Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Chromosome Fragments in DICTYOSTELIUM DISCOIDEUM Obtained from Parasexual Crosses between Strains of Different Genetic Background

The first aneuploid strains of Dictyostelium discoideum have been unambiguously characterized, using cytological and genetic analysis. Three independently isolated, but genetically similar, fragment chromosomes have been observed in segregants from diploids formed between haploid strains derived from the NC4 and V12 isolates of D. discoideum. Once generated, the fragment chromosomes, all of which have V12-derived centromeres, can be maintained in a NC4 genetic background. Genetic evidence is consistent with the view that all three fragment chromosomes studied encompass the region from the centromere to the whiA locus of linkage group II and terminate in the interval between whiA and acrA. From cytological studies, one of the fragment chromosomes consists of approximately half of linkage group II.—We observed no deleterious effect on viability or asexual fruiting-body formation in either haploid or diploid strains carrying an additional incomplete chromosome and hence are disomic or trisomic, respectively, for part of linkage group II. The incomplete chromosome is lost at a frequency of 2 to 3% from disomic and trisomic strains, but surprisingly this loss is not increased in the presence of the haploidizing agent, benlate. A new locus (clyA), whose phenotype is altered colony morphology, is assigned to the region of linkage group II encompassed by the fragment chromosome.     

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.     

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.     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

 Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100 kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved. Received: 21 December 1995/Accepted: 10 June 1996     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

Genetic analysis of the polyauxotrophy of the early mitotic progeny of Saccharomyces cerevisiae zygotes. III. The behavior of the chromosome-III markers in polyauxotrophic clones and their mitotic and meiotic segregants

Cloning and segregation analysis of polyauxotrophic (PA) progeny, as well as the study of diploid segregants, revealed an unusual state of the diploid genome in these strains. Analysis of linkage markers of chromosome III in all crosses may be expected to illuminate relationships between the homologous chromosomes. All PA strains were assigned to two major classes. In the class of PA strains with recombinant chromosome III, the effect of approachment of third linkage markers was noted. In other strains, transposition of genetic marker metA1 to chromosome III and its linkage to leu2 were discovered. The present study has demonstrated that the unusual state of the diploid genome of PA strains induces multiple nonspecific alterations in chromosome relationship and structure. It is likely that these alterations are the consequence of disturbance in mitotic apparatus of cell.     

Genetic and Morphological Study of Aggregation in the Cellular Slime Mold POLYSPHONDYLIUM VIOLACEUM

A system for genetic analysis in the cellular slime mold P. violaceum has been developed. Two growth-temperature-sensitive mutants were isolated in a haploid strain and used to select rare diploid heterozygotes arising by spontaneous fusion of the haploid cells. A recessive mutations to cycloheximide resistance in one strain enables selection of segregants, which often appear to be aneuploid.—Aggregation-defective (ag^- ) mutants having a wide range of phenotypes were isolated in both temperature-sensitive strains after nitrosoguanidine treatment, and complementation tests were performed between pairs of these mutants. Of 380 diploids isolated, 32 showed defective aggregation and were considered to contain 2 noncomplementing ag^- mutations. Among noncomplementing mutants interallelic complementation is common. Noncomplementing mutants fall into 4 complementation groups, and those within each complementation group are phenotypically similar. Statistical analysis of the results suggests that the number of complementation units involved in aggregation is about 50.     

Mutations specific to spore maturation in the asexual fruiting body of dicytostelium discoideum

Three mutations affecting spore maturation in the asexual fruiting body of Dictyostelium discoideum are assigned to a new locus, sprJ, on linkage group IV. Strains carrying mutations at the sprJ locus do not form mature spores, yet the cell patterning (spore, stalk and disc cell ratios) is apparently normal. These mutations will be useful to delineate branch points between the cell patterning and spore maturation pathways. There are some unusual features of the sprJ-containing mutants. In particular each of the parent strains of the three mutants has incomplete spore maturation as determined by colony-forming ability after heat shocking at 45°C. A mutation allowing growth in the presence of benlate (600 μg/ml), benA351, is mapped to linkage group I.     

Genetic and cytological characterisation of fusion chromosomes of Dictyostelium discoideum

Abnormally large chromosomes which appear to result from the fusion of 2 chromosomes of the normal karyotype have been found in diploids of Dictyostelium discoideum formed by parasexual fusion of haploid strains HU483 (n=7) and HU245 (n=7). These fusion chromosomes appear to be the products of the tandem translocation of most, if not all, of one acrocentric chromosome to the telomere of a second acrocentric. Thus the chromosome number of the diploids is reduced from the normal 2n=14 to 2n=13 with the formation of an abnormally large acrocentric fusion chromosome. Experimental haploidisation of such diploids results in two types of products, those with a normal 7 chromosome karyotype and those with an abnormal 6 chromosome karyotype which contains the fusion chromosome. Genetic analysis of haploid segregants indicates that linkage groups II and VII are involved in this fusion. Phenotypes of recombinant diploids obtained following mitotic crossing-over establishes that linkage group II is proximal to linkage group VII. Cytological examination of the karyotypes of haploid strains bearing the fusion chromosome suggest that chromosome 2 may correspond to linkage group II and chromosome 3 to linkage group VII. Haploid strains bearing the fusion chromosome grow and develop normally so little or no genetic information can have been lost in the fusion event. While the nature of this event is unknown it may have involved aberrant recombinational DNA repair since the parental haploid strain HU483 bears the radB13 DNA repair mutation.     

Parasexual Genetic Analysis of the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM A3

Haploid strain A3 of the cellular slime mold Dictyostelium discoideum is valuable for biochemical studies because it is capable of axenic growth. Mutants of A3 temperature-sensitive for growth and resistant to the drugs cycloheximide, acriflavin, or methanol were isolated.--Heterozygous diploid recombinants, formed at low frequency by cell and nuclear fusion, were isolated by selecting temperature-resistant progeny of mixed cultures of two nonallelic temperature-sensitive haploids (LOOMIS 1969). Each drug-resistant mutation was found to be recessive. Two independently isolated methanol-resistant mutants were in one complementation group.--Diploids of A3 heterozygous for drug resistance formed drug-resistant segregants with a frequency of approximately 10(-4). Segregants selected for resistance to a single drug were either haploid or diploid; the fraction which was haploid varied from 0.11 to 0.86, depending on the selected marker. Segregants selected for resistance to two or three drugs were almost all haploid.--Using this parasexual cycle of diploid formation and haploidization, linkage of these temperature-sensitive and drug-resistance mutations to each other and to mutations studied by KATZ and SUSSMAN (1972) and by WILLIAMS, KESSIN and Newell (1974b) was analyzed. The methanol-resistant mutants were found to be partially resistant to acriflavin, and unlinked to the mutant selected for acriflavin resistance, which was methanol-sensitive. Of the expected seven linkage groups in D. discoideum, five, and a possible sixth, have been marked.--Linkage analysis of a mutant abnormal in morphogenesis showed that its phenotype results from two unlinked chromosomal mutations.     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100?kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.     

Riboflavin Overproduction in 4-Aminopyrazolo[3,4-d]pyrimidine-Resistant Mutants of Yeast Pichia guilliermondii

More than 90 mutants resistant to the adenine analogue 4-amino-pyrazolo[3,4-d]pyrimidine (4-APP), were isolated from a wild-type strain of yeast Pichia guilliermondii. Some of the App ^rmutants accumulated noticeable amounts of products absorbing at 260 nm in the culture medium, probably nucleotides and their derivatives. In comparison to the parent strain, the mutant App ^r-27 synthesized greater amounts of xanthine and uracil suggesting the presence of defects in the regulation of de novo biosynthesis of purines and pyrimidines. The regulatory mutations rib80 and rib81 are known to cause riboflavin (RF) overproduction and derepression of synthesis of corresponding enzymes in P. guilliermondii. The mutant App ^r-27 was crossed to the rib81 strain. The yield of RF biosynthesis in some meiotic segregants was significantly higher than that in segregants from the diploid rib81/RIB81. Apparently,rib81 and app ^r mutations were combined in a single genome on the favorable genetic background. An increase in RF production was also found in strains with app ^r mutations induced directly in the genome of the RF oversynthesizing strain rib80 rib81. These results indicate that introduction of app ^r mutations into the genome of P. guilliermondii can intensify their RF overproduction.     

Effect of Mutations for the ruvABC Genes on Recombination between Direct DNA Repeats in Escherichia coli Strains Carrying Extended Tandem Duplications

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants forruvABCgenes. Homologous recombination in duplications of rec ⁺ strains and in recBC sbcB, recQand recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.     

Isolation and characterization of cAMP unresponsive (frigid) aggregation-deficient mutants of Dictyostelium discoideum

To find mutants of Dictyostelium discoideum that are unable to respond to exogenous cAMP signals (frigid mutants), amoebae of 218 independent aggregation-deficient mutants were treated in suspension with artificial pulses of cAMP and screened for the capacity to form EDTA-resistant cohesion sites. Eleven frigid mutants were identified and further characterized. Using parasexual genetic techniques, these strains were assigned to five complementation groups (fgdA-E) and the fgd loci were mapped in three linkage groups: fgdA and D in group II, fgdC in group III, and fgdB and E in group VII. Biochemical and physiological experiments with these strains indicated that fgd mutants are of two general types. When starved, strains in groups fgdB, D, and E failed to produce detectable levels of membrane-associated cAMP phosphodiesterase, surface cAMP receptors, or extracellular phosphodiesterase inhibitor, and the cells continued to respond chemotactically to folate. Thus, these strains are probably arrested in the vegetative phase or very early in development. In contrast, strains in groups fgdA and C produced low levels of cAMP receptors and secreted phosphodiesterase inhibitor. Moreover, after starvation, some of these mutants elicited a weak chemotactic response to cAMP. Therefore, unlike the former group of mutants, these strains appear to initiate development when starved, but the process is blocked at an early stage.     

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.     

Parasexual Genetic Analysis of Cell Proportioning Mutants of DICTYOSTELIUM DISCOIDEUM

Eight independently isolated mutants of Dictyostelium discoideum that differentiate exclusively into stalk cells make up one complementation group and carry single recessive mutations at the stalky locus, stkA, located on linkage group II. KY19, a previously described strain that differentiates into spores, but not stalk cells, was found to possess a recessive mutation defining the stalkless locus, stlA, located on linkage group VI. An analysis of the properties of these mutants, together with the phenotype of a haploid double mutant carrying stkA and stlA indicates that stlA results in poorly organized stalk tubes and incomplete stalk cell differentiation, while stkA causes all of the cells to differentiate into stalk cells, even when not enclosed in the stalk tube. The significance of these results is discussed in relation to current theories of pattern formation in D. discoideum.     

Non-linkage of Induced Mutations in Chinese Hamster Cells

The relatively rapid loss of human chromosomes from human-rodent somatic cell hybrids has allowed the determination of linkage relationships between several human genes^1–4. Cells that have segregated out most of the human chromosomes are analysed for the presence or absence of particular human gene products; when two gene products are always found to be retained together, they are assumed to be linked. Little has been done to extend these genetic techniques to cell hybrids formed between two different mutants of the same cell line. A linkage analysis would provide a valuable means of interpreting the gene function altered in such mutants. The principal obstacle to such an approach has been the fact that homospecific cell hybrids are rather stable, losing chromosomes at only a low rate^5–7. Nevertheless, by using suitably marked strains, it is possible to select rare segregants from a homospecific hybrid population^7,8. I have applied such a system to test for linkage between several chemically induced mutations in a Chinese hamster cell line.     

Investigation of the het genes that control heterokaryon incompatibility between members of heterokaryon-compatibility (h-c) groups A and G1 of Aspergillus nidulans

A chromosome assay method was used to determine the heterokaryon compatibility relationships between strains belonging to heterokaryon-compatibility (h-c) groups A and G1 of Aspergillus nidulans. A hybrid strain (RD15) was isolated following protoplast fusion of strains 65-5 (h-cA) and 7-141 (h-cG1). The morphology of RD15 was severely abnormal compared to diploid strains of A. nidulans produced from heterokaryon-compatible haploid parents. Inocula of RD15 were induced to haploidize on medium containing Benlate and a parasexual progeny sample of 291 haploid segregants was obtained. The progeny strains were genotyped for standard markers. Allelic ratios and pairwise marker segregations were determined. Pairs of progeny strains that carried different alleles for the standard markers on each linkage group in turn were tested for compatibility. Strain pairs that possessed different alleles for the markers on linkage groups II, III, V, VI and VII were incompatible indicating the presence of heterokaryon-incompatible (het) genes on these linkage groups. Backcrosses to an h-cGl strain showed that two het genes were located on linkage group III and confirmed a total of six het gene differences between the h-cA and h-cGl strains.