Isolation and mapping of ribosomal RNA genes of Caulobacter crescentus

Ribosomal DNA fragments of 1.0, 3.4, 3.7 and 6.1 kb² produced by EcoRI digestion of the Caulobacter crescentus genome were identified by hybridization to a labeled ribosomal RNA probe. These genomic sequences were further characterized by the isolation of 13 hybrid λ Charon 4 phages with rDNA inserts, and two of the recombinant phages, Ch4Cc773 and Ch4Cc1880, were examined extensively. The Cc773 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb and the Cc1880 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 6.1 kb that hybridized to ³²P-labeled rRNA. Thus, the two clones contain different DNA inserts which together account for all of the rDNA fragments detected in digests of the C. crescentus genome. Hybridization with isolated transfer RNA and individual rRNA species indicated that the arrangement of genes in both units is 16 S-spacer tRNA(s)-23 S-5 S, tRNA(s). Homology between the DNA inserts is largely restricted to the rRNA coding regions, which suggests that the two rDNA units are located in different regions of the chromosome. Results of quantitative hybridization experiments are most consistent with a single Cc1880 and Cc773 unit per genome equivalent of 2.7 × 10⁹ daltons. The relatively simple organization of rDNA sequences in the C. crescentus chromosome compared to Escherichia coli is discussed.     

Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×10⁶ d and two SalI ribosomal fragments of 3.8 and 1.5×10⁶ d. The ribosomal DNA (rDNA) units, 5.3×10⁶ d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA ribosomal RNA - rDNA ribosomal DNA d, dalton     

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.     

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

Transfer RNA genes associated with the 16S and 23S rRNA genes of Euglena chloroplast DNA

Hybridization studies of Euglena chloroplast ¹²⁵I-labeled tRNAs to restriction fragments of Euglena chloroplast DNA have shown that the spacer between the 16S and 23S rRNA genes, in two and possibly all three of the ribosomal DNA units, contains genes for tRNA^Ile and tRNA^Ala, whereas a tRNA gene (for either tRNA^Trp or tRNA^Glu) is located before probably all four 16S rRNA genes present on the chloroplast DNA molecule.     

A comparison of the structural organization of amplified ribosomal DNA from Xenopus mulleri and Xenopus laevis.

Secondary structure maps of long single strands of amplified ribosomal DNA from two closely related species of frogs, Xenopus laevis and X. mulleri, have been compared. The secondary structure pattern of the gene region is identical in both ribosomal DNAs while the patterns in the non-transcribed spacers² differ. In X. mulleri, the spacer shows an extended region without any secondary structure adjacent to the 28 S ribosomal RNA sequence. In contrast, the same region in the X. laevis spacer has extensive secondary structure. A comparison of secondary structure maps and denaturation maps of these two ribosomal DNAs (Brown et al., 1972) reveals that the portion without secondary structure in the X. mulleri spacer corresponds to an early melting A + T-rich region. As in X. laevis ribosomal DNA, Escherichia coli restriction endonuclease (EcoRI) makes two cuts in each repeating unit of amplified ribosomal DNA from X. mulleri. The position of the cleavage sites is identical in the two species as judged from secondary structure mapping of the two classes of EcoRI fragments generated. The small fragments of X. mulleri ribosomal DNA are homogeneous in size with a duplex molecular weight of 3.0 × 10⁶, and contain about 85% of the 28 S ribosomal RNA gene and about 17% of the 18 S ribosomal RNA gene. The large fragments are heterogeneous in size with molecular weights ranging from 4.2 to 4.9 × 10⁶, and contain the remaining portions of the gene regions and the nontranscribed spacer. Heteroduplexes made between large fragments of different lengths show only deletion loops. The position of these loops indicates that the length heterogeneity resides in the non-transcribed spacer region. Electrophoretic analysis of EcoRI digests of chromosomal ribosomal DNA from X. mulleri demonstrates that this DNA is heterogeneous in length as well.     

Characterization of cloned rat ribosomal DNA fragments

Summary Two Charon 4A lambda bacteriophage clones were characterized which contain all and part of the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA region were in the same places in mouse and rat DNA but the restriction sites in the 5-spacer regions were different.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Isolation and some properties of DNA coding for tRNA1met from Xenopus laevis

DNA containing the reiterated genes for tRNA₁^met has been partially purified from Xenopus laevis by centrifugation in actinomycin C₁-CsCl and Ag⁺-Cs₂SO₄ gradients. These gradients separate the tRNA₁^met genes from those coding for tRNA₂^met and tRNA^val, thus confirming our earlier suggestion that these genes are not intermingled with each other (Clarkson, Birnstiel, and Purdom, 1973). The gradients also demonstrate the existence of a minor 5S DNA fraction which appears to differ from that previously isolated by Brown, Wensink, and Jordan (1971).When the enriched tDNA₁^met is digested to completion with either of the restriction endonucleases EcoRI or Hpa I, the tRNA₁^met genes are predominantly found within DNA fragments that are about 3100 base pairs long. A partial digestion with EcoRI shows that these fragments arise from the regular spacing of the enzyme restriction sites. The 3100 base pair EcoRI fragments are cleaved by Hpa I into fragments of two size classes, one of which is about 2200 base pairs long and contains the tRNA₁^met genes. The shorter fragments are about 700 base pairs long, and they appear to contain genes coding for at least one other kind of tRNA species. X. laevis tDNA₁^met thus comprises tandemly repeated DNA whose component parts show little if any length heterogeneity.     

Ribosomal DNA in Drosophila melanogaster. II. Heteroduplex mapping of cloned and uncloned rDNA.

Sequences in the cloned Drosophila melanogaster rDNA fragments described by Dawid et al. (1978) were compared by heteroduplex mapping. The nontranscribed spacer regions in all fragments are homologous but vary in length. Deletion loops were observed at variable positions in the spacer region suggesting that spacers are internally repetitious.Many rDNA repeats in D. melanogaster have a 28 S gene interrupted by a region named the ribosomal insertion. Insertions of 0.5, 1 and 5 kb were found in repeat-length EcoRI fragments. These DNA regions, named type 1 insertions, are homologous at their right ends. Although 1 kb insertions are quite precisely twice as large as 0.5 kb insertions they do not represent a duplication of the shorter sequence. Some insertions have at least one EcoRI site and therefore yield EcoRI fragments which are only part of a repeat. The sequences in two cloned right-hand partial insertion sequences are homologous, but the sequences in two lefthand partial insertions are not. None of the EcoRI-restrictable insertion sequences has any homology to any part of type 1 insertions; they are thus grouped together as type 2. Evidence for insertion sequences of at least two types in uncloned rDNA was obtained by annealing a cloned fragment with a 1 kb insertion to genomic rDNA. About 15% of the rDNA repeats show substitution type loops between the 1 kb type 1 insertion derived from the cloned fragment and type 2 insertions in the rDNA.     

Organization of the ribosomal RNA gene cluster in the yeast Saccharomyces cerevisiae

The chromosomal organization of the ribosomal RNA gene cluster from Saccharomyces cerevisiae was investigated. 18 S rRNA R-loops were formed with unfractionated high molecular weight DNA crosslinked once per 2.7 × 10³ bases with trioxsalen and observed in the electron microscope. Almost all the R-loops were found in very long continuous 9.34 ± 0.18 × 10³ base repeating units. In addition, molecules were found at a frequency of one to two per genome equivalent of rDNA where several rRNA genes were linked to long stretches of non-rDNA. These results suggest that rDNA is arranged in a single tandem repetitive cluster of 100 to 140 genes flanked on one or both sides by non-rDNA.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

Isolation and some properties of a mammalian ribosomal DNA

The DNA coding for 28 S and 18 S ribosomal RNA, including the spacer regions, has been isolated from calf (Bos taurus) thymus gland. The method used included shearing of the total DNA to a highly homogeneous size population, selective heat denaturation and S 1 nuclease treatment to remove single stranded DNA. Repeated centrifugation on density gradients yields a 140-fold purified rDNA fraction with a GC content of 61.2%. Eco RI nuclease cleaves this DNA into two fragments of 16.4 and 4.9×10⁶ daltons. Hybridization of these fragments with 28 S and 18 S rRNA shows that the 28 S coding sequence is located mostly on the 4.9×10⁶ dalton fragment, while both the 16.4 and 4.9×10⁶ dalton fragments contain the 18 S sequence. The data indicate that the ribosomal RNA gene has a repeat unit of 21.3×10⁶ daltons which includes a nontranscribed spacer of about 12.5×10⁶ daltons.     

A detailed physical map of HeLa cell mitochondria DNA and its alignment with the positions of known genetic markers

Twenty-one fragments have been identified among the products of digestion of HeLa cell mtDNA with the restriction enzyme Hpa II. The sum of the molecular sizes of these fragments, estimated from their mobility relative to that of known markers, accounts, within experimental error, for the total length of HeLa cell mtDNA. The 21 fragments have been ordered in a physical map by two approaches: (1) sequential digestion with Hpa II of the fragments produced by Eco RI, Hind III, andHpa I enzymes, and (2) fragment-primed DNA synthesis. The Hpa II map has been aligned with the maps constructed with the other three enzymes and with the unique cutting site produced by Bam I. The combined map thus obtained has resolved HeLa cell mtDNA into 27 recognizable segments in the molecular size range between 75 and 1950 base pairs. This physical map has been aligned with the known positions of the rRNA and 4 S RNA genes on the two mtDNA strands by RNA-DNA hybridization experiments utilizing purified ³²P-labeled 12 and 16 S rRNA.     

Characterization of yeast ribosomal DNA fragments generated by EcoR1 restriction endonuclease

Summary The action of Escherichia coli restriction endonuclease R1 (EcoR1) on DNA isolated from Saccharomyces cerevisiae (strain MAR-33) generates three predominent homogenously sized DNA fragments (species of 1.8, 2.2 and 2.5 kilo nucleotide base pairs (KB). Many DNA species of molecular weight greater than 2 million daltons can be recognized upon incomplete EcoR1 digestion of yeast DNA. Four additional DNA species ranging from 0.3–0.9 KB can be identified as the second major class of EcoR1-yeast DNA products.Hybridization with radioactive ribosomal RNA (rRNA) and competition with nonradioactive rRNA show that of the three predominent EcoR1-yeast DNA species, the 2.5 KB species hybridizes only with the 25S rRNA while the lighter 1.8 KB species hybridizes with the 18S rRNA. The intermediate DNA species of 2.2 KB hybridizes to a small extent with the 25S rRNA and could be a result of the presence of the 2.5 KB DNA species. The mass proportions and hybridization values of these 3 DNA species account for about 60% of the total ribosomal DNA (rDNA).The 5EcoR1-yeast DNA species of less than 0.9 KB (4 major and 1 minor species) hybridize to varying degrees with the 2 rRNA and can be grouped in two classes. In one class there are 3 DNA species that hybridize exclusively with the 18S rRNA. In the second class there are 2 DNA species that besides hybridizing predominently with the 25S rRNA also hybridize with the 18S rRNA. The 7 EcoR1-yeast DNA species (excluding the 2.2 KB DNA species) that hybridize with the two rRNA account for nearly a 5 million dalton DNA segment, which is very close to the anticipated gene size of rRNA precursor molecule. If the 2.2 KB DNA species is a part of the rDNA that is not transcribed or 5 sRNA then the cistron encoding the rRNA in S. cerevisiae has at least 8 EcoR1 recognition sites resulting in 8 DNA fragments upon digestion with the EcoR1. Consideration is given to the relationship of the rRNA species generated by EcoR1 digestion and the chromosomes containing ribosomal cistrons.     

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Fragments of rDNA3 from Drosophila melanogaster produced by the restriction endonuclease EcoRI were cloned in the form of recombinant plasmids in Escheriehia coli. Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single EcoRI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by EcoRI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.