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.     

Isolation and Characterization of Bacterial Ribosomal RNA Cistrons

The DNA sequences which code for ribosomal DNA have been isolated and purified. The technique used has general application for RNA:DNA hybridization studies and enables the isolation of any gene for which sufficient gene product can be obtained. Experiments with isolated ribosomal RNA cistrons demonstrated that (a) the majority of the ribosomal cistrons are similar to one another; (b) the cistrons which are similar to one another are virtually identical to one another; (c) ribosomal cistrons of different bacterial species are closely related to one another.     

Differential replication of ribosomal DNA during larval development in Calliphora (Diptera)

The hybridization of ribosomal RNA with DNA extracted from whole insects has been compared at six stages during the development of the blowfly Calliphora stygia. Total DNA from hatching larvae contains approximately 20% more rDNA than does total DNA from either mid-embryonic or adult insects. DNA from late third-instar larvae close to pupariation contains about 30% less rDNA than that from hatching larvae. The proportion of rDNA to total DNA in larvae of intermediate ages falls between these two extremes. Ribosomal RNA cistrons are thus replicated more rapidly than the remainder of the genome in the late embryonic or earliest larval stages, but more slowly during the later cycles of DNA synthesis in those larval tissues which develop high levels of polyteny.     

The location of 5S (ribosomal) RNA genes in Drosophila hydei

The location of the 5S ribosomal RNA cistrons in band 2-23B1,2 of the polytene (salivary gland) chromosomes of Drosophila hydei was indicated by in situ hybridization of tritiated low molecular weight RNA fractionated from total in vivo synthesized larval RNA or from in vitro synthesized salivary gland RNA and competition of the hybridization of this RNA by 5S RNA obtained from calf lens ribosomes. -- At the submicroscopic level, band 2-23B1,2 in salivary gland chromosomes shows a compact organization. The adjacent region, 23B2, is slightly puffed and displays typical RNP particles, some of which may be observed close to band 2-23B1,2.     

The properties of hybrids formed between the P-group plasmid RP1 and various plasmids from Pseudomonas aeruginosa

Summary Ribosomal DNA content has been determined in several adult and larval tissues of Drosophila melanogaster. Underreplication of rRNA genes was observed in polytenic salivary glands of larvae. On the contrary, polytenic/polyploid ovaries showed no decrease in rDNA. It is concluded that polyteny is not necessarily associated with underreplication of rDNA. No other tissue examined displayed any change in rDNa redundancy. Third-instar-larvae showed a decrease in rDNA amount which might be partly accounted for by underreplication of rDNA in salivary glands. No such decrease was seen in pupae. Bobbed genotypes were essentially similar to wild type in all tissues except salivary glands. In this case, it was found that the extent of underreplication is less in bobbed as compared to wild genotypes.Ribosomal DNA activity was examined in various tissues of Drosophila melanogaster. The rates of rRNA synthesis vary greatly between various tissues. It is concluded that a control at the level of gene activity operates as differences in the amount of precursor rRNA synthesized can be observed both in flies of varying rDNA contents as well as in various tissues of the same genotype.     

Mutants of Chlamydomonas reinhardtii with physical alterations in their chloroplast DNA

We have isolated nonphotosynthetic (acetate-requiring) mutants with physical alterations in chloroplast DNA following growth of haploid cells in the chloroplast specific mutagen 5-fluorodeoxyuridine (FdUrd) or treatment of FdUrd-grown diploid cells with X rays. About one-third of the nonphotosynthetic mutations resulting from FdUrd treatment alone show simple deletions. All eight of the mutants examined so far which were obtained with FdUrd plus X rays have deletions that are accompanied by rearrangements, including inversions or duplications. All the alterations extend into one of the two inverted repeat regions of the chloroplast genome which contain the ribosomal RNA cistrons. However, Southern hybridization experiments reveal that the rRNA cistrons are not deleted but instead are contained in new fragments. The relocated rRNA cistrons appear to be functional, since the mutants have normal levels of chloroplast ribosomes. In most cases the deletions and rearrangements are symmetrical and affect both inverted repeats in a similar fashion. An exception is the mutant ac-u-c-2–43, which lacks one inverted repeat region almost completely, including an entire set of rRNA genes. Three additional mutants, which fail to recombine with ac-u-c-2–43 to give photosynthetically competent cells, have smaller deletions in the same region of the genome. These physical mapping studies have allowed us to place the ac-u-c locus itself in a region of unique sequence DNA in a fragment, Ba10, which also includes the right-hand end of one inverted repeat.     

Regulation of ribosomal RNA cistron number in a strain of Neurospora crassa with a duplication of the nucleolus organizer region

Some progeny from a cross of the translocation mutant T(VL→IVL)AR33 with wild-type Neurospora crassa are double nucleolus organizer (DNO) strains, usually displaying two distinct nucleolus organizer regions. The DNO strain is sterile but displays the same growth response as normal laboratory strains of Neurospora. We used DNA-DNA hybridization techniques to quantify the number of rRNA cistrons in the DNO mutant and its vegetative progeny. Comparisons of the rate of hybridization of genomic DNA from the parental AR33 strain and from the DNO strain showed that hybridization was more rapid for the DNO strain than for the parental strain. Successive vegetative progeny of the DNO strain displayed hybridization rates intermediate to those of the original DNO strain and the parental single nucleolus strain, indicating that the number of rRNA cistrons had decreased during vegetative propagation. Estimates of rRNA cistron number obtained from comparisons of the amount of single copy DNA and rDNA hybridized to genomic DNO and AR33 DNA at saturation indicate that the parental AR33 strain contains 225 copies of the rRNA repeat unit, while the DNO strain has approx. 440 copies. The number of rRNA cistrons decreases gradually in the successive vegetative progeny, approximating the parental haploid value by the eleventh vegetative transfer.     

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.     

Localization of ribosomal DNA within the proximal X heterochromatin of Sciara coprophila (Diptera,Sciaridae)

By means of several reciprocal translocations in Sciara coprophila, each having a break-point in the proximal X heterochromatin, it has been possible in the salivary gland nucleus to bring about separation of specific regions of this heterochromatin and then, by means of in situ hybridization, to determine the relative number of ribosomal RNA cistrons in each. The three blocks of heterochromatin delineated by the translocation break-points have been designated H1, H2, and H3; H1 is the most proximal, lying immediately to the right of the X centromere, and H3 is the most distal, constituting the very end (right) of the chromosome. The distribution of ribosomal RNA cistrons is as follows: 10% are located in H1; 50% in H2; and 40% in H3. For the first time it has been possible to confirm by grain count our previous biochemical estimate of a 60% deletion of rRNA cistrons in the proximal heterochromatin of the X _W homologue. The grain count data also support the conclusion of our previously published cytological analysis, that the exchange points in the X heterochromatin are identical in translocations T29 and T32 (between H1 and H2), also in translocations T23 and T70 (between H2 and H3). The coincidence of break-points in the X heterochromatin is considered in relation to the chromomere make-up of the region. Also, the occurrence of ribosomal RNA cistrons in all three heterochromomeres is discussed in relation to the functional significance of chromomeres.     

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.     

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.     

Relative ribosomal RNA cistron multiplicity in oocytes and postembryonic stages of the eutelic nematodePanagrellus silusiae

Summary The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematodePanagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous¹²⁵I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplification alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.Supported by the National Research Council of Canada     

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.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A chromosomal region subjected to position effect variegation was analysed for possible DNA under-replication. DNA clones from the vicinity of the euheterochromatin junction and from a distance of hundreds of kilobase pairs were used as probes. Formation of compact blocks of chromatin is regarded as a characteristic feature of position effect variegation. It was shown that in T (1;2) dor ^var7 males undergoing position effect variegation clones representing the DNA nearest to the breakpoint in 2B7 hybridized normally in situ to the compact blocks, providing evidence against DNA underreplication. In females the same clones did not hybridize to the compact blocks. These variations in hybridization may be related to different degrees of compaction of chromosome regions in males and females. A correlation between the degree of underreplication and the level of cell polyteny was shown by Southern-blot hybridization of a DNA probe from the 2B region to DNA from an X/O strain carrying Dp (1;1)pn2b displaying position effect variegation and compaction in 94% of salivary gland cells. Almost complete underreplication of the DNA of this region was found in salivary gland cells (with a maximal degree of polyteny), intermediate underreplication was found in fat body cells (with an intermediate degree of polyteny), and replication was not disturbed in diploid cells of the larval cephalic complex.by W. Beermann     

Increased number of nucleoli in the salivary gland cells of Drosophila melanogaster under conditions of rDNA dose compensation

A considerable increase in the number of nucleoli non-associted with the nucleolar organizer (NO) was shown in the salivary gland cells of Drosophila melanogaster mutants, heterozygous for a deficiency of NO. The frequency of formation of additional nucleoli increased with the raising of the chromosome polyteny level. By means of in situ hybridization we showed that in the mutant and the wildtype polytene cells the ribosomal DNA (rDNA) of these unlawful nucleoli included ribosomal gene repeats (18S+28S) with two types of insertions: ivs-I and ivs-II Such additional nucleoli can be attached to varying sites of the polytene chromosomes containing type I insertion sequences.     

SOURCES OF LARVAL SALIVARY GLAND SECRETION IN THE DIPTERAN CHIRONOMUS TENTANS

The soluble proteins in the hemolymph, the salivary gland, and the salivary secretion of fourth instar Chironomus tentans were examined by disc electrophoresis in acrylamide gels. Of the 11 protein fractions detected in buffered saline extracts of the gland, 10 are present also in the hemolymph. Amino acid isotope incorporation experiments indicate that the protein fractions shared by the salivary gland and the hemolymph are not synthesized in the gland but are synthesized in other larval tissues. Immunochemical studies show that most of these proteins eventually are secreted from the gland. The salivary gland in vivo and in vitro is active in de novo protein synthesis. The protein synthesized tends to form large molecular weight aggregates. As demonstrated by radioautography, at least 80% of this protein is secreted from the 30 large cells forming most of the gland. The proteins synthesized in the salivary gland cannot be detected in the hemolymph. The results of this investigation are consistent with a mechanism of secretion formation involving both de novo synthesis of some secretion proteins and the selective uptake, transport, and secretion of hemal proteins by the salivary gland.     

Localization of rRNA genes in the nuclear space of Calliphora erythrocephala Mg. nurse cells during polytenization

Multicolor 3D fluorescence in situ hybridization was used to study arrangement of rRNA genes in Calliphora erythrocephala nurse cell nuclei with different levels of polyteny. It has been shown that the rRNA genes are exclusively localized to chromosome 6, suggesting that chromosome 6 is the only C. erythrocephala chromosome responsible for nucleolar formation. We have also described changes in localization of ribosomal genes within the chromosome territory during polytenization, namely, that rDNA signals are detected in the peripheral region of chromosome territory starting from the stage of polytene chromosomes. In addition, it has emerged that large nucleolus associated with chromosome 6 starts to develop in the central nuclear region in the C. erythrocephala nurse cell nuclei at the stage of a primary reticular structure. The central position and nucleolar structure are retained at the stages when chromosome 6 occupies the central position, that is, at the stages of polytene and bloblike chromosomes. When the nucleus restores a reticular structure but at a higher polyteny level, the displacement of chromosome 6 to the nuclear periphery is accompanied by disruption of the large nucleolus into micronucleoli. The micronucleoli are distributed in the nuclear space retaining their association with the nucleolar-organizing regions of chromosome 6. Thus, our data suggest that the large-scale alterations in the organization of chromosome 6 and the nucleolus during polytenization are the correlated processes directly dependent on the rRNA gene activity. The earlier described dynamics of nucleolar-organizing chromosome territory and nucleolus in the nuclear space is likely to be associated with the change in the total expression activity of the nucleus, which complies with the hypothesis on the correlation between spatial nuclear organization and expression regulation of genetic material.     

Nucleolar relationships in some Australian Chironomus species

Members of a group of Australian Chironomus species in the pseudothummi complex show wide variation in number and location of nucleolar organizing regions (NORs). The structure of these regions has been examined by phase contrast microscopy and silver banding of salivary gland polytene chromosomes. Presence of nucleoli was also checked on other types of chromosomes in some species. The contribution of the silver banding technique to nucleolar studies in these chironomid chromosomes is discussed. Nucleoli often seem to emerge from groups of (up to 9) bands. Further studies are necessary to confirm the presence of rRNA cistrons in all of these bands. Banding differences, in particular absence of bands from homologous regions of some species which have smaller nucleoli or lack particular nucleoli, have been found. In the case of Ch. tepperi, however, little banding difference is apparent in the 16B region between the N(IV)⁺ and N(IV)^– chromosomes, although in situ hybridization (Eigenbrod 1978) shows a deletion of rRNA cistrons in the N(IV)^– stock. Differences in heterochromatin amount have also been observed at different NORs. A scheme for the evolution of nucleolar-producing regions in this Chironomus group in terms of these and other known chromosomal changes is presented and discussed.