Nuclear DNA content and chromosome numbers in the myxomycete Physarum polycephalum

An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum. It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein.Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20–25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35–40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein:DNA and RNA:DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker.A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined plasmodial growth medium, containing 2% agar. Plasmodia are visible in 2–4 days.     

Ultrastructural localization of β-galactan in the nuclei of the myxomycete Physarum polycephalum

The acellular slime mold Physarum polycephalum produces an extracellular sulfated and phosphorylated β-D-galactan which was recently isolated from the nuclei of this organism. This polysaccharide has now been localized in the nuclei ofP. polycephalum by electron microscopy using a specific “sandwich” technique: thin sections of P. polycephalum microplasmodia were incubated with the Ricinus communis lectin specific for D-galactose residues. The bound lectin was then localized with gold granules labeled with a galactose-terminated glycoprotein (desialylated ceruloplasmin). The galactin was found in the nuclei mainly associated with chromatin and, also, but to a smaller extent, in the cytoplasma and in some vacuoles. The specificity of the method was assessed by marking under the same condition the galactomannan present in the cell wall of the yeast Schizosaccharomyces pombe.     

Replication of nucleolus-associated DNA during "G2 phase" in Physarum polycephalum

In the myxomycete, Physarum polycephalum, the bulk of nuclear DNA replication occurs during a period of a few hours immediately following upon mitosis. During the remainder of the intermitotic period, incorporation of thymidine-³H continues at a low rate in the region of the nucleolus (radioautographs). A few nuclei incorporated thymidine-³H into the extranucleolar chromatin at a high rate at all times of the intermitotic period. These nuclei were exceptionally large and they frequently contained several small nucleoli of different sizes rather than the one, central nucleolus which is characteristic of a normal interphase nucleus.     

A small nuclear precursor of messenger RNA in the cellular slime mold Dictyostelium discoideum

Previous work (Firtel et al., 1972) showed that messenger RNA from the cellular slime mold Dictyostelium discoideum, like that from mammalian cells, contains a sequence of about 100 adenylic acid residues at the 3′ end. We show here that Dictyostelium nuclei, labeled under a variety of conditions, do not contain material analogous to the large nuclear heterogeneous RNA found in mammalian cells. Rather, the majority of pulse-labeled nuclear RNA that is not a precursor of ribosomal RNA does contain at least one sequence of polyadenylic acid; this RNA, with an average molecular weight of 500,000, appears to be only 20% larger than cytoplasmic messenger RNA.Pulse-labeling experiments show that the nuclear poly(A)-containing RNA is a material precursor of messenger RNA. Whereas previous work showed that over 90% of messenger RNA sequences are transcribed from non-reiterated DNA, we show here that about 25% of nuclear poly (A)-containing RNA is transcribed from reiterated DNA sequences and only 75% from single-copy DNA. We present evidence that a large fraction of the nuclear poly(A)-containing RNA contains, at the 5′ end, a sequence of about 300 nucleotides that is transcribed from repetitive DNA, and which is lost before transport of messenger RNA into the cytoplasm.Based on these and other results, we present a model of arrangement of repetitive and single-copy DNA sequences in the Dictyostelium chromosome.     

A method for isolation of cytoplasmic RNA from a slime mold,Physarum polycephalum

A procedure for fast and simple preparation of cytoplasmic ribonucleic acid from Physarum polycephalum microplasmodia is described. Microplasmodia are homogenized in a high-magnesium-high-ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid buffer and nuclei are pelleted. The supernatant is extracted with sodium dodecyl sulfate-phenol-chloroform and crude RNA is precipitated. This is further purified by selective ethanol precipitation from 6 m guanidinum hydrochloride. This RNA preparation is suitable for direct use in hybridization studies.     

The nature of the changes in the pattern of RNA synthesis by the juvenile hormone analogue altosid

The effect of various concentrations of Altosid and actinomycin D under defined conditions on housefly metamorphosis was investigated with three strains of houseflies. The morphogenetic response varied with the strains and the length of time which the larvae were exposed to the juvenile hormone analogue. De novo RNA synthesis was studied with (2-¹⁴C)-glycine. Methods were developed for the isolation of nuclear, soluble, and ribosomal RNA. The procedure presented provides a DEAE-cellulose chromatographic method for the removal of high molecular weight RNA from DEAE at a neutral pH. Labelling of the RNAs was increased in the presence of the juvenile hormone analogue indicating an increase in the rate of RNA synthesis. The higher incorporation of the labelled precursor into nuclear RNA demonstrates that cytoplasmic RNA is derived from the nuclei.     

Structure of a β-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated β-D-galactan ([α]_D + 8°) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 → 4)-, but also of (1 → 3)-, and (1 → 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extra-cellular polysaccharide produced by P. polycephalum.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.