Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997     

Novel nematode amber suppressors

Nine amber suppressor mutations were isolated in the nematode Caenorhabditis elegans by reverting amber alleles of a sex-determining gene, tra-3. One suppressor maps to a known locus, sup-5 III , but the other eight map to three new loci, sup-21 X (five alleles), sup-22 IV (two alleles) and sup-23 IV (one allele). Amber alleles of tra-3 and of a dumpy gene, dpy-20, were used to measure the efficiency of suppression; the sup-21 and the sup-22 alleles were both shown to be heterogeneous and generally weaker suppressors than sup-5 alleles, which are homogeneous. The spectrum of mutations suppressed by a strong sup-21 allele, e1957, was investigated and compared to the spectra for the amber suppressors sup-5 III and sup-7 X, using amber alleles in 13 assorted genes. Some of the differences between these spectra may be due to limited tissue specificity in sup-21 expression.—Suppression of dpy-20 was used to show that the sex-linked suppressors sup-7 and sup-21 are not dosage compensated in male (XO) relative to hermaphrodite (XX).—Several uses of amber suppressors are critically discussed: for identifying null mutations, for varying levels of gene activity and for detecting maternal mRNA.     

Genetics and mode of expression of temperature-sensitive mutations arresting embryonic development in Caenorhabditis elegans

Eleven temperature-sensitive mutations causing arrest of embryogenesis in Caenorhabditis elegans have been mapped. The mutations define nine genes (emb-1 to emb-9) on four chromosomes. The functions of six genes seem to be required exclusively for embryogenesis. Mutants in these genes have no other detectable phenotype at the permissive (16°C) or nonpermissive (25°C) temperature. The function of the other three genes is also required for postembryonic development. As shown by progeny tests for parental effects, for seven genes, maternal gene expression is necessary and sufficient for normal embryogenesis; for one gene, emb-2, either maternal or zygotic expression is sufficient; for one gene, emb-9, zygotic expression is necessary and sufficient. The high proportion of emb genes with maternal expression is consistent with the model of intracellular preprogramming of the egg of C. elegans (U. Deppe, E. Schierenberg, T. Cole, C. Krieg, D. Schmitt, B. Yoder, and G. von Ehrenstein, 1978; Proc. Nat. Acad. Sci. USA75, 376–380). Two developmental stages have been defined by temperature-shift experiments: (1) the normal execution stage indicating the time of execution of the normal event at the permissive temperature; (2) the defective execution stage indicating the time of the execution of an irreversible defect at the nonpermissive temperature. The classes of mutants defined by the progeny tests have corresponding execution stages, but the maternal necessary and sufficient class is subdivided into mutants executing during oogenesis or embryogenesis.     

Identification and Characterization of Genes That Interact with Lin-12 in Caenorhabditis Elegans

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.     

Extragenic Suppressors of Mutations in the Cytoplasmic C Terminus of Sec63 Define Five Genes in Saccharomyces Cerevisae

Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function.     

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. V. Isolation and Genetic Properties of Nongroup-Specific Suppressors

Two classes of frameshift suppressors distributed at 22 different loci were identified in previous studies in the yeast Saccharomyces cerevisiae. These suppressors exhibited allele-specific suppression of +1 G:C insertion mutations in either glycine or proline codons, designated as group II and group III frameshift mutations, respectively. Genes corresponding to representative suppressors of each group have been shown to encode altered glycine or proline tRNAs containing four base anticodons.—This communication reports the existence of a third class of frameshift suppressor that exhibits a wider range in specificity of suppression. The suppressors map at three loci, suf12, suf13, and suf14, which are located on chromosomes IV, XV, and XIV, respectively. The phenotypes of these suppressors suggest that suppression may be mediated by genes other than those encoding the primary structure of glycine or proline tRNAs.     

Isolation and Characterization of Suppressors of Two Escherichia Coli Dnag Mutations, Dnag2903 and Parb

The dnaG gene of Escherichia coli encodes the primase protein, which synthesizes a short pRNA that is essential for the initiation of both leading and lagging strand DNA synthesis. Two temperature-sensitive mutations in the 3'' end of the dnaG gene, dnaG2903 and parB, cause a defect in chromosome partitioning at the nonpermissive temperature 42°. We have characterized 24 cold-sensitive suppressor mutations of these two dnaG alleles. By genetic mapping and complementation, five different classes of suppressors have been assigned: sdgC, sdgD, sdgE, sdgG and sdgH. The genes responsible for suppression in four of the five classes have been determined. Four of the sdgC suppressor alleles are complemented by the dnaE gene, which encodes the enzymatic subunit of DNA polymerase III. The sdgE class are mutations in era, an essential GTPase of unknown function. The sdgG suppressor is likely a mutation in one of three genes: ubiC, ubiA or yjbI. The sdgH class affects rpsF, which encodes the ribosomal protein S6. Possible mechanisms of suppression by these different classes are discussed.     

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. II. Genetic Properties of Group II Suppressors

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. One of these groups (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) appears to include a set of informational suppressors in which the vehicle of suppression is glycyl-tRNA. Some of the genetic properties of Group II suppressors are described in this communication.——Corevertants of the Group II frameshift mutations his4–519 and leu2–3 have been characterized to determine the spectrum of reversion events induced by the frameshift mutagen ICR-170. Seventythree ICR-induced corevertants were analyzed. With the exception of one corevertant, which carried an allele of SUF1, all carried alleles of SUF3 or SUF5. SUF1, SUF3, SUF4 and SUF6 were represented among spontaneous and UV-induced corevertants. In the course of these experiments one of the suppressors was mapped. SUF5, the probable structural gene for tRNA^GLY1, is located between ade2 and ade9 on chromosome XV.——SUF1, SUF4 and SUF6 have novel properties and comprise a distinct subset of suppressors. Although these suppressors show no genetic linkage to each other, they share several common features including lethality in haploid pairwise combinations, reduced tRNA^GLY3 isoacceptor activity and increased efficiency of suppression in strains carrying the cytoplasmically inherited [PSI] element. In addition, strains carrying SUF1, SUF4 or SUF6 are phenotypically unstable and give rise to mitotic Suf⁺ segregants at high frequency. These segregants invariably contain a linked, second-site mutation that maps in or adjacent to the suppressor gene itself. Strains carrying any of these suppressors also give rise to mitotic segregants that exhibit enhanced efficiency of suppression; mutations responsible for this phenotype map at two loci, upf1 and upf2. These genes show no genetic linkage to any of the Group II suppressors.——Methods that permit positive selection for mutants with decreased or enhanced efficiency of suppression have been devised in order to examine large numbers of variants. The importance of these interacting mutants is underscored by their potential utility in studying suppressor function at the molecular level.     

emb-1 encodes the APC16 subunit of the Caenorhabditis elegans anaphase-promoting complex

In the nematode Caenorhabditis elegans, temperature-sensitive mutants of emb-1 arrest as one-cell embryos in metaphase of meiosis I in a manner that is indistinguishable from embryos that have been depleted of known subunits of the anaphase-promoting complex or cyclosome (APC/C). Here we show that the emb-1 phenotype is enhanced in double mutant combinations with known APC/C subunits and suppressed in double mutant combinations with known APC/C suppressors. In addition to its meiotic function, emb-1 is required for mitotic proliferation of the germline. These studies reveal that emb-1 encodes K10D2.4, a homolog of the small, recently discovered APC/C subunit, APC16.     

Characterization of α1(IV) Collagen Mutations in Caenorhabditis elegans and the Effects of α1 and α2(IV) Mutations on Type IV Collagen Distribution

Type IV collagen is a major component of basement membranes. We have characterized 11 mutations in emb-9, the α1(IV) collagen gene of Caenorhabditis elegans, that result in a spectrum of phenotypes. Five are substitutions of glycines in the Gly-X-Y domain and cause semidominant, temperature-sensitive lethality at the twofold stage of embryogenesis. One is a glycine substitution that causes recessive, non–temperature-sensitive larval lethality. Three putative null alleles, two nonsense mutations and a deletion, all cause recessive, non–temperature-sensitive lethality at the threefold stage of embryogenesis. The less severe null phenotype indicates that glycine substitution containing mutant chains dominantly interfere with the function of other molecules. The emb-9 null mutants do not stain with anti–EMB-9 antisera and show intracellular accumulation of the α2(IV) chain, LET-2, indicating that LET-2 assembly and/or secretion requires EMB-9. Glycine substitutions in either EMB-9 or LET-2 cause intracellular accumulation of both chains. The degree of intracellular accumulation differs depending on the allele and temperature and correlates with the severity of the phenotype. Temperature sensitivity appears to result from reduced assembly/secretion of type IV collagen, not defective function in the basement membrane. Because the dominant interference of glycine substitution mutations is maximal when type IV collagen secretion is totally blocked, this interference appears to occur intracellularly, rather than in the basement membrane. We suggest that the nature of dominant interference caused by mutations in type IV collagen is different than that caused by mutations in fibrillar collagens.     

Patterns of Genetic and Phenotypic Suppression of lys2 Mutations in the Yeast SACCHAROMYCES CEREVISIAE

A total of 358 lys2 mutants of Saccharomyces cerevisiae have been characterized for suppressibility by the following suppressors: UAA and UAG suppressors that insert tyrosine, serine or leucine; a putative UGA suppressor; an omnipotent suppressor SUP46; and a frameshift suppressor SUF1–1. In addition, the lys2 mutants were examined for phenotypic suppression by the aminoglycoside antibiotic paromomycin, for osmotic remediability and for temperature sensitivity. The mutants exhibited over 50 different patterns of suppression and most of the nonsense mutants appeared similar to nonsense mutants previously described. A total of 24% were suppressible by one or more of the UAA suppressors, 4% were suppressible by one or more of the UAG suppressors, while only one was suppressible by the UGA suppressor and only one was weakly suppressible by the frameshift suppressor. One mutant responded to both UAA and UAG suppressors, indicating that UAA or UAG mutations at certain rare sites can be exceptions to the specific action of UAA and UAG suppressors. Some of the mutants appeared to require certain types of amino acid replacements at the mutant sites in order to produce a functional gene product, while others appeared to require suppressors that were expressed at high levels. Many of the mutants suppressible by SUP46 and paromomycin were not suppressible by any of the UAA, UAG or UGA suppressors, indicating that omnipotent suppression and phenotypic suppression need not be restricted to nonsense mutations. All of the mutants suppressible by SUP46 were also suppressible by paromomycin, suggesting a common mode of action of omnipotent suppression and phenotypic misreading.     

A New Kind of Informational Suppression in the Nematode Caenorhabditis Elegans

Independent reversions of mutations affecting three different Caenorhabditis elegans genes have each yielded representatives of the same set of extragenic suppressors. Mutations at any one of six loci act as allele-specific recessive suppressors of certain allels of unc-54 (a myosin heavy chain gene), lin-29 (a heterochronic gene), and tra-2 (a sex determination gene). The same mutations also suppress certain alleles of another sex determination gene, tra-1, and of a morphogenetic gene, dpy-5. In addition to their suppression phenotype, the suppressor mutations cause abnormal morphogenesis of the male bursa and the hermaphrodite vulva. We name these genes smg-1 through smg-6 (suppressor with morphogenetic effect on genitalia), in order to distinguish them from mab (male abnormal) genes that can mutate to produce abnormal genitalia but which do not act as suppressors (smg-1 and smg-2 are new names for two previously described genes, mab-1 and mab-11). The patterns of suppression, and the interactions between the different smg genes, are described and discussed. In general, suppression is recessive and incomplete, and at least some of the suppressed mutations are hypomorphic in nature. A suppressible allele of unc-54 contains a deletion in the 3' noncoding region of the gene; the protein coding region of the gene is apparently unaffected. This suggests that the smg suppressors affect a process other than translation, for example mRNA processing, transport, or stability.     

Single Site Suppressors of a Fission Yeast Temperature-Sensitive Mutant in cdc48 Identified by Whole Genome Sequencing

The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48⁺ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.     

Extragenic Suppressors of Saccharomyces Cerevisiae Prp4 Mutations Identify a Negative Regulator of Prp Genes

The PRP4 gene encodes a protein that is a component of the U4/U6 small nuclear ribonucleoprotein particle and is necessary for both spliceosome assembly and pre-mRNA splicing. To identify genes whose products interact with the PRP4 gene or gene product, we isolated second-site suppressors of temperature-sensitive prp4 mutations. We limited ourselves to suppressors with a distinct phenotype, cold sensitivity, to facilitate analysis of mutants. Ten independent recessive suppressors were obtained that identified four complementation groups, spp41, spp42, spp43 and spp44 (suppressor of prp4, numbers 1-4). spp41-spp44 suppress the pre-mRNA splicing defect as well as the temperature-sensitive phenotype of prp4 strains. Each of these spp mutations also suppresses prp3; spp41 and spp42 suppress prp11 as well. Neither spp41 nor spp42 suppresses null alleles of prp3 or prp4, indicating that the suppression does not occur via a bypass mechanism. The spp41 and spp42 mutations are neither allele- nor gene-specific in their pattern of suppression and do not result in a defect in pre-mRNA splicing. Thus the SPP41 and SPP42 gene products are unlikely to participate directly in mRNA splicing or interact directly with Prp3p or Prp4p. Expression of PRP3-lacZ and PRP4-lacZ gene fusions is increased in spp41 strains, suggesting that wild-type Spp41p represses expression of PRP3 and PRP4. SPP41 was cloned and sequenced and found to be essential. spp43 is allelic to the previously identified suppressor srn1, which encodes a negative regulator of gene expression.     

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.     

Indirect Suppression in CAENORHABDITIS ELEGANS

Two cases of indirect suppression have been characterized. One case involves suppressors compensating for defects in muscle structure. Nine independent suppressor mutations were judged to lie in a single suppressor gene, sup-3. Suppression is dominant, but dose dependent, and results in improved locomotion, as well as in an increase in the ability of mutant animals to lay eggs. Mutations in six genes known to affect muscle structure were tested for suppression by representative sup-3 mutations. Alleles of three of the six genes are suppressed, two of which are known to code for thick filament proteins. One suppressor allele was identified as a deletion by genetic criteria. A second case of indirect suppression is not associated with muscle defects, but involves two mutant genes producing uncoordinated phenotypes very similar to one another. As in the first case, suppression is dominant but dose dependent and is not allele specific.     

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.     

New positive regulators of lin-12 activity in Caenorhabditis elegans include the BRE-5/Brainiac glycosphingolipid biosynthesis enzyme

Screens for suppressors of lin-12 hypermorphic alleles in C. elegans have identified core components and modulators of the LIN-12/Notch signaling pathway. Here we describe the recovery of alleles of six new genes from a screen for suppressors of the egg-laying defect associated with elevated lin-12 activity. The molecular identification of one of the new suppressor genes revealed it as bre-5, which had previously been identified in screens for mutations that confer resistance to Bt toxin in C. elegans. bre-5 is the homolog of D. melanogaster brainiac. BRE-5/Brainiac catalyzes a step in the synthesis of glycosphingolipids, components of lipid rafts that are thought to act as platforms for association among certain kinds of membrane-bound proteins. Reducing the activity of several other genes involved in glycosphingolipid biosynthesis also suppresses the effects of constitutive lin-12 activity. Genetic analysis and cell ablation experiments suggest that bre-5 functions prior to ligand-induced ectodomain shedding that activates LIN-12 for signal transduction.     

Dominant Suppressors of a Muscle Mutant Define an Essential Gene of CAENORHABDITIS ELEGANS

The sup-11 I locus of C. elegans was defined by rare dominant suppressors of unc-93(e1500) III, a mutation that affects muscle structure. All ten of these dominant suppressors have a recessive "scrawny" phenotype. Two additional classes of sup-11 alleles were identified. One class, null alleles, was obtained by reversion of the dominant suppressor activity. These null alleles are recessive embryonic lethals, indicating that sup-11 is an essential gene. Members of the second class, rare semidominant revertants of the "scrawny" phenotype, are partial suppressors of unc-93(e1500). The genetic properties of the dominant suppressor mutations suggest that they are rare missense mutations that confer a novel activity to the sup-11 protein. We consider some of the ways that sup-11 alleles might suppress unc-93(e1500), including the possibilities that the altered sup-11 proteins restore function to a protein complex or are modified products of a gene that is a member of an unc-93 gene family.