Charles E. Holt

Mating type and the differentiated state in Physarum polycephalum

Abstract In the acellular slime mold, Physarum polycephalum, the differentiation of amoebae into plasmodia is controlled by a mating type locus, mt. Amoebae carrying heterothallic alleles usually do not differentiate within clones; plasmodia form when two amoebae carrying different alleles fuse and undergo karyogamy. In this paper, we show that amoebae heterozygous for heterothallic alleles can be isolated and maintained as amoebae; the amoebae form plasmodia in clones without a change in ploidy. Plasmodia were also found to be formed, infrequently, by heterothallic amoebae of a single mating type. The plasmodia are healthy and are also formed without a change in ploidy. Thus, the presence of two different heterothallic mating type genes in a single nucleus is compatible with the amoebal state and one heterothallic mating type gene is compatible with the plasmodial state, once established.

Cell fusion competence and its induction in Physarum polycephalum and Didymium iridis

Abstract In heterothallic Myxomycetes, diploid plasmodia arise when haploid amoebae of two different mating types are cultured together. In this mating process, the amoebae fuse in pairs, and the resulting zygotes develop directly into plasmodia. It has been shown previously that plasmodia start to form in this fashion only when the growing amoebae in a mixed culture reach a critical density. We have investigated the cellular basis of this phenomenon by growing amoebae of different mating type separately from one another and then mixing them to test their mating ability. Amoebae from cultures above and below the critical density were, respectively, competent and incompetent to mate. Furthermore, both partners had to be competent in order for mating to occur. No binucleate cells were formed in mixtures of incompetent amoebae, indicating that they failed to fuse with one another. Incompetent amoebae growing at low density on filters with 0.2-μm pores became competent when the filters were placed on dense cultures of amoebae. We suggest that amoebae release a filter-transmissible material that accumulates during growth and induces the cells to become fusion competent.

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

SummaryMutant (“APT”) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of reconstruction experiments show that the procedure enriches only for mutants blocked early in the pathway from amoeba to plasmodium. Mutants were isolated from four parents, two of which produce plasmodia asexually because they carry the allele mth of the mating type locus, and two because they carry gad (greater asexual differentiation) mutations. The APT mutants varied widely in the frequency of residual plasmodium formation, which occurred, in some cases, by reversion. The mutants, called apt (amoeba to plasmodium transition), were recessive in diploids and linked to the mating type (mt) locus. Mutants derived from the gad parents, unlike the parents themselves, crossed readily with heterothallic amoebae. Progeny analysis from such crosses indicates that both gad mutations are linked to mt. The mutants derived from one of the mth parents fell into two groups on the basis of their ability to cross with the mutants derived from the mt2 gad-8 parent. The result suggests that the mth-derived mutants represent two or more complementation groups. Mutants derived from the mt2 gad-8 parent cross with mt2 amoebae and hence display an altered mating specificity.

Molecular weight of mitochondrial DNA in Physarum polycephalum

Abstract The mitochondrial DNAs from a wide variety of organisms have a common molecular weight of approximately 10 × 106 daltons. Most of the molecules are isolated in a circular form ( Piko, et al. , 1968 ; Sinclair et al. , 1967 ; Dawid and Wolstenholme, 1967 ; Van Bruggen et al. , 1966 ) except in the case of yeast where only a small fraction is circular and the remainder linear ( Shapiro et al. , 1968 ). In contrast, Suyama and Miura (1968) have reported that mitochondrial DNAs from Tetrahymena , Paramecium and mung bean hypocotyl are up to 4 times larger than those of the first class of organisms. The molecules were in each case linear and no circles were observed. It thus appears that these is a second class of mitochondrial DNA, found so far in two protozoa and one plant. We report here that the mitochondrial DNA of the acellular slime mold, Physarum polycephalum , also falls in the higher molecular weight category. It is heterogeneous, and no closed circles could be detected. This study was facilitated by the ability to label specifically Physarum mitochondrial DNA (density 1.686 g/cc) by exposing a culture to radioactive thymidine during the G2 portion of the cell cycle ( Evans, 1966 ; Guttes, Hanawalt and Guttes, 1967 ; Holt and Gurney, 1968 ). Sedimentation of labeled, crude lysates provided a rapid assay of particular lytic conditions and led to the procedure described under Materials and Methods.

Isolation of ribosomal RNA precursors from Physarum polycephalum

Ribosomal RNA synthesis in Physarum polycephalum was studied by labeling intact microplasmodia with [3H]uridine. Labeled, high-molecular-weight RNA species were found in a 30,000 S structure released by phenol extraction at room temperature. RNA was released from the structure by further phenol extraction at 65–70 °C. If the labeling period was 15 min or longer, the labeled RNA was seen by polyacrylamide gel electrophoresis to be of two major types, a heterodisperse collection of 45-35 S molecules and a 26 S species. If the labeling was carried out for 30 min in the presence of cycloheximide, the major labeled species had an electrophoretic mobility corresponding to 40 S. Studies of the labeling kinetics, methylation, and base composition of these RNA molecules indicate that they are precursors to ribosomal RNA. The molecular weights of the homogeneous 40 and 26 S precursors are 3.0 × 106 and 1.45 × 106 daltons, respectively, in comparison with molecular weights of 1.29 × 106 and 0.68 × 106 daltons for the completed ribosomal RNAs.

Analysis of an inducer of the amoebal-plasmodial transition in the Myxomycetes Didymium iridis and Physarum polycephalum

Recent studies with amoebae of the Myxomycetes Didymium iridis and Physarum polycephalum reveal that competence to undergo sexual fusion or to differentiate to plasmodia apogamically can be induced by a diffusible factor. Suspension cultures of Didymium amoebae have proven to be a good source of the inducer activity. Cell-free culture supernatants accelerated mating of Didymium and Physarum amoebae and also apogamic differentiation of the Physarum strain Colonia . Characterization and purification of the inducer activity is being conducted with a semiquantitative bioassay based on the induction of zygote formation in suspensions of mating-compatible Didymium amoebae.

Ion fluxes in photoreception in Limulus polyphemus ventral eye. I. The response of potassium efflux to light.

Abstract 1. 1. Radioactive tracers were used to study the effect of light on the efflux of K + from Limulus polyphemus ventral eye. Eyes were labeled in artificial sea water containing 42 KCl and then perfused with non-radioactive artificial sea water. 2. 2. After the first 2 min of perfusion, 42 K in the eye decreased with time as the sum of two exponential terms of half-times 0.3 and 2 h. 3. 3. Illumination of an eye labeled with 42 K caused an abrupt increase in the rate of release of 42 K. The additional 42 K released in response to light comes from the photoreceptor cell bodies rather than from the axons. 4. 4. The proportional increase in efflux rate upon illumination did not vary with the length of time that an eye was perfused. This suggests that the two exponential terms above reflect two classes of photoreceptor cells rather than photoreceptor cells and glial cells. 5. 5. By collection of perfusate at intervals of less than one second, it was shown that the instantaneous rate of 42 K release increases up to 20-fold at the onset of illumination. With continuous illumination, the rate remains above the dark rate, but by less than a factor of 2. Thus, the response of potassium efflux to light has transient and steady phases like the receptor potential. The increased driving force on internal potassium resulting from the receptor potential is not sufficient to explain the flux changes, if one makes the constant field assumption for the photoreceptor membrane. 6. 6. A double label method with 42 K and 42 K was used to show that the average rate of influx over a 10-min period is not affected by illumination. 7. 7. In eyes cooled in the dark to 2–3 °C, light still elicited an increase in the rate of 42 K release and large, prolonged receptor potentials. Thus, the photoreception mechanism of the ventral eye is not, as has been previously suggested, inactive at low temperatures.