Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants at both loci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An anlysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show verly little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104^D1 and mei-41^D5 are located in th eregion 14B13±?14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101^D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103^D1 is between bands 12A1,2 and 12A6,7, and mus(1)-109^A1 is in section 8F3-9A2.     

The Isolation and Characterization of Mutants Defective in Nitrate Assimilation in NEUROSPORA CRASSA

The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described. The mutants isolated can be subdivided into five classes on the basis of growth tests that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing co-factor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-7, nit-8 and nit-9), all of which are genetically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar to the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1, 7, 8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one locus (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.     

Glucosamine Resistance in Yeast. I. a Preliminary Genetic Analysis

Mutants of the yeast Saccaromyces cerevisiae which can grow on glycerol medium in the presence of 0.05% D (+) glucosamine have been isolated. Genetic analysis of 13 of these glucosamine resistant (GR) mutants demonstrated two modes of inheritance. One group of mutants (GR 5, 6, 7, 8, 9 and 10) gave results characteristic of non-Mendelian inheritance and it is suggested that these mutants represent one or more new mitochondial loci. Four of the remaining mutants showed clear-cut Mendelian inheritance. These mutants fell into two complementation groups and subsequent mapping experiments demonstrated that two independent loci, gay 1 and gay 2, unlinked to each other or to the centromeres of chromosomes I, II, IV, VIII or IX, were responsible for conferring glucosamine resistance in these mutants.     

Genetic Fine Structure of the Y Chromosome of DROSOPHILA HYDEI

A genetic map of the Y chromosome of Drosophila hydei has been constructed from deletion/complementation experiments, with the aid of male sterile mutants of the Y chromosome. A central conclusion of our experiments is that not more than a single complementation group can be detected in each of the lampbrush loop forming sites. Additional complementation groups, functionally independent of lampbrush loops, reside between these loci. Six complementation groups have been defined by several methods of mapping. An additional ten complementation groups are indicated, but their exact definition requires further investigation. The "synthetic sterility" of mutations in these ten loci contributes to the difficulty in unequivocally establishing their individual boundaries. Mapping problems also arise from the instability of certain mutants.     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Isolation and Characterization of X-Linked Mutants of DROSOPHILA MELANOGASTER Which Are Sensitive to Mutagens

Thirteen X-linked mutants have been isolated in Drosophila melanogaster which render male and homozygous female larvae sensitive to the mutagen methyl methanesulfonate. Their characterization and preliminary assignment to functional groups is described. Four of these mutants are alleles of mei-41 (Baker and Carpenter 1972). Like previously isolated alleles of this locus, these mutants reduce fertility and increase loss and nondisjunction of the X-chromosome in homozygous females. The remaining mutants have been tentatively assigned to six functional groups (two mutants to the mus(1)101 locus, two to mus(1)102 , two to mus(1)103, and one each to mus(1)104, mus(1)105 , and mus(1)106). Several of the complementation groups can be distinguished on the basis of nondisjunction and cross sensitivity to mutagens. Females homozygous for the mei-41, mus(1)101 and mus(1)102 mutants exhibit elevated levels of nondisjunction. Mutants belonging to complementation groups mei-41, mus(1)101, and mus(1)104 are sensitive to nitrogen mustard (HN2) in addition to their MMS sensitivity. Among these mutants there is currently a direct correlation between sensitivity to HN2, sensitivity to 2-acetylaminofluorene and a deficiency in post-replication repair ( Boyd and Setlow 1976). Only the mei-41 mutants are hypersensitive to UV radiation, although several of the mutants exhibit sensitivity to gamma-rays. Semidominance is observed in female larvae of the mei-41, mus(1)104, and mus(1)103 mutants after exposure to high concentrations of MMS. The properties of the mutants generally conform to a pattern which has been established for related mutants in yeast. Additional properties of these mutants are summarized in Table 9.     

Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE

Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular.—Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes.—Part of this work has been reported in preliminary form (Culbertson and Henry 1974).     

Pathway analysis of radiation-sensitive meiotic mutants ofCoprinus cinereus

We have isolated 37 radiation-sensitive mutants of the basidiomyceteCoprinus cinereus. Each mutation is recessive, and the collection defines at least ten complementation groups for survival of gamma irradiation. Four complementation groups define the genesrad3, rad9, rad11 andrad12, which are required both for survival of gamma irradiation and for meiosis. Mutants in each of these four groups fail to complete meiosis and produce mushrooms with greatly reduced numbers of viable spores. Propidium iodide staining of meiotic nuclei showed a characteristic terminal appearance for each mutant: few cells of any of the meiotic mutants progress beyond prophase I, and both condensation and fragmentation or dispersal of meiotic chromatin are frequently observed. Scanning electron micrographs showed that the meiotic mutants make varying numbers (0–6) of basidiospore initials and that few of these initials develop into mature spores. When initials are present they are always symmetrically arrayed on the basidium, regardless of initial number. In quantitative measurements of gamma ray sensitivity, double mutants of every tested combination ofrad3, rad9, rad11 andrad12 consistently showed the same gamma ray sensitivity as the more sensitive single mutant parent of the cross. Therefore, these four genes are in the same pathway for the repair of gamma radiation damage, and this pathway also represents one or more functions essential for meiosis.     

Complementation Analysis of Metabolite-Resistant Mutations with Forced Heterokaryons of NEUROSPORA CRASSA

The double mutant strain pyr-3 arg-12^s is a prototroph because a common precursor of arginine and pyrimidine is supplied by the arginine pathway. Growth of this strain is inhibited by exogenous citrulline or arginine. Citrulline-resistant mutants of this strain were selected, and they resulted from modifier mutations at other loci. Forced heterokaryons were used to study complementation among these modifiers. Since the complementation test requires the scoring of non-growth as the positive result, there was concern that variations in nuclear ratios could give erroneous results. This possibility does not seem significant, since groups of mutants established by complementation correspond with groups established by physiological, enzymatic, and recombinational measurements.—The technique has revealed that the most frequently mutated loci are arg-1 and what is probably un-3. Arg-1 mutations affect the conversion of citrulline to argininosuccinate, while un-3 mutations reduce the citrulline uptake rate. Since most of these mutations are of the intracistronic complementing type, a complementation map was constructed for most of the affected loci. The high proportion of complementors in each map can be explained by assuming that partially functioning gene products are more likely to complement with each other than are those which are nonfunctional.     

Isolation of yeast mutants sensitive to the bifunctional alkylating agent nitrogen mustard

Summary Mutants of Saccharomyces cerevisiae with enhanced sensitivity to the DNA cross-linking agent nitrogen mustard (HN2) have been isolated and partially characterized with respect to their phenotypic and genetic properties. The screening technique, based on HN2-sensitivity as sole criterion, yields approximately 1 sensitive isolate in 200 clones when applied to an intensively mutagenized population of a resistant parent strain. Mutants characterized so far are all due to recessive nuclear genes and represent at least seven complementation groups. They exhibit different degrees as well as different patterns of sensitivity towards monofunctional and bifunctional alkylating agents, and ultraviolet light.     

Interaction of Colicins with Bacterial Cells III. Colicin-tolerant Mutations in Escherichia coli

Mutants that adsorb certain colicins without being killed, i.e., tolerant mutants (tol), were isolated from Escherichia coli K-12 strains. Selection was done either with colicin K or E2. Several groups of mutants showing different phenotypes were found, and some of them showed tolerance to both K and E colicins, which have different receptors. Many of these mutants mapped near gal. Typical mutants from group II, III, and IV were studied in more detail. The mutant loci were contransducible with gal by phage P1. The linkage order was deduced to be tol-gal-λ. In partially diploid strains, these mutant loci are recessive to wild-type alleles. Temperature-dependent conditionally tolerant mutants were also isolated. Two groups were found: the first was tolerant to E2 and E3 at 40 C, but sensitive at 30 C; the second was tolerant to E2 at 30 C, but sensitive at 40 C. Experiments done with these mutants suggest that these mutations affect the heat lability of some protein that is necessary for the response of cells to colicins. Conditionally lethal tolerant mutants were isolated which at 40 C were tolerant to E2 and E3 and could not grow, but which at 30 C were fully sensitive and grew normally. The mutation mapped near malA. The tolerance at 40 C is not due to a consequence of an inactivation of general cellular metabolism, but presumably is a cause of the subsequent inhibition of cellular growth. The results suggest that some protein components involved in the response to colicin are also vital to normal cellular growth.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

Gene Expression During the Development of Bacteriophage φ29 III. Analysis of Viral-Specific Protein Synthesis with Suppressible Mutants

Fifty-four suppressible mutants of bacteriophage phi29 have been isolated with a variety of mutagens and assigned to eight complementation groups. Viral-specific protein synthesis in UV light-irradiated, nonsuppressing Bacillus subtilis 60084 was analyzed with exponential acrylamide gels. Four additional phi29 proteins which were undetected on ordinary acrylamide gels are reported in this paper. Five phage phi29 proteins have been unambiguously assigned to specific cistrons. Two cistrons had pleiotropic effects on viral protein synthesis. Mutants in cistrons I or II were unable to synthesize DNA in nonsuppressing bacteria. Mutants in cistron I were unable to attach viral chromosomes to the host cell membrane, and the protein responsible for this function has been identified. The other viral protein playing a role in phage phi29 DNA synthesis is also identified and assigned to cistron II. Mutants in cistron II can attach viral chromosomes to membrane, but cannot synthesize DNA in nonsuppressing bacteria.     

Osmotic Avoidance Defective Mutants of the Nematode CAENORHABDITIS ELEGANS

A wild-type strain of the nematode Caenorhabditis elegans has been shown to avoid high concentrations of a number of sugars and salts. Individual and population assays for this response were developed and mutants were selected for their inability to avoid high concentrations of fructose or NaCl. Seven nonavoiding mutants representing six complementation groups were isolated and characterized. Genetic studies indicate that the mutants each carry a single recessive mutation responsible for the defective osmotic avoidance behavior. The map locations of the six complementation groups identified by these mutations have been determined. Mutants isolated for their inability to avoid fructose are also unable to avoid NaCl and vice versa. The mutants move normally, exhibit normal touch sensitivity, and, like wild type, follow isotherms in a radial thermal gradient. All of the mutants are at least partially defective in the attraction to sodium chloride exhibited by wild type. None of the mutants is temperature sensitive, and all exhibit defective osmotic avoidance behavior as young L1 larvae. Preliminary anatomical studies indicate selective sensory neuron changes in at least one mutant.     

Behavioral Mutants in PARAMECIUM CAUDATUM

Mutants of Paramecium caudatum with abnormal swimming behavior or responses to cations were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Some of the mutants, like pawn in P. tetraurelia, cannot swim backward and are called CNR. Seven independently obtained CNR mutants belonged to three complementation groups, designated as cnrA, cnrB and cnrC. Some characteristics of double homo- and heterozygotes were compared with single homo- and heterozygotes. Other behavioral mutants shown to have a genic basis included K⁺-sensitive, temperature-shock behavioral and slow swimmer. All those mutants except for slow swimmer had lesions in the membrane because Triton-extracted models of them show almost the same swimming behavior as wild type.     

Genetic Analysis of the Antennapedia Gene Complex (Ant-C) and Adjacent Chromosomal Regions of DROSOPHILA MELANOGASTER. II. Polytene Chromosome Segments 84A-84B1,2

The existence of a gene complex in the proximal right arm of chromosome 3 of Drosophila melanogaster involved in the development of the head and thorax was originally suggested by the phenotypes of several dominant homoeotic mutations and their revertants. A screen for mutations utilizing Df(3R) Antp^Ns+R17 (proximally broken in salivary region 84B1,2) yielded, among 102 recovered mutations, 17 localized by deficiency mapping to the putative homoeotic cluster. These fell into four complementation groups, two of which were characterized by homoeotic phenotypes. To explore the limits of the Antennapedia gene complex (ANT-C) more proximally, a second screen has been undertaken utilizing Df(3R)Scr, a deficiency of 84A1–B1,2.—Of 2832 chromosomes screened, 21 bearing alterations localized to polytene interval 84A–84B1,2 have been recovered. Sixteen are recessive lethals, and five showing reduced viability display a visible phenotype in surviving individuals. Complementation and phenotypic analyses revealed four complementation groups proximal to those identified in the previous screen, including two new alleles of the recessive homoeotic mutation, proboscipedia (pb). Ten of the new mutations correspond to complementation groups defined previously in the Df(3R)Antp^Ns+R17 screen four to the EbR11 group, two to the Scr group and four to the Antp group.—On the basis of the phenotypes of the 39 mutations localized to this region, plus their interactions with extant homoeotic mutations, we postulate that there are at least five functional sites comprising the ANT-C. Three have been demonstrated to be homoeotic in nature. The specific homoeotic transformations thus far observed suggest that these loci are critical for normal development of adult labial, maxillary and thoracic structures.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

Genetics of Extracellular Protease Production in SACCHAROMYCOPSIS LIPOLYTICA

Mutants of Saccharomycopsis lipolytica with reduced ability to produce zones of clearing on skim-milk agar plates were isolated and their properties studied. For 18 mutants it was possible to score unambiguously segregants of crosses between these mutants and wild type for extracellular protease production. These mutants all produce reduced levels of extracellular protease in liquid culture. The mutations are recessive and are in nuclear genes. The 18 mutations define 10 or 11 complementation groups, no two of which are closely linked. Mutants in four of the complementation groups also produced reduced levels of extracellular RNAse, and the reduced levels of extracellular protease and RNAse production segregate together. Five of the mutants exhibited reduced mating frequency, and one mutant was osmotic remedial for extracellular protease production. These results demonstrate that many genes can affect extracellular protease production. Besides mutations in the structural gene and in regulatory genes, mutations are likely to be in genes involved in steps common to the production of several extracellular enzymes or in genes coding for cell wall or membrane components necessary for extracellular enzyme production.