Ribosomal RNA Cistrons Research Articles

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.

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.

Intrageneric and intergeneric similarities of ribosomal RNA cistrons of the genus Pseudomonas and the implications for taxonomy.

Intrageneric and intergeneric similarities of ribosomal RNA cistrons of the genus Pseudomonas and the implications for taxonomy.

Intrageneric and intergeneric similarities of ribosomal RNA cistrons of free-living nitrogen-fixing bacteria and the implications for their taxonomy

Intrageneric and intergeneric similarities of ribosomal RNA cistrons of free-living nitrogen-fixing bacteria and the implications for their taxonomy

Replication of the linear mitochondrial DNA of Tetrahymena pyriformis

1. Electron micrographs of the linear mtDNA from Tetrahymena pyriformis strain GL show linear molecules with a duplex ‘eye’ of variable size in the middle. This indicates that replication of this DNA starts near the middle of the molecule and proceeds bidirectionally to the ends, as previously shown for the mtDNA of strain ST (Arnberg, A.C., Van Bruggen, E.F.J., Clegg, R.A., Upholt, W.B. and Borst, P. (1974) Biochim. Biophys. Acta 361, 266–276). The mtDNAs of these two strains have little base sequence homology beyond the ribosomal RNA cistron (Goldbach, R.W., Bollen-De Boer, J.E., Van Bruggen, E.F.J. and Borst, P. (1978) Biochim. Biophys. Acta 521, 187–197). 2. Electron micrographs of mtDNA from strain ST, spread under non-denaturing conditions, contain only molecules with fully duplex ends. mtDNA spread under conditions of early denaturation contains duplex loops on one end (40% of all molecules) or both ends (37%). The loops are stable to partial denaturation and vary in size from 0.15 to approximately 1.0 μm, most loops measuring 0.25–0.40 μm. No loops are formed with single-stranded DNA under analogous conditions and we conclude from this result that loop formation is based on the presence of straight, rather than inverted, duplications near the ends. 3. When full-length 3H-labelled mtDNA from strain ST, 32P-labelled at the 5′-termini with T 4 polynucleotide kinase, was sedimented in alkaline sucrose gradients, > of the 3H and <30% of the 32P cosedimented with full-length molecules; the remaining 32P sedimented heterogeneously and predominantly with the DNA less than 10% the size of intact single strands. Brief incubations of full-length mtDNA with DNA polymerase I from Escherichia coli and labelled dNTPs at 15°C did not lead to preferential labelling of terminal EcoRI fragments of the DNA. From these results we infer that the DNA contains nicks or gaps near the termini and that these are not bordered by free 3′-OH groups. 4. A model is presented in which straight sequence repetitions at the termini of Tetrahymena pyriformis mtDNA are involved in the later stages of replication. This model can also account for the pronounced terminal heterogeneity previously observed in this DNA.

X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (diptera, sciaridae)

The so-called controlling element (CE), which normally programs the curious behavior of the sex chromosome in this genus, has been localized in the short right arm of the polytene X in S. coprophila. The localization was accomplished by use of five X-autosome translocations whose break points define three blocks of heterochromatin (“heterochromomeres”) extending from the X centromere to the very end (right) of the chromosome. The behavior of the translocation chromosomes at the crucial second spermatocyte division was examined and the “precocious” chromosome identified in all five cases. Then, knowing the heterochromomere make-up of each chromosome, the position of the CE could be mapped; it is located in heterochromomere H2, the same block of heterochromatin that contains 50% of the ribosomal RNA cistrons. — The question of whether the CE can manipulate any centromere in the nucleus has been only partially answered. It can manipulate translocation chromosomes which possess the centromere of the metacentric autosome (salivary chromosome IV) or that of the shorter rod (salivary chromosome II); but the longer rod (salivary chromosome III) whose proximal end, as seen in the polytene nucleus, is heavily laden with heterochromatin of its own, has not been brought under CE control. — In one of the translocations, T23, the precocious chromosome is a very large metacentric chromosome which resembles the “peculiar” V-shaped X of S. pauciseta. This peculiarity is not observed in the J-shaped precocious chromosome of T29. These points are discussed.

The number, physical organization and transcription of ribosomal RNA cistrons in an archaebacterium: Halobacterium halobium.

Because it is now clear that archaebacteria may be as distinct from eubacteria as either group is from eukaryotic cells, and because a specifically archaebacterial ancestry has been proposed for the nuclear-cytoplasmic component of eukaryotic cells, we undertook to characterize, for the first time, the ribosomal RNA cistrons of an archaebacterium (Halobacterium halobium). We found these cistrons to be physically linked in the order 16S-23S-5S, and obtained evidence that they are also transcribed from a common promoter(s) in the order 5'-16S-23S-5S-3'. We showed that, although slightly larger immediate precursors of 16S and 23S are readily seen, no common precursor of both 16S and 23S can be easily detected in vivo. In all these respects the archaebacterium H. halobium is like a eubacterium and unlike the nuclear-cytoplasmic component of eukaryotic cells. We found, however, that it differs from eubacteria of comparable (large) genome size in having only one copy of the rRNA gene cluster per genome.

In vitro transcripts from the rrn B ribosomal RNA cistron originate from two tandem promoters

The functional structure of the promoter region of a bacterial ribosomal operon is analyzed by in vitro transcription of linear DNA fragments derived from the hybrid Col EI plasmid pGG1. This plasmid contains the promoter region of the rrn B ribosomal cistron present in the lambdarifd18. When transcripts arising from this promoter region are terminated by restriction endonuclease cleavage of DNA, two RNA chains are resolved by gel electrophoresis that differ in length by about 100 bases. Evidence is presented indicating that these two transcripts arise from different initiation sites, each directing tandem transcription on the sense strand of the early portion of the ribosomal cistron.

Conservation of the sequence and position of the ribosomal RNA genes in Tetrahymena pyriformis mitochondrial DNA

1. We have done cross-hybridizations between the mitochondrial ribosomal RNAs and DNAs from strains ST and PP of Tetrahymena pyriformis. DNA · ribosmal RNA hybrid formation can be completely prevented by an excess of the heterologous ribosomal RNA and the heterologous hybrids melt 6°C below the homologous hybrids. This shows that the ribosomal RNA cistrons can account for the 5% cross-hybridization previously observed between the mtDNAs of strains PP and ST (Goldbach et al. (1977) Biochim. Biophys. Acta 477, 37–50). 2. By electron microscopy of DNA · ribosomal RNA hybrids we have determined the position of the ribosomal RNA cistrons on the mtDNA of strain GL, a mtDNA which we have shown to contain a sub-terminal 1 μm duplication-inversion and a terminal palindrome at one end which varies in length from 0 to 5 μm and which includes the 1 μm duplication-inversion (Arnberg et al. (1977) Biochim. Biophys. Acta 477, 51–69). The 21 S ribosomal RNA cistron overlaps the 1 μm duplication-inversion and as a result two or three cistrons are present, depending on the size of the terminal palindrome. Only one 14 S ribosomal RNA cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron and with the same polarity as this cistron. 3. We conclude from these results and those in the preceding paper that the sequence of the ribosomal RNAs and the position of the ribosomal RNA genes in the mtDNA is strongly conserved in Tetrahymena. Possible reasons for the duplication of 21-S ribosomal RNA genes and the terminal heterogeneity of Tetrahymena mtDNA are discussed.

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S 1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359–369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA · RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage λ-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5′-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.

Nucleoli and ribosomal RNA cistron numbers in Hordeum species and interspecific hybrids exhibiting suppression of secondary constriction

The interspecific hybrids of Hordeum exhibit selective suppression of secondary constriction formation in the chromosome(s) contributed by one of the two parents. A comparison of the number of SAT (secondary constriction) chromosomes in the metaphase cells and the maximum number of nucleoli in interphase cells revealed that the chromosomes capable of organising nucleoli were not always reflected through secondary constriction formation. — The rDNA (DNA complementary to rRNA) amounts were estimated by DNA-rRNA filter hybridisation in diploid and polyploid species of Hordeum and their hybrids. While similar rDNA proportions were present in diploid and autotetraploid lines of H. bulbosum, there were up to threefold differences between H. vulgare and allohexaploids. Furthermore, differences were also apparent between species of same ploidy level. Ribosomal RNA (18S+5.8S+26S) cistron numbers in each of the five experimental hybrids exhibiting the selective suppression of secondary constriction formation revealed no selective loss of rDNA. — The presence of a higher number of nucleoli than the number of SAT chromosomes seen and the presence of expected number of rRNA cistrons suggest that the suppression of secondary constriction formation is not due to selective loss of rDNA.

Trisomic analysis of ribosomal RNA cistron multiplicity in barley (Hordeum vulgare L.)

Ribosomal RNA cistron numbers in all the seven primary trisomics of diploid barley (Hordeum vulgare L.) were determined by DNA-rRNA filter hybridisation. Trisomies for the nucleolus organiser (NO) chromosomes 6 and 7 showed the highest levels of rDNA (DNA complementary to rRNA) indicating the localisation of rRNA cistrons on the NOs. Chromosomes 6 and 7 possessed 1,580 and 2,690 rRNA (18S + 5.8S + 26S) cistrons respectively. Trisomics for the other chromosomes (except for 3) also displayed levels of rDNA significantly higher (22–32%) than the diploid controls although the dosage of NOs was not altered. These non-specific increases were also present in trisomics for 6 and 7 (NOs) which showed further increases equivalent to their respective contributions. The nonspecific increases due to trisomy is indicative of rDNA compensation. Such increases did not persist in diploid sibs of the trisomics, demonstrating the nonheritable nature of the compensation.

Ribosomal RNA cistrons in Allomyces arbuscula

Bulk and nuclear DNA have been fractionated by preparative neutral CsCl equilibrium density gradient centrifugation and each fraction hybridized to labeled rRNA ( 25 + 18 S ). The cistrons coding for rRNA appeared on the light side of the main peak. Hybridization of the nuclear DNA fractionated by preparative Ag +—Cs 2SO 4 gradients at different pHs showed that the banding profile did not change as compared to the CsCl pattern. In Hg 2+—Cs 2SO 4 gradients, however, the peak of the rRNA · DNA hybrids shifted on the heavier side of the profile. This indicates that the ribosomal RNA cistrons in Allomyces are A · T-rich. Hybridization with homologous rRNA showed that, at saturation, 3.25% of the DNA is complementary to rRNA. With the genome size of 1.7 · 10 10 daltons, the multiplicity of rRNA cistrons has been found to be close to 270.

Environmentally induced changes in ribosomal RNA cistron number: purported lack of correlation with phenotype—A reply

Environmentally induced changes in ribosomal RNA cistron number: purported lack of correlation with phenotype—A reply

Environmentally induced changes in ribosomal RNA cistron number: purported lack of correlation with phenotype

Environmentally induced changes in ribosomal RNA cistron number: purported lack of correlation with phenotype

Increased RNA polymerase activity in isolated liver nucleoli from thyroidectomized rats treated with triiodothyronine.

In isolated liver nucleoli from thyroidectomized rats the activity of the two RNA polymerase I populations, one of which is active and the other inactive towards the endogenous chromatin template, is greatly enhanced 10 and 24h after a single ip injection triiodothyronine (T3). When the nucleolar enzyme is solubilized and assayed with exogenous DNA as template, it retains, after T3 treatment, the same increase in activity as observed in intact nucleoli. On the contrary, the template availability, as judged by the binding capacity of isolated nucleoli for [3H]actinomycin D, does not appear to be modified by the hormone. These observations support the conclusion that the enhanced nucleolar RNA synthesis following T3 administration is due to an increased activity of the RNA polymerase I itself rather than to a greater availability of ribosomal RNA cistrons. The hormonal stimulation of both nucleolar RNA polymerase activities depends on continuous protein synthesis since it is almost completely abolished by the administration of cycloheximide.

A model for switching on ribosomal RNA synthesis by creating a palindromic DNA sequence in the promoter region of the ribosomal RNA cistron: the “structon”

We propose here a new model in which the formation of a symmetrical sequence by replication of a cistron to form a linear palindrome creates an active promoter site and thereby activates transcription. The name we propose for this model is “the structon”, a condensation of “structural turn on”, this being the element that distinguishes it from the classical repressor-operator type of regulation. The experimental system from which these ideas arose is the nucleolar DNA (rDNA) of the ciliated protozoan Tetrahymena pyriformis. We propose this as a model for gene regulation, which might explain the high occurrence of palindromic sequences in eukaryotes. A short review of the significance of palindromes in DNA-protein interactions is presented.

Molecular aspects of the environmental induction of heritable changes in flax

The conditions for the induction and stabilisation of environmentally induced changes in flax, and the characterisation of these changes for six characters have been reviewed. A model for the induction and expression of these changes has been proposed. The environmental conditions for the successful induction of heritable changes comprise at least two components; a specific inducing component, for example a particular fertiliser treatment, which is required to induce the change, and a general inducing component, providing for vigorous healthy growth, which is necessary for the induced changes to be inherited. Stable induced changes require appropriate environments for their maintenance, for example heated greenhouses for the first 5 weeks of seedling growth and adequate pot size. Under certain growth conditions the induced changes can themselves be modified. DNA differences induced in the different genotrophs can occur at a number of sites within the genome, including the ribosomal RNA cistrons. Environmental induction causes changes in the isozyme band patterns of peroxidase, acid phosphatase and esterase isozymes. The genotrophs behave as distinct genetic types in crosses, with a complex pattern of inheritance of the hairy septa character. The association between the different induced characters is examined. All the genotrophs can be distinguished from each other on at least one of the six characters. A model for the induction of the DNA changes, involving a type of IS-element has been proposed. The expression of the induced changes, in terms of altered numbers of copies of particular sequences at specific sites within the genome, has been considered.

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.

In vitro transcription of three different ribosomal RNA cistrons of E. coli; heterogeneity of control regions

Ribosomal RNA synthesis from three different rRNA cistrons of E. coli, located on different phage DNAs was compared and found to have the same characteristics as regards chain length, salt and temperature dependence and the effect of ppGpp. However, some clear and reproducible quantitative differences between rRNA synthesis from the different templates both in presence and absence of ppGGpp were found. Rifampicin and heparin experiments showed that these differences were located at the initiation sites. We propose that heterogeneity exists in the RNA polymerase binding regions of the rRNA prmoters in E. coli.