Analysis of Mutations in the Sqt-1 and Rol-6 Collagen Genes of Caenorhabditis Elegans

Different mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans can cause diverse changes in body morphology and display different genetic attributes. We have determined the nucleotide alterations in 15 mutant alleles of these genes. Three mutations in sqt-1 and one in rol-6 that cause dominant right-handed helical twisting (RRol) of animals are arginine to cysteine replacements. These mutations are all within a short conserved sequence, on the amino terminal side of the Gly-X-Y repeats, that is found in all C. elegans cuticle collagens. A recessive RRol mutation of rol-6 is a replacement of one of the same conserved arginines by histidine. In contrast, three sqt-1 mutations that cause recessive left-handed helical twisting (LRol) are replacements of a conserved carboxyterminal cysteine residue with either tyrosine or serine. These results suggest that disulfide bonding is important in collagen organization and that a deficit or surplus of disulfides may cause cuticle alterations of opposite handedness. In contrast to other collagens, glycine replacement mutations in the Gly-X-Y repeats of sqt-1 cause very mild phenotypes. Nonsense mutations of both sqt-1 and rol-6 cause nearly, but not totally, wild-type phenotypes. A nonsense mutation in sqt-1 suppresses the phenotype of rol-6 RRol mutations, suggesting that rol-6 collagen function is dependent on the presence of sqt-1 collagen. Mutations of sqt-1 are not suppressed by a rol-6 nonsense mutation, however, indicating that sqt-1 collagen can function independently of rol-6.     

The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.

The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration. The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.     

Ultrastructural analyses of the Caenorhabditis elegans sqt-1(sc13) left roller mutant

The sqt-1 gene is 1 of the several loci in Caenorhabditis elegans that primarily affects organismal morphology. Certain mutations in the sqt-1 gene can produce left roller animals, i.e., they rotate around their long axis and move in circular paths. We describe the morphological alterations seen in the cuticle of the left roller sqt-1(sc13). Deep-etched replica analyses showed that the fibrous layer is composed of a unique strand of parallel fibers, instead of the 2 meeting at an angle of 60 degrees as observed in the wild-type strain. In addition, honeycomb elements, fibers organized in a pentagonal fashion above the fibrous layer, completely fill the intermediate layer that is empty spaces in the wild type. These morphological alterations are likely to be involved in generating the helical twist of the sqt-1(sc13) left roller mutant.     

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.     

Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology.

We have identified and cloned the Caenorhabditis elegans dpy-2 and dpy-10 genes and determined that they encode collagens. Genetic data suggested that these genes are important in morphogenesis and possibly other developmental events. These data include the morphologic phenotypes exhibited by mutants, unusual genetic interactions with the sqt-1 collagen gene, and suppression of mutations in the glp-1 and mup-1 genes. The proximity of the dpy-2 and dpy-10 genes (3.5 kilobase) and the structural similarity of their encoded proteins (41% amino acid identity) indicate that dpy-2 and dpy-10 are the result of a gene duplication event. The genes do not, however, appear to be functionally redundant, because a dpy-10 null mutant is not rescued by the dpy-2 gene. In addition, full complementation between dpy-2 and dpy-10 can be demonstrated with all recessive alleles tested in trans. Sequence analysis of several mutant alleles of each gene was performed to determine the nature of the molecular defects that can cause the morphologic phenotypes. Glycine substitutions within the Gly-X-Y portion of the collagens can result in dumpy (Dpy), dumpy, left roller (DLRol), or temperature-sensitive DLRol phenotypes. dpy-10(cn64), a dominant temperature-sensitive DLRol allele, creates an Arg-to-Cys substitution in the amino non-Gly-X-Y portion of the protein. Three dpy-10 alleles contain Tc1 insertions in the coding region of the gene. dpy-10(cg36) (DRLol) creates a nonsense codon near the end of the Gly-X-Y region. The nature of this mutation, combined with genetic data, indicates that DLRol is the null phenotype of dpy-10. The Dpy phenotype results from reduced function of the dpy-10 collagen gene. Our results indicate that a variety of molecular defects in these collagens can result in severe morphologic changes in C. elegans.     

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster

Summary The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.     

Proteolytic processing of Caenorhabditis elegans SQT-1 cuticle collagen is inhibited in right roller mutants whereas cross-linking is inhibited in left roller mutants.

The sqt-1 gene encodes a C. elegans cuticle collagen that when defective can cause dramatic alterations of organismal morphology. Specific antisera were used to examine the assembly of wild-type and mutant SQT-1 in the cuticle. Wild-type SQT-1 chains associate into dimer, tetramer, and higher oligomers that are cross-linked by non-reducible, presumably tyrosine-derived, covalent bonds. The SQT-1 pattern differs from the bulk of cuticle collagens which are found in trimer and larger forms. sqt-1 mutations that cause left-handed helical twisting of animals remove a conserved carboxyl-domain cysteine and inhibit formation of these non-reducible bonds. SQT-1 monomers accumulate and novel trimer-sized products form. A conserved tyrosine immediately adjacent to the affected cysteine suggests that disulfide bond formation is required for this tyrosine to form a cross-link. sqt-1 mutations that cause right-handed helical twisting affect conserved arginines in a predicted cleavage site for a subtilisin-like protease. These mutant SQT-1 molecules retain residues on the amino side of the predicted cleavage site and are larger than wild-type by the amount expected if cleavage failed to occur. The conservation of this site in all nematode cuticle collagens indicates that they are all synthesized as procollagens that are processed by subtilisin-like proteases.     

Characterization of New Punch Mutations: Identification of Two Additional Mutant Classes

The Punch locus of Drosophila melanogaster which encodes the pteridine biosynthetic enzyme, GTP cyclohydrolase, is genetically complex. Lethal alleles of the locus resolve into an array of interallelic complementation groups, and at least one class of mutations is developmentally specific, affecting GTP cyclohydrolase activity only in the heads of adults. All previously isolated Punch alleles were identified on the basis of a mutant eye color phenotype. By screening mutagenized chromosomes over Punch region deficiencies, we have now isolated new alleles on the basis of lethal and visible phenotypes. Most of these alleles fall into previously identified genetic classes, but two new classes of mutations were also found. One class contains two alleles that behave as dominant lethal mutations in some genetic backgrounds. The other class represents a second developmentally specific set of alleles that affect the function of the Punch locus only during embryogenesis.     

Genetics analysis of mouse mutations Abnormal feet and tail and rough coat, which cause developmental abnormalities and alopecia

Mutations in the mouse Brachyury (T) gene are characterized by a dominant reduction of tail length and recessive lethality. Two quantitative trait loci, Brachyury-modifier 1 and 2 (Brm1 and Brm2) are defined by alleles that enhance the short-tail Brachyury phenotype. Here we report on a genetic analysis of a visible dominant mutation Abnormal feet and tail (Aft) located in the vicinity of Brm1. Affected animals display kinky tails and syndactyly in the hindlimbs, both likely resulting from a defect in apoptosis. We observed an unusual genetic incompatibility between Aft and certain genetic backgrounds. We show that Aft and T are likely to interact genetically, since some double heterozygotes are tailless. In addition to the tail and hindlimb phenotypes, Aft-bearing mutants display characteristic late-onset skin lesions. We therefore tested for allelism between Aft and a closely linked recessive mutation rough coat (rc) and found that these two mutations are likely nonallelic. Our results provide a valuable resource for the study of mammalian skin development and contribute to the genetic analysis of Brachyury function.     

Genetic Interactions at the Fla10 Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas Reinhardtii

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.     

Properties of a Class of Genes Required for Ray Morphogenesis in Caenorhabditis Elegans

We have identified eight mutations that define at least five terminal differentiation genes (ram genes) whose products are required during the extension of the male-specific ray sensilla in Caenorhabditis elegans. ram gene mutations result in morphological abnormalities in the sensory rays but do not appear to interfere with ray functions. A similar ray morphology phenotype was observed in males harboring mutations in three previously defined genes, dpy-11, dpy-18 and sqt-1, that also affect body shape. One of these genes, sqt-1, is known to encode a collagen. Mutations in different ram genes failed to complement, from which we infer that their gene products functionally interact. For one ram gene, failure to complement was shown to result from haploinsufficiency. Intergenic noncomplementation did not extend to the body morphology genes. The temperature-sensitive periods of both ram and body morphology mutations corresponded to the period of development in which ray extension occurs. We propose that ram gene products act together in a critical interaction between the rays and the cuticle required for wild-type ray morphology.     

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.     

Genetic and Phenotypic Characterization of Roller Mutants of CAENORHABDITIS ELEGANS

Eighty-eight mutants of C. elegans that display a roller phenotype (a helically twisted body) have been isolated and characterized genetically and phenotypically. The mutations are located in 14 different genes. Most genes contain a number of alleles. Their distribution among the chromosomes appears nonrandom, with seven of the genes being located on linkage group II, some very closely linked. The phenotypes of the mutants suggest that there are five different classes of genes, each class representing a set of similar phenotypic effects: Left Roller (four genes), Right Roller (one gene), Left Squat (one gene), Right Squat (two genes) and Left Dumpy Roller (six genes). The classes of mutants differ with respect to a number of characteristics that include the developmental stages affected and the types of aberrations observed in cuticle structure. A variety of gene interactions were found, arguing that these genes are involved in a common developmental process. The presence of alterations in cuticle morphology strongly suggests that these genes are active in the formation of the nematode cuticle.     

Two Recessive Suppressors of SACCHAROMYCES CEREVISIAE CHO1 That Are Unlinked but Fall in the Same Complementation Group

Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus. The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine. One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis. Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV. Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously. The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.     

Fs(1)yb Is Required for Ovary Follicle Cell Differentiation in Drosophila Melanogaster and Has Genetic Interactions with the Notch Group of Neurogenic Genes

Phenotypic and genetic analyses demonstrate that fs(1)Yb activity is required in the soma for the development of a subset of ovarian follicle cells and to support later stages of egg maturation. Mutations in fs(1)Yb cause a range of ovarian phenotypes, from the improper segregation of egg chambers to abnormal dorsal appendage formation. The mutant phenotypes associated with fs(1)Yb are very similar to the ovarian aberrations produced by temperature-sensitive alleles of Notch and Delta. Possible functional or regulatory interactions between fs(1)Yb and Notch are suggested by genetic studies. A duplication of the Notch locus partially suppresses the female-sterility caused by fs(1)Yb mutations, while reducing Notch dosage makes the fs(1)Yb mutant phenotype more severe. In addition, fs(1)Yb alleles also interact with genes that are known to act with or regulate Notch activity, including Delta, daughterless, and mastermind. However, differences between the mutant ovarian phenotype of fs(1)Yb and that of Notch or Delta indicate that the genes do not have completely overlapping functions in the ovary. We propose that fs(1)Yb acts as an ovary-specific factor that determines follicle cell fate.     

Genetic Analysis of Rough Sheath1 Developmental Mutants of Maize

Maize Rough sheath1 (Rs1) mutants are dominant and cause a proliferation of sheath-like tissue at the base of the blade and throughout the ligular region. They also cause ligule displacement, a chaotic pattern of vasculature and abnormal cellular structure of vascular bundles. The affected region of Rs1-O leaves displays genetic and morphological attributes of both sheath and auricle, suggesting an overlap of these genetic programs. The rs1 locus maps approximately 26 map units distal to opaque2 (o2) on chromosome 7S, defining a new distal-most locus on the genetic map. Three mutant alleles, Rs1-O, Rs1-1025 and Rs1-Z, all display similar phenotypes. The mutations are completely dominant and the Rs1-O phenotype is not affected by dosage of the chromosome arm carrying the rs1(+) allele, indicating that these alleles are neomorphic. Analysis of genetic mosaics showed that the Rs1-O phenotype is non-cell-autonomous, suggesting that intercellular signals convey the phenotype. Rs1 mutant phenotypes are affected by modifiers present in particular genetic backgrounds. An enhancer of Rs1-O was identified; segregation data imply a single recessive gene, ers1. Rs1 mutants were also found to enhance the expression of unlinked rs2 and Rs4 mutants, suggesting that these mutations affect similar developmental processes. We discuss the phenotypic and genetic similarities between Rs1 and Knotted1 (Kn1) mutants that led to the identification of rs1 as a kn1-like homeobox gene (unpublished data).     

The Hsp70 and Hsp40 chaperones influence microtubule stability in Chlamydomonas

Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70-Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex.     

Modifications of the Notch Function by Abruptex Mutations in Drosophila Melanogaster

The function of the Notch gene is required in cell interactions defining alternative cell fates in several developmental processes. The Notch gene encodes a transmembrane protein with 36 epidermal growth factor (EGF)-like repeats in its extracellular domain. This protein functions as a receptor that interacts with other transmembrane proteins, such as Serrate and Delta, which also have EGF repeats in their extracellular domain. The Abruptex mutations of the Notch locus are associated with amino acid substitutions in the EGF repeats 24-29 of the Notch protein. We have studied, in genetic combinations, the modifications of Notch function caused by Abruptex mutations. These mutations lead to phenotypes which are opposite to those caused by Notch deletions. The Abruptex phenotypes are modified by the presence of mutations in other loci, in particular in the genes Serrate and Delta as well as Hairless, and groucho. The results suggest that all Abruptex mutations cause stronger than normal Notch activation by the Delta protein. Some Abruptex alleles also display an insufficiency of N function. Abruptex alleles which produce stronger enhancement of Notch activation also display stronger Notch insufficiency. This insufficiency could be due to reduced ability of Abruptex proteins to interact with Notch ligands and/or to form functional Notch dimers.     

Genetic interaction of BBS1 mutations with alleles at other BBS loci can result in non-Mendelian Bardet-Biedl syndrome

Bardet-Biedl syndrome is a genetically and clinically heterogeneous disorder caused by mutations in at least seven loci (BBS1-7), five of which are cloned (BBS1, BBS2, BBS4, BBS6, and BBS7). Genetic and mutational analyses have indicated that, in some families, a combination of three mutant alleles at two loci (triallelic inheritance) is necessary for pathogenesis. To date, four of the five known BBS loci have been implicated in this mode of oligogenic disease transmission. We present a comprehensive analysis of the spectrum, distribution, and involvement in non-Mendelian trait transmission of mutant alleles in BBS1, the most common BBS locus. Analyses of 259 independent families segregating a BBS phenotype indicate that BBS1 participates in complex inheritance and that, in different families, mutations in BBS1 can interact genetically with mutations at each of the other known BBS genes, as well as at unknown loci, to cause the phenotype. Consistent with this model, we identified homozygous M390R alleles, the most frequent BBS1 mutation, in asymptomatic individuals in two families. Moreover, our statistical analyses indicate that the prevalence of the M390R allele in the general population is consistent with an oligogenic rather than a recessive model of disease transmission. The distribution of BBS oligogenic alleles also indicates that all BBS loci might interact genetically with each other, but some genes, especially BBS2 and BBS6, are more likely to participate in triallelic inheritance, suggesting a variable ability of the BBS proteins to interact genetically with each other.