Richard Braun

Structure of ribosomal DNA in Physarum polycephalum.

Abstract The sequences coding for Physarum ribosomal RNA are localized on idependently replicating, linear DNA molecules of a discrete size, M r = 37 × 10 6 . Restriction endonucleases Eco RI and Hin dIII each cut ribosomal DNA into one large and two small fragments. The latter are represented twice per intact molecule, once at each end. Sedimentation and electron microscopic analyses of intact rDNA † that has been neutralized from alkaline solution indicate that the entire rDNA molecule has a rotational axis of symmetry near the center. Blocks of short, inverted repetitious sequences appear to be located at the center of the native rDNA and also 3.7 × 10 6 to 11 × 10 6 daltons flanking the center.

Eimeria maxima gametocyte antigens: potential use in a subunit maternal vaccine against coccidiosis in chickens

Affinity-purified gametocyte antigens (APGA) from Eimeria maxima, emulsified in Freunds adjuvant, were injected intramuscularly into breeding hens on two or three occasions. As a result, progeny of the immunized hens were partially immune to infection with E. maxima, Eimeria tenella and Eimeria acervulina (with a reduction in total oocyst output of 45-63% as compared with progeny of untreated hens). Thus, APGA in Freunds adjuvant appears to have great potential as part of a maternally applied vaccine against coccidiosis. The ability of APGA to induce partial cross-species protection is most probably due to the existence of conserved epitopes in the different species as indicated by comparative Western blots of E. maxima and E. tenella. Surprisingly, Freunds adjuvant by itself also induced significant levels of maternal immunity to coccidiosis (with a 12-35% reduction in oocyst output in the progeny). In contrast to the purified antigens or Freunds alone, crude extracts from gametocytes as well as other developmental stages, induced little if any significant maternal immunity despite provoking the production of large amounts of parasite-specific IgG, including antibodies to APGA. This result indicates that a successful maternal vaccine against coccidiosis requires, in addition to good recognition of protective antigens, the exclusion of irrelevant antigens from the vaccine preparation.

Time sequence of DNA replication in Physarum

Abstract The time sequence of DNA replication in successive mitotic cycles was investigated using synchronous cultures of the slime mold Physarum polycephalum . DNA molecules replicated in a small subfraction (about one fifth) of the S-period of one interphase were again replicated in the corresponding subfraction of the S-period in the following interphase. This suggests that the time of replication of a specific DNA molecule is genetically fixed in eukaryotic cells.

Replication of nuclear satellite and mitochondrial DNA in the mitotic cycle of Physarum

Abstract In synchronous plasmodia of the myxomycete Physarum polycephalum, the rate of replication of mitochondrial DNA and “nuclear” satellite DNA during the mitotic cycle was investigated. Mitochondrial DNA (density in CsCl approx. 1.686 g·cm−3) and nuclear satellite DNA (density approx. 1.714 g·cm−3) accounted for about 7 and 1% respectively of the total DNA. Both of these DNAs differed from the major nuclear DNA (density approx. 1.700 g·cm−3) in their narrow band width in CsCl gradients and in their selective extraction by a procedure involving precipitation with 1 M NaCl in the presence of sodium dodecyl sulfate. These observations indicated that both satellite DNAs were relatively homogeneous molecular species and that they had either a different association with cellular structures or a different size than the majority of chromosomal DNAs. Each of the satellite DNAs were made at a rather constant rate throughout the interphase, while the majority of nuclear DNA replicated only during a subfraction of the mitotic cycle, the S-period. The implication of this finding is discussed with respect to the control of chromosomal DNA replication in eukaryotic cells.

Maternal transfer of antibodies induced by infection with Eimeria maxima partially protects chickens against challenge with Eimeria tenella

Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and partial immunity to infection with another important species, Eimeria tenella. As an example, in an experiment using hatchlings from eggs collected between days 28 and 39 after infection of the hens with 20,000 sporulated E. maxima oocysts, control chicks (challenged with 100 sporulated oocysts) excreted 6.8 +/- 1.2 million (mean +/- S.E., n = 10) or 5.8 +/- 1.2 million (n = 8) oocysts of E. maxima or E. tenella, respectively, compared to 0.9 +/- 0.4 million (n = 5) E. maxima oocysts or 2.2 +/- 0.4 million (n = 9) E. tenella oocysts excreted by hatchlings of infected hens. This represents an 87% reduction in oocyst excretion with regard to E. maxima and a 62% reduction in oocyst excretion with regard to E. tenella in the progeny of the infected hens. In another experiment, eggs were collected from days 28 to 37 and again from days 114 to 123 after infection of the hens with E. maxima and hatchling oocyst excretion rates were 82% and 62%, respectively, reduced for E. maxima and 43% and 41%, respectively, reduced for E. tenella in the progeny of hens infected with E. maxima compared to the progeny of uninfected hens. ELISA and Western blot analyses of maternally-derived IgG revealed a high degree of cross-reactivity to antigens of E. maxima and E. tenella.(ABSTRACT TRUNCATED AT 250 WORDS)

Ribosomal genes of Physarum: Their isolation and replication in the mitotic cycle

Abstract In plasmodia of the myxomycete Physarum polycephalum more than 95% of the 10 8 nuclei go through mitosis at the same time. In cesium chloride gradients three fractions of Physarum DNA can be distinguished: main nuclear DNA; mitochondrial DNA (about 10% of total DNA) and nuclear satellite DNA (about 2% of total DNA). The main nuclear DNA replicates in the first 3 hours (S-phase) of the 9-hour intermitotic time. Both satellite DNA fractions replicate in the G2-phase as well as in the S-phase, but no isotope incorporation into nuclear satellite DNA is seen in the first hour after mitosis, a time during which nucleoli are reformed following their apparent disintegration in mitosis. It is shown here that preparations of nucleoli contain most of the nuclear satellite DNA and that contaminating main band DNA can be removed in preparative cesium chloride gradients. The isolated nuclear satellite DNA hybridizes to an extent of about 30% with excess ribosomal RNA. Thus most of this DNA consists of genes for ribosomal RNA. It can be calculated that ribosomal genes are re-iterated 1000 to 2000 times per nucleus and that each gene is transcribed, on average, two to four times per minute.

A ribonuclease from Physarum: Biochemical properties and synthesis in the mitotic cycle

Abstract A ribonuclease from the myxomycete Physarum polycephalum has been partially purified and characterized; it is called Physarum ribonuclease I. It is an extracellular enzyme, has a pH optimum of 4.0, is heat labile, stable over a wide range of pH values; and has a molecular weight of about 31 000 daltons, as determined by gel filtration. No metal ions are required for its activity. RNA serves as substrate, while native or denatured DNA are resistant. Poly U and poly A are rapidly degraded to cyclic mononucleotides and slowly to 3′-mononucleotides. Poly C is very slowly degraded to cyclic cytidine monophosphate. Dinucleotide phosphates are rapidly hydrolyzed, except those of the form CpN. After prolonged incubation, RNA is degraded primarily to 3′-AMP, 3′-UMP, 3′-GMP and cyclic 2′,3′-CMP. The enzyme is an endonuclease. It may be useful for sequence studies of RNA, particularly for further partial degradation of T1 fragments. The activity of ribonuclease I was studied in the highly synchronous mitotic cycle of Physarum. Enzyme activity doubles stepwise shortly before the middle of each interphase. This rise depends on protein synthesis de novo .

Repeated structure of chromatin in metaphase nuclei of Physarum

Several lines of evidence suggest that eucaryotic chromatin is organized in a repetitive structure. Digestion of nuclei with endogenous nuclease [ 11, or with appropriate concentrations of micrococcal nuclease [2] leaves most of the DNA in fragments of about 200 nucleotide pairs, or multiples thereof. Two molecules each of histones F2a, F2b, F3, and F4 appear to condense with each 200 nucleotide pair length of DNA to form one ‘nucleosome’ of chromatin [3,4]. These nucleosomes can be visualized as contiguous beads of approximate diameter 60130 A in the electron microscope [3,5-71. The roles of histone Fl and of other proteins in the organization of chromatin structure are not yet clear. Among the macromolecular changes associated with mitosis are cessation of transcription [8,9] and modifications of histones [ 10-131. In particular, phosphorylation of histone Fl has been implicated in chromatin condensation [ 141. Although the total histone content of mitotic and interphase chromatin is the same, it is not known in these cases whether or not the histones are arranged in the same supramolecular structure. In the experiments presented, we addressed ourselves to the question, are the nucleosomes characteristic of interphase also present at mitosis.

Pools of deoxyribonucleoside triphosphates in the mitotic cycle of Physarum

Abstract The pool sizes of ribonucleoside and deoxyribonucleoside triphosphates were determined in the synchronous mitotic cycle of the acellular slime mold Physarum polycephalum . Averaged over the whole mitotic cycle the pool of all four ribonucleoside triphosphates was found to be 32.0 μmoles/g protein and that of the deoxyribonucleoside triphosphates 0.63 μmoles/g protein. For the eight nucleoside triphosphates the following average pool sizes were found, expressed in μmole/g protein: ATP 18.6, GTP 5.37, CTP 2.77, UTP 5.20, dATP 0.028, dGTP 0.077, dCTP 0.18, dTTP 0.33. At the beginning of the S phase and shortly before it, the pools of all four deoxyribonucleoside triphosphates are higher than in the middle of the G2 period. This pattern is particularly noticeable for dATP and dCTP, for which the pools expand by a factor of more than five.

RNA of Physarum: I. Preparation and properties

Abstract Methods of preparing RNA and DNA from the myxomycete Physarum polycephalum were modified in such a way as to allow RNA/DNA hybridization studies to be carried out. Purified DNA, denatured subsequently by alkali and by heat, could be fixed to nitrocellulose membranes by filtering at 60 °C, giving a fixation yield of 97%. Extraction of pulse-labelled RNA was quantitative by combining cold and hot phenol extractions; the product showed little or no degradation. As judged by gel electrophoresis, the two ribosomal RNA species have molecular weights of 1.37 × 106 daltons (26S) and 0.76 × 106 D (19S). Three stable RNA species of low molecular weight could be observed: 4S transfer RNA and two species with approximate S-values of 5 and 6. Base compositions of all five types of stable RNA are given. Hybridization of ribosomal RNA with DNA showed that 1.3 ± 0.2% of total Physarum nuclear DNA hybridizes at saturation with homologous rRNA. DNA fractionated in a cesium chloride gradient was also hybridized with rRNA. The densest DNA fraction hybridized about four times more with rRNA than any other DNA fraction. Thus the genes for rRNA are concentrated in the densest part of the DNA.