Functional Analysis of Cytoplasmic Dynein Heavy Chain in Caenorhabditis elegans with Fast-acting Temperature-sensitive Mutations

Cytoplasmic dynein, a minus-end–directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of a Caenorhabditis elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of Saccharomyces cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems.

The Dominant Temperature-Sensitive Lethal DTS7 of Drosophila melanogaster Encodes an Altered 20S Proteasome β-Type Subunit

Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation. The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 α-type and 7 β-type. DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29° that also acts as a recessive lethal at ambient temperatures. DTS7 maps to cytological position 71AB. Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a β-type subunit gene, β2. A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another β-type subunit gene, β6. These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1. We propose that these mutants might act as “poison subunits,” disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.

A Limited Number of Caenorhabditis elegans Genes Are Readily Mutable to Dominant, Temperature-Sensitive Maternal-Effect Embryonic Lethality

Dominant gain-of-function mutations can give unique insights into the study of gene function. In addition, gain-of-function mutations, unlike loss-of-function alleles, are not biased against the identification of genetically redundant loci. To identify novel genetic functions active during Caenorhabditis elegans embryogenesis, we have collected a set of dominant temperature-sensitive maternal-effect embryonic lethal mutations. In a previous screen, we isolated eight such mutations, distributed among six genes. In the present study, we describe eight new dominant mutations that identify only three additional genes, yielding a total of 16 dominant mutations found in nine genes. Therefore, it appears that a limited number of C. elegans genes mutate to this phenotype at appreciable frequencies. Five of the genes that we identified by dominant mutations have loss-of-function alleles. Two of these genes may lack loss-of-function phenotypes, indicating that they are nonessential and so may represent redundant loci. Loss-of-function mutations of three other genes are associated with recessive lethality, indicating nonredundancy.

Cytogenetic analysis of chromosome region 73AD of Drosophila melanogaster.

The 73AD salivary chromosome region of Drosophila melanogaster was subjected to mutational analysis in order to (1) generate a collection of chromosome breakpoints that would allow a correlation between the genetic, cytological and molecular maps of the region and (2) define the number and gross organization of complementation groups within this interval. Eighteen complementation groups were defined and mapped to the 73A2-73B7 region, which is comprised of 17 polytene bands. These complementation groups include the previously known scarlet (st), transformer (tra) and Dominant temperature-sensitive lethal-5 (DTS-5) genes, as well as 13 new recessive lethal complementation groups and one male and female sterile locus. One of the newly identified lethal complementation groups corresponds to the molecularly identified abl locus, and another gene is defined by mutant alleles that exhibit an interaction with the abl mutants. We also recovered several mutations in the 73C1-D1.2 interval, representing two lethal complementation groups, one new visible mutant, plucked (plk), and a previously known visible, dark body (db). There is no evidence of a complex of sex determination genes in the region near tra.

TEMPERATURE-SENSITIVE MUTATIONS IN DROSOPHILA MELANOGASTER XII. THE GENETIC AND DEVELOPMENTAL EFFECTS OF DOMINANT LETHALS ON CHROMOSOME 3

ABSTRACT Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29°C but survive at 22°C were recovered. Seven of the eight mutations mapped were tested for complementation; these mutants probably define eight loci. Only DTS-2 survived in homozygous condition at 22°C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic phenotype with respect to the time of death at 29°C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.