Isolation and sequence organization of human ribosomal DNA.

The genes coding for 28 S and 18 S ribosomal RNA have been purified from leukemic leukocytes of one human individual by density gradient centrifugation. The purified ribosomal DNA was analyzed by restriction endonuclease digestion and electron microscopy. The location of cleavage sites for the restriction endonuclease EcoRI was established by R-loop mapping of restriction fragments by electron microscopy. The results are in agreement with gel analysis and gel transfer hybridization. One type of ribosomal DNA repeating unit contains four cleavage sites for EcoRI. Two of these cuts are located in the genes coding for 28 S and 18 S rRNA, while the other two are in the non-transcribed spacer. Thus, one of the restriction fragments generated contains non-transcribed spacer sequences only and is not detected by gel transfer hybridization if labeled rRNA is used as the hybridization probe. A second type of repeating unit lacks one of the EcoRI cleavage sites within the non-transcribed spacer. This indicates that sequence heterogeneity exists in human rDNA spacers. R-loop mapping of high molecular weight rDNA in the electron microscope reveals that the majority of repeats are rather uniform in length. The average size of 22 repeats was 43.65(±1.27) kb. Two repeats were found with lengths of 28.6 and 53.9 kb, respectively. This, and additional evidence from gels, indicates that some length heterogeneity does exist in the non-transcribed spacer. The structure of the human rDNA repeat is summarized in Figure 10.     

Length heterogeneity of amplified circular rDNA molecules in oocytes of the house cricket Acheta domesticus (Orthoptera: Gryllidae)

Amplification of the genes coding for rRNA occurs in the oocytes of a wide variety of organisms. The amplification process appears to be mediated through a rolling-circle mechanism. The approximate molecular weight of the smallest rDNA circles is equivalent to the estimated combined molecular weight of DNA which codes for a single ribosomal RNA precursor molecule and an associated non-transcribed spacer DNA sequence. RNA-DNA hybridization studies carried out on oocytes of the house cricket, Acheta domesticus, suggest that DNA coding for rRNA accounts for only a small fraction of the rDNA satellite, all of which is amplified in the oocyte. In order to test the possibility that the remainder of the amplified rDNA represents spacer and to determine whether a rolling-circle mechanism might also be involved in amplification in A. domesticus oocytes, rDNA was isolated from ovaries of A. domesticus and spread for electron microscopy. A large proportion of the rDNA isolated from ovaries is circular, while main-band DNA and rDNA prepared from other tissues demonstrates few if any circles. The mean size of the smallest rDNA circles is approximately 8 times longer than the length estimated for DNA which codes for 18 S and 28 S rRNA. Denaturation mapping shows the rDNA circles to contain two major readily denaturing regions located about equidistant from one another on the circle. Each readily denaturing region accounts for 4–6% of the total DNA in the circle. The fact that only 12% of the average molecule is required to code for A. domesticus 18 S and 28 S rRNA is consistent with the hybridization data. Considerable size heterogeneity exists in the length of the smallest class of rDNA molecules. In the rDNA of other species such heterogeneity has been shown to reside in the non-transcribed spacer.     

Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units,inverted sequences,pseudogenes and chromosome loci

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements.     

Human ribosomal RNA gene spacer sequences are found interspersed elsewhere in the genome

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolated varied by not more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.     

Number and sites of rDNA loci of Guizotia abyssinica (L. f.) Cass. as determined by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was employed on somatic chromosome preparations of Guizotia abyssinica using a DNA probe of the 18S-5.8S-26S rRNA genes including the transcribed and non-transcribed spacer sequences. A maximum of six major and two minor signal sites of rDNA was observed. However, the minor signals were not persistently detected. The positions of the FISH signals coincided with the sites of the nucleolar organizer regions and their adjacent C-banded heterochromatin when present.     

Structural analysis of ribosomal DNA homologues in nucleolus-less mutant of Xenopus laevis

Sequences homologous to the ribosomal DNA (rDNA) in a Xenopus anucleolate (nucleolus-less) mutant were analyzed by Southern blot analysis. The mutant was found to possess a variety of sequences homologous to non-transcribed spacer (NTS) and/or coding region of rDNA. 65 rDNA-homologous clones were isolated from a genomic DNA library of the mutant. All the clones showed only partial homology to the normal rDNA unit and their restriction maps differed from that of the normal rDNA unit. Based on the hybridization patterns, the rDNA-homologous clones were divided into four groups (I-IV). Structure of group IV, which most strongly hybridized to normal rDNA probe, was analyzed by nucleotide sequencing. The group IV sequence was found to contain a part of the rDNA, including Bam island, enhancer element, promoter region, external transcribed spacer, and a portion of 18S rRNA gene. The blotting analysis suggested that the group IV sequence is specific for a particular strain of Xenopus.     

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.     

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.     

Chromosomal localization of 5S and 18S–5.8S–25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.     

Study of ribosomal deoxyribonucleic acid of sea urchin

The structure of ribosomal DNA (rDNA) satellite of sea urchin (Lytechinus variegatus) sperm has been re-examined after purification by two widely used methods. Saturation hybridization experiments indicate that about 28–32% of the rDNA contain sequences complementary to rRNA. Results of hyperchromic spectral analysis reveal that the rDNA melts as two distinct but unequal components. The early transition presumably corresponds to the melting of most of the transcribed part and the late transition is suggestive of the melting of a G+C-rich segment of the rDNA, which may be a non-transcribed spacer.Abbreviations F_AT fraction of all A+T pairs denatured - F_GC fraction of all G+C pairs denatured - SSC standard saline citrate - rDNA template for ribosomal RNA This work is a part of the author's Ph.D. thesis (U.N.C., Chapel Hill).     

Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct non-transcribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

Polymorphism of untranscribed spacer rDNA as a molecular genetic marker in population studies of cattle]

Variable polymorphic patterns were detected using EcoRI-SalI fragment of bovine rDNA, including 3'-end of 28S rRNA gene with the adjacent portion of the transcribed spacer, as a probe for hybridization. Some features of these polymorphic patterns are similar to DNA fingerprints detected with the M13 probe. Bovine rDNA spacer polymorphism was used as a molecular genetic marker for population analysis of individual specific patterns of 4 cattle breeds with the help of the Jeffreys' method. It was supposed that the probability of identical fingerprints appearance could be the characteristics of heterogeneity of cattle populations. The observed length of polymorphic gragments ranged from 2000 to 6000 bp. The mean number of fragments per individual for all breeds was 15.05. The probability of identical patterns appearance was very high: from 1.18 x 10(-5) in ajshir's breed to 1.43 x 10(-7) in "white and black"s' breed. So, high probability seems to be dependent on the high allelic frequency and the way of breeding.     

A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes

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. Ribosomal DNA fragments were detected in electrophoretically fractionated digests of total nuclear DNA by in situ hybridization with radioiodinated rRNAs or with complementary RNA synthesized directly on rRNA templates. A map of the rDNA which includes 13 restriction sites was constructed from the sizes of rDNA fragments and their labeling by different probes The map indicates that the rRNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long. At least two, and possibly four, classes of repeating unit can be distinguished, the heterogeneity probably residing in the very large nontranscribed spacer region. The 5.8S rRNA gene lies in the transcribed region between the 18S and 28S genes.     

An electron microscope heteroduplex study of the ribosomal DNAs of Xenopus laevis and Xenopus mulleri

The arrangement of the genes and spacers has been analyzed in ribosomal DNA of Xenopus laevis and Xenopus mulleri by heteroduplex mapping and visualization of ribosomal RNA-DNA hybrids. Heterologous reassoeiated molecules show a characteristic pattern in which two perfectly duplexed regions, whose lengths are those predicted by the known lengths of the 18 S and 28 S genes, are separated by a small substitution loop of about 0.23 × 10⁶ daltons and a large region of partial homology which averages 3.24 × 10⁶ daltons. These mismatched regions are entirely consistent with the known sequence divergence previously described (Brown et al., 1972) for the transcribed and non-transcribed spacer regions of the two rDNAs, respectively. Hybrids of X. laevis rDNA with 18 S and 28 S rRNA contain two duplex regions of the expected lengths for the 18 S and 28 S genes separated by 0.49 × 10⁶ daltons of single-strand DNA. This latter value is the length of the transcribed spacer region between the 18 S and 28 S RNAs that has been measured within the 40 S RNA precursor molecule by secondary structure mapping (Wellauer &; Dawid, personal communication). There is also a longer single-strand region separating one 18 S + 28 S gene set from the next; this is considered to be mainly non-transcribed spacer.We conclude that the 18 S and 28 S genes are separated by about 0.5 × 10⁶ daltons of DNA of which about half is homologous in the two Xenopus species. This region is part of the transcribed spacer. In addition, the longer non-transcribed spacer can be seen to have some homology between the two species; the location of this homology is fairly reproducible between molecules and has been carefully documented by contour length measurements.     

Isolation and organization of calf ribosomal DNA.

Ribosomal DNA (rDNA) from calf was isolated by three density gradient centrifugations. The first centrifugation in Cs2S04/BAMD was used to obtain partially resolved dG+dC-rich fractions from total DNA. The second and third centrifugations, in Cs2S04/Ag+, led to the isolation of an rDNA fraction characterized by a symmetrical band in CsCl, p = 1.724 g/cm3. This new procedure appears to be generally suitable for the isolation of rDNA and other dG+dC-rich repeated genes. The organization of isolated calf rDNA has been studied by restriction enzyme digestion and by hybridization with cloned rDNA from Xenopus laevis. The repeat unit of calf rDNA has a molecular weight of 21x10(6) and is split by EcoR1 into two fragments, 16x10(6) and 5.0x10(6), and by BamHI into seven fragments. EcoRI and BamHI sites have been mapped. Most of the 18S and 28S RNA genes and the transcribed spacer are contained in the small EcoRI fragment, while the non-transcribed spacer is localized in the large EcoRI fragment. This spacer showed length heterogeneity within a single individual; such heterogeneity is limited to two regions of the spacer.