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.     

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.     

Replicon size and rate of DNA replication fork movement are correlated in grasses

In eukaryotes, most nuclear DNA replication proceeds bidirectionally from multiple origins of replication. A unit of DNA, replicated by two replication forks from a single origin, is termed a replicon. Using results from DNA fiber autoradiography we show a novel positive correlation between replicon size and the rate of replication fork movement in root meristem nuclei of 13 grass species. Although there is interspecific variation in replicon size, it is balanced by similar variation in the rate of replication fork progression.     

Requirements for DNA replication preceding cell division in Tetrahymena pyriformis

Populations of Tetrahymena pyriformis were synchronized by 30 min heat shocks at 34 °C separated by 160 min intervals at the normal growth temperature. The cells initiate DNA synthesis immediately after the cellular division, and the S period of the population lasts about 80 min. It was found that DNA replication is a prerequisite for the following synchronous division. Inhibition of the DNA synthesis in early S by starvation of the cells for thymidine prevents the forthcoming division. However, inhibition in the latter half of S does not prevent the subsequent division. Thus the cells have synthesized enough DNA to permit cell division before the end of a normal S period. These results are discussed in relation to the organization of the genome replication in the highly polyploid macronucleus.     

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.     

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.     

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.     

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.     

DNA elimination and its relation to quantities in the macronucleus of Tetrahymena

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminate. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes. The result indicates that in a given cell line at least one of the DNA molecules becoms extinct after 60 generations which we expect would cause the cell's extinction and restrict a clone's life to 60 generations. As this does not happen in nature, there must be some control of the copy numbers preventing their extinction during vegetative multiplication. Whether elimination increases or decreases the imbalance of genes remains to be investigated. © 1992 Wiley-Liss, Inc.     

Replicon size of yeast ribosomal DNA

Summary The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9Kbp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 m long, on chromosome XII. Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined. We have used immobilised rRNA as a probe to examine the size of radioactively labelled rDNA replicons resolved on alkaline sucrose gradients. The replicons were found to be uniformly sized, about 5 repeat units in length, and groups of 4 adjacent replicons may be activated simultaneously. These observations suggest that replicon initiation events are not determined solely by the recognition of specific DNA sequences that function as origins of replication.     

DNA elimination and its relation to quantities in the macronucleus of Tetrahymena.

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies. Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T. pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminated. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of two DNA polymerases in Tetrahymena pyriformis.

Two DNA polymerases were detected in Tetrahymena pyriformis, strain GL. One (enzyme I) was sensitive to N-ethylmaleimide, while the other (enzyme II) was insensitive. The molecular weight of the enzymes, as determined by glycerol gradient centrifugation analysis, were approximately 130,000 and 70,000, respectively. Optimal concentration of MgCl2 was 10mM for enzyme I and 18mM for enzyme II. KCl inhibited enzyme I but stimulated enzyme II. Poly (dA-dT) served effectively as a template for enzyme I, while poly(dA).(dT)12-18 was an effective template for enzyme II. Enzyme I activity increased with cell growth and sharply declined after the cells reached the stationary phase. On the other hand, enzyme II activity appeared only at the end of log phase. In cells synchronized by starvation-refeeding technique enzyme I was markedly stimulated in correspondence to the rate of DNA synthesis, whereas the level of enzyme II activity changed to lesser extent. By ethidium bromide treatment, only enzyme I activity was induced.