Organization of ribosomal genes in Paramecium tetraurelia

The macronuclear ribosomal DNA (rDNA) of the ciliated protozoan Paramecium tetraurelia (stock 51) was analyzed by digestion with restriction endonucleases. The fragments which contained ribosomal RNA (rRNA) coding sequences and spacer sequences were identified. The spacer sequences exhibited some heterogeneity in size. The genes coding for 5.8S RNA, but not for 5S RNA, are linked to the 17S and 25S rRNA genes. Complementary RNA, synthesized from rDNA of stock 51, was hybridized with restriction digests of whole cell DNA from six other allopatric stocks of this species. The restriction patterns of the rDNA from these seven stocks were, in general, very similar, and the sizes of the coding sequences were identical in all seven stocks. Only the restriction pattern of rDNA from stock 127 differed significantly from that of stock 51. The rDNA from stock 127 was isolated and characterized, and with the exception of the restriction pattern of its spacer, it resembled the rDNA from stock 51. It is concluded that the rDNA repeat in Paramecium, including the spacer, has, in general, been conserved during the course of evolution. It is suggested that in some species, even in the absence of genetic exchange among geographically separated populations, selection pressure may act to conserve spacers of tandemly repeated rDNA. The conservation may be related to the number of rDNA copies in the germinal nucleus.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with ³H 18S+28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA the DNA sequences coding for 18S+28S rRNA plus the intervening spacer sequences - SSC 0.15 M sodium chloride, 0.015 sodium citrate, pH 7     

Ribosomal RNA genes from Prevotella bryantii: organization and heterogeneity

FiveP. bryantii B₁4 16S rRNA gene copies and their flanking regions were cloned and analyzed. A genomic library was constructed and screened with oligonucleotide DNA probe specific for 16S rRNA gene ofP. bryantii. Five out of six different copies of 16S RNA gene were recovered and sequenced. Only minor differences (0.3–1.2%) between copies were detected within the 1541 bp long sequence. The impact of the sequence variability of 16S rRNA gene copies on phylogenetic positioning ofP. bryantii was determined. All five sequences from clonedP. bryantii B₁4 16S rRNA genes were placed in the same operational taxonomy unit. Control regions of all five analyzed rRNA operatons were almost identical and three candidate for promoter sequences were identified by Neutral Network Promoter Prediction. Spacer regions between 16S-rRNA and 23S rRNA genes in all five cloned copies were 543 bp long and genes for tRNA^lle and tRNA^Ala were identified inside this regions.     

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.     

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.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.     

A temperature sensitive mutant of Saccharomyces cerevisiae defective in pre-rRNA processing.

A recessive temperature sensitive mutant has been isolated that is defective in ribosomal RNA processing. By Northern analysis, this mutant was found to accumulate three novel rRNA species: 23S', 18S' and 7S', each of which contains sequences from the spacer region between 25S and 18S rRNA. 35S pre-rRNA accumulates, while the level of the 20S and 27S rRNA processing intermediates is depressed. Pulse-chase analysis demonstrates that the processing of 35S pre-rRNA is slowed. The defect in the mutant appears to be at the first processing step, which generates 20S and 27S rRNA. 7S' RNA is a form of 5.8S RNA whose 5' end is extended by 149 nucleotides to a position just 5 nucleotides downstream of the normal cleavage site that produces 20S and 27S rRNA. 7S' RNA can assemble into 60S ribosomal subunits, but such subunits are relatively ineffective in joining polyribosomes. A single lesion is responsible for the pre-rRNA processing defect and the temperature sensitivity. The affected gene is designated RRP2.     

tRNA genes are found between 16S and 23S rRNA genes in Bacillus subtilis.

There are at least nine, and probably ten, ribosomal RNA gene sets in the genome of Bacillus subtilis. Each gene set contains sequences complementary to 16S, 23S and 5S rRNAs. We have determined the nucleotide sequences of two DNA fragments which each contain 165 base pairs of the 16S rRNA gene, 191 base pairs of the 23S rRNA gene, and the spacer region between them. The smaller space region is 164 base pairs in length and the larger one includes an additional 180 base pairs. The extra nucleotides could be transcribed in tRNAIIe and tRNA Ala sequences. Evidence is also presented for the existence of a second spacer region which also contains tRNAIIe and tRNA Ala sequences. No other tRNAs appear to be encoded in the spacer regions between the 16S and 23S rRNA genes. Whereas the nucleotide sequences corresponding to the 16S rRNA, 23S rRNA and the spacer tRNAs are very similar to those of E. coli, the sequences between these structural genes are very 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.     

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     

Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3′ end of 5.8S rRNA and the 5′ end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

5 S DNAs of Xenopus laevis and Xenopus mulleri: evolution of a gene family

The 5 S DNA which contains the genes for 5 S RNA has been purified from the frog Xenopus mulleri and compared with the 5 S DNA of Xenopus laevis. Both DNAs contain highly repetitive sequences in which the gene sequence that codes for 5 S RNA alternates with a spacer sequence. The 5 S DNAs of X. laevis and X. mulleri comprise about 0.7% of the total DNA or about 24,000 and 9000 repeating sequences, respectively. The average repeat length within native X. laevis and X. mulleri 5 S DNA is about 0.5 to 0.6 and 1.2 to 1.5 × 10⁶ daltons, respectively, each repeat of which contains a single gene sequence for 5 S RNA (0.08 × 10⁶ daltons). The two DNAs differ in the average length of their spacers and no cross homology can be detected by heterologous hybridization of the two DNAs, except within the 5 S RNA gene regions. Despite their differences, the spacer sequences of X. laevis and X. mulleri 5 S DNA resemble each other enough to conclude that they have diverged from a common ancestral sequence.The multiple repeating sequences of 5 S DNA in each species have evolved as a family of similar, but not identical sequences. It is known that 5 S DNA is located at the ends (telomeres) of the long arms of most, if not all, X. laevis chromosomes. It is proposed that multiple gene sequences located on the ends of many chromosomes can evolve together as a family if there is extensive and unequal exchange of DNA sequences between homologous and non-homologous chromosomes at their ends.