Preparation of chromatin containing ribosomal deoxyribonucleic acid from the macronucleus of Tetrahymena pyriformis.

A method is described that enables a chromatin fraction containing ribosomal DNA (DNA containing sequences coding for rRNA) to be prepared from the macronuclei of growing or stationary cultures of Tetrahymena pyriformis. This material is obtained in yields of between 25 and 75% of the theoretical maximum. The DNA in this fraction was identified as ribosomal DNA on the basis of its density and molecular weight, and it appears not to be appreciably contaminated by other DNA. The method relies on the approximate assumption that ribosomal DNA is the smallest species of DNA in chromatin in the nucleus, and avoids the use of mechanical force, or enzyme action, to fractionate chromatin.     

Autonomous rDNA molecules containing single copies of the ribosomal RNA genes in the macronucleus of Tetrahymena pyriformis

The DNA containing the genes for rRNA (commonly called rDNA) of $\text{Tetrahymena}$ sediments in sucrose density gradients considerably slower than the main part of the DNA when DNA from gently lysed whole cells or isolated nuclei are fractionated by this method. In rDNA purified by CsCl gradient centrifugation about 20% of the DNA (40% of the bases in one strand) consists of sequences homologous to 25S and 17S rRNA as determined by DNA-RNA hybridization. The purified rDNA co-sediments in sucrose gradients with Ø29 phage DNA (M.W. = 11 × 10⁶). Examination by electron microscopy of the rDNA demonstrates that the molecules are linear with a length of 5.65 ±0.6 μm corresponding to a molecular weight of 11 × 10⁶.     

Extrusion of DNA from the macronucleus of Tetrahymena pyriformis GL.

Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.     

Induced elimination of DNA from macronucleus of Tetrahymena pyriformis

The effect of thymidine starvation on replicating DNA in Tetrahymena pyriformis is described. Cells were pulse-labelled with ³H-thymidine just prior to inhibition of replication. Autoradiographic analysis has shown that the fraction of labelled DNA—which was in replication at the time of inhibition—is eliminated from the macronucleus prior to the following cell division and is located in the cytoplasm during the following cell generation.     

Mitochondrial deoxyribonucleic acid from Tetrahymena pyriformis and its kinetic complexity

1. Mitochondrial DNA from Tetrahymena pyriformis strain T has a buoyant density (rho) of 1.685 compared with rho1.688 for whole cell DNA. Mitochondrial preparations from T. pyriformis strain W show an enrichment of a light satellite (rho1.686), although this is not obtained free from nuclear DNA (rho1.692). 2. T. pyriformis mitochondrial DNA renatures rapidly and the kinetics of this process indicate a complexity of approx. 3x10(7) daltons. 3. The base-pairing in the renaturation product is of a precise nature, since the ;melting' temperature (80.5 degrees C) is indistinguishable from that of the native DNA (80.5 degrees C). 4. Centrifugation of mitochondrial DNA in an alkaline caesium chloride density gradient gives two bands, implying the separation of the complementary strands.     

Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila.

We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila. It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine. Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3'. A specific pattern of doubly methylated GATC sequences was found; hemimethylated sites were not detected. The patterns and levels of methylation of these sites did not change significantly in different physiological states. A molecular form of the rDNA found in the newly developing macronucleus and for several generations following the sexual process, conjugation, contained no detectably methylated GATC sites. However, both the bulk macronuclear DNA and palindromic rDNA from the same macronuclei were methylated. Possible roles for N-6-methyladenine in macronuclear DNA are discussed in light of these findings.     

Base composition of deoxyribonucleic acid isolated from mycobacteria

Guanine plus cytosine values of deoxyribonucleic acid derived from 30 cultures representing 14 mycobacterial species or varieties are presented. These data provide impressive reasons for maintaining the separation between the genera Corynebacterium and Mycobacterium; no conclusions can be arrived at from these data with respect to the Nocardia-Mycobacterium relationship. A bimodal clustering, in terms of guanine plus cytosine composition, is apparent within the genus Mycobacterium. In general, all members of any single phenetic species appear to fit into one or another of these clusters. The phenetic separation of species is, in some cases, confirmed by separation in terms of guanine plus cytosine values. The bimodal separation of guanine plus cytosine values within the genus Mycobacterium does not correspond to a division of the species into slow and rapid growers; it thus provides no justification for splitting Mycobacterium into two genera, composed of slow and rapid growers. This is not to say that such a split would not be useful, only that these data do not contribute to such a decision. Any further attempts to correlate phenetic classification with properties of mycobacterial deoxyribonucleic acid will require more specific techniques, such as molecular hybridization.     

A dolichyl phosphate-cleaving acid phosphatase from Tetrahymena pyriformis.

Tetrahymena pyriformis contains an enzyme which hydrolyzed dolichyl phosphate. This activity was solubilized from lyophilized samples of this organism and was relatively stable when stored frozen. The soluble enzyme preparation had an acid pH optimum and hydrolyzed both dolichyl and phytanyl phosphates at equivalent rates. The polyprenylphosphate phosphatase activity was compared with the acid phosphatases which hydrolyzed p-nitrophenyl phosphate and marked differences were found. Dolichyl phosphate hydrolysis required Mg2+ for maximum activity while the bulk of the phosphatase activity was not effected by the absence of this ion. Other differences were that the polyprenylphosphate phosphatase was relatively insensitive to inhibitors such as tartrate and vanadium oxide sulfate which had a pronounced effect on the rate of p-nitrophenyl phosphate hydrolysis. The two activities also appeared to have different subcellular distributions. The polyprenylphosphate phosphatase was markedly inhibited by ethoxy formic anhydride, a reagent which is active against enzymes containing a histidine residue at their active site, while p-nitrophenyl phosphate hydrolysis was unaffected. The polyprenylphosphate phosphatase may be important in regulating the level of dolichyl phosphate in T. pyriformis and thus the rate of glycoprotein synthesis. It is also a useful tool which is capable of liberating dolichol from dolichyl phosphate under mild conditions which will permit the further characterization of the polyprenols.     

Replicon size and rate of DNA replication in the macronucleus of Tetrahymena pyriformis]

The size of replication units (or replicons) measured in Tetrahymena pyriformis GL macronuclear DNA reaches 20--30 microns, according to the two independent methods: DNA fiber autoradiography, and alkaline isokinetic sucrose gradient centrifugation. The synthesis of new DNA fragments--replicons and their subsequent assembly are separated by time intervals (30 min). The rate of DNA synthesis for one fork averaged 0.6--0.7 microns/min. These data were obtained for cells of cultures being both in the expotential phase of growth, and those synchronized by starvation-refeeding. The generation time of T. pyriformis cells, calculated by the increase of the part of labeled nuclei, is almost 2 hours; the synthesis lasts 1 hour. Total amount of replication units in polyploid (polygenomic) Tetrahymena macronucleus is about 3000. Their initiation during S-period is presumably asynchronous.     

Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis.

A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively.     

Studies of the catalytic properties of an endonuclease isolated from Tetrahymena pyriformis.

An acid endonuclease hydrolyzing both DNA and RNA was purified from Tetrahymena pyriformis, strain E. The enzyme is distributed in all major subcellular compartments and is excreted into the growth medium towards the middle of the logarithmic phase. It hydrolyzes DNA to penta or hexanucleotides, on the average, bearing the monoesterified phosphate at the 3'-position. Particularly in early phases of the reaction it shows a very pronounced specificity for bases with a keto group at position 4 of the pyrimidine ring, such as guanine and thymine.