Nucleotide sequence of the Physarum polycephalum small subunit ribosomal RNA as inferred from the gene sequence: secondary structure and evolutionary implications.

The nucleotide sequence of the Physarum polycephalum small subunit ribosomal RNA (SSU rRNA) gene has been determined. Sequence data indicate that the mature 19S SSU rRNA is 1,964 nucleotides long. A complete secondary structure model for P. polycephalum SSU rRNA has been constructed on the basis of the Escherichia coli 16S rRNA model and data from comparative analyses of 28 different eukaryotic sequences. A "four-helix" model is presented for the central domain variable region. This model can be applied both to vertebrate and most lower eukaryotic SSU rRNAs. The increased size of P. polycephalum SSU rRNA relative to the smaller SSU rRNAs from such other lower eukaryotes, as Dictyostelium, Tetrahymena or Saccharomyces is due mainly to three G+C-rich insertions found in two regions known to be of variable length in eukaryotes. In a phylogenetic tree constructed from pairwise comparisons of eukaryotic SSU rRNA sequences, the acellular myxomycete P. polycephalum is seen to diverge before the appearance of the cellular myxomycete Dictyostelium discoideum.

Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus.

A synthetic oligodeoxynucleotide probe was used to clone the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. The sequence of a 2050-bp restriction fragment containing the gene was determined. Analysis of the gene-derived amino acid (aa) sequence showed that this exoenzyme is probably synthesized as a 283-aa precursor with a 24-aa signal peptide and a 14-aa propeptide. The mature, secreted enzyme comprises 245 aa residues. Sonicates of Escherichia coli HB101 carrying the gene on a multicopy plasmid showed phospholipase C activity. This activity was inhibited by Tris, a known inhibitor of the B. cereus enzyme and also by antiserum raised against pure B. cereus phospholipase C. We conclude therefore that the gene is expressed in E. coli. The cloning and sequencing described here complete the first step toward using in vitro mutagenesis for investigations of the structure-function relationships of B. cereus phospholipase C.

Muliple sites of action of cycloheximide in addition to inhibition of protein synthesis in Physarum polycephalum.

The specificity action of cycloheximide was tested using a cycloheximide resistant mutant of Physarum polycephalum. This resistance has previously been shown to reside with the ribosomes, making cytoplasmic protein synthesis refractile to the action of the drug. We show here that cycloheximide in the mutant strain causes specific alterations in metabolism without influencing the growth rate. These are: 1. lowered specific activity of glutamate dehydrogenase during starvation, 2. alteration of the molecular weight of glutamate dehydrogenase, 3. inhibition of uptake of amino acids from the medium into the internal pools. Possible explanations for these effects of cycloheximide outside of protein synthesis per se are considered. We conclude that cycloheximide may not be considered a specific inhibitor of protein synthesis, and that a causal relationship between protein synthesis and any biological process cannot be claimed unless such specificity is demonstrated in each case, preferably by use of mutants.

NAD turnover in microplasmodia of physarum polycephalum.

The rate of NAD turnover in microplasmodia of Physarum polycephalum was investigated using a double labeling technique with (14C)-adenine or adenosine and (3H)-nicotinamide. The half-life of an NAD molecule in Physarum was estimated to be 25 min, which is shorter than in either E. coli or human cell lines. The half-life of NAD in the presence of an inhibitor of NADase and poly ADPR synthase, 5-methylnicotinamide, was also investigated, but found to be indistinguishable from controls. The possible reasons for this and for the rapid turnover is discussed in the light of the known functions for NAD in prokaryotes and eukaryotes.

Thymidine kinase-deficient mutants of Physarum polycephalum; relationships between enzyme activity levels and ploidy.

Thymidine (TK) and deoxycytidine (dCK) kinase levels, chromosome counts and whole culture protein:DNA ratios have been determined in the TK- plasmodial strains of P. polycephalum--TU84 and TU63--and on several wild type (wt) plasmodial and amoebal strains from Wis 1 and Colonia backgrounds. It was found that (a) dCK levels and protein:DNA ratios were similar in all plasmodia, regardless of ploidy or genetic background; (b) TU84 and 63 were haploid; (c) TK specific activity was similar in all wt plasmodia but lower in amoebae; (d) TK activities of mutants and wt were approximately additive in heterokaryons. It was concluded that TK deficiency is due to a defect in a structural gene and that wt strains are TK+/TK+ and mutants hemizygous TK-.

The presence of vitamin A in human tryptophan-rich prealbumin.

Human tryptophan-rich prealbumin has been isolated from serum and its homogeneity evaluated by immunoelectrophoresis and polyacrylamide-gel electrophoresis. The green fluorescence of the protein when exposed to ultraviolet light was shown to be due to the presence of vitamin A. It is suggested that trytophanrich prealbumin represents the hitherto unknown specific transport protein for vitamin A.