Qualitative and quantitative studies on DNA and RNA synthesis in Loxodes striatus nuclei.

In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei and differentiated morphologically into a clearly recoginzable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a 3H-labeled precursor ([3H]thymidine for DNA and [3H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [3H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [3H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macronucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components synthesis can occur in adult macronuclei. The possible explantion of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.     

Macro- and Micronuclei of Tetrahymena pyriformis: A Model System for Studying the Structure and Function of Eukaryotic Nuclei*†

The macro- and micronucleus of Tetrahymena pyriformis are formed from a common diploid synkaryon during conjugation. Shortly after the 2nd postzygotic division, distinct morphologic and physiologic differences develop between the 2 nuclei. Micronuclei remain small, presumably diploid, and electronmicroscopic observations indicate that micronuclear DNA is contained in a dense, fibrous, chromosome-like coil. Macronuclei contain considerably more DNA than micronuclei, and the DNA of the macronucleus is found largely in the chromatin bodies typical of ciliate nuclei. The functional differences between macro- and micronuclei in vegetative cells also are striking. The template activity of DNA in the micronucleus is highly restricted compared to that in the macronucleus. Micronuclei synthesize and contain little RNA, and do not contain either nucleoli or ribonucleoprotein granules. Macronuclei, on the other hand, synthesize and contain large amounts of RNA and have many nucleoli and ribonucleoprotein granules. Macro- and micronuclei also have distinct differences in the timing of DNA synthesis during the cell cycle and in the timing and mechanism of nuclear division. Finally, during conjugation the macronucleus becomes pycnotic and disappears while the micronucleus undergoes meiosis and fertilization, ultimately giving rise to new macro- and new micronuclei. In short, the macro- and micronuclei of Tetrahymena provide an excellent system for studying the molecular mechanisms by which the same (or related) genetic information is maintained in different structural and functional states. Methods have been devised to isolate and purify macro- and micronuclei of Tetrahymena in the hope of correlating differences in the nucleoprotein composition of these nuclei with differences in their structure and function. The DNAs of macro- and micronuclei have been found to differ markedly in their content of a methylated base, N⁶-methyl adenine, and major differences in the histones of the 2 nuclei have been observed. Macronuclei contain histones similar to those found in vertebrate nuclei, while 2 major histone fractions seem to be missing in micronuclei. In addition, histone fraction F2A1 which is found in multiple, acetylated forms in macronuclei, is present only as a single, unacetylated form in micronuclei.     

DNA Damage and Repair in Stylonychia lemnae (Ciliata,Protozoa)1

ABSTRACT Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-³H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.     

Localization of genes for ribosomal RNA in the nuclei of Oxytricha fallax

The location of ribosomal RNA (rRNA) genes in the nuclei of the ciliated protozoan, Oxytricha fallax, was analysed by in situ hybridization. The micronuclear genome of O. fallax has typical chromosomal DNA organization. Macronuclei, although derived from micronuclei, lack chromosomes and instead contain short pieces of DNA ranging from 500 to 20 000 base pairs in length. In situ hybridization was carried out to determine if specific DNA sequences are limited to certain locations within the macronucleus, or if sequences are randomly arranged. Cells were fixed, squashed and then hybridized with ³H-labelled RNA synthesized in vitro using cloned O. fallax rDNA as a template. After autoradiography, silver grains were found to be distributed uniformly over the entire macronucleus without any detectable localization to specific regions. The uniformity of hybridization indicates that rDNA molecules are randomly dispersed throughout the macronucleus and suggests that the macronuclear genetic apparatus lacks any substantial multimolecular organization. S phase macronuclei also showed a uniform distribution of rDNA molecules, irrespective of the position of the replication band at which DNA synthesis takes place. The micronuclei, in contrast, did not show any hybridization, even in cells in which macronuclei were heavily labelled. Macronuclear anlagen, in which the micronuclear chromosomes are polytenized, also do not hybridize. This absence of hybridization indicates a much lower concentration of rDNA in the micronucleus than in the macronucleus. The change in rDNA concentration of rRNA genes presumably occurs during the complicated process of development of a macronucleus from a micronucleus.     

DNA damage and repair in Stylonychia lemnae (Ciliata, Protozoa)

Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-3H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Cytochemical studies on the problem of macronuclear subnuclei in tetrahymena

DNA amounts in macronuclei and micronuclei of Tetrahymena pyriformis were measured by Feulgen microspectrophotometry. Assuming that the unreplicated micronucleus is diploid, the unreplicated macronucleus was found to contain approximately 45 times the haploid DNA amount. The relationship of these findings to the 45 independently assorting genetic subunits characterized by Allen and Nanney and their collaborators is discussed. The pattern of DNA synthesis in macro- and micronuclei during the cell cycle is also described.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : I. RNA Synthesis in Macro- and Micronuclei

Tetrahymena in the log phase of growth were pulse labeled with uridine-³H, fixed in acetic-alcohol, extracted with DNase, and embedded in Epon. 0.5-µ sections were cut, coated with Kodak NTB-2 emulsion, and developed after suitable exposures. Grains were counted above macronuclei, above 1000 micronuclei, and above 1000 micronucleus-sized "blanks" which were situated next to micronuclei in the visual field by means of a camera lucida. An analysis of grain counts showed that micronuclei were less than ½₀₀₀ as active as macronuclei on the basis of grains per nucleus. Since micronuclei contained, on the average, about ½₀ as much DNA as macronuclei, micronuclear DNA had less than 1% of the specific activity of macronuclear DNA in RNA synthesis. However, even this small amount of apparent incorporation was not significantly different from zero. Comparisons of the frequency distributions of labeled micronuclei with those of micronuclear "blanks" showed no evidence of a small population of labeled nuclei such as might be expected if micronuclei synthesized RNA for only a brief portion of the cell cycle. We conclude from these studies that there is no detectable RNA synthesis in Tetrahymena micronuclei during vegetative growth and reproduction.     

Numbers of 5S and tRNA genes in macro- and micronuclei of Tetrahymena pyriformis

Macronuclei of Tetrahymena pyriformis contain approximately 200 copies of the genes for 25S and 17S ribosomal RNA (rRNA) per haploid genome. Micronuclei, however, contain only a few copies of the rRNA genes per haploid complement. Since macronuclei develop from, products of meiosis, fertilization and division of micronuclei, we suggested that the multiple copies of the rRNA genes in macronuclei are generated by amplification of the small number of genes in micronuclei (Yao et al., 1974). This process provides a simple mechanism for maintaining the homogeneity of the repeated rRNA genes. To test if amplification is a general mechanism operating on all repeated genes in Tetrahymena, we have examined the numbers of 5S RNA and tRNA genes in macro- and micronuclei. 5S RNA was purified by polyacrylamide gel electrophoresis and hybridized to saturation against macro- and micronuclear DNA. Approximately 0.013–0.014% of macronuclear DNA and about 0.009% of micronuclear DNA is complementary to 5S RNA. After correcting for the differences in the DNA sequence complexities between the two nuclei, we calculate that there are 300–350 5S genes per haploid macro- or micronuclear genome. From these data we conclude that there is little or no detectable amplification of the 5S genes in macronuclei relative to micronuclei. Similar studies using tRNA indicate that these genes are also highly repeated in both nuclei; about 800 genes are present per haploid genome. Thus, amplification from a small number of genes can be excluded as the mechanism for generating the repeated copies of the 5S and tRNA genes in Tetrahymena and it is likely that another, as yet unidentified, mechanism operates to maintain the homogeneity of these genes.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones α, β, γ, and H3^F. Of these histones, only α appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, α is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14–16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14–16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides β and γ in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that β and γ are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10–16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3^S into H3^F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3^S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3^S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena. The disappearance of specific histones also raises the possibility that developmentally regulated proteolytic processing of specific histones plays an important (and previously unsuspected) role in this system.     

Comparison of the sequences of macro- and micronuclear DNA of Tetrahymena pyriformis

Macro- and micronuclei were isolated from Tetrahymena pyriformis (Syngen 1, strain WH-6) and their DNAs compared by isopycnic centrifugation in neutral and alkaline CsCl, by analysis of thermal denaturation properties and by molecular hybridization. Unlike the situation observed in Stylonychia the buoyant densities and thermal denaturation patterns of Tetrahymena macro- and micronuclear DNAs were virtually identical—the only observable differences bordering on the limits of resolution of these techniques. DNA was isolated from the two nuclei which had been labelled with different radioactive isotopes (i.e. ¹⁴C-thymidine and ³H-thymidine), and the renaturation kinetics of mixtures of macro- and micronuclear DNA were examined using a single-strand specific deoxyribonuclease (S₁). Renaturation kinetics obtained using varying ratios of macro- and micronuclear DNA suggested that 80–90% of the sequences present in micronuclei were present in similar amounts in macronuclei. However, careful analyses of the renaturation kinetics indicate that approximately 10–20% of the sequences found in micronuclei are probably absent in macronuclei, and that most of these sequences are probably moderately repetitive (100 copies per genome or less). These findings place severe constraint on possible models concerning the structure of the Tetrahymena macronucleus, and are very different from the situation observed in Stylonychia where it has been suggested that only a small percentage of the sequences in micronuclei are present in significant amounts in macronuclei. Nonetheless, these results along with those in Stylonychia can be taken as an indication that the loss or under-replication of some DNA sequences accompanies macronuclear differentiation in ciliates.     

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.     

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.