Ribosomal RNA cistrons in Euglena gracilis

Euglena gracilis chloroplasts contain about 12 fg DNA of average density 1.686 g cm^?3 and 1.7 pg RNA. The large (1.1 × 10⁶ mol. wt) and small (0.56 × 10⁶ mol. wt) ribosomal RNA components are coded for by separate cistrons, both of which band at a density of 1.696 g cm^?3 in a CsCl gradient. About 6% of the chloroplast DNA codes for rRNA indicating that there are 240 cistrons for rRNA in each chloroplast or about three to six cistrons per chloroplast genome. Similar studies with rRNA from cytoplasmic ribosomes indicate that the cistrons for cytoplasmic rRNA band at a density of 1.716 g cm^?3, denser than that of the main-band DNA, and that there are 1000 cistrons for cytoplasmic rRNA per cell. Fractionation of E. gracilis DNA on CsCl gradients and subsequent hybridization experiments, as well as melting curves of DNA-RNA hybrids, show that chloroplast rRNA does not anneal specifically with either the cistrons for cytoplasmic rRNA or any DNA in the dark-grown cell, in contrast to those results found in some higher plants.     

Ribosomal RNA Cistron Multiplicity and Nucleolar Organizers in Hexaploid Wheat

The ribosomal DNA (DNA complementary to ribosomal RNA) content of twelve different wheat genotypes has been studied. Some of these genotypes are aneuploids with deletions or additions of chromosomes bearing nucleolar organisers. The rDNA contents of these genotypes provide several examples of a clear departure from a correlation between the number of rRNA cistrons and the number of nucleolar organisers. Thus the number of rRNA cistrons per nucleolar organiser is not constant in wheat. Wheat DNA was found to have a mean buoyant density of approximately 1.702 g/cc for all genotypes studied and rRNA hybridized selectively to DNA of buoyant density approximately 1.710 g/cc. The thermal stabilities of all the rRNA/DNA hybrids were essentially identical.     

Individual quantitative rDNA variation in three species of the Cucurbitaceae family

Individual quantitative variation in rDNA content within three species of the Cucurbitaceae family has been studied by rRNA/DNA filter hybridization experiments. The results showed a 2.3-fold range of variation in the number of ribosomal cistrons per diploid cell in an Ecballium elaterium natural population. This range of variation is compared with the smaller range observed in three Cucumis sativus and in two Cucurbita pepo varieties obtained as F₁ hybrids between pure lines.This work was supported by CNR Contract No. 74/0267.     

Differentiation of metaxylem cell line in the root ofAllium cepa

Summary DNA was extracted from three root segments ofAllium cepa: i) an apical portion 500 m long from the tip (meristem); ii) a second portion 4 mm long (I root segment containing metaxylem cells in the initial stages of differentiation); iii) a third portion 6 mm long (II root segment containing metaxylem cells in further stages of differentiation). A mixture of homologous 18 S and 25 S³H-rRNA was used for invitro DNA-rRNA hybridization. The following percent saturation values were detected in the three samples: 0.08 in meristem DNA (samplea), 0.129 in I root segment DNA (sampleb), and 0.105 in II root segment DNA (samplec).Thermal denaturation of DNA and the derivative curves of the melting profiles evidenced five DNA families which were differently represented in the three DNA samples. DNA elution by thermal chromatography on hydroxyapatite followed by hybridization with³H-rRNA, revealed that ribosomal cistrons melt between 90 and 91 °C, corresponding to a G-C content of 50.7%. Moreover, the amount of the DNA family containing ribosomal cistrons was greater in sampleb andc, in sampleb to a greater extent, as compared with samplea. On the other hand, one DNA family melting at a higher temperature (92–93 °C) was drastically increased in samplec.Buoyant density profiles of unsonicated DNA showed no peaks in the three DNA samples. Upon somcation, a heavy shoulder was observed in the profile of sampleb. As the density of ribosomal cistrons and that of shoulder were very similar, it seems possible that the two fractions contain many DNA sequences in common.The present studies demonstrate that the proportion of ribosomal cistrons and other DNA families does not keep constant during the development of the metaxylem cell line.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast

The colorless alga Polytoma obtusum has been found to possess leucoplasts, and two kinds of ribosomes with sedimentation values of 73S and 79S. The ribosomal RNA (rRNA) of the 73S but not the 79S ribosomes was shown to hybridize with the leucoplast DNA (rho - 1.682 g/ml). Nuclear DNA of Polytoma (rho = 1.711) showed specific hybridization with rRNA from the 79S ribosomes. Saturation hybridization indicated that only one copy of the rRNA cistrons was present per leucoplast genome, with an average buoyant density of rho = 1.700. On the other hand, about 750 copies of the cytoplasmic rRNA cistrons were present per nuclear genome with a density of rho = 1.709. Heterologous hybridization studies with Chlamydomonas reinhardtii rRNAs showed an estimated 80% homology between the two cytoplasmic rRNAs, but only a 50% homology between chloroplast and leucoplast rRNAs of the two species. We conclude that the leucoplasts of Polytoma derive from chloroplasts of a Chlamydomonas-like ancestor, but that the leucoplast rRNA cistrons have diverged in evolution more extensively than the cistrons for cytoplasmic rRNA.     

Use of disomic strains to study the arrangement of ribosomal cistrons in saccharomyces of ribosomal cistrons in Saccharomyces cerevisiae

Summary Disomic strains ofSaccharomyces cerevisiae were studied by DNA-rRNA hybridization to examine the arrangement of rRNA cistrons on yeast chromosomes as well as to identify a disomic strain which was enriched for rRNA cistrons. Four of the five disomic strains tested showed a per cent hybridization lower than wild type. Two of these strains were found to be disomic for more than one chromosome. A slight increase in the per cent hybridization was observed with DNA isolated from one disomic strain. It was concluded that some chromosomes inSaccharomyces cerevisiae had few if any rRNA cistrons suggesting that the rRNA cistrons are non randomly distributed over the genome. From DNA-tRNA hybridization experiments, evidence for the presence of tyrosine tRNA genes on chromosomes VI was obtained.     

Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides.

Reduction in the number of nucleoli/nucleus and increase in their size were usually observed in rat liver after partial hepatectomy. These changes of nucleoli were greatest 16–18 h after the operation, when RNA biosynthesis in the nucleoli is reported to be highest. Approx. 50% of the nuclei had one enlarged nucleolus at this time but after the increase in nuclear DNA synthesis less than 15% of the nuclei had one nucleolus, as in normal liver. Before the next peak of nuclear DNA synthesis, nucleolar changes appeared again, though less conspicuously.The enlarged nucleoli of regenerating liver were separated from smaller ones by discontinuous sucrose gradient centrifugation and the contents of nucleic acid and ribosomal cistrons in different-sized nucleoli were measured. The large nucleoli in regenerating liver were found to have increased DNA content, whereas smaller ones had the normal content. The total number of ribosomal cistrons in the enlarged nucleoli from regenerating liver was also increased roughly in proportion to the DNA content. No significant difference was found between the percentages of ribosomal cistrons in whole nuclear DNAs from regenerating and normal liver. Small but reproducible [³H]TdR incorporation into nucleolar DNA was observed and this was similar in normal liver and regenerating liver 12 h after partial hepatectomy. Therefore, the nucleolar changes in regenerating liver were not accompanied by any particular DNA synthesis in the nucleolus itself. These results suggest that in the nuclei of regenerating liver nucleolar chromatins may be redistributed and assembled into large nucleoli, rather than that any amplification of ribosomal cistrons occurs.     

Indications for Changes in DNA Composition Correlated with Early Embryonic Differentiation (Triturus vulgaris,Urodela)*

During early embryogenesis of the newt Triturus vulgaris (early gastrula to early neurula) the DNA was characterized by various methods of genome analysis. By preparative CsCl density gradient centrifugation an AT-rich satellite fraction (about 10% of total DNA) was found in all developmental stages studied. An additional GC-rich fraction, with a portion of about 3% of the genome, could be visualized only in the yolk plug stages. Filter hybridization experiments with labelled ribosomal RNA indicate that the number of rRNA cistrons is by a factor of 1.69±0.17 higher in the DNA of the mid-gastrula than of the tailbud stage. In ethidium bromide-CsCl gradients this additional (possibly amplified) rDNA bands at the same density as linear DNA. The analytical cleavage of DNA with ten restriction endonucleases reveals an extreme heterogeneity of the Triturus genome. The methylation pattern of DNA, studied with the aid of the isoschizomers Hpa II and Msp I, remains constant during early development. The reassociation kinetics of DNA, recorded spectrophotometrically, show that the portion of DNA that reassociates until Cot 10 increases significantly from 20% in the gastrula stages to 30% in the early neurula stage.     

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

Ribosomal gene amplification does not occur in the oocytes of Locusta migratoria

It has been suggested that Locusta migratoria amplifies its ribosomal RNA genes in the growing oocytes (Kunz (1967) Chromosoma20, 332–370). Cloned ribosomal DNA of L. migratoria was used to analyze rDNA structure and number. The rDNA is localized on three chromosome pairs in six nucleolus organizers. It was found that all structural variants of the rRNA genes which have been described previously are represented in the same relative amounts in DNA from isolated oocytes as in somatic cells. Furthermore, the rRNA gene number is not increased in oocyte DNA, i.e., amplification does not occur. Therefore, the large number of multiple nucleoli seen in the growing oocytes has to be interpreted as the fully extended and fully active set of chromosomal rRNA genes. The total rRNA gene number was determined by dot blot hybridization to be about 3300 genes/haploid genome.     

Amount of DNA complementary to ribosomal RNA in polyploid series of Scilla autumnalis L. and Urginea maritima (L.) Baker

Abstract

The percentage of rDNA in polyploid series of Scilla autumnalis and Urginea maritima has been determined by hybridization experiments rRNA/DNA. Previous reports indicate that a regulation of the number of rRNA cistrons exists in polyploid plants, but other works are in contrast with this assertion. We find approximately the same percentage of rDNA in species with different ploidy. We conclude that in the species examined does not exist a regulation of the ribosomal RNA genes.     

The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus

When DNA from blood or liver of Plethodon c. cinereus is centrifuged to equilibrium in cesium chloride it separates out into 2 components. The smaller or satellite component is relatively rich in G + C and is therefore heavy, and it amounts to about 2% of the total DNA. The heavy satellite does not include the ribosomal cistrons, and it is unrelated to the nucleolar organizer. When squash preparations of cells from the testis of P. c. cinereus are incubated in synthetic E³RNA complementary to the satellite DNA, the RNA anneals specifically to the centromeric heterochromatin of spermatogonia, spermatocytes, and spermatids, and to the centromeric regions of all discernible chromosomes. RNA/DNA hybrids were located by autoradiography. H³RNA complementary to the major component of the DNA anneals to all nuclei and to all parts of the chromosomes. H³RNA complementary to nucleolar DNA from Xenopus laevis anneals specifically to the chromatin associated with nucleoli in nuclei at various stages of the meiotic divisions. The nature of the centromeric heterochromatin and its role in the meiotic divisions are discussed.     

Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora erythrocephala

We have examined the possibility that the intron-containing (intron⁺) rDNA cistrons of Dipteran flies are active in the germ-line derived polyploid nurse cell nuclei of the ovarian follicles. Using the organism, Calliphora erythrocephala, we describe here a procedure which yields very pure nurse cell nuclei and compare the intron-free (intron^–) and intron⁺ rDNA cistron contents of nurse cell nuclei prepared by this procedure to those of 2–18 h embryo nuclei and 3 day pupal nuclei. DNA from three preparations of each nuclear type was examined and the intron^– and intron⁺ cistron contents quantitated using a Southern transfer procedure. The number of intron^– and intron⁺ rDNA cistrons per haploid genome in the presumed diploid 2–18 h embryo DNA was first established, and then the intron^– and intron⁺ cistron contents of nurse cell nuclear DNA and 3 day pupal DNA were determined relative to these values.The intron^– cistron content of nurse cell nuclear DNA was indistinguishable from that of embryonic DNA but the intron⁺ cistrons showed an 8-fold under-replication relative to the presumed diploid DNA. A slight under-representation of the intron^– cistrons and 3-fold under-replication of the intron⁺ cistrons were demonstrated for 3 day pupal DNA. These findings strongly suggest that intron⁺ rDNA cistrons are non-functional in nurse cell nuclei and substantiate the generality of this implication for the whole organism during early pupal life.     

Synthesis and characterization of amplified DNA in oocytes of the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

At a time in the life cycle when a large proportion of the oocytes of Acheta incorporate ³H-thymidine into an extrachromosomal DNA body, synthesis of a satellite or minor band DNA, the density of which is greater than main band DNA, is readily detected. Synthesis of the satellite DNA is not detectable in tissues, the cells of which do not have a DNA body, or in ovaries in which synthesis of extrachromosomal DNA by the oocytes is completed. The DNA body contains the amplified genes which code for ribosomal RNA. However, less than 1 percent of the satellite DNA, all of which appears to be amplified in the oocyte, is complementary to ribosomal 18S and 28S RNA. In situ hybridization demonstrates that non-ribosomal elements, like the ribosomal elements of the satellite DNA, are localized in the DNA body.Abbreviations used rRNA ribosomal RNA, includes 18S and 28S RNA - rDNA gene sequences complementary to rRNA - cRNA complementary RNA synthesized in vitro     

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10⁶ daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.     

Differences in the number of nucleolus organizers in Chironomus tepperi shown by in situ hybridization

Two stocks of Chironomus tepperi could be isolated. One stock, N(IV)⁺, contains nucleolus organizers in chromosome I and IV, whereas the other one, N(IV)^–, shows only one nucleolus in chromosome I. It is demonstrated by in situ hybridization with radioactive rRNA that the absence of the nucleolus in chromosome IV of stock N(IV)^– is not related to an inactivation of the nucleolar DNA, as might have been suggested, but is due to the lack of ribosomal cistrons.     

Number and DNA content of nuclei in the free-living nematode Panagrellus silusiae at each stage during postembryonic development

Nuclei from the four major tissues of the nematode Panagrellus silusiae were enumerated and examined using Feulgen microspectrophotometry at each stage during postembryonic development. The number of nuclei in the hypodermis, nerve, and intestine remains fairly constant during maturation, but there is a slight increase (57%) in the number of muscle nuclei. Thus, this organism is not stringently eutelic. The total number of somatic nuclei is about 600. DNA values of hypodermis and nerve nuclei were unimodal and adult nuclei had 2C amounts of DNA. The DNA distribution of muscle nuclei reflects the pattern expected for a tissue in which a portion of the nuclei are undergoing DNA synthesis. Intestinal nuclei accumulated DNA in the absence of nuclear division and in the adult the nuclei fall into discrete DNA classes which correspond to a geometric series of the 2C value. It is concluded that chromatin diminution does not occur in this species. In addition, the relationship in the different tissues of nuclear DNA content to nuclear volume and cell size is discussed.The study was supported by the National Research Council of Canada (Grant A-3491).