Point mutations in awdKpn which revert the prune/Killer of prune lethal interaction affect conserved residues that are involved in nucleoside diphosphate kinase substrate binding and catalysis.

The awd gene of Drosophila melanogaster encodes a nucleoside diphosphate kinase. Killer of prune (Kpn) is a mutation in the awd gene which substitutes Ser for Pro at position 97 and causes dominant lethality in individuals that do not have a functional prune gene. This lethality is not due to an inadequate amount of nucleoside diphosphate (NDP) kinase activity. In order to understand why the prune/Killer of prune combination is lethal, even in the presence of an adequate NDP kinase specific activity level, and to understand the biochemical basis for the conditional lethality of the awdKpn mutation, we generated second site mutations which revert this lethal interaction. All of the 12 revertants we recovered are second site mutations of the awdKpn gene. Three revertants have deletions of the awdKpn protein coding region. Two revertants have substitutions of the initiator methionine and do not accumulate KPN protein. Seven revertants have amino acid substitutions of conserved residues that are likely to affect the active site: five of these have no enzymatic activity and two have a very low level of specific activity. These data suggest that an altered NDP kinase activity is involved in the mechanism underlying the conditional lethality of the awdKpn mutation.

Multiple Allele Approach to the Study of Genes in DROSOPHILA MELANOGASTER That Are Involved in Imaginal Disc Development.

The phenotypes of five different lethal mutants of Drosophila melanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. To examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromosome containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no significant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were compared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles at these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect significant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold-sensitive mutants.

Genetic analysis of two allelic temperature-sensitive mutants of Drosophila melanogaster both of which are zygotic and maternal-effect lethals.

After fertilization, the development of a zygote depends upon both gene products synthesized by its maternal parent and gene products synthesized by the zygote itself. To analyze genetically the relative contributions of these two sources of gene products, several laboratories have been isolating two classes of mutants of Drosophila melanogaster: maternal-effect lethals and zygotic lethals. This report concerns the analysis of two temperature-sensitive mutants, OX736hs and PC025hs, which were isolated as alleles of a small-disc mutant, l(3)1902. These alleles are not only zygotic lethals, but also maternal-effect lethals. They have temperature-sensitive periods during larval life and during oogenesis. Mutant larvae exposed continuously to restrictive temperature have small discs. One-or two-day exposures to the restrictive temperature administered during the third larval instar lead to a homeotic transformation of the midlegs and hindlegs to the pattern characteristic of the forelegs. Mutant females exposed to the restrictive temperature during oogenesis produce eggs that can develop until gastrulation, but do not hatch.--The existence of these mutants, and one that was recently described by another group, implies that there may be a class of genes, heretofore unrecognized, whose products are synthesized during oogenesis, are essential for embryogenesis and are also synthesized during larval stages within imaginal disc cells.