Identification of nonsense mutations in Neurospora: Application to the complex arg-6 locus

Summary The arg-6 locus of Neurospora crassa encodes two enzymes of arginine synthesis, acetylglutamate kinase and acetylglutamyl phosphate reductase. Mutants lacking one or the other enzyme fall into two different complementation groups; a large non-complementing group lacks both enzymes. We wished to survey over 50 alleles for suppressibility by a nonsense suppressor. We compared two methods of assessing suppressibility. One, based on trans-action of a nonsense suppressor, was simple, but not efficient in detecting all suppressible alleles. The other, based on crosses involving a marker linked to the locus surveyed was very efficient in methodology and is suited to all cases, such as the arg-6 locus, in which allelic crosses are sterile. The data indicate that the arg-6 locus encodes a bifunctional protein.     

Developmental genetic analysis of lethal alleles at the ewg locus and their effects on muscle development in Drosophila melanogaster

The recessive X-linked mutation erect wing (ewg), in Drosophila melanogaster, was characterized as a flightless behavioral mutant which specifically lacked the dorsal longitudinal flight muscles [1]. This mutation was mapped distal to the X chromosomal locus yellow, and further to the cytological segment 1 A 1 to 1 B2-3 [2]. Several lethal complementation groups have been mapped to this interval [3]. Our complementation tests show that ewg is allelic to one lethal complementation group in the region 1 A 1 to 1 B2-3. A further analysis of ewg and several lethal alleles isolated at this locus was undertaken in the present investigation. Most of the lethal alleles at this locus lead to a late embryonic or early larval lethal phase, indicating that the ewg⁺ gene product is necessary for the development of more than just the dorsal longitudinal flight muscles. Intragenic complementation was observed for some of the ewg lethal alleles. Genetic mosaics with ewg lethal alleles showed that mutant cell clones in cuticular structures are viable. Mosaic analysis is consistent with a mesodermal defect associated with the locus.     

Co-Localization to Chromosome Bands 99e1-3 of the Drosophila Melanogaster Myosin Light Chain-2 Gene and a Haplo-Insufficient Locus That Affects Flight Behavior

Using overlapping synthetic deficiencies, we find that a haplo-insufficient locus affecting flight behavior and the myosin light chain-2 gene co-map to the Drosophila melanogaster polytene chromosome interval 99D9-E1 to 99E2-3. From screening over 9000 EMS-treated chromosomes, we obtained alleles of two complementation groups that map to this same interval. One of these complementation groups lfm(3)99Eb, exhibits dominant flightless behavior; thus, flightless behavior of the deficiency is in all likelihood due to hemizygosity of this single locus. Rescue of flightless behavior by a duplication indicates that the single allele, E38, of the Ifm(3)99Eb complementation group is a hypomorph. Based upon its map position and a reduction in concentration of myosin light chain-2 mRNA in heterozygotes, we propose that Ifm(3)Eb(E38) is a mutant allele of the myosin light chain-2 gene. Our genetic analysis also resulted in the identification of four dominant flightless alleles of an unlinked locus, l(3)nc99Eb, that exhibits dominant lethal synergism with Ifm(3)99Eb.     

Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen

We have performed an F₂ genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. Received: 13 July 1999 / Accepted: 4 November 1999     

Mutants of Escherichia coli with temperature-sensitive lesions in membrane phospholipid synthesis: genetic analysis of glycerol-3-phosphate acyltransferase mutants

Summary Escherichia coli mutants possessing temperature-sensitive lesions in glycerol-3-phosphate acyltransferase, the enzyme catalysing the first step in phospholipid biosynthesis, have been characterized genetically. By recombinational and complementation tests, the mutants have been found to map in a single locus, called plsA, which is cotransducibile with the purE locus and lies between the purE and proC loci at minute 13 on the E. coli genetic map.     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

Omnipotent Suppressors Effective in psi Strains of SACCHAROMYCES CEREVISIAE: Recessiveness and Dominance

We have characterized recessive and dominant omnipotent suppressor mutations obtained by conversion of the leu2-1 UAA mutation and the met8-UAG mutation in a ψ⁺ strain of Saccharomyces cerevisiae. The suppressors that act recessively upon these markers fell into two complementation groups; the sup47 and sup36 suppressors show linkage to the tyr1 locus and the aro1 locus, respectively. Of the suppressors acting dominantly upon both markers, those linked to the tyr1 locus are alleles of the SUP46 ribosomal mutation. The sup47 suppressors differ from the SUP46 suppressors not only in their suppressor activities in heterozygous diploids but also in their map positions relative to the tyr1 locus and their effects on the S11 ribosomal protein. The remaining dominant suppressors are not alleles of sup36 as judged by linkage analysis. The recessive suppressors and the dominant suppressors also differ in their effects on cell growth.     

The evolutionary fate of heterogeneous gene duplications: A precarious overdominant equilibrium between environment,sublethality and complementation

Gene duplications occur at a high rate. Although most appear detrimental, some homogeneous duplications (identical gene copies) can be selected for beneficial increase in produced proteins. Heterogeneous duplications, which combine divergent alleles of a single locus, are seldom studied due to the paucity of empirical data. We investigated their role in an ongoing adaptive process at the ace‐1 locus in Culex pipiens mosquitoes. We assessed the worldwide diversity of the ace‐1 alleles (single‐copy, susceptible S and insecticide‐resistant R, and duplicated D that pair one S and one R copy), analysed their phylogeography and measured their fitness to understand their early dynamics using population genetics models. It provides a coherent and comprehensive evolutionary scenario. We show that D alleles are present in most resistant populations and display a higher diversity than R alleles (27 vs. 4). Most appear to result from independent unequal crossing‐overs between local single‐copy alleles, suggesting a recurrent process. Most duplicated alleles have a limited geographic distribution, probably resulting from their homozygous sublethality (HS phenotype). In addition, heterozygotes carrying different HS D alleles showed complementation, indicating different recessive lethal mutations. Due to mosaic insecticide control practices, balancing selection (overdominance) plays a key role in the early dynamics of heterogeneous duplicated alleles; it also favours a high local polymorphism of HS D alleles in natural populations (overdominance reinforced by complementation). Overall, our study shows that the evolutionary fate of heterogeneous duplications (and their long‐term role) depends on finely balanced selective pressures due to the environment and to their genomic structure.     

N-Ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse Chromosome 7

Two loci [l(7)5Rn and l(7)6Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the albino (c) locus in Chromosome (Chr) 7. Both loci were found to map to the subregion of the Mod-2-sh-1 interval that contains the eed (embryonic ectoderm development) locus. eed has been defined by the inability of embryos homozygous for certain c deletions to develop beyond the early stages of gastrulation. Evidence for at least two loci necessary for normal prenatal development, rather than one locus, that map within the eed interval came from the observation that two prenatally lethal mutations, 3354SB [l(7)5Rn ^3354SB ] and 4234SB [l(7)6Rn ^4234SB ], could complement each other in trans, but could not each be complemented individually by c deletions known to include the eed locus. A somewhat leaky allele of l(7)5Rn [l(7)5Rn ^1989SB ] was also recovered, in which hemizygotes are often stillborn and homozygotes exhibit variable fitness and survival. The mapping of the loci defined by these mutations is likely to be useful for genetic, molecular, and phenotypic characterization of the eed region, and mutations at either locus (or both loci) may contribute to the eed phenotype.     

A method for rapid mapping of mutations by plasmid rescue strategy inSaccharomyces cerevisiae

The products ofPRP17 andPRP18 genes are required for the second step of pre-mRNA splicing reactions inSaccharomyces cerevisiae. Temperature-sensitive mutants at either of these loci accumulate products of the first splicing reaction at nonpermissive temperature. To characterize functional regions in these proteins the mutations in three temperature-sensitive alleles ofPRP17 and two temperature-sensitive alleles ofPRP18 were mapped by the plasmid rescue strategy, One of the procedures adopted in the past is plasmid rescue of the mutant allele followed by sequencing of the entire gene. In this work we describe an adaptation of the above procedure that allows, first, rapid mapping of chromosomal segments bearing the mutations, followed by sequence characterization of the minimal segment. The strategy adopted was to integrate a wild-type copy of the gene at the homologous mutant chromosomal locus, followed by recovery of the chromosomal fragments from these integrants as plasmids inE. coli. The recovered plasmids were screened by a complementation assay for those that contained in them the chromosomal mutation. The mutations in all the three alleles ofPRP17 map to a small region in the N-terminal half of the protein, whereas the temperature-sensitive mutations in the two alleles ofPRP18 map to different regions of the PRP18 protein. The recovered mutant plasmids from all five alleles at the two loci were sequenced and the nucleotide changes were found to result in missense mutations in each case. Our strategy is therefore a rapid method to map chromosomal mutations and is of general use in structure-function analysis of cloned genes.     

Genetic and Physiological Characterization of met15 Mutants of SACCHAROMYCES CEREVISIAE: a Selective System for Forward and Reverse Mutations

One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus.—It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.     

An analysis of xanthine dehydrogenase negative mutants of the rosy locus in Drosophila melanogaster.

Eighteen alleles of the rosy locus in Drosophila melanogaster were characterized to identify putative nonsense mutants. Seven alleles exhibited no evidence of intragenic complementation, no evidence of immunological complementation, no evidence of immunological cross-reactivity to antibodies elicited by wild type xanthine dehydrogenase (XDH), and of course were completely deficient in measurable XDH activity. It is possible that one or more of these highly negative ry alleles are nonsense mutants. The remaining eleven ry alleles code for XDH molecules that retain some antigenic similarities to the wild type enzyme as assessed by immunoelectrophoresis and six of these eleven were capable of intragenic complementation.     

Immune response of mice to the Thy-1.1 antigen: intra-H-2 mapping of the complementary Ir-Thy-1 loci.

The primary immune response to the Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes. F1 hybrids which were b/d heterozygous at the entire H-2 complex or at only some of its regions were studied. All F1 hybrids which were b/d heterozygotes at the K, IA and possibly IB regions displayed a complementary effect of the alleles at the Ir-Thy-1A and Ir-Thy-1B loci. The complementary effect was indicated by the significantly higher responses of the F1 hybrids than the responses of their parental strains. No complementary effect could be demonstrated in F1 hybrids which were b/d heterozygous at the IB, IC, S and D regions, but not at the K and IA regions. The results excluded participation of the IC, S and D regions in the phenomenon of the complementation. Furthermore, one of the Ir-Thy-1 loci could be mapped to the left of the IB subregion, most likely in the IA subregion of the H-2 complex. The second Ir-Thy-1 locus could be mapped to the left of the IC subregion in either the IA or IB subregion.     

Nonsense-missense suppression in yeast

Summary A dominant suppressor has been discovered which suppresses polarity type mutations, noncomplementing ones and those belonging to short nonpolar complementation groups at the ad ₂ locus of S. cerevisiae. The suppressor translates two types of nonsense previously established at ad ₂. These data together with theoretical consideration of relationships between nonsense-translating anticodons and different codons make it possible to suggest a mechanism for the observed suppression. According to this mechanism two nonsense types of ad ₂ are identified as amber and ochre codons.     

Temperature-Sensitive Mutations of the Notch Locus in DROSOPHILA MELANOGASTER

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)N^ts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax^59d5 is found to be temperature-sensitive, while fa ^g, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fa^g, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fa^g, spl, and l(1)N cause defects at specific, non-overlapping times in development.—We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.     

Genetic Analysis of the Dumpy Complex Locus in DROSOPHILA MELANOGASTER: Complementation, Fine Structure and Function

An extensive genetic analysis of the dumpy locus is presented. This study includes complementation, fine structure mapping and allelic interaction. A number of complementing recessive lethals of the dp complex have been genetically mapped. Two alleles of the ol(v) type that complement l alleles map to the left portion of the locus. A number of olv alleles that complement both l and lv lethals map within the right portion of the locus.——Fine-structure analysis demonstrated that both olv and o alleles are distributed among various subloci. Evidence for spacer regions between subloci is presented.——An extensive discussion of the data considers whether the locus is unicistronic or multicistronic. The conclusion reached is that the locus is not a single functional cistron. The possibility of a single cistron encoding a multifunctional polypeptide is discussed.——The hypothesis is proposed that the left portion of the map and the l mutations function as regulatory sequences and that the right portion of the map encodes structural sequences.     

Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas

Summary We have carried out a molecular and genetic analysis of the chloroplast ATPase in Chlamydomonas reinhardtii. Recombination and complementation studies on 16 independently isolated chloroplast mutations affecting this complex demonstrated that they represent alleles in five distinct chloroplast genes. One of these five, the ac-u-c locus, has been positioned on the physical map of the chloroplast DNA by deletion mutations. The use of cloned spinach chloroplast ATPase genes in heterologous hybridizations to Chlamydomonas chloroplast DNA has allowed us to localize three or possibly four of the ATPase genes on the physical map. The beta and probably the epsilon subunit genes of Chlamydomonas CF₁ lie within the same region of chloroplast DNA as the ac-u-c locus, while the alpha and proteolipid subunit genes appear to map adjacent to one another approximately 20 kbp away. Unlike the arrangement in higher plants, these two pairs of genes are separated from each other by an inverted repeat.     

Genetic Fine Structure Analysis of the AMYLOSE-EXTENDER Locus in ZEA MAYS L

On the basis of interallelic recombination frequencies measured in diallelic crosses of the 5 amylose-extender alleles in maize, ae, B1, B3, M2 and i1, it was possible to construct a unique linear genetic map ordering all 5 alleles within the locus. The reciprocal diallelic crosses each gave comparable frequency estimates. The relative order is ae, i1, B3, B1, M2 or the reverse. Even though F1 endosperms resulting from all possible diallelic crosses were phenotypically mutant, therefore non-complementing, no decision as to whether or not these alleles exhibit functional complementation should be made without biochemical characterization of the starches from the various heteroallelic genotypes.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998