Structure of melon rDNA and nucleotide sequence of the 17–25S spacer region

Summary Restriction enzyme and hybridization analysis of melon nuclear DNA suggests a homogenous rDNA population with a repeat unit of 10.2 kb. Several full length Hind III rDNA repeat units were cloned and one of these is described in detail. The regions coding for 25S, 17S and 5.8S rRNAs were located by crossed-contact hybridization and R-loop mapping. Introns were not observed. The nucleotide sequence of the internal transcribed spacer and flanking regions was determined and compared with the corresponding region from rice rDNA by dot matrix analysis. In addition, the extent of gross sequence homology between cloned melon and pea rDNA units was determined by heteroduplex mapping.     

Isolation and characterization of rat ribosomal DNA clones

Four EcoRI fragments, which contain the transcribed portion of the rat rDNA repeat, have been isolated from a rat genome library cloned in lambda Charon 4A vector. Three of the fragments, 9.6, 6.7, and 4.5 kb, from clones lambda ChR-B4, lambda Nr-42, and lambda ChR-C4B9, contained part of the 5'-NTS, the 5'-ETS, 18S rDNA, ITS-1, 5.8S rDNA, 28S rDNA and approximately 3.5 kb of the 3'-NTS. Two EcoRI fragments, from clones lambda ChR-B4 and lambda ChR-B7E12, which coded for the 5'-NTS, the ETS, and most of the 18S rDNA, differed by 1 kb near the EcoRI site upstream of the 5' terminus of 18S rRNA. Restriction maps of the cloned DNA fragments were constructed by cleavage of the fragments with various restriction endonucleases and Southern hybridization with 18S, 5.8S, and 28S rRNA. These maps were confirmed and extended by subcloning several regions of the repeat in pBR322.     

Internal transcribed spacer rDNA can be used to infer the phylogenetic relationships of species within the genus Nematodirus (Nematoda: molineoidea)

Sequences of the first internal transcribed spacer rDNA were characterised for four veterinary important species of gastrointestinal nematodes from the genus Nematodirus. The sequence data were combined with previously published data of the second internal transcribed spacer to determine whether these rDNA regions provided a suitable number of informative characters to determine the phylogenetic relationships of species within the genus. A total of 32 alignment positions of the first internal transcribed spacer data set and 33 characters from the second internal transcribed spacer data set were informative in phylogenetic analyses. Irrespective of whether the data from each spacer were analysed separately or combined, only one most parsimonious tree was produced, with the relationships of the four species fully resolved. In addition, several regions of conservatism in the first internal transcribed spacer sequence among the four Nematodirus species suggests that this rDNA region may also provide phylogenetic information for higher taxonomic levels within the Molineoidea.     

Structure of the Syrian hamster ribosomal DNA repeat and identification of homologous and nonhomologous regions shared by human and hamster ribosomal DNAs

We have cloned and partially characterized Bam HI fragments of Syrian hamster DNA containing most of the ribosomal RNA-coding region. Several restriction site polymorphisms within the transcribed portions of the hamster rDNA repeats have been noted. Approximately three-fifths of the repeats contain a Bam HI site upstream of the 18 S coding sequences. Approximately four-fifths of the repeats contain a Bam HI site very close to the 5' end of the 28 S coding sequences. This microheterogeneity has been maintained in the DNA of baby hamster kidney (BHK)-21 cells, a cell line established nearly 20 years ago. R-loop analysis with homologous hamster rRNAs has established the size of the coding regions and the internal transcribed spacer. Heterologous R-loop analyses with cloned hamster rDNAs and human rRNAs reveal several well-defined regions within the 28 S gene where the homology between human and hamster RNAs is greatly reduced. These regions are not detectable in heteroduplexes of hamster and human rDNAs. Sequences encoding the 18 S gene do not exhibit such reduced homology.     

Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila

Using [32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia.     

Nuclear ribosomal spacer regions in plant phylogenetics: problems and prospects

The nuclear ribosomal locus coding for the large subunit is represented in tandem arrays in the plant genome. These consecutive gene blocks, consisting of several regions, are widely applied in plant phylogenetics. The regions coding for the subunits of the rRNA have the lowest rate of evolution. Also the spacer regions like the internal transcribed spacers (ITS) and external transcribed spacers (ETS) are widely utilized in phylogenetics. The fact, that these regions are present in many copies in the plant genome is an advantage for laboratory practice but might be problem for phylogenetic analysis. Beside routine usage, the rDNA regions provide the great potential to study complex evolutionary mechanisms, such as reticulate events or array duplications. The understanding of these processes is based on the observation that the multiple copies of rDNA regions are homogenized through concerted evolution. This phenomenon results to paralogous copies, which can be misleading when incorporated in phylogenetic analyses. The fact that non-functional copies or pseudogenes can coexist with ortholougues in a single individual certainly makes also the analysis difficult. This article summarizes the information about the structure and utility of the phylogenetically informative spacer regions of the rDNA, namely internal- and external transcribed spacer regions as well as the intergenic spacer (IGS).     

Defining orthologous groups among multicopy genes prior to inferring phylogeny,with special emphasis on the Triticeae (Poaceae)

Sequence information from multicopy genes has been widely used for phylogenetic inference. Among those sequences analyzed, nuclear 5S rRNA genes, the two internal transcribed spacer regions (ITS1 and ITS2) of the 18S-26S rDNA genes, and the intergenic spacer (IGS) regions of the same 18S-26S rDNA genes have all been used at the specific, generic, familial and tribal levels. Many investigations have used direct sequencing of PCR products to generate sequence data. The merits of an alternate approach, namely, cloning prior to sequencing followed by careful alignment of numerous cloned sequences to discern groups of putative orthologous sequences that may then be useful for the inference of relationships among species and genera, are examined and discussed. This process discerns patterns resulting from several cycles of careful alignment followed by manual editing conducted by eye--an exacting operation especially when sequences are unequal in length due to the presence of additions/deletions. Based upon examples taken from our work on the sequencing of individual 5S rDNA clones from several wheat and barley species (Triticum and Hordeum respectively), and the re-analysis of data of others taken from several studies using the nuclear genes mentioned above, we are able to identify groups of putative orthologous sequences that we have named "unit classes". Furthermore, comparisons between provisional orthologous sequences isolated from different species are required for the inference of phylogenetic relationships between them. Paralogous sequences from different unit classes can be compared to infer evolutionary relationships among repeat types only, i.e. among unit classes. In several cases, the analysis of the sequence diversity obtained from different clones permitted the assignment of unit classes to specific haplomes.     

Cloning and comparative characterization of the rIGS regulatory regions in humans and the pygmy chimpanzee Pan paniscus

Investigation of randomly cloned genomic and chromosome-specific sequences of ribosomal DNA (rDNA) from different organisms show that different regions of this long repeat unit evolve at different rates. This proves to be true not only with regard to evolutionary variability of transcribed and nontranscribed intergenic (spacer) regions of rDNA. The intergenic spacer (rIGS) of human ribosomal DNA contains both highly variable and more conservative regions with putative regulatory functions. In the present study a comparative analysis of some segments of the rIGS pre-promoter (regulatory) region in human and pygmy chimpanzee (Pan paniscus) was carried out. For these purposes, the corresponding DNA fragments were amplified in PCR with oligonucleotide primers specific to human rIGS and sequenced. Our results show that at the background of substantial structural similarity of these regions in man and chimpanzee, i.e., the presence of highly homologous sequences and similar repetitive units, there are substantial differences between them. These differences are associated with point mutations, insertions, deletions, and complex structural rearrangements.     

Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa.

We have cloned and characterized Neurospora crassa ribosomal deoxyribonucleic acid (rDNA). The rDNA is found as a tandemly repeated 6.0-megadalton sequence. We have mapped a portion of the rDNA repeat unit with respect to its sites for 13 restriction endonucleases and defined those regions coding for the 5. 8S, 17S, and 26S ribosomal ribonucleic acids (rRNA's). We have also isolated several clones containing 5S rRNA sequences. The 5S rRNA coding sequences are not found within the rDNA repeat unit. We found that the sequences surrounding the 5S rRNA coding regions are highly heterogeneous.     

Cloning and Comparative Characterization of the rIGS Regulatory Regions in Human and Pygmy Chimpanzee Pan paniscus

Investigation of randomly cloned genomic and chromosome-specific sequences of ribosomal DNA (rDNA) from different organisms show that different regions of this long repeat unit evolve at different rates. This proves to be true not only with regard to evolutionary variability of transcribed and nontranscribed intergenic (spacer) regions of rDNA. The intergenic spacer (rIGS) of human ribosomal DNA contains both highly variable and more conservative regions with putative regulatory functions. In the present study a comparative analysis of some segments of the rIGS pre-promoter (regulatory) region in human and pygmy chimpanzee (Pan paniscus) was carried out. For these purposes, the corresponding DNA fragments were amplified in PCR with oligonucleotide primers specific to human rIGS and sequenced. Our results show that at the background of substantial structural similarity of these regions in man and chimpanzee, i.e., the presence of highly homologous sequences and similar repetitive units, there are substantial differences between them. These differences are associated with point mutations, insertions, deletions, and complex structural rearrangements.     

Comparison of the nuclear ribosomal units of five Chlamydomonas species

The organization of the nuclear ribosomal units of five different species of Chlamydomonas has been examined by hybridization of their nuclear DNA fragments produced by several restriction endonucleases with a radioactively labelled probe consisting of the two cloned BamHI ribosomal fragments of C. reinhardii. The results indicate that a) the ribosomal units of these five species are structurally related, b) changes in the non transcribed spacer occur in C. eugametos and in C. globosa, c) the rDNA unit of C. intermedia contains either an enlarged internal transcribed spacer or a ribosomal intervening sequence, d) the rDNA units of C. reinhardii and C. callosa are indistinguishable.     

Ribosomal DNA of the fly Sciara coprophila has a very small and homogeneous repeat unit

Summary In this report we show by hybridization of restriction fragments and by Miller spreads that the unit repeat of the fly Sciara coprophila is only 8.4 kb which is the smallest known for a multicellular eukaryote. The 8.4 kb EcoR1 fragment containing a complete unit of Sciara rDNA was cloned in pBR322, and mapped by the method of Parker (1977) and also by double digestion. The coding regions for 28S, 18S, and 5.8S RNA were localized by the method of Berk and Sharp (1977). From these data we conclude that the nontranscribed spacer, external transcribed spacer, and internal transcribed spacer are all shorter than in other organisms, thereby giving rise to the shorter overall rDNA repeat unit of Sciara.At least 90% of the Sciara rDNA repeats are homogeneous, with a length of 8.4 kb, but a 700 bp ladder of minor bands can also be found in digestions of total genome DNA. This profile of major and minor bands is identical between the X and X chromosomes, as seen by a comparison of several genotypes.There are only 45 rRNA genes per X chromosome of Sciara (Gerbi and Crouse, 1976). These can easily be counted by low magnification Miller speads which show that virtually all gene copies are actively being transcribed in the stage of spermatogenesis examined. This is the first demonstration for any reiterated gene family where all copies are shown to be simultaneously active.Present address same as last author     

Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila.

The free, linear macronuclear ribosomal RNA genes (rDNA) of Tetrahymena are derived from a unique copy of micronuclear rDNA during development. We have injected cloned copies of the micronuclear rDNA that have been altered in vitro into developing macronuclei and obtained transformants that express the paromomycin-resistant phenotype specified by the injected rDNA. In most cases, these transformants contain almost exclusively the injected rDNA which has been accurately processed into macronuclear rDNA. Mutants with a 119 bp insertion at three points in the transcribed spacers and at two points in the 26S rRNA coding region were tested. Cells containing these spacer mutant rDNAs are viable, although one of them grows slowly. This slow-growing line contains the insertion between the 5.8S and 26S rRNA coding regions and accumulates more rRNA processing intermediates than control lines. One of the 26S rRNA mutants failed to generate transformants, but the other did. These transformants grew normally, and produced 26S rRNA containing the inserted sequence. A longer insertion (2.3 kb) at the same four points either abolished transformation or generated transformants that retained at least some wild-type rDNA. This study reveals that some rRNA sequences can be altered without significantly affecting cell growth.