A Member of the Phylogenetically Conserved CAD Family of Transcriptional Regulators Is Dramatically Up-Regulated during the Programmed Cell Death of Skeletal Muscle in the Tobacco HawkmothManduca sexta

Abstract

The intersegmental muscles (ISMs) of the tobacco hawkmothManduca sextaparticipate in the emergence behavior of the adult moth at the end of metamorphosis and then die during the subsequent 30-hr period. The trigger for this death is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone (20-HE). Previous work has demonstrated that the ability of the ISMs to die is dependent on new gene expression. Using a differential hybridization cloning strategy, a cDNA library made from the ISMs committed to die was screened, and four up-regulated clones were isolated. One clone, 18-56, was selected for this study. Northern and Western analysis demonstrated that while clone 18-56 was expressed in all tissues examined and during every stage of ISM development, there was a dramatic increase in expression at both mRNA and protein levels when the ISMs became committed to die. If ISM death was delayed by an injection of 20-HE on the day proceeding adult emergence, 18-56 expression remained at basal levels. Immunocytochemistry demonstrated that 18-56 protein was located predominantly in nuclei prior to the commitment of the ISMs to die and then accumulated to high levels in cytoplasm at the time of cell death. DNA sequence analysis revealed that 18-56 protein shares 74% identity with yeast SUG1 and 92% with human Trip1, both of which are members of the conserved CAD (Conserved ATPase-containing Domain) family of putative transcriptional regulators. To verify that these genes shared functional as well as sequence homology,Manducaclone 18-56 was transformed into a yeast mutant for SUG1 function.Manduca18-56 was able to both complement the lethal SUG1 phenotype and to suppress the transcriptional activity of a SUG1 mutation in yeast. Taken together, these data support the hypothesis that members of the phylogenetically conserved CAD family participate in important basal and developmental processes.