abstracts). We have identified 27 suppressor mutations: eight alleles of
sup-9, 13 alleles of
unc-93, one
sup-18 allele and five alleles of
sup-10. We have shown that one of the
unc-93 alleles (
e1500n1415) has a very closely linked Tc1 insertion. Of recombinants in the interval between
daf-2 and
dpy-17 (7mu), the extra Tc1 is present in all nine
unc-93 (0) recombinants and absent in all 17
unc-93 (+) recombinants (p<0.7mu to the left and p<0.8mu to the right within 95% confidence limits; note that this gene is on an arm). We have cloned a 7 Kb EcoRI restriction fragment containing this Tc1 into a phage vector. We have examined one
sup-9 allele and found no linked Tc1, Tc3 or Tc4. Other backcrossed
sup-9 alleles are being examined. The C. elegans 'regulatory' MLC gene family consists of two gene copies
mlc-1 and
mlc-2, that are separated by 2.7 Kb. (see Cummins and Anderson, 1987 C. elegans meeting abstracts). Cosmid clones that cover
mlc-1 and
mlc-2 were identified by J. Sulston and A. Coulson. In situ hybridization of these clones to metaphase chromosomes indicated that these genes are located near the right end of the X chromosome. To define more precisely the position of the
mlc-1 and
mlc-2 genes, a
mlc-1/mlc-2 plasmid clone was used to probe Southern blots of total genomic DNA from strains heterozygous for deficiencies in this region. Hybridizations were quantitated and the results indicated that
mlc-1 and
mlc-2 are located within the deletion interval defined by mnDf8 and mnDf42.
sup-10 and nine other genes have been mapped within this interval. These results suggested the possibility that
sup-20 encodes a myosin light chain protein. To test whether
sup-10 encodes a myosin light chain protein, we have screened for MLC gene alterations in
sup-10 mutants. A
mlc-1/mlc-2 clone was used to probe Southern blots of total genomic DNA from 13
sup-10 alleles. Two of five
sup-10 alleles isolated in a
mut-2 background and three of eight gamma ray-induced alleles (seven isolated by I. Greenwald) exhibit altered Southern blot patterns (two insertions, one deletion, one apparent tandem duplication and one complex rearrangement). Each of the rearrangements affects the
mlc-1 gene but not the
mlc-2 gene. The C. elegans 'essential' MLC gene family consists of at least two members, only one of which has been cloned. The cloned gene,
mlc-3, was mapped by in situ hybridization to linkage group III, 34-41% from one end. Both
unc-93 and
sup-18 are located in this region. We have examined 11
unc-93 alleles (nine isolated in a
mut-2 background, including
unc-93(
e1500n1415) and two gamma ray-induced alleles). All of these have a wild-type hybridization pattern when probed with a cosmid that includes the
mlc-3 gene. Thus, we think it unlikely that
unc-93 encodes the
mlc-3 gene product, but we will examine additional
unc-93 alleles. We are currently investigating whether
sup-18 is mlc- 3 and whether
unc-93, nd to other members of the C. elegans 'essential' MLC gene family.