Nucleosome Phasing in Tetrahymena Macronuclei1

Core-protected DNA can drive only 60% of the Tetrahymena thermophila macronuclear genome into duplexes in hybridization experiments. This core-protected DNA therefore contains only a subset of the genome complexity. We interpret this to mean that a large fraction, if not all, of the genome is phased with respect to nucleosome placement. Among the sequences present in total DNA and absent from core-protected DNA are most of the sequences containing N⁶-methyladenine (MeAde) residues, consistent with our previous demonstration that most of these residues lie in linker DNA. We show that these results are not due to artifacts resulting from the small size of the DNA driver, nor are they due to any sequence preferences exhibited by staphylococcal (staph) nuclease. This is the first evidence that nucleosome phasing may be a bulk genome characteristic.     

Isocitrate dehydrogenase of Tetrahymena pyriformis

Summary We have studied the isocitrate dehydrogenase ofTetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance.Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg²⁺ and Mn²⁺ will serve as cofactors but the latter is more effective than the former.It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of theTetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 23µm and 18µm.     

Isolation and Properties of Macronuclei from Tetrahymena thermophila1

A simple and efficient method is described for the isolation of macronuclei from Tetrahymena thermophila (7B). The steps involved are deciliation and removal of the mucocysts’ contents by dibucaine treatment, digitonin mediated lysis, differential centrifugations, and finally isopyenic sucrose density gradient centrifugation. Judging from the distribution of marker enzymes and electron microscopy, the macronuclei obtained were free of cytoplasmic and paniculate contamination and were highly active in endogenous RNA-synthesis (1.5 pmol UTP incorporation/ng DNA min at 30°C). The ratio of protein: RNA: DNA was 2.0:0.33:1.0 (weight) and each macronucleus contained an average of 17 pg DNA. The average yield of isolation was 50%.     

Metabolism of extracellular phospholipids in Tetrahymena pyriformis

We studied the metabolism of phospholipids exogenously added to cultures of the protozoan, Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated via endocytosis of phospholipid vesicles, as judged from the following facts: Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. Phospholipid vesicles were incorporated into a phagosome-like structure in Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with 14C and 3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with Tetrahymena cells, the glycerol moiety and fatty acyl chain at the sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in Tetrahymena (J. Biochem. 99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells via endocytosis were hydrolyzed through the action of lysosomal phospholipases A1 and C, and also that one of the products, sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol via the microsomal "monoacylglycerol pathway."     

Metabolism of cytosine arabinoside in Tetrahymena pyriformis

The metabolism of cytosine arabinoside (araC) in Tetrahymena pyriformis amicronucleate strain W was studied. araC inhibited cell multiplication and protein synthesis at concentrations higher than 0.1 and 0.25 M respectively. araC had no effect on protein synthesis. araC was converted to araCMP, araCDP and araCTP by homogenized cell preparations. A deaminase activity converted araC to uracil arabinoside. The deaminase activity totally inhibited by tetrahydrouridine (THU) at a concn of 4 X 10(-6) M. The Ki for THU was 8 X 10(-8) M.     

Induction of DNA polymerase in Tetrahymena pyriformis

A number of different treatments will induce DNA polymerase in Tetrahymena pyriformis. In the present paper we have studied the induction of DNA polymerase by the inhibitor of DNA synthesis methotrexate plus uridine (M+U) and UV irradiation (after incorporation of bromodeoxyuridine (BUdR) into DNA) in synchronized Tetrahymena populations. We have found that M+U must be present in the nuclear S-period in order to induce the polymerase, and that a dose of UV irradiation which is too low to induce DNA polymerase will do so if the damaging effect of the irradiation is enhanced by incorporation of BUdR into DNA.     

Intracellular phospholipase activities of Tetrahymena pyriformis

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.     

Site of tubulin synthesis in Tetrahymena pyriformis

Free and membrane-bound polysomes were isolated from the protozoa Tetrahymena pyriformis, and the contribution of these two types of polysomes to tubulin synthesis were studied using immunoprecipitation of the 35S-translational products in a rabbit reticulocyte lysate. One-dimensional electrophoretic analysis shows that tubulin is synthesized by polyadenylated RNA isolated from free and membrane-bound polysomes. Non-polyadenylated RNAs of free polysomes are also able to direct tubulin synthesis. Two-dimensional electrophoretic analysis using O'Farrell's system confirms these results and also reveals the existence of the alpha- and beta-tubulin subunits.