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Showing papers on "Ribosomal DNA published in 1977"


Journal ArticleDOI
01 Feb 1977-Cell
TL;DR: In this article, the authors analyzed Drosophila melanogaster embryos by two different methods: gel electrophoresis after digestion with restriction endonucleases and by electron microscopy.

293 citations


Journal ArticleDOI
TL;DR: Cricket oocyte chromosomes were stained with silver at pachytene when certain chromosome regions are active in rDNA amplification and rRNA transcription and it was revealed that the silver binds neither to the rDNA nor transcribed rRNA, but rather to proteins which rapidly associate with the freshly-transcribed rRNA.
Abstract: Cricket oocyte chromosomes were stained with silver at pachytene when certain chromosome regions are active in rDNA amplification and rRNA transcription. The silver preferentially stained the known locations of 18S + 28S ribosomal cistrons. Cytochemical tests revealed that the silver binds neither to the rDNA nor transcribed rRNA, but rather to proteins which rapidly associate with the freshly-transcribed rRNA. As rRNA transcription proceeds, the quantity of silver stainable proteins progressively increases. The silver procedure can be used to visualize gene activity at the rDNA sites with conventional light microscopy.

229 citations


Journal ArticleDOI
TL;DR: A diploid strain of yeast was found to be heterozygous for two forms of the highly repetitious ribosomal DNA, finding that all of the approximately 100 copies of the ribosome DNA genes of the yeast cell are located on one chromosome and that meiotic recombination within these genes is suppressed.
Abstract: A diploid strain of yeast (Saccharomyces cerevisiae) was found to be heterozygous for two forms of the highly repetitious ribosomal DNA. These forms could be distinguished by the pattern of fragments produced after digestion with the site-specific restriction endonuclease EcoRI. The mode of inheritance of ribosomal DNA was determined by tetrad analysis. Of 14 tetrads analyzed, 12 clearly showed the ribosomal DNA forms segregating as a single Mendelian unit. The simplest interpretation of this result is that all of the approximately 100 copies of the ribosomal DNA genes of the yeast cell are located on one chromosome and that meiotic recombination within these genes is suppressed. Two of the 14 tetrads showed the segregation patterns expected as the result of mitotic recombination within the ribosomal DNA.

213 citations


Journal ArticleDOI
TL;DR: The organization of the ribosomal DNA repeating unit from Saccharomyces cerevisiae has been analyzed and the polarity of the 35 S ribosome RNA precursor has been established by a combination of hybridization analysis and DNA sequence determination.

172 citations


Journal ArticleDOI
01 Sep 1977-Cell
TL;DR: Evidence is presented that the germinal micronucleus of the protozoan Tetrahymena contains a single gene for rRNA integrated into the chromosomal DNA.

143 citations


Journal ArticleDOI
01 Jun 1977-Cell
TL;DR: Studies on mouse-human cell hybrids suggest that some spacer classes are present on more than one of the five human nucleolus organizers, which indicates that ribosomal DNA in man is also heterogeneous.

122 citations


Journal ArticleDOI
TL;DR: Silver-staining results indicate that during mouse embryogenesis ribosomal RNA genes start to function at the 2-cell stage, which supports the view that the polar body genome remains inactive.
Abstract: The Ag-AS method, developed by Goodpasture and Bloom (1975) stains transcriptionally active nucleolus organizer regions (NORs) on the chromosomes and in the interphase nuclei. Metaphases and interphase nuclei of early mouse embryos (unfertilized eggs, pronucleus stages, 2-, 4-, 8-cell stages, and morulae) were subjected to silver-staining. First staining of a single chromosome bearing an NOR was observed at the 2-cell stage. At the 4-cell stage 4–6 chromosomes, and at the 8-cell stage invariably all the 6 chromosomes known to bear NORs, respond positively to silver-staining. These results indicate that during mouse embryogenesis ribosomal RNA genes start to function at the 2-cell stage. The polar body does not respond to silver-staining, which supports the view that the polar body genome remains inactive.

88 citations


Journal ArticleDOI
TL;DR: A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described, using avidin-biotin interaction for enriching Drosophila ribosomal DNA in high yield and 42 to 80% pure.
Abstract: A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.

77 citations


Journal ArticleDOI
TL;DR: A DNA fragment containing the structural gene for the 5 S ribosomal RNA and intergenic regions before and after the 35 S ribOSomal RNA precursor gene of Saccharomyces cerevisiae has been amplified in a bacterial plasmid and physically mapped by restriction endonuclease cleavage and hybridization to purified yeast 5 S ribsomal RNA.

75 citations


Journal ArticleDOI
01 Aug 1977-Cell
TL;DR: The organization of the 18S, 28S and 5.8S rRNA genes in the mouse has been elucidated by mapping with restriction endonucleases Eco RI, Hind III and Bam HI and indicates that the r RNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long.

74 citations


Journal ArticleDOI
TL;DR: Results suggest a preferential transcription of ribosomal DNA from the D. melanogaster genome when nucleolus organizers from both species are present in conjunction, a phenomenon termed nucleolar dominance.

Journal ArticleDOI
TL;DR: It is reported here that members of the same sequence family occur outside the ribosomal locus, suggesting that non-rDNA insertion sequences are linked to DNA of different nucleotide composition.
Abstract: Many repeating units of Drosophila melanogaster rDNA contain a DNA sequence within the gene for 28S rRNA that does not code for rRNA. This sequence has been called the ribosomal insertion [Wellauer, P. K. & Dawid, I. B. (1977) Cell 10, 193-212]. We report here that members of the same sequence family occur outside the ribosomal locus. "Non-rDNA insertion DNA" was separated from rDNA by density gradient centrifugation, and sequences homologous to the ribosomal insertion were detected by hybridization with restriction endonuclease fragments derived from a cloned rDNA repeating unit. Pure insertion sequences from cloned rDNA separated from main band DNA and behaved like a component with high G + C content. Non-rDNA components hybridizing to the insertion also separated from main band DNA but less so than pure insertion sequences, suggesting that non-rDNA insertion sequences are linked to DNA of different nucleotide composition. Restriction endonuclease analysis of non-rDNA insertion DNA showed many fragments of different sizes. The patterns obtained were similar in embryonic, larval, pupal, and adult DNA and DNA from cultured cells (Schneider cell line 3). Non-rDNA insertion sequences account for about 0.2% of the genome or about 400 kbases of DNA per haploid complement.

Journal ArticleDOI
TL;DR: Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with 3H 18S+28S rRNA.
Abstract: Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with 3H 18S + 28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.

Journal ArticleDOI
TL;DR: Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124, resulting in three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes.
Abstract: Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.

Journal ArticleDOI
TL;DR: Using hybridization techniques nuclei from both amoebae and plasmodia of Physarum polycephalum were found to contain 275 genes each coding for 58-S, 19-S and 26-S rRNA.
Abstract: 1 Using hybridisation techniques nuclei from both amoebae and plasmodia of Physarum polycephalum were found to contain 275 genes each coding for 58-S, 19-S and 26-S rRNA, 685 genes for 5-S rRNA and 1050 genes for tRNA 2 Hybridisation of these RNA species to both amoebal and plasmodial DNA fractionated on CsCl gradients reveal that the 58-S, 19-S and 26-S rRNA genes are located at a satellite position (Q= 1714 g/cm3) with respect to the main band of DNA, whereas 4-S and 5-S RNA genes are located exclusively in the main band of DNA (Q= 1702 g/cm3) 3 This result was confirmed by demonstrating that only the 58-S, 19-S, and 26-S rRNA species hybridise to purified plasmodial ribosomal DNA 4 The 19-S and 26-S rRNA genes of amoebae are located on extrachromosomal DNA molecules of a discrete size (Mr= 38 × 106) with identical properties to plasmodial ribosomal DNA

Journal ArticleDOI
08 Apr 1977-Science
TL;DR: Recombinant phages have been constructed in vitro by joining the smaller fragment of ribosomal DNA and the outer arms of DNA from the vector lambdagtWES-lambdaC.
Abstract: The ribosomal RNA genes (ribosomal DNA) of chicken are present in approximately 200 copies and are cleaved into two fragments of molecular weight 5 X 10(6) and 12 to 14 X 10(6) by restriction endonuclease Eco RI. Recombinant phages have been constructed in vitro by joining the smaller fragment of ribosomal DNA and the outer arms of DNA from the vector lambdagtWES-lambdaC. In one of the recombinants, the coding strand of the cloned fragment is in the proper orientation for transcription with lambda "early" genes; in the other two the orientation is reversed, with the coding strand in the proper position for transcription with lambda "late" genes.

Journal ArticleDOI
TL;DR: The results show that actinomycin-CsCl gradients can be used for the purification of plant ribosomal DNA.

Journal ArticleDOI
TL;DR: The analysis of ribosomal DNA purified from ovaries indicates that, during oogenesis, amplification involves preferentially, if not exclusively, only some size classes of genes.

Journal ArticleDOI
TL;DR: Two structurally reconstructed karyotypes of Hordeum vulgare which contain three chromosome pairs with nucleolus organizing activity have been studied to reveal quantitative relationships between the size of the secondary constriction and the number and size of nucleoli being formed.
Abstract: Two structurally reconstructed karyotypes of Hordeum vulgare which, due to appropriate reciprocal translocations, contain three chromosome pairs with nucleolus organizing activity (chromosomes 57, 6 and 77 in translocation line T 21, chromosomes 36, 63, and 7 in translocation line T 627) have been studied with respect to the position and function of rDNA as inferrred from the pattern of nucleolus formation. The results obtained reveal quantitative relationships between the size of the secondary constriction (the amount of rRNA cistrons) and the number and size of nucleoli being formed. Quantitative and qualitative aspects of rDNA location and expression are being discussed.

Journal ArticleDOI
TL;DR: For the first time it has been possible to confirm by grain count the previous biochemical estimate of a 60% deletion of rRNA cistrons in the proximal heterochromatin of the X′W homologue, and the grain count data support the conclusion of the previously published cytological analysis.
Abstract: 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.

Journal ArticleDOI
TL;DR: The study of a highly fluorescent chromosome indicates that the ribosomal DNA does not contribute to the fluorescent nature of the secondary constrictions, and the chromosomal inheritance of these genes is explored by in situ hybridization.
Abstract: Summary: The genetic sequence complementary to ribosomal RNA was separated from human DNA and its characteristics studied. Both 18S and 28S sequences were located on the same isolated strand. DNA rRNA hybrids have a melting temperature of 80° or 4° less than that of native ribosomal DNA. Ribosomal genes behave as a satellite when complexed with Ag+ in the Cs2SO4 gradient. With in situ hybridization, these rRNA genes can be shown to chromosomally translocate. The study of a highly fluorescent chromosome indicates that the ribosomal DNA does not contribute to the fluorescent nature of the secondary constrictions. This study examines the molecular organization of human ribosomal genes. The chromosomal inheritance of these genes is explored by in situ hybridization. Speculation: Human ribosomal DNA represents a well defined repetitious DNA which can vary quantitatively in normal and abnormal ways. This DNA should be representative for studying the inheritance patterns of repetitious DNA.

Journal ArticleDOI
TL;DR: Results support the model that requires the formation of rDNA extra-copies for rDNA magnification (Ritossa, 1972) and indicate that most of these circular molecules are DNA complementary to rRNA.

Journal ArticleDOI
TL;DR: A cloned 14.3-kbase segment of Drosophila melanogaster rDNA (Dm207) is described in which only a 4-Kbase region is homologous to a cloned 17-k base rDNA repeating unit, Dm103; this 4- kbase region consists of part of the 28S rRNA gene and most but not all of the adjacent transcribed spacer that normally connects the 18S and 28S genes.
Abstract: A cloned 14.3-kbase segment of Drosophila melanogaster rDNA (Dm207) is described in which only a 4-kbase region is homologous to a cloned 17-kbase rDNA repeating unit, Dm103; this 4-kbase region consists of part of the 28S rRNA gene and most but not all of the adjacent transcribed spacer that normally connects the 18S and 28S genes. The transcribed spacer in Dm207 is interrupted by a 2.2-kbase stretch of DNA that does not contain any 18S gene sequences. At the other end of the 4-kbase homology, the 28S gene is interrupted by an 8.1-kbase stretch of DNA at a position equivalent to the site of the 28S insertion found in the 17-kbase units. The question of whether the 2.2-kbase and 8.1-kbase interrupter segments in Dm207 derive from longer insertions into the transcribed spacer and 28S genes of a very long repeating unit (greater than or equal to 22 kbases) or represent a region of the chromosomal DNA into which a 4-kbase fragment of rDNA has been inserted is discussed.

Journal ArticleDOI
TL;DR: The results are consistent with either a transcriptional activation of the ribosomal genes or an increased rRNA stability during developmental, and quantitative estimates of the cellular rRNA and rDNA gene content were made.
Abstract: The cellular content and transcription program of the chloroplast ribosomal RNA genes of Euglena gracilis Z have been determined during the light-induced development of chloroplasts by hybridization of total cell DNA or RNA to purified 3H-labeled chloroplast ribosomal DNA ([3H]ctrDNA). Pancreatic DNase activated, partially purified chloroplast rDNA was enzymatically labeled in vitro by E. coli DNA polymerase I with [3H]TTP as a substrate. The [3H] DNA was denatured and hybridized with a vast excess of purified chloroplast 16 and 23S rRNA. The rRNA-[3H]ct rCNA hybrid was isolated by chromatography on hydroxylapatite. The [3H]ct rDNA was purified and characterized by the kinetics of its renaturation with chloroplast DNA and rRNA, and by the thermal stability of [3H]DNA-DNA and [3H]DNA-RNA hybrids. [3H]ct rDNA was hybridized in trace amounts to cellular RNA or DNA isolated from Euglena cells 0,4,8,12,24,48, and 72 h after the onset of chloroplast development. From a comparison of the kinetics of hybridization with hybridization of standards of known kinetic complexity quantitative estimates of the cellular rRNA and rDNA gene content were made. Chloroplast rRNA increases from 2 to 26% of the cellular RNA during development, while the percentage of cellular DNA represented by ct rDNA increases two- to threefold. Correcting for the change in cellular RNA and DNA content during development, the number of copies of the rRNA gene increases less than twofold, while the number of copies of rRNA per cell increases sixfold. The results are consistent with either a transcriptional activation of the ribosomal genes or an increased rRNA stability during developmental.

Journal ArticleDOI
TL;DR: Nucleolar chromatin could be separated into its various topographical components to improve knowledge of the structure/function relationships in chromatin as the fine structure of the nucleolus is very sensitive to changes in ribosomal RNA synthesis.
Abstract: A stepwise physico-chemical dissection of nucleoli isolated from Chinese hamster ovary cells has allowed the isolation of actively transcribed regions of chromatin, highly enriched in ribosomal genes. Nucleolar chromatin could be separated into its various topographical components. This separation should improve our knowledge of the structure/function relationships in chromatin as the fine structure of the nucleolus is very sensitive to changes in ribosomal RNA synthesis. The spatial distribution of ribosomal genes among the various chromatin areas of the nucleolus was assayed by hybridization with rRNA. In exponentially growing Chinese hamster ovary cells ribosomal genes are predominantly located in the intranucleolar stretches of nucleolus-associated chromatin. However, ribosomal sequences represent but a very minor part (less than 1 %) of intranucleolar DNA. In an attempt to improve the analysis of the actively transcribed chromatin areas, intranucleolar chromatin was submitted to additional fractionation steps involving, among others, isopycnic separation in metrizamide gradients. In these latter conditions a small subset of intranucleolar chromatin banded with preribosomal ribonucleoprotein. In this subfraction, about one-third of the DNA sequences corresponded to preribosomal RNA matrix units. The yield of recovery of this intranucleolar chromatin subfraction being closely related to the level of transcriptional activity of ribosomal genes, it appears likely that this separating procedure depends on the presence of ribosomal RNA transcription complexes and could, therefore, represent a general procedure for the isolation of actively transcribed areas from chromatin.

Journal ArticleDOI
TL;DR: Hybridization of purified, 32p-labeled 5.8S ribosomal RNA from Xenopus Laevis to fragments generated from X. laevis rDNA by the restriction endonuclease, EcoRI, demonstrates that the 5.7S rRNA cistron lies within the transcribed region that links the 18S and 28S r RNA cistrons.
Abstract: Hybridization of purified, 32p-labeled 5.8S ribosomal RNA from Xenopus laevis to fragments generated from X. laevis rDNA by the restriction endonuclease, EcoRI, demonstrates that the 5.8S rRNA cistron lies within the transcribed region that links the 18S and 28S rRNA cistrons.

Journal ArticleDOI
TL;DR: The hypothesis that the nucleolus, in this organism, contains several distinct globular subunits each containing ribosomal DNA as a key component appears to account for the unusual behaviour of theucleolus during the cell cycle.
Abstract: The present study has been mainly focused on the nucleolar cycle in the slime mould Physarum polycephalum. The ultrastructural characteristics of the interphase nucleolus, in this species, are quite similar to those of nucleoli in other organisms: it is essentially constituted of large particulate zones surrounding denser regions which are predominantly fibrillar in texture. The latter nucleolar zones, following fixation with osmium tetroxide, are characterized by the presence of opaque granules approximately 25 nm in diameter. Contrary to the situation which generally prevails in other eukaryotes, the late prophase nucleolus fragments into numerous globular bodies which are recognizable by the presence of opaque particles. These fibrillogranular nucleolar fragments persist during mitosis and are observed to become incorporated in the newly formed nucleolus. High-resolution radioautographic observations reveal that these nucleolar remnants contain DNA. The present observations together with recent biochemical data from other authors on the characteristics and mode of duplication of nucleolar DNA in P. polycephalum have led us to the hypothesis that the nucleolus, in this organism, contains several distinct globular subunits each containing ribosomal DNA as a key component. The existence of such morphological subunits appears to account for the unusual behaviour of the nucleolus during the cell cycle.

Journal ArticleDOI
TL;DR: An attempt to relate differences with functional and ecological characteristics indicates that evolutionary variation of rDNA proportions is not casual and can help to define systematic affinities and circumscribe infrageneric taxa.
Abstract: Eight species ofAllium subgen.Allium sect.Allium have been studied at the cytological level by means of karyological analyses and at the biochemical level with regard to the proportions of ribosomal DNA. All the species have a basic genome of x = 8.A. sativum, A. commutatum, A. ampeloprasum, andA. vineale possess approximately 0.050% rDNA and two nucleolus organizer regions per basic chromosome set.A. sphaerocephalon andA. arvense have two nucleolus organizers, andA. amethystinum three nucleolus organizers per haploid (n = x) genome: the three species possess approximately 0.075% rDNA.A. acutiflorum has five nucleolus organizer regions per haploid genome and 0.121% rDNA. An attempt to relate these differences with functional and ecological characteristics indicates that evolutionary variation of rDNA proportions is not casual. Such data also can help to define systematic affinities and circumscribe infrageneric taxa.

Journal ArticleDOI
01 Jun 1977-Cell
TL;DR: Sucrose gradient analysis of DNA from detergent-pronase lysates of whole adult flies has been used to examine a variety of genotypes for the presence of ribosomal genes not integrated into the DNA of the chromosome to induce unintegrated genes.

Journal ArticleDOI
TL;DR: These experiments were performed in view of the hypothesis that there was a polyploidization event, when acraniate-like organisms emerged from tunicate-like animals during early chordate evolution.
Abstract: 1. 1. The relative genomic proportion of genes coding for ribosomal RNA has been determined in Ciona intestinalis (Tunicata) and Branchiostoma lanceolatum ( Acrania). 2. 2. These experiments were performed in view of the hypothesis that there was a polyploidization event, when acraniate-like organisms emerged from tunicate-like animals during early chordate evolution. 3. 3. Ciona intestinalis, however, has a rDNA content six times that of Branchiostoma lanceolatum.