Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

Genetic Analysis of the Reciprocal Translocation T2(I;VIII) of Aspergillus Using the Technique of Mitotic Mapping in Homozygous Translocation Diploids

A UV-induced sulphite-requiring mutant (sD50) consistently shows mitotic linkage to groups I and VIII in haploids from heterozygous mapping diploids. This linkage was found to be due to a reciprocal translocation T2(I;VIII) which could not be separated from the sulphite requirement in about 100 tested progeny from heterozygous crosses, and both may well have been induced by the same mutational event. T2(I;VIII) is the first case of a reciprocal translocation in Aspergillus which showed meiotic linkages between markers of different linkage groups, and, in addition, involved chromosome arms containing markers suitable for complete mapping by the technique of mitotic recombination in homozygous translocation diploids.-Using various selective markers, haploid segregants and diploid crossovers of all possible types were isolated from homozygous translocation diploids. (1) Haploid segregants showed new linkage relationships in T/T diploids: all available markers of VIII now segregated as a group with the majority of the markers of I, except for the markers of the left tip of I. These formed a separate linkage group and are presumably translocated to VIII. (2) Diploid mitotic crossovers confirmed this information and showed that the orientation of the translocated segments was unchanged. These findings conclusively demonstrate that T2(I;VIII) is a reciprocal translocation due to an exchange of the left tip of group I with the long right arm of group VIII.-Since the position of the break on VIIIR was found to be at sD50 this marker could be used to map the break on IL by meiotic recombination in heterozygous crosses. In addition, such crosses showed reduced recombination around the breaks, so that it was possible to sequence markers which normally are barely linked.     

The Structural Gene for α-Mannosidase-1 in DICTYOSTELIUM DISCOIDEUM

We have isolated 4 independent mutations affecting alpha-mannosidase-1, a developmentally regulated activity in Dictyrostelium discoideum. Three of these result in a thermolabile alpha-mannosidase-1 activity. One mutation also affects the substrate affinity (Km) of the activity. In diploids these mutations show a gene dosage effect and are all alleles. The structural gene for alpha-mannosidase-1, as defined by these mutations, defines a new linkage group, linkage group VI. alpha-mammosidase 1 is probably a homopolymer with subunits of 54,000 daltons. We have also mapped two temperature-sensitive-for-growth mutations onto two previously defined linkage groups.     

Choice of microsatellite markers for identifying homozygosity of mitotic gynogenetic diploids in Japanese flounder Paralichthys olivaceus

A set of 72 microsatellite markers distributed evenly among 24 linkage groups were selected from the published genetic linkage maps of Japanese flounder Paralichthys olivaceus. In two normal diploid full‐sib families, the test for Mendelian inheritance showed that genotypic segregation deviations were not significant at all analysed loci. To estimate microsatellite‐centromere map distances, four meiotic gynogenetic diploid lines were produced by the activation of eggs using UV irradiated sperm of red seabream Pagrus major and cold‐shock treatment to block the extrusion of the second polar body. Under the assumption of complete interference, 21 markers were located in the centromeric region, 39 in the telomeric region and the rest in the intermediate region of linkage groups. A total of 192 mitotic gynogenetic diploids from one spawn were identified by these markers. Genotype analysis showed that the number of homozygous individuals decreased as microsatellite‐centromere map distance increased on each linkage group.     

New Loci in DICTYOSTELIUM DISCOIDEUM Determining Pigment Formation and Growth on BACILLUS SUBTILIS

Seventeen independently isolated pigmentless (white) mutations in Dictyostelium discoideum are all recessive and fall into three complementation groups identifying two new whi loci in addition to the previously characterized whiA locus. whiB and whiC map to linkage groups III and IV, respectively. In addition, it was discovered that our laboratory stock of NC4, the wild-type strain from which these mutants were derived, has spontaneously lost the ability to grow on Bacillus subtilis. This new mutation, bsgB500, maps to linkage group VII and is not allelic to bsgA. bsgB500 is the first spontaneously derived mutation in D. discoideum that can be used to select heterozygous diploids, and for the first time allows genetic analysis to be routinely performed on strains derived from an unmutagenized background.     

The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast

In yeast, the functions controlled by radiation-repair genes RAD6, RAD50, RAD52 and RAD57 are essential for normal meiosis; diploids with lesions in these genes either fail to sporulate (rad6) or sporulate but produce inviable spores (rad50, 52, 57). Since RAD genes may control aspects of DNA metabolism, we attempted to define more precisely the role of each gene in meiosis, especially with regard to possible roles in premeiotic DNA replication and recombination. We constructed diploids singly homozygous for each of the four rad mutations, heteroallelic at his1 and heterozygous for a recessive canavanine-resistance marker. Each strain was exposed to sporulation-inducing conditions and monitored for (1) completion of mitotic cell cycles, (2) cell viability, (3) utilization of acetate for mass increases, (4) premeiotic DNA synthesis, (5) intragenic recombination at his1, and (6) formation of viable haploid spores. Control strains heterozygous for the rad mutations completed mitosis, metabolized acetate, replicated their DNA, and showed typically high levels of gene conversion and viable-spore formation. The mutant diploids also completed mitosis, utilized acetate, and carried out premeiotic DNA replication. The mutants, however, showed little or no meiotic gene conversion. The rad50, 52 and 57 strains sporulated, but the spores were inviable. The rad6 strain did not sporulate. The rad50, 52 and 57 strains exhibited viability losses that coincided with the period of DNA synthesis, but not with later meiotic events; the rad6 strain did not lose viability. We propose that the normal functions specified by RAD50, 52 and 57 are not essential for either the initial or terminal steps in meiosis, but are required for successful recombination. The rad6 strain may be recombination-defective, or it may fail to progress past DNA replication in the overall sequence leading to formation and recovery of meiotic recombinants.     

Translocations in DICTYOSTELIUM DISCOIDEUM

Fourteen translocations of independent origin were identified in Dictyostelium discoideum on the basis of segregation anomalies of diploids heterozygous for these chromosome rearrangements, all of which led to the cosegregation of unlinked markers. Many of these translocations were discovered in strains mutagenized with MNNG or in strains carrying mutations affecting DNA repair; however, spontaneous translocations were also obtained. Haploid mitotic recombinants of the rearranged linkage groups were produced from diploids heterozygous for the translocations at frequencies of up to 5% of viable haploid segregants; this is at least a ten-fold higher frequency than that seen with diploids not heterozygous for translocations (approximately 0.1%). These haploid recombinants included both translocated and nontranslocated strains. The T354(II, VII) translocation and possibly the T357(IV, VII) translocation reduce the chromosome number to n = 6; haploids carrying 11 other translocations all have karyotypes with n = 7. Genetic characterization of the T357(IV, VII) translocation showed that the bwnA and whiC loci normally found on linkage group IV were physically linked to the linkage group VII loci couA, phgA, bsgB and cobA.     

Molecular and Genetic Analysis of Rec103, an Early Meiotic Recombination Gene in Yeast

In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination. A new early recombination gene REC103 is described in this paper. It was initially defined by the rec103-1 mutation found in a selection for mutations overcoming the spore inviability of a rad52 spo13 haploid strain. Mutations in REC103 also rescue rad52 in spo13 diploids. rec103 spo13 strains produce viable spores; these spores show no evidence of meiotic recombination. rec103 SPO13 diploids produce no viable spores, consistent with the loss of recombination. Mutations in REC103 do not affect mitotic recombination, growth, or repair. These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., REC102, REC104, REC114, MEI4, MER2, and SPO11). REC103 maps to chromosome VII between ADE5 and RAD54. Cloning and sequencing of REC103 reveals that REC103 is identical to SKI8, a gene that depresses the expression of yeast double-stranded (``killer') (ds)RNA viruses. REC103/SKI8 is transcribed in mitotic cells and is induced ~15-fold in meiosis. REC103 has 26% amino acid identity to the Schizosaccharomyces pombe rec14(+) gene; mutations in both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.     

Biochemical and genetic analysis of a mutant with altered alkaline phosphatase activity in Dictyostelium discoideum

Alkaline phosphatase is one of several enzymes that accumulate in a temporally regulated sequence during the development of Dictyostelium discoideum. These enzymes can be used to monitor specific gene expression; moreover, isolation and analysis of mutations in the structural gene(s) can serve to indicate some of the essential steps in programmed synthesis and morphogenesis. A mutation (alpA) which affects the activity and substrate affinity of alkaline phosphatase was isolated in D discoideum using a procedure for screening large numbers of clones. Alkaline phosphatase activity at all stages of vegetative growth and development was altered by the mutation. Several physical properties of the enzyme from growing cells and developed cells were compared and found to be indistinguishable. It is likely that a single enzyme is responsible for the majority of alkaline phosphatase activity in growth and development. The mutation is coexpressed in diploids heterozygous for alpA and maps to linkage group III. One of the haploid segregants isolated from these diploids carries convenient markers on each of the six defined linkage groups and can be used for linkage analysis of other genetic loci.     

Reciprocal Translocations and Translocation Disomics of Aspergillus and Their Use for Genetic Mapping

Two new techniques are described for genetic mapping of reciprocal translocations in A. nidulans, which can be used to locate centromeres and meiotically unlinked markers. They both make use of unbalanced disomics from heterozygous translocation crosses. These are mainly hyperhaploids of two classes: either typical-looking n + 1 with a normal chromosome in addition to a haploid set containing the translocation, or translocation disomics. When large chromosome segments are involved, such disomics, as well as stable aneuploids and duplication types, show characteristic phenotypes and can be classified visually. The first method maps translocation breaks qualitatively, since translocated markers can be identified when translocation disomics are analyzed for heterozygous markers. The second method measures meiotic linkage of any marker to the translocation breaks when allele ratios in the balanced haploid sectors of either or both classes of disomics are determined: linked markers show reciprocal deviations from 1:1—In addition, it can be shown that frequencies of nondisjunction and recovery of specific translocation disomics both depend on the relative position of the break within a chromosome arm. Such information can provide a rough estimate of the positions of breaks for a new translocation.—Using these techniques, as well as mitotic mapping in homo- and heterozygous translocation diploids, four reciprocal translocations were mapped. From these results, information on the sequence and orientation of most of the "meiotic fragments" of the current maps (groups III, VI, VII and VIII) was obtained, and the position of the centromeres of groups VI and VII were identified. Translocation disomics are also used to map meiotically unlinked single genes, e.g. oliA of group VII, to specify chromosome segments.     

The large loop repair and mismatch repair pathways of Saccharomyces cerevisiae act on distinct substrates during meiosis

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of <16 nucleotides in length. We also find that the MMR pathway can act on loops up to 17, but not >19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.     

Linkage analysis of developmental mutations in aggregation-deficient mutants of Dictyostelium discoideum

Summary Simple parasexual genetic techniques have been employed to extend the linkage analysis initiated in an earlier study (Coukell, 1975) of developmental mutations (agg mutations) in 40 independently isolated aggregation-deficient mutants of Dictyostelium discoideum. Using these techniques, agg mutations in 28 of the 40 mutants have been assigned to 4 linkage groups: 16 in group II, 1 in group III, 10 in group IV, and 1 in group VI. None of the agg mutations analyzed appear to map in linkage group I. In addition, a new temperature-sensitive growth locus, designated tsgJ, was mapped in group III. It was also found that diploid strains of D. discoideum are readily induced to undergo haploidization when grown on 0.1% p-fluorophenylalanine (PFP) at 25.5 °C. Growth of diploid strains on PFP had no effect on the type of segregant classes obtained (i.e., PFP does not induce mitotic crossing-over), the subsequent growth and/or development of the segregants, or the ability of the segregants to reform stable diploids.     

Mutations Affecting Sexual Conjugation and Related Processes in SACCHAROMYCES CEREVISIAE. II. Genetic Analysis of Nonmating Mutants

Rare diploids formed by sterile mutants have been studied by tetrad analysis. Sixteen classes of mutants representing at least five distinct genetic loci have been defined. One group of mutations, isolated only in alpha, maps at the mating-type locus, while none of the others shows any linkage to mating type. Some of the mutations are nonspecific for mating type, while others act only on a or alpha. In addition, mutations were found that prevent sporulation when heterozygous in diploids. These appear to be mutations of the mating-type alleles.     

Genetic Mapping in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

A method of meiotic segregation analysis based on recombinant selection in the homothallic basidiomycete Phanerochaete chrysosporium was developed. Using this method, we were able to reveal linkage relationships and to estimate recombination frequencies between seven mutations to auxotrophy. We detected two linkage groups, the first containing four and the second three of the seven mapped mutations.     

Genetics of Aspergillus flavus: linkage of aflatoxin mutants

Eight aflatoxin (afl) mutants of Aspergillus flavus were induced with N-methyl-N'-nitro-N-nitrosoguanidine. Heterozygous diploids formed between afl mutants and tester strains revealed that each afl mutant was recessive. Haploids selected from these heterozygous diploids indicated the linkage of all eight afl mutants to markers on group VII. These include previously mapped arg-7 (arginine), leu (leucine), dominant afl-1, and nor which accumulates norsolorinic acid that is visible as an orange-red pigment. Diploid complementation tests indicated that all but two afl mutants were nonallelic. Diploids homozygous for nor, resulting from crossing-over, were isolated and used to map new afl genes.     

A Genetic Map of DICTYOSTELIUM DISCOIDEUM Based on Mitotic Recombination

A genetic map of the cellular slime mold Dictyostelium discoideum is presented in which 42 loci are ordered on five of the seven linkage groups. Although most of the loci were ordered using standing mitotic crossing-over techniques in which recessive selective markers were employed, use was also made of unselected recombined haploid strains. Consistent with cytological studies in which the chromosomes appear to be acrocentric, only a single arm has been found for each of the five linkage groups studied. The mating-type locus, matA, has been located in the tsgE-sprA interval on linkage group I on the basis of studies on diploids formed between strains of opposite mating type that have escaped from vegetative incompatibility.     

Isolation and characterisation of nuv11, a mutation affecting meiotic and mitotic recombination in Aspergillus nidulans

A mutant of Aspergillus nidulans, designated nuv11, has been isolated as hypersensitive to the monofunctional alkylating agent MNNG and the quasi-UV-mimetic mutagen 4-NQO. The mutation was recessive, resulting from mutation of a single gene which mapped to chromosome IV, and was non-allelic to the previously characterised repair-deficient mutations uvsB and uvsH which are also located on this linkage group. The nuv11 mutation results in slow growth, deficient intragenic and intergenic meiotic recombination, increased spontaneous chromosome instability, and increased intragenic and intergenic mitotic recombination in homozygous diploids. By screening a wild-type gene bank of A nidulans, a clone (pNUV11A40) has been isolated which complements the nuv11 mutation, restoring wild-type responses to both MNNG and 4-NQO.     

Initiation of sporulation in Saccharomyces cerevisiae. Mutations causing derepressed sporulation and G1 arrest in the cell division cycle

Mutants of Saccharomyces cerevisiae that are derepressed for meiosis and spore formation have been isolated and characterized genetically. All are the result of single, recessive nuclear mutations that fall into four linkage groups. Three of these groups are represented by spd1, spd3 and spd4 mutations, which in homozygous diploids confer poor growth and extensive sporulation on a range of non-fermentable media. Haploids carrying any of these mutations are arrested under these conditions in the G1 phase of the cell division cycle as large unbudded cells. The alleles of the spd2 mutation complemented all other mutations but were very closely linked to the spd1 locus. The fourth linkage group was represented by a mutation conferring temperature-sensitive growth and derepressed sporulation on homozygous diploids grown between 25 degrees C and 30 degrees C on media containing galactose or glycerol, but not glucose, as energy source. Above 30 degrees C this mutant lysed on all media. The mutation it carried failed to complement available cdc25 mutations. These data bring to five the number of loci at which mutation can lead to derepressed sporulation (spd1, spd3, spd4, cdc25 and cdc35). The spd1 locus has been mapped 13.9 cM to the left of the centromere on chromosome XV, adjacent to the SUP3 gene. Diploid strains homozygous for spd mutations are genetically unstable, giving rise to asporogenous mutants at high frequency, usually as the result of a second mutation unlinked to the spd mutation. Diploids homozygous for these mutations, and for spd mutations, show an altered regulation of the formulation of at least three polypeptides normally subject to carbon source repression.     

Mutations in Chlamydomonas reinhardtii Conferring Resistance to the Herbicide Sulfometuron Methyl

Chlamydomonas reinhardtii mutants resistant to the herbicide sulfometuron methyl (SM) were isolated and characterized. Growth of C. reinhardtii is sensitive to inhibition by SM at a concentration of 1 micromolar. Four mutants resistant to 10- to 100-fold higher concentrations were isolated. All possess a form of acetolactate synthase (ALS) whose specific activity in cell extracts is 100- to 1000-fold more resistant to SM than is the specific activity of wild-type enzyme. Only one mutant had abnormally low ALS specific activity in the absence of SM. All mutations were inherited as single lesions in the nuclear genome and were expressed in heterozygous diploids. The mutations in two strains mapped to linkage group IX about 30 centimorgans from streptomycin resistance and on the same side of the centromere, and in genetic crosses between mutants no segregation was observed. Accordingly, all mutations are tentatively assigned to gene smr-1. Herbicide resistance appears to be suitable as a selectable marker for molecular transformation in this organism.     

Intracellular cAMP in Dictyostelium discoideum: Characterization of an aggregation-deficient mutant with elevated cAMP pools

Forty aggregation-deficient mutants of Dictyostelium discoideum were screened for changes in intracellular cAMP during the first 10 hr of starvation. The pools in 39 of the mutants remained low and relatively static during this period. However, amoebae of one mutant, strain HC151, exhibited significantly elevated levels of intracellular cAMP during vegetative growth and for several hours after starvation. A more detailed analysis of this mutant indicated that the elevated cAMP pools in these cells are a consequence of the premature appearance and partial activation of an adenylate cyclase. The mutation(s) altering adenylate cyclase regulation in this strain appears to map in linkage group IV. Complementation tests between strain HC151 and another mutant, HH201, which has recently been shown to produce an adenylate cyclase activity precociously [1], indicated that the mutations affecting adenylate cyclase activity in these strains map at different loci. Although both of these mutations behave recessively in heterozygous diploids with respect to gross development, an examination of early cAMP metabolism and terminal spore differentiation in these diploids suggest that these mutations are at least partially expressed during some stage(s) of the developmental cycle.