Conditional dominant mutations in the Caenorhabditis elegans gene act-2 identify cytoplasmic and muscle roles for a redundant actin isoform

Animal genomes each encode multiple highly conserved actin isoforms that polymerize to form the microfilament cytoskeleton. Previous studies of vertebrates and invertebrates have shown that many actin isoforms are restricted to either nonmuscle (cytoplasmic) functions, or to myofibril force generation in muscle cells. We have identified two temperature-sensitive and semidominant embryonic-lethal Caenorhabditis elegans mutants, each with a single mis-sense mutation in act-2, one of five C. elegans genes that encode actin isoforms. These mutations alter conserved and adjacent amino acids predicted to form part of the ATP binding pocket of actin. At the restrictive temperature, both mutations resulted in aberrant distributions of cortical microfilaments associated with abnormal and striking membrane ingressions and protrusions. In contrast to the defects caused by these dominant mis-sense mutations, an act-2 deletion did not result in early embryonic cell division defects, suggesting that additional and redundant actin isoforms are involved. Accordingly, we found that two additional actin isoforms, act-1 and act-3, were required redundantly with act-2 for cytoplasmic function in early embryonic cells. The act-1 and -3 genes also have been implicated previously in muscle function. We found that an ACT-2::GFP reporter was expressed cytoplasmically in embryonic cells and also was incorporated into contractile filaments in adult muscle cells. Furthermore, one of the dominant act-2 mutations resulted in uncoordinated adult movement. We conclude that redundant C. elegans actin isoforms function in both muscle and nonmuscle contractile processes.     

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle.     

Roles for two partially redundant alpha-tubulins during mitosis in early Caenorhabditis elegans embryos

The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.     

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.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER Xii. the Genetic and Developmental Effects of Dominant Lethals on Chromosome 3

Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29 degrees C but survive at 22 degrees 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 degrees 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 degrees C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.     

Mutations in Polycombeotic, a Drosophila Polycomb-Group Gene, Cause a Wide Range of Maternal and Zygotic Phenotypes

The polycomb-group genes, a set of genes characterized by mutations that cause similar phenotypes and dosage-dependent interactions, are required for the normal expression of segment-specific homeotic loci. Here we report that polycombeotic (formerly 1(3)1902), originally identified by a lethal mutation that causes a small-disc phenotype, is also a member of this group of essential genes. Adults homozygous for temperature-sensitive pco alleles that were exposed to the restrictive temperature during larval life display the second and third leg to first leg transformation characteristic of polycomb-group mutants. Adult females homozygous for temperature-sensitive alleles exposed to the restrictive temperature during oogenesis produce embryos that show anterior segments with structures normally unique to the eighth abdominal segment, another transformation characteristic of polycomb-group mutants. Mutations in the polycombeotic gene also cause defects not reported for mutations in other polycomb-group genes. Females homozygous for the most extreme temperature-sensitive allele are sterile, and larvae homozygous for null alleles have small imaginal discs and reduced frequencies of mitotic figures in the brain. Dominant mutations originally identified as enhancers or suppressors of zeste are gain-of-function alleles of polycombeotic. The type and variety of defects displayed by different mutations in this gene indicate that the product might be involved in chromosome structure and/or function.     

Cycloheximide-Resistant Temperature-Sensitive Lethal Mutations of Saccharomyces Cerevisiae

We describe the isolation and preliminary characterization of a set of pleiotropic mutations resistant to the minimum inhibitory concentration of cycloheximide and screened for ts (temperature-sensitive) growth. These mutations fall into 22 complementation groups of cycloheximide resistant ts lethal mutations (crl). None of the crl mutations appears to be allelic with previously isolated mutations. Fifteen of the CRL loci have been mapped. At the nonpermissive temperature (37°), these mutants arrest late in the cell cycle after several cell divisions. Half of these mutants are also unable to grow at very low temperatures (5°). Although mutants from all of the 22 complementation groups exhibit similar temperature-sensitive phenotypes, an extragenic suppressor of the ts lethality of crl3 does not relieve the ts lethality of most other crl mutants. A second suppressor mutation allows crl10, crl12, and crl14 to grow at 37° but does not suppress the ts lethality of the remaining crl mutants. We also describe two new methods for the enrichment of auxotrophic mutations from a wild-type yeast strain.     

Dominant Maternal-Effect Mutations Causing Embryonic Lethality in Caenorhabditis Elegans

We undertook screens for dominant, temperature-sensitive, maternal-effect embryonic-lethal mutations of Caenorhabditis elegans as a way to identify certain classes of genes with early embryonic functions, in particular those that are members of multigene families and those that are required in two copies for normal development. The screens have identified eight mutations, representing six loci. Mutations at three of the loci result in only maternal effects on embryonic viability. Mutations at the remaining three loci cause additional nonmaternal (zygotic) effects, including recessive lethality or sterility and dominant male mating defects. Mutations at five of the loci cause visible pregastrulation defects. Three mutations appear to be allelic with a recessive mutation of let-354. Gene dosage experiments indicate that one mutation may be a loss-of-function allele at a haploin sufficient locus. The other mutations appear to result in gain-of-function "poison" gene products. Most of these become less deleterious as the relative dosage of the corresponding wild-type allele is increased; we show that relative self-progeny viabilities for the relevant hermaphrodite genotypes are generally M/+/+ greater than M/+ greater than M/M/+ greater than M/Df greater than M/M, where M represents the dominant mutant allele.     

Identification of genes that interact with glp-1, a gene required for inductive cell interactions in Caenorhabditis elegans

The glp-1 gene functions in two inductive cellular interactions and in development of the embryonic hypodermis of C. elegans. We have isolated six mutations as recessive suppressors of temperature-sensitive (ts) mutations of glp-1. By mapping and complementation tests, we found that these suppressors are mutations of known dumpy (dpy) genes; dpy genes are required for development of normal body shape. Based on this result, we asked whether mutations previously isolated in screens for mutants defective in body shape could also suppress glp-1(ts). From these tests, we learned that unselected mutations of eight genes required for normal C. elegans morphogenesis, including the four already identified, suppress glp-1(ts). All of these suppressors rescue all three mutant phenotypes of glp-1(ts) (defects in embryonic induction of pharyngeal tissue, in embryonic hypodermis development, and in induction of germline proliferation). However, they do not rescue putative glp-1 null mutants and therefore do not bypass the requirement for glp-1 in development. In the light of current ideas about the molecular nature of the glp-1 and suppressor gene products, we propose an interaction between the glp-1 protein and components of the extracellular matrix and speculate that this interaction may impose spatial constraints on the decision between mitosis and meiosis in the germline.     

Use of a psoralen-induced phenocopy to study genes controlling spermatogenesis in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, spermatogenesis represents one of two alternative developmental pathways open to premeiotic germ cells. At least two genes, fem-1 and fem-2, control the initiation of spermatogenesis in XX (hermaphrodite) worms, and the entire spectrum of male differentiation in XO animals. Low-dose irradiation of worms treated with the light-activated DNA crosslinking drug trimethylpsoralen, at levels that do not affect cell division or growth rates, blocks spermatogenesis in C. elegans hermaphrodites and produces an identical phenotype to that of temperature-sensitive mutations in the fem genes. Psoralen treatment does not, however, produce corresponding phenotypes of these mutants in XO animals. The developmental age for phenocopy production is the same as the hermaphrodite temperature-sensitive period of the two mutants. The effects of pulses of restrictive temperature and psoralen treatment on fem-2 mutant hermaphrodites are additive, suggesting that psoralen crosslinking may reduce the level of the fem-2 gene product. Microbeam experiments localize the target for the psoralen effect to the primary germ cells in the first stage larvae, indicating that a critical step occurs in a small number of precursor cells prior to their commitment to spermatogenesis.     

Comparative proteomic analysis reveals differentially expressed proteins in Caenorhabditis elegans pgl-1 mutants grown at 20°C and 25°C

PGL-1 is an RNA-binding protein component of germ granules and essential for fertility in Caenorhabditis elegans. To clarify the molecular function of PGL-1, we performed comparative proteomic analysis using 2-D DIGE and LC-MS/MS. Five groups of synchronized adult hermaphrodites were analyzed: (1) wild-type N2 grown at 20°C, (2) pgl-1(bn101) mutants grown at 20°C, (3) pgl-1(bn101) mutants grown at 20°C then upshifted to 25°C after the L1 stage, (4) pgl-1(ct131) mutants grown at 20°C, and (5) pgl-1(ct131) mutants grown at 20°C then upshifted to 25°C after the L1 stage. The five groups were divided into two experimental sets for 2-D DIGE: set A included N2 and pgl-1(bn101) mutants, and set B included N2 and pgl-1(ct131) mutants. Dunnett's test indicated 90 and 100 specific spots, respectively, with significantly different expression levels from the rest of the experimental set (q≤0.1). Among them, 69 and 58 spots, respectively, were analyzed by LC-MS/MS. Finally, we identified 19 proteins from 24 specific spots common to both the experimental sets. RNAi analysis indicated that decreased eef-1G expression is strongly associated with the temperature-sensitive sterile phenotype of pgl-1. Our results suggest that PGL-1 is closely involved in translational processes during C. elegans germline development.     

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae.

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.     

Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii

A series of conditional mutants of the algal, biflagellate Chlamydomonas reinhardtii with temperature-sensitive defects in flagellar assembly and function were isolated. The genetics and phenotypes of 21 mutants displaying a rapid alteration in flagellar function upon shift from the permissive (20 degrees C) to the restrictive (32 degrees C) temperatures are described. These mutants designated as "drop-down" or dd-mutants have been placed in four categories on the basis of their defective phenotypes: (a) dd-assembly mutants - the preformed flagella are resorbed at 32 degrees C and reassembly of flagella is inhibited; (b) dd-fragile flagella mutants - the flagella are lost by detachment at 32 degrees C, but can be reassembled; (c) dd-motility mutants - the flagella are retained at 32 degrees C, but are functionally defective; (d) dd-lethal mutants - display combined defects in flagellar function and cell growth. Tetrad analysis of the mutants back-crossed to wild-type, recombination analysis of intermutant crosses, and complementation tests in the construction of heterozygous diploid strains indicate that at least 14 nuclear genetic loci are represented among 21 mutants. The availability of temperature-sensitive mutations affecting the assembly and function of the flagellum suggests that the morphogenesis of this complex eukaryotic organelle is amenable to genetic dissection.     

Behavioral and Biochemical Defects in Temperature-Sensitive Acetylcholinesterase Mutants of DROSOPHILA MELANOGASTER

Temperature-sensitive (ts) mutants of the Ace gene, which codes for acetylcholinesterase (AChE) in Drosophila melanogaster, were analyzed for defects in viability, behavior and function of the enzyme. The use of heat-sensitive and cold-sensitive mutations permited the function of AChE in the nervous system to be analyzed temporally. All ts mutations were lethal, or nearly so, when animals expressing them were subjected to restrictive temperatures during late embryonic and very early larval stages. Heat treatments to Ace-ts mid- and late larvae had little effect on the behavior of these animals or on the viability or behavior of the eventual adults. Heat-sensitive mutants exposed to nonpermissive temperatures as pupae, by contrast, had severe defects in phototaxis and locomotor activity as adults. AChE extracted from adult ts mutants that had developed at a permissive temperature were abnormally heat labile, and they had reduced substrate affinity when assayed at restrictive temperatures. However, enzyme activity did not decline during exposure of heat-sensitive adults to high temperatures even though such treatments caused decrements in phototaxis (29°) and, eventually, cessation of movement (31°). The cold-sensitive mutant also produced readily detectable levels of AChE when exposed to a restrictive temperature during the early developmental stage when this mutation causes almost complete lethality. We suggest that the relationship among the genetic, biochemical and neurobiological defects in these mutants may involve more than merely temperature-sensitive catalytic functions.     

Isolation of Temperature-Sensitive Mutants of Arabidopsis thaliana That Are Defective in the Redifferentiation of Shoots

Three temperature-sensitive mutants of Arabidopsis thaliana that were defective in the redifferentiation of shoots were isolated as tools for the study of organogenesis. M3 lines were constructed by harvesting M3 seeds separately from each M2 plant. Comparative examination of shoot redifferentiation in root explants of 2700 M3 lines at 22[deg]C (permissive temperature) and at 27[deg]C (restrictive temperature) led to the identification of seven temperature-sensitive mutant lines. Genetic tests of three of the seven mutant lines indicated that temperature-sensitive redifferentiation of shoots in these three lines resulted from single, nuclear, recessive mutations in three different genes, designated SRD1, SRD2, and SRD3. The morphology of root explants of srd mutants cultured at the restrictive temperature suggests that the products of these SRD genes function at different stages of the redifferentiation of shoots.     

Maternal-Effect Lethal Mutations on Linkage Group II of Caenorhabditis Elegans

We have analyzed a set of linkage group (LG) II maternal-effect lethal mutations in Caenorhabditis elegans isolated by a new screening procedure. Screens of 12,455 F1 progeny from mutagenized adults resulted in the recovery of 54 maternal-effect lethal mutations identifying 29 genes. Of the 54 mutations, 39 are strict maternal-effect mutations defining 17 genes. These 17 genes fall into two classes distinguished by frequency of mutation to strict maternal-effect lethality. The smaller class, comprised of four genes, mutated to strict maternal-effect lethality at a frequency close to 5 X 10(-4), a rate typical of essential genes in C. elegans. Two of these genes are expressed during oogenesis and required exclusively for embryogenesis (pure maternal genes), one appears to be required specifically for meiosis, and the fourth has a more complex pattern of expression. The other 13 genes were represented by only one or two strict maternal alleles each. Two of these are identical genes previously identified by nonmaternal embryonic lethal mutations. We interpret our results to mean that although many C. elegans genes can mutate to strict maternal-effect lethality, most genes mutate to that phenotype rarely. Pure maternal genes, however, are among a smaller class of genes that mutate to maternal-effect lethality at typical rates. If our interpretation is correct, we are near saturation for pure maternal genes in the region of LG II balanced by mnC1. We conclude that the number of pure maternal genes in C. elegans is small, being probably not much higher than 12.     

Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tail spike protein: III. Inactive polypeptide chains synthesized at 39 °C

Salmonella typhimurium cells infected by temperature-sensitive mutants in gene 9 of bacteriophage P22 at the restrictive temperature (39 °C) fail to accumulate functional tail spike protein. We report here studies of the inactive mutant tail spike polypeptide chains synthesized at 39 °C by temperature-sensitive mutants at 15 different sites of gene 9. For all 15 mutants, the gene 9 polypeptide chains were synthesized at 39 °C at rates similar to wild type. The mutant polypeptide chains were stable within the infected cells.The inactive polypeptide chains were tested for three functions displayed by the mature tail spike protein: irreversible binding to phage heads, endorhamnosidase activity, and reaction with anti-tail antibody. The 15 mutant proteins that accumulated at 39 °C lacked all three functions. Since the amino acid substitutions do not affect these functions of the mature protein, the mutant polypeptide chains synthesized at 39 °C have a conformation very different from the wild type, and different from the same proteins when matured at 30 °C. The fact that amino acid substitutions throughout the 76,000 M_r polypeptide chain prevent all three functions suggests that the mutations prevent the correct folding of the gene 9 polypeptide chain at restrictive temperature. Thus, these mutations identify sites in the polypeptide chain critical for protein maturation.Many of the mutant proteins could be activated in the absence of new protein synthesis by shifting infected cells from restrictive to permissive temperature before cell lysis. For these mutants, the immature chains accumulating at high temperature must be reversibly related to intermediates in protein folding or subunit assembly.     

DNA supercoiling in gyrase mutants.

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.     

An improved integration replacement/disruption method for mutagenesis of yeast essential genes

We improved the integration replacement/disruption method (Shortle, D., Novic, P., and Botstein, D. Proc. Natl. Acad. Sci. USA 81: 4889-4893, 1984) for isolating mutants in any of essential genes of the yeast Saccharomyces cerevisiae by integrating mutagenized DNA into the wild type gene of interest. We adopted this method to isolate temperature-sensitive mutants of the MPC1 gene encoding the YLL031C ORF. To facilitate integration of the mutagenic plasmid at a site near the 5' end of the ORF, a BamHI site was created at 300 bp downstream of the 5' end of the truncated ORF to be mutagenized. The MPC1 gene was disrupted in the wild type haploid strain by integrating a 5'-truncated derivative of the gene with mutations induced by in vitro mutagenesis. Transformants thus obtained were subjected for diagnosis of conditional lethality by replica-plating onto an appropriate selection medium to detect mutants. A primary mutant isolated by this method reverted in a high frequency due to a tandem repeat created by mutagenic integration. We deviced a method to obtain a stable temperature-sensitive strain by disrupting the tandem duplication. Two stable temperature-sensitive mutants thus obtained were found to be remedial either with 1 M sorbitol or with 0.1 M Mg2+ and to be sensitive to local anestheticum, tetracaine, at 25 degrees C.