Genetic analysis of rolled, which encodes a Drosophila mitogen-activated protein kinase.

Genetic and molecular characterization of the dominant suppressors of D-raf(C110) on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rl(Su23), was found to bear the same molecular lesion as rl(Sem), which has been reported to be dominant female sterile. However, rl(Su23) and the current stock of rl(Sem) showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.     

A genetic screen for maternal-effect suppressors of decapentaplegic identifies the eukaryotic translation initiation factor 4A in Drosophila

The Dpp signaling pathway is essential for many developmental processes in Drosophila and its activity is tightly regulated. To identify additional regulators of Dpp signaling, we conducted a genetic screen for maternal-effect suppressors of dpp haplo-insufficiency. We screened approximately 7000 EMS-mutagenized genomes and isolated and mapped seven independent dominant suppressors of dpp, Su(dpp), which were recovered as second-site mutations that resulted in viable flies in trans-heterozygous with dpp(H46), a dpp null allele. Most of the Su(dpp) mutants exhibited increased cell numbers of the amnioserosa, a cell type specified by the Dpp pathway, suggesting that these mutations may augment Dpp signaling activity. Here we report the unexpected identification of one of the Su(dpp) mutations as an allele of the eukaryotic translation initiation factor 4A (eIF4A). We show that Su(dpp)(YE9) maps to eIF4A and that this allele is associated with a substitution, arginine 321 to histidine, at a well-conserved amino acid and behaves genetically as a dominant-negative mutation. This result provides an intriguing link between a component of the translation machinery and Dpp signaling.     

A Genetic Analysis of Suppressors of the Pf10 Mutation in Chlamydomonas Reinhardtii

A mutation at the PF10 locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pf10 motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the pf10 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XII and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-1) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another.     

Mutations Affecting the Meiotic and Mitotic Divisions of the Early Caenorhabditis Elegans Embryo

We describe interactions between maternal-effect lethal mutations in four genes of Caenorhabditis elegans whose products appear to be involved in the meiotic and mitotic divisions of the one-cell embryo. Mitosis is disrupted by two dominant temperature-sensitive gain-of-function maternal-effect lethal mutations, mei-1(ct46) and mel-26(ct61), and by recessive loss-of-function maternal-effect lethal mutations of zyg-9. The phenotypic defects resulting from these mutations are similar. Doubly mutant combinations show a strong enhancement of the maternal-effect lethality under semipermissive conditions, suggesting that the mutant gene products interact. We isolated 15 dominant suppressors of the gain-of-function mutation mei-1(ct46). Thirteen of these suppressors are apparently intragenic, but 11 of them suppress in trans as well as cis. Two extragenic suppressors define a new gene, mei-2. The suppressor mutations in these two genes also result in recessive maternal-effect lethality, but with meiotic rather than mitotic defects. Surprisingly, most of these suppressors are also able to suppress mel-26(ct61) in addition to mei-1(ct46). The products of the four genes mei-1, mei-2, zyg-9 and mel-26 could be responsible for some of the specialized features that distinguish the meiotic from the mitotic divisions in the one-cell embryo.     

Dominant Mutations of Drosophila Map Kinase Kinase and Their Activities in Drosophila and Yeast Map Kinase Cascades

Eight alleles of Dsor1 encoding a Drosophila homologue of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppressed mutations for receptor tyrosine kinases (RTKs); torso (tor), sevenless (sev) and to a lesser extent Drosophila EGF receptor (DER). Furthermore, the Dsor1 alleles showed no significant interaction with gain-of-function mutations of DER. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrated that Dsor1 can activate yeast MAP kinase homologues if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes revealed that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors.     

Intragenic and Extragenic Suppressors of Temperature Sensitive Mutations in the Replication Initiation Genes dnaD and dnaB of Bacillus subtilis

Background

The Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.

Methodology/Principal Findings

We isolated second site suppressors of the temperature sensitive phenotypes caused by one dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid (alanine).

Conclusions/Significance

The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function.     

Suppressors of a host range mutation in the rabbitpox virus serpin SPI-1 map to proteins essential for viral DNA replication

The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.     

A novel gain-of-function mutant of the cyclic GMP-dependent protein kinase egl-4 affects multiple physiological processes in Caenorhabditis elegans

cGMP-dependent protein kinases are key intracellular transducers of cell signaling. We identified a novel dominant mutation in the C. elegans egl-4 cGMP-dependent protein kinase (PKG) and show that this mutation causes increased normal gene activity although it is associated with a reduced EGL-4 protein level. Prior phenotypic analyses of this gain-of-function mutant demonstrated a reduced longevity and a reduced feeding behavior when the animals were left unperturbed. We characterize several additional phenotypes caused by increased gene activity of egl-4. These phenotypes include a small body size, reduced locomotion in the presence of food, a pale intestine, increased intestinal fat storage, and a decreased propensity to form dauer larvae. The multiple phenotypes of egl-4 dominant mutants are consistent with an instructive signaling role of PKG to control many aspects of animal physiology. This is among the first reported gain-of-function mutations in this enzyme of central physiological importance. In a genetic screen we have identified extragenic suppressors of this gain-of-function mutant. Thus, this mutant promises to be a useful tool for identifying downstream targets of PKG.     

A Tripartite Interaction among Alleles of Notch, Delta, and Enhancer of Split during Imaginal Development of Drosophila Melanogaster

A dramatic example of a phenotypic interaction that involves neurogenic loci during Drosophila imaginal development is the synergistic impact of split (spl), a recessive allele of the Notch locus, and E(spl)D, a dominant gain-of-function allele of the Enhancer of split locus, on morphogenesis of the compound eye. Screens for mutations that relieve the enhancing effect of E(spl)D on spl have yielded three classes of mutations: intragenic revertants of the E(spl)D allele, extragenic suppressors that are allelic to the neurogenic gene Delta (Dl) and unlinked extragenic modifiers. Analysis of the suppression of the spl-E(spl)D interaction by various Dl alleles indicates that this modification is sensitive to the dosage of the Dl locus. This tripartite interaction illustrates the combinatorial action of N, Dl and E(spl) during imaginal development.     

Mammalian Raf-1 is activated by mutations that restore Raf signaling in Drosophila.

An interaction with the Ras proto-oncogene product is a requirement for Raf-1 activation in many signaling cascades. The significance of this interaction is demonstrated by the fact that a mutation preventing the Ras-Raf interaction severely impairs the function of both mammalian (Raf-1) and Drosophila (D-Raf) Raf proteins. In D-Raf, however, dominant intragenic mutations have been identified that suppress the effect of the Ras-binding site (RBS) mutation. To address the mechanism by which these mutations restore Raf signaling, we have introduced the suppressor mutations into the analogous residues of mammalian Raf-1. Here, we show that rather than compensating for the RBS mutation by restoring the Ras-Raf-1 interaction, the suppressor mutations increase the enzymatic and biological activity of Raf-1, allowing Raf-1 to signal in the absence of Ras binding. Surprisingly, we find that while one of the suppressor mutations (P181L) increases the basal kinase activity of Raf-1, it also abolishes the ability of wild-type Raf-1 to become activated by Ras. This mutation occurs in the cysteine-rich domain (CRD) of Raf-1 and demonstrates the importance of this region for a productive Ras-Raf interaction. Finally, we present evidence that the most activating suppressor mutation (G498S) increases Raf-1 activity by introducing a novel phosphorylation site into the L12 activation loop of the Raf-1 kinase domain.     

Characterization of Suppressor of Fused, a Complete Suppressor of the Fused Segment Polarity Gene of Drosophila Melanogaster

fused (fu) is a maternal effect segment polarity gene of Drosophila melanogaster. In addition, fu females have tumorous ovaries. Two ethyl methanesulfonate mutageneses were carried out in order to isolate suppressors of the fu phenotype. A new gene, Suppressor of fused (Su(fu)), was identified. It is located in the 87C8 region of the third chromosome. Su(fu) displays a maternal effect and is also expressed later in development. Although Su(fu)LP is a complete loss-of-function mutation, it is homozygous viable and has no phenotype by itself. Su(fu) fully suppresses the embryonic and adult phenotypes of fu mutants. Su(fu) mutations are semidominant and a Su(fu)+ duplication has an opposite effect, enhancing the fused phenotype. It is proposed therefore that the Su(fu)+ product is involved in the same developmental step as the Fu+ kinase. Thus, a new gene interacting with the segment polarity pathway was identified using an indirect approach.     

Gene interactions in Caenorhabditis elegans define DPY-31 as a candidate procollagen C-proteinase and SQT-3/ROL-4 as its predicted major target

Zinc metalloproteases of the BMP-1/TOLLOID family (also known as astacins) are extracellular enzymes involved in important developmental processes in metazoans. We report the characterization of the Caenorhabditis elegans gene dpy-31, which encodes the first essential astacin metalloprotease identified in this organism. Loss-of-function mutations in dpy-31 result in cuticle defects, abnormal morphology, and embryonic lethality, indicating that dpy-31 is required for formation of the collagenous exoskeleton. DPY-31 is widely expressed in the hypodermal cells, which are responsible for cuticle secretion. We have investigated the dpy-31 function through reversion analysis. While complete reversion can be obtained only by intragenic suppressors, reversion of the Dpy-31 lethal phenotype also can be caused by dominant extragenic suppressors. Nine extragenic suppressors carry mutations in the uniquely essential collagen gene sqt-3, which we show is the same gene as rol-4. Most mutations exhibit the unusual property of exclusively dominant suppression and all affect the sequence of the SQT-3 collagen C terminus. This suggests that DPY-31 is responsible for C-terminal proteolytic processing of collagen trimers and is therefore a structural and functional homolog of vertebrate BMP-1. The results also demonstrate the critical importance of the collagen C-terminal sequence, which is highly conserved among all 49 members of the SQT-3 subfamily.     

Genetic modifiers of the Drosophila NSF mutant, comatose, include a temperature-sensitive paralytic allele of the calcium channel alpha1-subunit gene, cacophony

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Our previous work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden our analysis of synaptic transmission to other gene products, we have performed a genetic screen for mutations that interact with comt. Here we report the isolation and analysis of four mutations that modify TS paralysis in comt, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel alpha1-subunit gene, cacophony (cac). Analysis of synaptic function in these mutants alone and in combination will further define the in vivo functions and interactions of specific gene products in synaptic transmission.     

Genetic Analysis of Bacteriophage P22 Lysozyme Structure

The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains. Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30 degrees; an additional seven were defective at 40 degrees. Revertants were isolated on the "missuppressing" hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants. It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host. Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13. Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype.     

Sequence Analysis of Mutations Arising during Prolonged Starvation of Salmonella Typhimurium

We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.     

Extragenic Suppressors of Nudc3, a Mutation That Blocks Nuclear Migration in Aspergillus Nidulans

Nuclear migration plays an important role in the growth and development of many organisms including the filamentous fungus Aspergillus nidulans. We have cloned three genes from A. nidulans, nudA, nudC, and nudF, in which mutations affect nuclear migration. The nudA gene encodes the heavy chain of cytoplasmic dynein. The nudC gene encodes a 22-kD protein. The nudF gene was identified as an extracopy suppressor of the temperature sensitive (ts(-)) nudC3 mutation. The nudC3 mutation substantially decreases the intracellular concentration of the nudF protein at restrictive temperature. This is restored toward the normal level by an extra copy of nudF. To identify other genes whose products interact directly or indirectly with the NUDC protein, we have isolated a set of extragenic suppressors of the nudC3 temperature-sensitive mutation. Genetic analysis of 16 such extragenic suppressors showed them to represent nine different genes, designated sncA-sncI (for suppressor of nudC). sncA-sncH were either dominant or semidominant in diploids homozygous for nudC3 and heterozygous for the snc mutations. All of the suppressors reversed the ts(-) phenotype of nudC3 by restoring the intracellular concentration of the NUDF protein.     

Drosophila Src42A is a negative regulator of RTK signaling

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. Here we show that an extragenic suppressor of Raf, Su(Raf)1, encodes a Drosophila Src family gene Src42A. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Our study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed.     

Genetic Analysis of the Gyrase a-like Domain of DNA Topoisomerase II of Saccharomyces Cerevisiae

We have undertaken a genetic analysis of heat-sensitive and cold-sensitive mutations in TOP2, the gene encoding yeast DNA topoisomerase II. Deletion mapping was used to localize 14 heat-sensitive and four cold-sensitive top2 mutations created by a method biased toward mutations in the 3' two-thirds of the gene. The mutations all appear to be located in the region of DNA topoisomerase II that shows homology to the "A" subunit of bacterial DNA gyrase. The heat-sensitive mutations and one cold-sensitive mutation lie in the center of the gene near the sequence that encodes the active site tyrosine. The three other cold-sensitive mutations map farther toward the 3' end of the gene. The cold-sensitive mutations exhibit intragenic complementation, and the complementation groups correspond to the physical map. We sequenced nine top2 mutations and found that the mutations are usually single missense mutations, frequently involve proline, and affect conserved regions of the protein. Suppressor analysis yielded two intragenic suppressors and seven independent isolates of an allele-specific extragenic suppressor we named tos1; tos1 is not allelic to any genes predicted to encode type I topoisomerase-related proteins. The two intragenic suppressors were tested for allele-specificity; the results revealed a complex pattern of suppression between heat-sensitive and cold-sensitive top2 alleles. These top2 mutations may have compensatory effects on the general stability of the protein.     

Drosophila-raf acts to elaborate dorsoventral pattern in the ectoderm of developing embryos.

In the early Drosophila embryo the activity of the EGF-receptor (Egfr) is required to instruct cells to adopt a ventral neuroectodermal fate. Using a gain-of-function mutation we showed that D-raf acts to transmit this and other late-acting embryonic Egfr signals. A novel role for D-raf was also identified in lateral cell development using partial loss-of-function D-raf mutations. Thus, we provide evidence that zygotic D-raf acts to specify cell fates in two distinct pathways that generate dorsoventral pattern within the ectoderm. These functional requirements for D-raf activity occur subsequent to its maternal role in organizing the anterioposterior axis. The consequences of eliminating key D-raf regulatory domains and specific serine residues in the transmission of Egfr and lateral epidermal signals were also addressed here.