Non-transcribed Spacer Region Research Articles

Structural organization of Acheta rDNA : Evidence for differential amplification of soma and germ-line-specific rDNA sequences

Amplification is one of the mechanisms whereby the expression of genes can be specifically reinforced. Ribosomal gene amplification in amphibian and insect oocytes is a particularly well documented case. We studied heterogeneity, amplification and size of Acheta domesticus (insects; Orthoptera) ribosomal DNA and characteristics of male and female somatic or germ line rDNAs by analysis of genomic clones from a conventional and a microclone library. The length of the Acheta rDNA repeat unit (transcription unit and non-transcribed spacer (NTS) varied from 47 × 10 3 to 60 × 10 3 base-pairs, with highest variability within the NTS region. Deletions, fragment length heterogeneity and size variability in small steps of individual NTS segments are responsible for the observed size variation. The number of rDNA repeat units per haploid genome of Acheta was determined as 190(±10%). The rDNA is amplified 14(±10%)-fold in the oocyte, producing about 10,000 gene copies per cell. Our results show that the amplification mechanism does not favor individual fragments within the repeat unit. Thus, it can be concluded that amplification does not change the chromosomal characteristics of the rDNA pool. Two fragments specific for oocyte rDNA suggest that the rearrangements accompanying amplification are preferentially located in one distinct EcoRI fragment. Certain regions of Acheta rDNA contain cell-type-specific fragments: it was thus possible to characterize one purely male fragment and a second one specific for male and female soma but not for germ line rDNA. We show that Acheta rDNA reveals a combination of many features reported from different organisms and novel tissue-specific alterations on an extremely large repeat unit. The tissue-specific alterations indicate sexual and soma/germ line differentiation events that are derived by as yet unknown mechanisms.

Restriction fragment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans

Differences in restriction fragment length polymorphisms (RFLPs) have been detected in isolates of Aspergillus fumigatus. Genomic DNA from 11 isolates was digested with EcoRI, separated by electrophoresis, Southern blotted and probed with DNA from the intergenic spacer or non-transcribed spacer region of the rRNA gene complex of Aspergillus nidulans. Three distinct RFLP patterns were detected which differed from the control patterns observed with A. nidulans, Aspergillus flavus and Aspergillus niger hybridized with the same probe. Furthermore, the differences in RFLP patterns in the A. fumigatus isolates were not detected when probed with DNA coding for the rRNA complex in Saccharomyces cerevisiae. These findings may be of use in the study of the epidemiology and pathogenesis of infections caused by A. fumigatus.

Spacer Promoters Are Orientation-Dependent Activators of Pre-rRNA Transcription in Drosophila melanogaster

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster.

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

Variation in the ribosomal DNA repeat unit within single-oospore isolates of the genus Pythium

Restriction analyses of the DNA repeat unit coding for the production of 5.8S, 17S, and 26S ribosomal RNA (rDNA) isolated from several different Pythium species revealed polymorphic forms within a single-oospore isolate. Restriction mapping of cloned rDNA from Pythium paroecandrum indicated that two major forms were present in approximately equal ratios (A and B, 11.6 and 11.9 kb in size, respectively), which differed in the nontranscribed spacer region for numbers and locations of restriction sites for HaeIII, SalI, and KpnI and an insertion–deletion adjacent to the 3′ end of the 26S RNA coding region. The insertion–deletion makes form B 0.27 kb larger than form A. An additional insertion–deletion in both forms of approximately 60 bp in the same region makes a total of four polymorphic types in the isolate investigated. Similar results also were obtained for Pythium spinosum following restriction digestion of purified rDNA. A survey of other Pythium species indicates that the presence of polymorphic forms in the same isolate, variation in the number or polymorphic forms in different isolates of the same species, and insertions–deletions in multiples of approximately 60 bp adjacent to the 3′ end of the 26S coding region are common in the genus.Key words: Pythium spp., rDNA.

Primary structure of the non-transcribed spacer region and flanking sequences of the ribosomal DNA of Neurospora crassa and comparison with other organisms

The non-transcribed spacer (NTS) region of the rDNA of Neurospora crassa contains the transcription regulatory sequences. We isolated a 3.4 kb EcoRI fragment from wild type N.crassa rDNA and cloned in the plasmid pBR325 at the EcoRI site. The insert contains the entire NTS region along with the flanking sequences. Nucleotide sequencing of 3592 nt shows many interesting features like: the NTS region is rich in G+C content (65% G+C); it contains the conserved rRNA processing site 6 (with the nucleotide sequence motif GGTGCGAGAACCCGG, from nt residue 226 to 240, a characteristic feature of most eukaryotic rDNA nontranscribed spacer region); and the NTS region also contains the transcription termination site with the representative Sal I ☐ (from nt residue 1469 to 1477). The potential sequences of transcription termination site are located 288 nt downstream from the end of 26S rRNA gene, and another sequence motif CTTCCT (from nt residue 512 to 517) shows similarity with the human transcription termination site T-2 of its pre-rRNA. Nucleotide sequence homology matrix analysis suggests its relatedness to Saccharomyces cerevisiae and not to human, mouse, rat, Drosophila, Xenopus, wheat, rice and cucumber NTS region. The phylogenetic implication of the NTS region and exploitation of N.crassa NTS rDNA clone to correlate the otherwise indistinguishable species of Neurospora and the correlation with other organisms has been discussed. To the best of our knowledge this is the first report where the nucleotide sequence of the entire NTS region of a filamentous fungus has been determined.

An unusual Y chromosome of Drosophila simulans carrying amplified rDNA spacer without rRNA genes.

The X and Y chromosomes of Drosophila melanogaster each contain a cluster of several hundred ribosomal RNA genes (rDNA). A nontranscribed spacer region separates adjacent rRNA genes and contains tandem copies of 240 bp repeats that include the initiation site for RNA polymerase I transcription. We show here that Drosophila simulans, a sibling species of D. melanogaster, contains few, if any, rRNA genes on its Y chromosome but carries instead a large block (3,000 kb or 12,500 copies) of 240 bp nontranscribed spacer repeats. The repeats are located at the tip of the long arm of the simulans Y chromosome, in contrast to their location among rRNA genes on the short arm of the Y chromosome of D. melanogaster. The bobbed mutation in homozygous females of D. melanogaster shortens and thins the bristles, owing to a partial deletion of rRNA genes on the X chromosome. The bristles of bobbed/Y males are normal owing to the presence of a full complement of rRNA genes on the Y chromosome. Peculiarly, in bobbed/Y males of D. simulans the short bristle phenotype does not return to normal but is enhanced by the presence of the Y chromosome. We propose that the 12,500 nontranscribed spacer repeats on the Y chromosome are responsible for this biological effect by competition for a protein factor(s) essential for normal levels of rDNA transcription at the X-linked locus.

Sequencing of the non-transcribed spacer region of the ribosomal RNA genes of Neurospora crassa

Sequencing of the non-transcribed spacer region of the ribosomal RNA genes of Neurospora crassa

Mosquito ribosomal RNA genes: Characterization of gene structure and evidence for changes in copy number during development

A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus, has been characterized by restriction mapping, Southern-blotting and S1-nuclease analyses. The 18S rRNA coding region measured 1800 bp and contained a conserved Eco RI site near the 3′-end. The 28S rRNA coding region was divided into α and β sequences, comprising 1750 and 2000 bp, respectively, which were separated by a 350 bp sequence that is removed from the rRNA precursor during processing. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region that contained a series of PvuI repeats upstream of the 18S rRNA coding sequence. During development of the mosquito, Aedes aegypti, the rRNA gene copy number per haploid genome increased from about 400 in larvae to about 1200 in adults.

Fibrillar center distribution in nucleoli of PHA-stimulated human lymphocytes

We have utilized acidic toluidine blue staining for RNA and immunofluorescence staining for RNA polymerase 1 to visualize the distribution of fibrillar centers (FCs) in nucleoli of PHA-stimulated human lymphocytes. At 0 h, there is a single large fibrillar center in each nucleolus which splits into smaller and more numerous FCs until the number of FCs reaches five, the number of nucleolus organizers in normal haploid human cells. With time, each FC then "unwinds" to form linear arrays of smaller FCs until the maximum number of FCs approaches the ribosomal gene copy number of 200 at 48 h in culture. It is hypothesized that in the most active state, each nucleolar FC visualized by RNA polymerase 1 staining actually represents a single transcription unit and the distance between adjacent FCs is occupied by the nontranscribed spacer region. We conclude that the number of fibrillar centers per nucleolus can be used as a direct quantitative measure of nucleolar transcriptional activity.

Restriction enzyme mapping of ribosomal DNA can distinguish between fasciolid (liver fluke) species

Recognition sites for nine different restriction endonucleases were mapped on rDNA genes of fasciolid species. Southern blots of digested DNA from individual worms were probed sequentially with three different probes derived from rDNA of Schistosoma mansoni and known to span between them the entire rDNA repeat unit in that species. Eighteen recognition sites were mapped for Fasciola hepatica, and seventeen for Fasciola gigantica and Fascioloides magna. Each fasciolid species had no more than two unique recognition sites, the remainder being common to one or both of the other two species. No intraspecific variation in restriction sites was noted in F. hepatica (individuals from 11 samples studied; hosts were sheep, cattle and laboratory animals; geographical origins, Australia, New Zealand, Mexico, U.K., Hungary and Spain), or in F. gigantica (two samples; Indonesia and Malaysia). Only one sample of F. magna was available. One specimen of Fasciola sp. from Japan (specific identity regarded in the literature as uncertain) yielded a restriction map identical to that of F. gigantica. Almost all recognition sites occurred in or near the putative rRNA coding regions. The non-transcribed spacer region had few or no cut sites despite the fact that this region is up to about one half of the entire repeat unit in length. Length heterogeneity was noted in the non-transcribed spacer, even within individual worms.

Proteins That Bind to the Yeast rDNA Enhancer

The transcription of ribosomal RNA genes differs from that of other genes in several respects: the use of a specialized polymerase, the generally high level of transcription, and the tandem arrangement of the genes. In the yeast Saccharomyces cerevisiae, we identified a nucleotide sequence in the "nontranscribed" spacer region that had many characteristics of an enhancer of transcription (8). More recently, it has become apparent that transcription of this sequence occurs (9) and that it may also be involved in some aspect of termination of 35 S rRNA transcription. The likelihood that there are protein factors involved in termination and activation of transcription and that these may participate in the coupling of the transcription of adjacent rRNA genes led us to search for proteins that might bind to the enhancer. We have identified two such proteins, termed REB1 and REB2, that bind to the enhancer and protect specific sequences from attack by chemical and enzymatic reagents. It is noteworthy that there is a second REB1 binding site approximately 210 base pairs upstream of the origin of transcription of rRNA and that binding of REB1 to this site alters the conformation of DNA adjacent to the site of initiation.

Genetic Diversity in Pearl Millet (Pennisetum glaucum [L.] R. Br.) at the DNA Sequence Level

We analyzed restriction-fragment-length polymorphisms among chloroplast, nuclear ribosomal RNA, and alcohol dehydrogenase (ADH) sequences in a group of 25 wild and 54 cultivated accessions of pearl millet (Pennisetum glaucum [L] R. Br.). We observed no polymorphism for chloroplast DNA sequences representing approximately 50% of the total chloroplast genome. Nuclear rRNA genes exhibited polymorphisms, the majority of which mapped to the nontranscribed spacer region. Most of the rRNA gene variants were observed among wild pearl millet accessions; the cultivars appeared to be highly uniform. Adh 1 sequences exhibited substantial sequence diversity, and we observed no marked differences in diversity between wild and cultivated pearl millet types. The highly outcrossing nature of pearl millet, the parallel geographic distribution of Adh1 variants, and the widespread occurrence of the most frequent Adh 1 and rDNA variants in wild and cultivated pearl millet genotypes preclude the identification of the precise pattern of domestication. This study, however, points to the need for more empirical studies to determine the level of genetic diversity associated with various molecular markers; it also indicates the need for additional germ plasm explorations, particularly in the eastern Sahel.

Interaction between the Yeast Mitochondrial and Nuclear Genomes Influences the Abundance of Novel Transcripts Derived from the Spacer Region of the Nuclear Ribosomal DNA Repeat

We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription.

Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat.

We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription.

Conserved organization of an avian histone gene cluster with inverted duplications of H3 and H4 genes

The organization of histone gene clusters of the duck Cairina moschata was studied in the DNA inserts of two recombinant phage that overlap and feature identical histone gene arrangements but differ in sequence details and in the extent of repetition of an AT-rich motif in one of the nontranscribed spacer regions. These few but substantial differences between otherwise nearly identical histone gene groups suggest that we have independently isolated alleles of the same site of the duck genome or that this gene arrangement occurs (with slight variations) more than once per haploid genome. Within the histone gene cluster described, H3 and H4 genes are duplicated (with inverted orientation), whereas one H1 gene is flanked by single H2A and H2B genes. The arrangement of duck histone genes described here is identical to a subsection of the chicken genome but differs from any other published histone gene cluster.

Cis-acting sequences from mouse rDNA promote plasmid DNA amplification and persistence in mouse cells: implication of HMG-I in their function

Searching for amplification promoting sequences within the murine rDNA cistrons, we isolated two elements from the nontranscribed spacer region. These 370 bp and 423 bp long cis-acting elements, referred to as muNTS1 and muNTS2, are localized 4.1 kb and 4.6 kb upstream the RNA polymerase I transcriptional start site. They contain ca. 50 bp long AT-rich sequences that strongly interact with a protein from nuclear extracts. The protein could be purified and identified as HMG-I. A synthetic oligonucleotide encompassing the AT-rich stretch from muNTS1 is able to substitute for the muNTS elements. A similar sequence from the nontranscribed spacer of rat has previously been reported to be important for the function of the RNA polymerase I enhancer (1). Therefore the interaction of HMG I with the muNTS elements may play a role both in the stimulation of DNA amplification and transcription.

Organization of replication of ribosomal DNA in Saccharomyces cerevisiae.

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

Organization of replication of ribosomal DNA in Saccharomyces cerevisiae.

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

Proximity of an ARS consensus sequence to a replication origin of pea (Pisum sativum)

The replication origin (ori-r9) of the 9.0 kb rDNA repeats of pea (Pisum sativum, cv. Alaska) was cloned and found to reside in a 1.5 kb fragment of the non-transcribed spacer region located between the 25 S and 18 S genes. Labeled rDNA rich in replication forks, from cells positioned at the G1/S phase boundary, was used to map ori-r9 by hybridization procedures. Ori-r9 is in a 210-base fragment that is 1.6 kb from the 5' end of the 18 S gene and about 1.5 kb from the 3' end of the 25 S gene. The same procedures, using labeled synthetic ARS consensus sequence as a probe, showed than an ARS consensus sequence is located 3' to ori-r9 in a 710-base fragment. An ARS consensus sequence is, therefore, adjacent to ori-r9 but not coincidental with it.