Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.     

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.     

The Distribution of Tetrahymena pyriformis1

SYNOPSIS. This paper is a brief account of both amicronucleate and sexually active strains of Tetrahymena pyriformis and their distribution with some comments on their possible evolution.     

RNA synthesis in starved deciliated Tetrahymena pyriformis.

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non-nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)-containing messenger RNA content of deciliated cells, and in particular, formation of beta-tubulin mRNA can be detected almost immediately after deciliation.     

Isolation of nucleoli from Tetrahymena pyriformis.

A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented.     

Cytochemical localization of peroxisomes in Tetrahymena pyriformis.

Pronounced staining of Tetrahymena pyriformis peroxisomes was demonstrated when glutaraldehyde-fixed cells were incubated in an alkaline medium containing 3,3'-diaminobenzidine and hydrogen peroxide. A variable amount of electron-opague deposit was observed when cells were incubated in diaminobenzidine and H2O2 for 1 hr while an intense deposit followed incubation for 4 hr in the same medium. The staining was abolished completely when 3-amino-1,2,4-triazole, KCN or NaN3 was added to the incubation medium. Based on these cytochemical observations and the morphologic identification by size, shape and other ultrastructural details, it is suggested that this study presents evidence for a conclusive morphologic identification of Tetrahymena peroxisomes.     

Dolichyl phosphate phosphatase from Tetrahymena pyriformis.

A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.     

The methylated purines of Tetrahymena pyriformis transfer ribonucleic acid.

By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N2-DMG. Also present were 1-MG, N2-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N6-methyladenines were detected.