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.     

DNA sequence organisation in avian genomes

By means of renaturation kinetics of DNA of the three avian species Cairina domestica, Gallus domesticus and Columba livia domestica the following major DNA repetition classes were observed: a very fast reannealing fraction comprising about 15% of the DNA, a fast or intermediate reannealing fraction that makes up 10%, and a slow reannealing fraction of about 70%, which apparently renatures with single copy properties. — Comparing the reassociation behaviour of short (0.3 kb) and long (>2 kb) DNA fragments of duck and chicken it becomes apparent that only 12% (duck) and 28% (chicken) of the single copy DNA are interspersed with repetitive elements on 2 to 3 kb long fragments. The lengths of the repetitive sequences were estimated by optical hyperchromicity measurements, by agarose A-50 chromatography of S₁ nuclease resistant duplexes and by electron microscopic measurements of the S₁ nuclease resistant duplexes. It was found that in the case of the chicken DNA the single copy sequences alternating with middle repetitive ones are at least 2.3 kb long; the interspersed moderate repeats have a length average of at least 1.5 kb. The sequence length of the moderate repeats in duck DNA is smaller. The results show that the duck and the chicken genomes do not follow the short period interspersion pattern of genome organisation, characteristic of the eucaryotic organisms studied so far.     

Emerging roles of macrosatellite repeats in genome organization and disease development

Abundant repetitive DNA sequences are an enigmatic part of the human genome. Despite increasing evidence on the functionality of DNA repeats, their biologic role is still elusive and under frequent debate. Macrosatellites are the largest of the tandem DNA repeats, located on one or multiple chromosomes. The contribution of macrosatellites to genome regulation and human health was demonstrated for the D4Z4 macrosatellite repeat array on chromosome 4q35. Reduced copy number of D4Z4 repeats is associated with local euchromatinization and the onset of facioscapulohumeral muscular dystrophy. Although the role other macrosatellite families may play remains rather obscure, their diverse functionalities within the genome are being gradually revealed. In this review, we will outline structural and functional features of coding and noncoding macrosatellite repeats, and highlight recent findings that bring these sequences into the spotlight of genome organization and disease development.     

Patterns of tandem repetition in plant whole genome assemblies

Tandem repeats often confound large genome assemblies. A survey of tandemly arrayed repetitive sequences was carried out in whole genome sequences of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the monocots rice and sorghum, and the dicots Arabidopsis thaliana, poplar, grapevine, and papaya, in order to test how these assemblies deal with this fraction of DNA. Our results suggest that plant genome assemblies preferentially include tandem repeats composed of shorter monomeric units (especially dinucleotide and 9–30-bp repeats), while higher repetitive units pose more difficulties to assemble. Nevertheless, notwithstanding that currently available sequencing technologies struggle with higher arrays of repeated DNA, major well-known repetitive elements including centromeric and telomeric repeats as well as high copy-number genes, were found to be reasonably well represented. A database including all tandem repeat sequences characterized here was created to benefit future comparative genomic analyses.     

DNA sequence organization in soybean investigated by electron microscopy

An electron microscopic analysis of the DNA sequence organization in the soybean genome is reported. This analysis employed the gene 32 proteinethidium bromide spreading technique, a procedure which produces striking contrast between double and single-stranded DNA regions. To investigate the arrangement of repetitive sequences differing in genomic frequency, three kinetic fractions of 5 kb DNA fragments were isolated by reassociation and hydroxyapatite chromatography. Renatured structures in each fraction were then visualized in the electron microscope. The majority of repeated sequences, irrespective of frequency, were shown to be relatively non-divergent, to exceed 1.5 kbp in length (number-average), and to be organized primarily into long regularly repeating tandem or clustered arrays. Duplex regions >5 kbp were commonly visualized. A small fraction of low frequency repeats (<100 copies per genome), however, was observed to have a distinctly different form of arrangement. These repeats averaged 0.2 kbp in length, contained divergent sequences, and were contiguous to single copy DNA sequences having an average length of 1.15 kbp. Repeats which flanked a given single copy sequence did not appear to be homologous. Neither short clustered permuted repeats nor interspersion of repeats which differed significantly in reiteration frequency were found to be major features of soybean genome organization.     

Repeat sequence interspersion in coding DNA of peas does not reflect that in total pea DNA

The pattern of sequence organization in the regions of the pea genome near sequences coding for mRNA differs significantly from that in total DNA. Interspersion of repeated and single copy sequences is so extensive that 85% of 1300 nucleotide-long fragments contain highly repetitive sequences (about 5000 copies per haploid genome). However, data presented here demonstrate that sequences which code for mRNA are enriched in the small fraction of fragments which do not contain these highly repetitive sequences. Thus, in contrast to the great majority of other sequences in the genome, most mRNA coding sequences are not located within 1300 nucleotides of highly repetitive elements. Moreover, our data indicate that those repeats (if any) which are closely associated with mRNA coding sequences belong to low copy number families characterized by an unusually low degree of sequence divergence.Abbreviations NT nucleotides - NTP nucleotide pairs - C_ot the product of molar concentration of DNA nucleotides and time of incubation (mol s/L) - T_m the temperature at which half of the nucleotides are unpaired - T_m,i the temperature at which half of the complementary strands are completely separated - PIPES 1, 4, Piperazinediethane sulfonic acid - PB an equimolar mixture of NaH₂PO₄ and Na₂HPO₄ (pH 6.8).     

Characterization of the nuclear genome of pearl millet.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.     

DNA sequence organization in theThysanura Thermobia domestica

Summary Cot and chemical analysis show that the haploid genome size of Thermobia domestica is 3–4×10⁹ nucleotide pairs. Of this DNA 33% is single copy sequences and 67% is repetitive sequences. The repetitive sequences are predominantly 300 nucleotides in length and are interspersed among the single copy sequences in a short period interspersion pattern similar to that observed in Xenopus and many other higher eucaryotes. The DNA sequence organization observed in Thermobia is compared with that of other more highly evolved insects.Abbrevations HAP-hydroxyapatite, Cot mole nucleotides × liter^–1 s. - N sodium phosphate, pH 6.8     

DNA sequence organization in the mollusc Aplysia californica.

The sequence organization of the DNA of the mollusc Aplysia californica has been examined by a combination of techniques. Close-spaced interspersion of repetitive and single copy sequences occurs throughout the majority of the genome. Detailed examination of the DNA of this protostome reveals great similarities to the pattern observed in the two deuterostome organisms previously examined in detail in this laboratory, Xenopus laevis and Strongylocentrotus purpuratus. Labeled and unlabeled Aplysia DNA were prepared from developing embryos and sheared to a fragment length of 400 nucleotides. The kinetics of reassociation were studied by means of hydroxyapatite chromatography, single-strand-specific S1 nuclease, and optical methods of assay. Aplysia DNA of this fragment length contains at least five resolvable kinetic fractions. One classification of these fractions, listed with their reassociation rate constants (l M-1 sec-1) is: single copy (0.00057), slow (0.047), fast (2.58), very fast (4000), and foldback (greater than 10(5)). Sequence arrangement was deduced from: the kinetics of reassociation of DNA fragments of length 400 or 2000 nucleotides; the hyperchromicity of reassociated fragments containing duplex regions; the size of duplex regions resistant to S1 nuclease; and the reassociation of labeled fragments of various lengths with short driver fragments. More than 80% of the single copy DNA sequences are interspersed with repetitive sequences. The maximum spacing of the repeats is about 2000 nucleotides, and the average less than 1000. The very fast fraction does not show interspersion with single copy sequences or with other kinetic fractions. The foldback fraction sequences are fairly widely interspersed. The slow fraction sequences are interspersed with the fast fraction, and possibly also with the single copy DNA. The fast fraction is the dominant interspersed repetitive fraction. Its sequences are adjacent to the great majority of the single copy sequences and have an average length of about 300 nucleotides.     

Evolutionary divergence and length of repetitive sequences in sea urchin DNA

Summary The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease Sl fromAspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form Sl resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with Sl nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive Sl nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9°C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.Also Staff Member, Carnegie Institution of Washington     

Contrasting patterns of DNA sequence arrangement in Apis mellifera (Honeybee) and Musca domestica (housefly)

We have examined the organization of the repeated and single copy DNA sequences in the genomes of two insects, the honeybee (Apis mellifera) and the housefly (Musca domestica). Analysis of the reassociation kinetics of honeybee DNA fragments 330 and 2,200 nucleotides long shows that approximately 90% of both size fragments is composed entirely of non-repeated sequences. Thus honeybee DNA contains few or no repeated sequences interspersed with nonrepeated sequences at a distance of less than a few thousand nucleotides. On the other hand, the reassociation kinetics of housefly DNA fragments 250 and 2,000 nucleotides long indicates that less than 15% of the longer fragments are composed entirely of single copy sequences. A large fraction of the housefly DNA therefore contains repeated sequences spaced less than a few thousand nucleotides apart. Reassociated repetitive DNA from the housefly was treated with S1 nuclease and sized on agarose A-50. The S1 resistant sequences have a bimodal distribution of lengths. Thirty-three percent is greater than 1,500 nucleotide pairs, and 67% has an average size about 300 nucleotide pairs. The genome of the housefly appears to have at least 70% of its DNA arranged as short repeats interspersed with single copy sequences in a pattern qualitatively similar to that of most eukaryotic genomes.     

Periodically interspersed repetitive sequences may govern higher-order DNA coiling in chromatin and chromosomes

The interspersed periodic arrangement of repetitive and unique sequences in eukaryotic DNAs is proposed as the underlying molecular basis for higher-order DNA coiling in chromatin and mitotic chromosomes. It is assumed that (i) two types of interspersed repetitive sequences are distributed strictly periodically throughout the genome, splitting the single copy DNA into short and long periods respectively in such a pattern that each long period is composed of a definite number of short periods and repeats (ii) the short and long periods make the turn lengths of the solenoid and supersolenoid structures respectively determing their diameters; (iii) specific proteins interact with each type of repeats making cross ties between nearby repeats of each class helping to form, constrain, and stabilize the solenoid and the supersolenoid structures; (iv) the long period may be equated with the basic chromomere unit. The model predicts: (i) splitting of contiguous genes by inserted repetitive sequences; and (ii) two types of genomes differing in the hierarchy of DNA coiling.     

Genome-Wide Analysis of Repetitive Elements in Papaya

Papaya (Carica papaya L.) is an important fruit crop cultivated in tropical and subtropical regions worldwide. A first draft of its genome sequence has been recently released. Together with Arabidopsis, rice, poplar, grapevine and other genomes in the pipeline, it represents a good opportunity to gain insight into the organization of plant genomes. Here we report a detailed analysis of repetitive elements in the papaya genome, including transposable elements (TEs), tandemly-arrayed sequences, and high copy number genes. These repetitive sequences account for ～56% of the papaya genome with TEs being the most abundant at 52%, tandem repeats at 1.3% and high copy number genes at 3%. Most common types of TEs are represented in the papaya genome with retrotransposons being the dominant class, accounting for 40% of the genome. The most prevalent retrotransposons are Ty3-gypsy (27.8%) and Ty1-copia (5.5%). Among the tandem repeats, microsatellites are the most abundant in number, but represent only 0.19% of the genome. Minisatellites and satellites are less abundant, but represent 0.68% and 0.43% of the genome, respectively, due to greater repeat length. Despite an overall smaller gene repertoire in papaya than many other angiosperms, a significant fraction of genes (>2%) are present in large gene families with copy number greater than 20. This repeat database clarified a major part of the papaya genome organization and partly explained the lower gene repertoire in papaya than in Arabidopsis.     

Sequence organization of the human genome

The organization of three sequence classes—single copy, repetitive, and inverted repeated sequences—within the human genome has been studied by renaturation techniques, hydroxylapatite binding methods, and DNA hyperchromism. Repetitive sequence classes are distributed throughout 80% or more of the genome. Slightly more than half of the genome consists of short single copy sequences, with a length of about 2 kb interspersed with repetitive sequences. The average length of the repetitive sequences is also small and approximates the length of these sequences found in other organisms. The sequence organization of the human genome therefore resembles the sequence organization found in Xenopus and sea urchin. The inverted repeats are essentially randomly positioned with respect to both sequence class and sequence arrangement, so that all three sequence classes are found to be mutually interspersed in a portion of the genome.     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Structural genes adjacent to interspersed repetitive DNA sequences

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. ³H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3–5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80% of that mRNA hybridizable with whole DNA (approximately 65%) had reacted with the repeat-contiguous DNA fraction (50%). This result shows that 80–100% of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.     

Organisation and properties of repeated DNA sequences in rice genome

Reassociation of high molecular weight rice DNA has revealed the occurrence of long stretches of repeated DNA which are not interrupted by single copy DNA even at a fragment length as high as 20 kilo base pairs (kbp). Majority of these repeated sequences are unusually G + C rich and show significant variations in their thermal stability. Homology studies indicate that short repeats may have evolved from long repeats in total repetitive DNA while they may be of different origin in highly repetitive DNA fraction. Restriction enzyme analysis shows the occurrence of Ava I and EcoR V repeat families.     

Extrachromosomal circular DNA derived from tandemly repeated genomic sequences in plants

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It was found in various non-plant organisms from yeast to humans. EccDNA is heterogeneous in size and contains sequences derived primarily from repetitive chromosomal DNA. Here, we report the occurrence of eccDNA in small and large genome plant species, as identified using two-dimensional gel electrophoresis. We show that eccDNA is readily detected in both Arabidopsis thaliana and Brachycome dichromosomatica , reflecting a normal phenomenon that occurs in wild-type plants. The size of plant eccDNA ranges from > 2 kb to < 20 kb, which is similar to the sizes found in other organisms. These DNA molecules correspond to 5S ribosomal DNA (rDNA), non-coding chromosomal high-copy tandem repeats and telomeric DNA of both species. Circular multimers of the repeating unit of 5S rDNA were identified in both species. In addition, similar multimers were also demonstrated with the B. dichromosomatica repetitive element Bdm29. Such circular multimers of tandem repeats were found in animal models, suggesting a common mechanism for eccDNA formation among eukaryotes. This mechanism may involve looping-out via intrachromosomal homologous recombination. The implications of these results on genome plasticity and evolutionary processes are discussed.     

The contribution of short repeats of low sequence complexity to large conifer genomes

The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC)₅ and (GACA)₄ are present in only low copy numbers. The variation in abundance of simple sequence motifs reflects a similar situation to that found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus. The hybridization pattern of Picea species, bald cypress and gingko were different from the patterns detected in the Pinus species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG)_n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three- needle pine genomes, such as slash pine (Pinus elliottii Engelm. var. elliottii), compared to P. strobus, Picea species, bald cypress and gingko. P. elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome.Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity, such to (CA)₈, (GA)₈, (GGAT)₄ and (GATA)₄, and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated. Received: 1 October 1999 / Accepted: 3 November 1999