Quantitative analysis of repetitive sequences in human genomic DNA and detection of an elevated ribosomal repeat copy number in patients with schizophrenia (the results of molecular and cytogenetic analysis)

A modified version of quantitating repetitive sequences in genomic DNA was developed to allow comparisons for numerous individual genomes and simultaneous analysis of several sequences in each DNA specimen. The relative genomic content of ribosomal repeats (rDNA) was estimated for 75 individuals, including 33 healthy donors (HD) and 42 schizophrenic patients (SP). The rDNA copy number in HD was 427 +/- 18 (mean SE) per diploid nucleus, ranging 250-600. In SP, the rDNA copy number was 494 +/- 15 and ranged 280-670, being significantly higher than in HD. The two samples did not differ in contents of sequences hybridizing with probes directed to a subfraction of human satellite III or to the histone genes. Cytogenetic analysis (silver staining of metaphase chromosomes) showed that the content of active rRNA genes in nucleolus organizer regions is higher in SP compared with HD. The possible causes of the elevated rRNA gene dosage in SP were considered. The method employed was proposed for studying the polymorphism for genomic content of various repeats in higher organisms, including humans.     

Distribution of 5S ribosomal RNA genes in somatic and germ cells of the house cricket,Acheta domesticus

In the house cricket,Acheta domesticus, the 110 genes per haploid genome encoding 18S and 28S rRNA are contained within rDNA repeats which are amplified during oogenesis. The 5S rRNA coding sequences of this cricket are found in two sizes of 5S DNA repeating units (measuring 2.1 and 3.0 kb). The 3.0 kb repeats account for more than 90% of the totalAcheta 5S DNA. We have determined the number of cricket 5S rRNA genes by RNA-DNA hybridization analysis: 310 5S DNA repeats/haploid genome clearly approximates the number of 18S and 28S rRNA genes. Because of the relatively low copy number of 5S rRNA genes the possibility of 5S DNA amplification in oocytes ofA. domesticus was also examined. Although amplification of rDNA is readily detectable, amplification of 5S DNA is not observed in oocytes ofA. domesticus. Unlike the genes coding for 18S and 28S rRNA which are localized at a single chromosomal site in the genome ofA. domesticus, the 5S rRNA genes occupy numerous sites distributed along the length of most chromosomes.     

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²–10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.     

Arrangement and size distribution of repeat and single copy DNA sequences in four species ofCucurbitaceae

DNA sequence organization patterns have been studied in fourCucurbitaceae plant species, namely,Luffa cylindrica (sponge gourd),L. acutangula (ridge gourd),Benincasa hispida (ash gourd) andCoccinia indica (ivy gourd). Extensive interspersion of repeat and single copy sequences has been observed in sponge gourd and ridge gourd. In ash gourd and ivy gourd, however, there is a limited interspersion of these sequences and a large portion of the single copy DNA remains uninterspersed. The interspersed repetitive sequences are composed of a major class (75–80%) of short repeats (300 base pairs long) and a minor class (15–20%) of long repeats (2 000–4 000 base pairs) in all the four species. The average length of single copy sequences dispersed among repeats is 1 800–2 900 base pairs. In spite of these gross similarities in the genome organization in the four species, the fraction of repeats and single copy sequences involved in short and long period interspersion patterns, and fraction of single copy sequences remaining uninterrupted by repeats are vastly different. The probable implications of these differences with respect to speciation events and rates of genome evolution are discussed.Molecular Analysis ofCucurbitaceae Genomes, III. — NCL Communication No.: 3595.     

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.     

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.     

Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon

The nucleotide sequences of the rRNA genes and the 5 flanking region were determined for R. salmoninarum ATCC 33209^T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5 leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S–23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S–5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.     

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number.     

Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA.

Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development. In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction. The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development. The breakage event is associated with DNA elimination and telomeric sequence addition. Similar results were also found previously for the 3' junction of this gene. These results could explain how the extrachromosomal rDNA is first generated. Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found. These sequences might serve as signals for site-specific breakage. In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene. The repeats are 42 nucleotides long and are separated by 28 nucleotides. The existence of this structure provides a simple explanation for the formation of the palindromic rDNA.     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

DNA sequence organization in the genomes of three related millet plant species

Summary A major portion of the genomes of three millet species, namely, barn yard millet, fox tail millet and little millet has been shown to consist of interspersed repeat and single copy DNA sequences. The interspersed repetitive DNA sequences are both short (0.15–1.0 kilo base pairs, 62–64% and long (>1.5 kilo base pairs, 36–38%) in barn yard millet and little millet while in fox tail millet, only long interspersed repeats (>1.5 kilo base pairs) are present. The length of the interspersed single copy DNA sequences varies in the range of 1.6–2.6 kilo base pairs in all the three species. The repetitive duplexes isolated after renaturation of 1.5 kilo base pairs and 20 kilo base pairs long DNA fragments exhibit a high thermal stability with Tms either equal to or greater than the corresponding native DNAs. The S1 nuclease resistant repetitive DNA duplexes also are thermally stable and reveal the presence of only 1–2% sequence divergence.The present data on the modes of sequence arrangement in millets substantiates the proposed trend in plants, namely, plants with 1C nuclear DNA content of less than 5 picograms have diverse patterns of sequence organization while those with 1C nuclear DNA content greater than 5 picograms have predominantly a short period interspersion pattern.Abbreviations kbp kilobase pairs NCL Communication No. 3606.     

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.     

The four ribosomal DNA units of the malaria parasite Plasmodium berghei. Identification, restriction map, and copy number analysis

The four ribosomal RNA genes (rDNA units) of the rodent malaria parasite, Plasmodium berghei, were identified and mapped by restriction enzyme analysis and Southern blot hybridization of genomic DNA. Although the four genes share common characteristics, they appear to be internally different from each other in expanse and sequence. One HindIII site near the 3' end of the coding region for the large rRNA is the only restriction site which we have detected that is conserved in all of the genes. The distance between the conserved HindIII site and the coding region for the small rRNA is at least 1-2 kilobases longer in two of the rDNA units than in a third unit. None of the four rDNA units were linked by restriction mapping where about 150 kilobases of the genome were accounted for. The copy number of two of the rDNA units was determined to be approximately 1 per haploid genome by quantitative analysis of cloned (plasmid) DNA versus genomic DNA digests on Southern blots. The other two genes also appear to be present in 1 copy. Restriction analysis confirms both the low copy number and that these genes are not in an easily recognizable tandem array. The low number of rDNA units requires that new copies of the genome produced during intraerythrocytic growth be active in RNA synthesis soon after their replication.     

Comparison of the ribosomal RNA genes in four closely relatedCucurbitaceae

Cucurbitaceae are characterized by a high copy number for nuclear ribosomal RNA genes. We have investigated the genomic ribosomal DNA (rDNA) of four closely related species of this family with respect to structure, length heterogeneity, and evolution. InCucumis melo (melon) there are two main length variants of rDNA repeats with 10.7 and 10.55kb.Cucumis sativus (cucumber) shows at least three repeat types with 11.5, 10.5, and 10.2kb.Cucurbita pepo (zucchini) has two different repeat types with 10.0 and 9.3kb. There are also two different repeat types inCucurbita maxima (pumpkin) of about 11.2 and 10.5kb. Restriction enzyme mapping of the genomic rDNA of these four plants and of cloned repeats ofC. sativus shows further heterogeneities which are due to methylation or point mutations. By comparison of the restriction enzyme maps it was possible to trace some evolutionary events in the family ofCucurbitaceae. Some aspects of regulation and function of the middle repetitive rRNA genes (here between 2000 and 10000 copies) are discussed.     

Arrangement of the rRNA sequences in the rDNA of Lytechinus variegatus

The arrangement of the 26S RNA and 18S RNA sequences of the ribosomal DNA (rDNA) from the sea urchin Lytechinus variegatus was investigated by an electron microscopic analysis of R-loops formed between the ribosomal RNA genes and the mature ribosomal RNAs. Ninety-eight percent of observed molecules contained R-loops clearly seen as a three-stranded complex. The size of DNA complementary to mature cytoplasmic 18S and 26S ribosomal RNA (rRNA) was calculated by measuring the double-strand (ds) and single-strand (ss) part of the R-loops separately. The values for the 18S R-loop are 1.75±0.24 kb¹ (ss) and 1.56±0.23 kb (ds). The 26S R-loop is 3.34±0.39 kb (ss) and 3.33±0.33 kb (ds). These measurements agree fairly well with the rRNA sizes measured on denaturing sucrose density gradients: 3.23±0.22 kb for the 26S and 1.93±0.10 kb for 18S. The short spacer between the 18S and 26S R-loops is 1.03±0.24 kb and the longer spacer is 5.36±0.53 kb. In long molecules a repeating pattern was observed. The average length of an rDNA repeat unit is 11.33±0.64 kb when computed using double-strand R-loop measurements and 11.50±0.72 when computed using R-loop single-strand lengths.Abbreviations kb kilobases, 1000 bases of RNA or single-strand DNA, and kilobase pairs, 1000 base pairs of duplex DNA or DNA/RNA hybrid - EDTA ethylenediaminetetraacetate - SSC 0.15 M NaCl, 0.015 M sodium citrate - PIPES piperazine-N,N-bis (2-ethanesulfonic acid)-Na_1.4     

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.