Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.     

Characterization of mouse ribosomal gene fragments purified by molecular cloning.

Four mouse ribosomal gene fragments cloned in lambda gtWES were studied by restriction enzyme mapping and Southern transfer experiments. These fragments were found to contain 18S DNA and transcribed as well as non-transcribed spacer DNA. Variation in the structure of these mouse DNA inserts was limited to one region of spacer DNA. This variation may reflect real structural differences found in mouse ribosomal genes or possibly deletion events which occurred during cloning. The transcribed regions of the inserts appear identical to one antoher and restriction enzyme fragments from this region correspond to fragments observed in digests of total mouse DNA. These clones will be useful in studying the structure of transcribed spacer DNA including the ribosomal gene promoter.     

More ribosomal spacer sequences from Xenopus laevis.

The base sequence analysis of a Xenopus laevis ribosomal DNA repeat (7) has been extended to cover almost the entire non-transcribed and external transcribed spacer. A compilation of these sequences is presented. All the repetitive and non-repetitive sequence elements of the spacer are identified and their evolution discussed. Comparison of the X.laevis and S.cerevisiae (25,26) ribosomal DNAs shows about 80% sequence conservation in the 18S gene but no sequence conservation, from the available data, in the external transcribed spacer. The sequence coding for the 3' terminus of the X.laevis 40S ribosomal precursor RNA is presented and its structural features analyzed.     

Evolutionary implication of heterogeneity of the nontranscribed spacer region of ribosomal DNA repeating units in various subspecies of Mus musculus

Genetic variability of the nontranscribed spacer (NTS) region within ribosomal DNA repeating units in the various subspecies of Mus musculus was determined. Mice belonging to several laboratory mouse strains were examined by means of Southern blot hybridization with a mouse ribosomal DNA probe. This probe encompasses the 3' end of the 28S ribosomal RNA (rRNA) gene and the following spacer. Restriction enzyme digestions of the liver DNAs from various wild mice revealed that each of the subspecies has a unique pattern in the spacer encompassing a distance approximately 10 kb downstream from the ribosomal gene. These restriction patterns permit the classification of mouse subspecies and also provide insights into the origin of the laboratory mouse strains.     

Rapid identification of Streptococcus pneumoniae by PCR amplification of ribosomal DNA spacer region

Abstract Streptococcus pneumoniae is one of the important human pathogens in clinical microbiology. A polymerase chain reaction assay was designed to detect and identify S. pneumoniae through amplification of the ribosomal DNA spacer regions between the pneumococcal 16S-23S ribosomal RNA genes. Thirty-two Streptococcus and non- Streptococcus strains were tested to verify the specificity of the assay, and only S. pneumoniae strains gave a positive reaction. This method is a powerful technique for the rapid identification of S. pneumoniae .     

Specific 5S ribosomal RNA primers for plant species i dentification in admixtures 3

The DNA sequences of the spacers between the 5S ribosomal RNA genes were determined for the cereals maize, barley, soghum, rye, rice, oat, and wheat. Species-specific primers were designed from the spacer region. PCR with these primers and a common primer from the conserved 5S ribosomal RNA gene sequence was investigated as a method for detection of the seven cereal species. DNA from these species could be specifically detected in mixtures. This technique could find application in the determination of the composition of admixtures or processed cereal products. The protocol described has potential for general application in the identification of plant species.     

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.     

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 molecular basis for discrete size variation in human ribosomal DNA.

The tandemly repeated human ribosomal RNA (rRNA) genes contain a region of size heterogeneity that is present in the nontranscribed spacer of every individual examined. This heterogeneity has been previously examined by Southern analysis of BamHI-digested human DNA. Using a ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene, at least four discrete sizes of BamHI fragments were seen in human populations. Molecular analysis of the cloned DNA from this region reveals tandem duplication of a segment of spacer rDNA located 388 base pairs (bp) 3' to the end of the 28S ribosomal RNA gene. Five hundred fifty bp of DNA, flanked on either side by a 150-bp repeated element, is either duplicated or deleted to produce a series of spacers that differ in size by 850 bp. These duplications/deletions appear to be the product of unequal homologous exchange, mediated by the small repeated element. Thus, human rDNA fragments cloned in lambda vectors and propagated in E. coli generate the same apparent size variation seen in genomic DNA. This study suggests that unequal homologous exchange is the molecular basis for the observed length heterogeneity in the spacer rDNA and may be a common mechanism for the generation of human genetic diversity.     

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.     

High intra-individual sequence variation in the nuclear rDNA LSU-5S intergenic spacer in the Sargassaceae (Fucales, Phaeophyceae)

Of four species of Sargassaceae, representing the genera Carpophyllum, Cystoseira, Landsburgia, and Sargassum, the intergenic spacer of the ribosomal cistron was amplified using a forward primer annealing at the 3′-end of the large subunit (LSU) of the ribosomal cistron and a reverse primer annealing at the 5S rRNA gene. The PCR products were cloned and the DNA sequences of multiple clones were determined. Almost each clone showed a unique DNA sequence. Intra-individual variation of this LSU-5S intergenic spacer was extremely high and was characterized by great length variation and a high number of short tandem repeats. Sequences were unalignable and therefore it was concluded that the LSU-5S intergenic spacer is unsuitable for phylogenetic and phylogeographic studies of sargassacean taxa.     

Electron microscope mapping of the distribution of ribosomal genes of the Bacillus subtilis chromosome

When high molecular weight Bacillus subtilis DNA is denatured, renatured and then examined by electron microscopy the main renatured product seen is a long duplex, usually with a single strand at each end, due to in-register renaturation. In addition, structures containing short duplex segments of length 4830 ± 250 base pairs, with two single-strand arms at each end, are seen at a low frequency. Several lines of evidence support the hypothesis that these short duplex segments are formed by out-of-register renaturation of the 16 S + 23 S ribosomal RNA genes (rDNA) of B. subtilis. They are of the correct length. Their formation is inhibited if homologous, but not if heterologous, ribosomal RNA is added to the hybridization mixture. The frequency of occurrence of the duplex structures is consistent with the rDNA hypothesis. Heteroduplex molecules are seen with two or three rDNA duplex segments separated by single-strand substitution loops with specific lengths for each of the two single-strand arms of any one loop. On the basis of these structures, linkage groups containing seven or nine rDNA sets (each set containing one 16 S and one 23 S rDNA gene) separated by spacer DNA's are proposed. All of the 16 S rDNA genes are linked to 23 8 rDNA and vice versa with little or no spacer DNA between a 16 S and 23 S sequence. If 5 S DNA is present in the set, any spacer between it and the other ribosomal RNA gene must also be short. The prophage SPO2 bacterial att site maps at a distance of 6200 bases away from a 16 S + 23 S rDNA set, which is itself separated by a very short spacer (less than 600 bases) from a second rDNA set.     

Distinct Transcription Products of Ribosomal Genes in Two Different Tissues

MOST organisms contain multiple copies of the genes which code for ribosomal RNA (rRNA), the number varying from about 160 to 28,000 per nucleus in different eukaryotic species¹; these genes are clustered in the nucleolus. The repeating unit is a DNA sequence containing the structural genes for the 18S and 28S rRNA together with spacer DNA, only a part of which is transcribed². Ribosomal RNA is transcribed from these genes as a polycistronic precursor molecule (pre-rRNA) which contains the rRNA sequences of the larger and smaller ribosomal subunits together with some additional sequences that are discarded during the maturation to rRNA³. The multiple gene copies are identical in the ribosomal regions within the limits detectable by present methods¹, although there is some evidence that regions of non-transcribed spacer DNA vary in length and may therefore not all be identical². We have suggested that the pre-rRNA may also be heterogeneous in molecular weight⁴.