The induction and characterization of recessive suppressors of arg-2 in Schizophyllum commune

Summary Genetic analyses have been made to detect recessive suppressor mutations in eight prototrophic strains derived by treating an arginine dependent strain with hydroxylamine. The results indicate that one strain possesses a recessive suppressor, su-1, which maps outside the arg-2 locus and is capable of suppressing auxotrophy conferred by the arg-2 mutation. This suppressor is incapable of suppressing auxotrophy conferred by eight other loci. Prototrophy in the remaining seven strains resulted from either intragenic suppression, reversion, or from a suppressor mutation that is closely linked to the arg-2 locus. The results of heterokaryotic allelic tests with the seven strains indicate that the mutation to prototrophy is recessive.     

Arg-7 mutant x wild-type crosses in Chlamydomonas reinhardi: Study of the enzyme produced in diploid strains

Summary The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL.Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size.The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1xpab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned.These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.     

Absence of suppressible alleles at the his 1 locus of yeast

Summary No suppressible alleles have been found at the his1 locus of Saccharomyces cerevisiae. Seventeen noncomplementing alleles have been tested against the strong ochre suppressor SUP2. These alleles, plus an additional 34 complementing alleles, were previously tested against a weaker suppressor SUP11. These results, which are in marked contrast to experience with other yeast loci where suppressible alleles are frequent, may be explained if the his1 product is transcribed from a polycistronic message so that nonsense mutations lead to loss of a second nonsupplementable function, or if the his1 protein serves as an essential subunit for an unknown enzyme complex.     

Acetylglutamate kinase-acetylglutamyl-phosphate reductase complex of Neurospora crassa. Evidence for two polypeptides

Mutations at the arg-6 locus in Neurospora crassa are divided into two complementation groups (A and B) and a third noncomplementing group. There are many suppressible nonsense mutations among mutants in complementation group B and one in the noncomplementing group; no nonsense mutations exist among mutants in complementation group A (Davis, R. H., and Weiss, R. L. (1983) Mol. Gen. Genet. 192, 46-50). We show here that the mutants are defective in either or both of two enzymes of arginine biosynthesis, acetylglutamate kinase and/or acetylglutamyl-phosphate reductase. Mutants in complementation group A lack acetylglutamate kinase, those in complementation group B lack acetylglutamyl-phosphate reductase, and those in the noncomplementing group lack both activities. Mutants in group B also have reduced levels of acetylglutamate kinase. The enzymes from purified mitochondria are readily separable by gel filtration and by Blue A dye affinity chromatography. Acetylglutamate kinase appears to be an octamer with a molecular weight of 400,000, whereas acetylglutamyl-phosphate reductase appears to be a dimer with a molecular weight of 93,000. This suggests that the two activities reside on distinct polypeptides. These results are best accommodated by the following model: the arg-6 locus encodes a single mRNA which is translated into a single polypeptide; the latter is then cleaved post-translationally to yield two physically separable enzymes.     

Triallelic complementation and hybrid enzyme formation in Chlamydomonas reinhardi

Summary In Chlamydomonas, the arg-7 cistron (linkage group I) is the structural gene for the multimeric (probably pentameric) enzyme argininosuccinate lyase. Most of the alleles of the cistron were previously shown to complement in some pair combinations, giving rise to phenotypically wild-type diploids.By crossing diploid (mt^-) and haploid (mt⁺) cells bearing different markers of auxotrophy, seven different presumptive triploid strains, phenotypically wild-type, were isolated. Each strain had 3 different arg-7 alleles or 2 mutant alleles associated with a wild one.The isolates were cytologically and biochemically analyzed: it could be concluded that they were triploid or ar least trisomic for the linkage group I.The specific activity and the thermosensitivity of the lyase were compared in the different triploids and in the diploids bearing two of the three corresponding arg-7 alleles. In most cases, the enzyme formed by triallelic complementation was more active and more heat resistant than the enzyme formed by diallelic complementation. These results can be interpreted by assuming that hybrid enzyme is formed by interaction between the products of the three different alleles. They provide a molecular basis for explaining the increased vigor often found in polyploids.     

The genetic fine structure of the complex locus aro3 involved in early aromatic amino acid biosynthesis in Schizosaccharomyces pombe.

Summary The complex locus aro3 of Schizosaccharomyces pombe was subjected to genetical fine structure analysis. By comparing the complementation map and the meiotic recombination map, the aro3 locus could be subdivided into the five adjacent subregions A, B, C, D and E. Out of 115 aro3 alleles, 26 nonsense alleles and 30 missense alleles could be identified by the criteria of nonsense suppressor sensitivity and leakiness, respectively. Most alleles with a pleiotropic complementation pattern are of the nonsense type. We conclude from the polarity of the complementation patterns characterising the nonsense alleles that the translation direction proceeds from subregion A to subregion E. Antipolar effects in complementation are more frequent than in the analogous system of the arom gene cluster of Neurospora crassa.This work formed part of a Ph.D. thesis submitted to the University of Bern     

Polarity of localised conversion in Streptococcus pneumoniae transformation

Summary Localised conversion in pneumococcal transformation is a process that spans a few nucleotides when the 5-ATTAAT/3-TAAGTA configuration occurs at the pairing step. It was first observed in two-point crosses between an amiA mutation (amiA36) carrying this sequence and other closely linked mutants of the locus. The yield of the amiA resistance allele conversion to wild type is 20%. In order to characterize this process, which differs from longpatch conversion by the length of DNA repair, gene requirements and sequence specificity, we devised expreriments to detect the reciprocal conversion, AmiA⁺ to AmiA^r. For this purpose we examined the suppressibility by a pneumococcal informational suppressor of several nonsense mutations at the locus. Amber (UAG) and ochre (UAA) mutations are suppressed whereas UGA is not suppressed. In this genetic background, where amiA36 is partly suppressed, it was possible to select for double mutants in a cross between amiA36 and a closely linked non-suppressible marker. Direct isolation of such double mutants was also performed without any screening in crosses between amiA36 and the same linked marker in cloned DNA. The frequency of double mutants was very low (1/175) suggesting that there is no conversion of wild-type to mutant alleles. Thus conversion is a polarized process changing specifically A to C.     

Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype

Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the RolA phenotype were observed in 65 000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wild-type rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425 000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.     

Dominant and recessive informational suppressors of a missense mutation in Coprinus

Summary Twenty-one suppressor gene mutations which suppress the met-5.1 missense mutation of Coprinus were separated into six groups (A-F) on the basis of dominance or recessiveness, linkage to the met-5 locus, comlementation in heterozygous cells and growth behaviour. The actual number of suppressor loci could not be determined because crosses between suppressed mutants were inviable. The allele specificity of group A, C, D and F suppressors was confirmed by appropriate crosses. Group B and E suppressors were not tested because of close linkage to the met-5 locus. No evidence for functional suppression of met-5 mutations was obtained thus it is likely that all the suppressors cause translational corelation of met-5.1. Suppressors in four groups (C-F) have properties expected of tRNA structural gene mutations: the group C mutation is dominant, the other mutations are recessive but do not complement in heterozygous cells. The relative efficiencies of the tRNA species involved was assessed by comparing the degree to which the different sup ⁺ mutations depressed the growth rate on methionine supplemented medium. The dominant mutation depressed growth to the greatest extent and is, therefore, the most efficient suppressor. The least efficient suppressors did not depress growth at all. When growth was compared on minimal medium it was found that the more efficient the suppressor the less well it restored growth. The mutations in groups A and B depressed growth more than the tRNA mutations but affect some other component in translation because they are recessive and complement normally. It is suggested that they may act to alter tRNA modifying enzymes.     

Independent localization and regulation of carbamyl phosphate synthetase A polypeptides of Neurospora crassa

Summary Carbamyl phosphate synthetase A is a two-polypeptide, mitochondrial enzyme of arginine synthesis in Neurospora. The large subunit is encoded in the arg-3 locus and can catalyze formation of carbamyl-P with ammonia as the N donor. The small subunit is encoded in the unlinked arg-2 locus and imparts to the holoenzyme the ability to use glutamine, the biological substrate, as the N donor. By using nonsense mutations of arg-3, it was shown that the small subunit of the enzyme enters the mitochrondrion independently and is regulated in the same manner as it is in wild type. Similarly, arg-2 mutations, affecting the small subunit, have no effect on the localization or the regulation of the large subunit. The two subunits are regulated differently. Like most polypeptides of the pathway, the large subunit is not repressible and derepresses 3- to 5-fold upon argininestarvation of mycelia. In contrast, the glutamine-dependent activity of the holoenzyme is fully repressible and has a range of variation of over 100-fold. In keeping with this behavior, it is shown here that the small polypeptide, as visualized on two-dimensional gels, is also fully repressible. We conclude that the two subunits of the enzyme are localized independently, controlled independently and over different ranges, and that aggregation kinetics cannot alone explain the unusual regulatory amplitude of the native, two-subunit enzyme. The small subunit molecular weight was shown to be approximately 45,000.     

mRNA surveillance mitigates genetic dominance in Caenorhabditis elegans

Nonsense mutant mRNAs are unstable in all eucaryotes tested, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. Functions of the seven smg genes are required for mRNA surveillance in Caenorhabditis elegans. In Smg(+) genetic backgrounds, nonsense-mutant mRNAs are unstable, while in Smg(−) backgrounds such mRNAs are stable. Previous work has demonstrated that the elevated level of nonsense-mutant mRNAs in Smg(−) animals can influence the phenotypic effects of heterozygous nonsense mutations. Certain nonsense alleles of a muscle myosin heavy chain gene are recessive in Smg(+) backgrounds but strongly dominant in Smg(−) backgrounds. Such alleles probably express disruptive myosin polypeptide fragments whose abundance is elevated in smg mutants due to elevation of mRNA levels. We report here that mutations in a variety of C. elegans genes are strongly dominant in Smg(−), but recessive or only weakly dominant in Smg(+) backgrounds. We isolated 32 dominant visible mutations in a Smg(−) genetic background and tested whether their dominance requires a functional NMD system. The dominance of 21 of these mutations is influenced by NMD. We demonstrate, furthermore, that in the case of myosin, the dominant-negative effects of nonsense alleles are likely to be due to expression of N-terminal nonsense-fragment polypeptides, not to mistranslation of the nonsense codons. mRNA surveillance, therefore, may mitigate potentially deleterious effects of many heterozygous germline and somatic nonsense or frameshift mutations. We also provide evidence that smg-6, a gene previously identified as being required for NMD, performs essential function(s) in addition to its role in NMD. Received: 10 June 1998 / Accepted: 21 July 1998     

Organization and mapping of legumin genes in Pisum

Summary The organization of three classes of legumin gene has been studied by exploiting restriction fragment length polymorphisms. Hybridization of cDNA probes, representing different sub-families of legumin gene, to DNA from F₂ and F₆ plants from selected crosses showed that there was a tight linkage of individual genes within each of two sub-families. One of these sub-families has been mapped to a locus on chromosome 7 close to r, while the other maps to a locus near a on chromosome 1. The results from one of the crosses analyzed indicated that a third class of legumin gene is also linked to a.     

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     

A single precursor protein for two separable mitochondrial enzymes in Neurospora crassa

The arg-6 locus of Neurospora crassa encodes two early enzymes of the arginine biosynthetic pathway, acetylglutamate kinase and acetylglutamyl-phosphate reductase. Previous genetic and biochemical analyses of this locus and its products showed that: 1) strains carrying polar nonsense mutations in the acetylglutamate kinase gene lacked both enzyme activities (Davis, R.H., and Weiss, R.L. (1983) Mol. Gen. Genet. 192, 46-50), and 2) the proteins isolated from mitochondria were completely separable (Wandinger-Ness, A., Wolf, E.C., Weiss, R.L., and Davis, R.H. (1985) J. Biol. Chem. 260,5974-5978). These data suggested that the two enzymes were initially synthesized as a single precursor which was subsequently cleaved into two distinct polypeptides. We report here the identification of a high molecular weight protein, synthesized in vitro from isolated N. crassa RNA, that contains sequences corresponding to acetylglutamate kinase as well as acetylglutamyl-phosphate reductase. An analogous precursor was identified in vivo by pulse-labeling experiments. The precursor was similar to other mitochondrial precursors in that its uptake and processing in vivo was rapid and required an intact mitochondrial electrochemical gradient. This represents the first report of a bifunctional protein precursor which gives rise to two mitochondrial enzymes.     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

Unidirectional gene conversions in the chloroplast of Chlamydomonas interspecific hybrids

Summary With the goal of studying directly the inheritance and recombination of physically mapped markers on the chloroplast genome, we have recently identified and localized physical differences between the chloroplast DNAs (cpDNAs) of the interfertile algae Chlamydomonas eugametos and C. moewusii. Here we report the inheritance patterns of 24 polymorphic loci mapping throughout the chloroplast genome in hybrids recovered from reciprocal crosses between the two algae. Most polymorphic loci were found to be inherited mainly from the mt ⁺ parent, with no apparent preference for one or the other parental alternatives in reciprocal crosses. Virtually all hybrids, however, inherited exclusively the long alleles of three loci; i.e. an intron in the large subunit ribosomal RNA gene of C. eugametos, a 21 kbp sequence addition in the inverted repeat of the C. moewusii cpDNA and a 5.8 kbp sequence addition in one of the single-copy regions of C. moewusii cpDNA. As these alleles are derived from opposite parental strains, their unidirectional inheritance in hybrids results necessarily from interspecific recombination of cpDNA molecules. We propose that gene conversion events led to the spreading of the long alleles of the three loci.     

Identification of alleles at the gliadin loci Gli-U1 and Gli-Mb1 in Aegilops biuncialis Vis.

The diversity of alleles at the gliadin loci Gli-U1 and Gli-M ^b 1 was studied in the tetraploid species Aegilops biuncialis (UUM^bM^b). The collection of 41 Ae. biuncialis accessions and F₂ seeds obtained from five crosses served as the material used in this study. Gliadins were separated by acid polyacrylamide gel electrophoresis. To determine genomic affiliation (U or M^b) of components of Ae. biuncialis gliadin pattern, accessions of Ae. umbellulata and Ae. comosa were analyzed. In Ae. biuncialis accessions, 14 alleles were identified at the locus Gli-U1 and 12 alleles, at the locus Gli-M ^b 1. The results testify to a high degree of allele diversity at major gliadin-coding loci of homeologous group 1 chromosomes of Ae. biuncialis.     

Instability at the y-1 locus of Chlamydomonas reinhardtii

Summary Twenty-three spontaneous yellow mutants were isolated from two stable green strains of the unicellular green alga Chlamydomonas reinhardtii. Genetic characterization indicated that 22 of 23 mutants had a mutation at the y-1 locus, and all 22 y-1 alleles were unstable. Crosses designed to follow the inheritance of instability at the y-1 locus showed that instability is caused by a single genetic factor located at the y-1 locus or very close to it.     

Genetics of gliding motility in Myxococcus xanthus: Molecular cloning of the mgl locus

Summary Wild-type Myxococcus xanthus cells move across solid surfaces by gliding. However no locomotory organelles for gliding have as yet been identified. Two sets of genes are required for gliding in M. xanthus: Gene System A is necessary for the gliding of isolated cells and Gene System S comes into play when cells are close together. The product of the mgl locus is required for both types of gliding and therefore may be a structural component of the gliding organelle. To begin to investigate the function of mgl in gliding a 12 kb segment of M. xanthus DNA containing the locus was cloned in Escherichia coli and returned to Myxococcus by specialized transduction with coliphage P1. In M. xanthus the chimeric plasmid integrates into the chromosome by recombination between the cloned segment and its homolog in the recipient chromosome forming a tandem duplication of the cloned segment with the vector sequences at the novel joint. The construction of partial diploids in this manner facilitated dominance tests and interallelic crosses with ten mgl alleles. We also describe a method for the analysis of tandem duplications that precisely maps alleles to a specific copy of the duplicated sequences. This method provides evidence for the dominance of mgl ⁺ over the mgl ^- alleles. It also reveals what appears to be gene conversion at this locus during recombination between a cloned mgl sequence and its homolog in the chromosome.