A thermal denaturation study of genomic DNAs from North American minnows (Cyprinidae: Teleostei)

Base compositions and differential melting rate profiles of genomic DNAs from twenty species of North American cyprinid fishes were generated via thermal denaturation. Base pair composition expressed as % GC values ranged among the twenty species from 36.1–41.3%. This range is considerably broader than that observed at comparable taxonomic levels in other vertebrate groups. Both the range and average difference in base pair composition between species in the diverse and rapidly evolving genus Notropis were considerably greater than those between species in other North American cyprinid genera. This may indicate that genomic changes at the level of base pair composition are frequent and possibly important events in cyprinid evolution. Compositional heterogeneity and asymmetry values among the twenty species were uniform and low, respectively, suggesting that most of the species lacked DNA components in their genomes which differed substantially from their main-band DNAs in base pair composition. The melting rate profiles revealed a prominent and distinct heavy or GC-rich DNA component in the genomes of three species belonging to the subgenus Cyprinella of Notropis. These and other data suggest that the heavy melting component may reflect a large, comparatively GC-rich family of highly repeated or satellite DNA sequences common to all three genomes.     

Satellite DNA and chromosomes in Neotropical fishes: methods,applications and perspectives

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C₀t–1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII‐DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.     

Dispersity of repeat DNA sequences in Oncopeltus fasciatus,an organism with diffuse centromeres

DNA of Oncopeltus fasciatus, an organism with diffuse centromeres, has been characterized by determination of its base composition, buoyant density, thermal stability, and reassociation kinetics; renatured DNA was characterized similarly. We conclude that repeated sequences are primarily short and scattered throughout the genome. This is in contrast to the extensive tandem repeats which are found in DNAs of organisms with discrete centromeres.     

Characterisation of rye B chromosome DNA by DNA/DNA hybridisation

The DNAs of wheat and rye plants with rye B chromosomes have been compared with wheat, rye and oats DNAs by DNA/DNA hybridisation. The presence of DNA from B chromosomes made no significant difference to the proportion of repeated sequence DNA. The repeated sequence fractions of these cereal DNAs were quantitatively divided into eight different groups on the basis of the amount of DNA/DNA hybridisation occurring between the different DNAs. Rye A and B chromosomes contained similar proportions of three of the groups. These results, together with estimates of the thermal stabilities of all the renatured DNA duplexes suggest that rye B chromosome DNA is very similar to rye A chromosome DNA in the proportion and heterogeneity of its repeated sequences.     

DNA condensation with polyamines. II. Electron microscopic studies

Approximately 75% of the wheat and rye genomes consist of repeated sequence DNA. Three-quarters of the non-repeated or few copy sequences in wheat are less than 1000 base-pairs long, whilst in rye approximately half of the non-repeated or few copy sequences are in this size class. Most of the remaining non-repeated or few copy sequences appear to be a few thousand base-pairs long.In this paper a somewhat novel approach has been used to quantitatively analyse the linear organisation of the large proportion of repeated sequence DNA as well as the non-repeated DNA in the wheat and rye genomes. Repeated sequences in the genomes of oats, barley, wheat and rye have been used as probes to distinguish and isolate four different groups of repeated sequences and their neighbouring sequences from the wheat and rye genomes. Radioactively labelled wheat or rye DNA fragments ranging from 200 to over 9000 nucleotides long were incubated separately with large excesses of denatured unlabelled oats, barley, wheat and rye DNAs to C_ot values which enable all the repeated sequences of the unlabelled DNA to renature. The following parameters were then determined from the proportions of total labelled DNA in fragments which had at least partially renatured. (1) The proportions of the repeated sequences in the labelled DNAs that were able to hybridise to each unlabelled DNA; (2) the mean distance apart of the hybridising sequences on the longer labelled fragments; and (3) the proportion of the genome in which the hybridising sequences were concentrated. Analysis of these results, together with those of separate experiments designed to quantitatively estimate the nature of sequences unable to reanneal with the repeated sequences of each of the probe DNAs, have enabled schematic maps to be drawn which show how the repeated and non-repeated sequences are arranged in the wheat and rye genomes.Both genomes are constructed from millions of relatively short sequences, most of them considerably shorter than 3000 base-pairs. This structure was recognised because adjacent sequences can be distinguished by their frequency of repetition (i.e. repeated or non-repeated) or by their evolutionary origin. Approximately 40 to 45% of the wheat genome and 30 to 35% of the rye genome consists of short non-repeated sequences interspersed between short repeated sequences. Approximately 50% of the wheat genome and 60% of the rye genome consists of tandemly arranged repeated sequences of different evolutionary origins. It is postulated that much of this complex repeated sequence DNA could have arisen from amplification of compound sequences, each containing repeated and non-repeated sequence DNA.Short repeated sequences with a number average length of around 200 base-pairs and which occupy about 20% of the wheat and rye genomes are related to repeated sequences also found in oats and barley. They are concentrated in 60 to 70% of the wheat and rye genomes, being interspersed with different short repeated sequences and a significant proportion of the short non-repeated sequences.Rye chromosomes contain more DNA than wheat chromosomes. This is principally, but not entirely, due to additional repeated sequence DNA. Many quantitative changes appear to have occurred in both genomes, possibly affecting most families of repeated sequences, since wheat and rye diverged from a common ancestor. Both species contain species-specific repeated sequences (24% of rye genome; 16% of wheat genome) but a large proportion of these are closely interspersed with repeated sequences found in both genomes.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Changes in DNA composition in the evolution of Vicia species

Summary The composition of nuclear DNA in 3 Vicia species are compared. The species V. eriocarpa, V. johannis and V. melanops are from three separate subgeneric sections of Vicia and show a fourfold variation in their amounts of nuclear DNA. DNA melting experiments, buoyant density gradient analysis and Cot reassociation experiments show that the quantitiative change in nuclear DNA between the three species is achieved by changes in the amounts of both repetitive and nonrepetitive DNA sequences. It is suggested that while the increase in the repetitive fraction is achieved by the proliferation of repetitive base sequences the increase in the nonrepetitive fraction is due to the steady accretion of highly diverged base sequences resulting from mutations, deletions, insertions and base sequence rearrangements among families of repetitive sequences.     

Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.     

Molecular Genetic Distinction of Pneumocystis carinii from Rats and Humans

Pneumocystis carinii from rats and from humans were compared with respect to electrophoretic karyotype, presence of DNA sequences known to be repeated in rat-derived P. carinii, overall DNA sequence homology, and the sequences at two genetic loci. The organisms from each host species were different in each respect. Neither of two repeated DNAs from rat-derived P. carinii was found in the genome of human-derived organisms, and total DNA from rat-derived P. carinii failed to hybridize to human-derived P. carinii DNA. The sequences of the α-tubulin genes from the two P. carinii were strikingly different and the base composition of the α-tubulin gene from rat-derived P. carinii was rich in adenine and thymine, while the base composition of this gene from human-derived P. carinii was rich in guanine and cytosine. The sequence from the 18S rRNA gene of human-derived P. carinii was twice as divergent from that of rat-derived P. carinii as the sequence from the corresponding region of Candida albicans was from that of Candida tropicalis. These data show that rats and humans can harbor distinct types of P. carinii that are sufficiently different to suggest that P. carinii from the two hosts could be different species.     

Detection and location of three simple sequence DNAs in polytene chromosomes from virilis group species of Drosophila

In vitro synthesized RNAs complementary to the three satellite DNAs of Drosophila virilis have been used in a series of in situ hybridization experiments with polytene chromosomes from virilis group species. Gall and Atherton (1974) demonstrated that each of the satellites of D. virilis is comprised of many repeats of a distinct, seven base pair long, simple sequence. With few exceptions, copies of each of these simple sequences are detected in the chromocenters of all virilis group species. This is true even in species which do not possess satellite DNAs at buoyant densities corresponding to those of the satellite DNAs of D. virilis. Small quantities of the three simple sequences are also detected in euchromatic arms of several different species. The same euchromatic location may contain detectable copies of one, two, or all three simple sequence DNAs. The amounts of simple sequences at each location in the euchromatin may vary between species, between different stocks of the same species, and even between individuals of the same stock. The simple sequences located in the euchromatin appear to undergo DNA replication during formation of polytene chromosomes unlike those in heterochromatin. The locations of the euchromatic sequences are not the results of single chromosomal inversion events involving heterochromatic and euchromatic breakpoints.     

Interspecies comparison of the highly-repeated DNA of Australasian Luzula (Juncaceae)

The woodrush genus Luzula is characterised by having holocentric chromosomes. DNA of nine related Australasian species shows similar satellite DNAs which re very similar in nucleotide sequence content and unit length. Differences between the repetitive DNAs are evident as either the presence or absence of particular restriction enzyme sites. Sequence variants have probably been introduced into the repeated DNA components of ancestral species and particular variants reamplified during the evolution of the genus. Sequence amplification appears to be restricted to sequences already present in the genome rather thant the de novo generation of repeats. The evolution of highly-repeated DNA sequences dispersed throughout the holocentric chromosomes of Luzula thus appears to be very similar to that known in eukaryotes with the more normal monocentric chromosome organisation.     

Different AT-rich satellite DNAs in Cucurbita pepo and Cucurbita maxima

Summary The AT-rich highly repeated satellite DNA of Cucurbita pepo (zucchini) and Cucurbita maxima (pumpkin) were cloned and their DNA structure was investigated. DNA sequencing revealed that the repeat length of satellite DNA in Cucurbita pepo is 349–352 base pairs. The percentage of AT-base pairs is about 61%. This satellite is highly conserved in restriction enzyme pattern and DNA sequence; sequence heterogeneity is about 10%. In contrast, the satellite DNA of Cucurbita maxima has a repeat length of 168–169 base pairs. This satellite is also rich in AT-base pairs (64%), existing in at least three different variants as revealed by restriction enzyme analysis and DNA sequencing. The sequence heterogeneity between these variants is about 15%. The two satellite DNAs showed no cross-hybridization to each other and sequence homology is only limited. Nevertheless, we found in the C. pepo genome a high amount of sequences resembling the satellite of C. maxima. In contrast, the satellite repeat of C. pepo is found in the C. maxima DNA only in a few copies. These observations were discussed with respect to satellite DNA evolution and compared to the data received from monocotyledonous species.     

Characterization of heterochromatin in different species of the Scilla siberica group (Liliaceae) by in situ hybridization of satellite DNAs and fluorochrome banding

Satellite DNAs have been isolated from the monocotyledonous plants Scilla siberica, S. amoena, S. ingridae (all are highly GC-rich), and S. mischtschenkoana by using the Ag⁺ –Cs₂SO₄ density centrifugation technique. Hybridization in situ has been performed with ₃H-cRNA to these satellite DNAs in all four species. In each species, the endogenous satellite DNA is located mainly in intercalary and major heterochromatin bands associated with terminal regions and nucleolar organizer regions (NORs) but not in centromeric regions. Patterns observed after cross-species hybridization show a high degree of satellite DNA homology between S. siberica, S. amoena, and S. ingridae. By contrast, satellite DNA of S. mischtschenkoana consists largely of different, non homologous DNA sequences, with two exceptions: (i) the NORs of all four species contain similar satellite sequences, and (ii) a strong homology exists between the satellite DNA of S. mischtschenkoana and centromeric DNA of S. siberica but not with those of S. amoena and S. ingridae. — Heterochromatin has also been characterized by the AT-specific fluorochromes quinacrine (Q) and DAPI and the GC-specific agent chromomycin A₃ (CMA₃), in combination with two counterstaining techniques. While CMA₃-fluorescence is largely in agreement with data on base composition and location of the specific satellite DNAs, the results with Q and DAPI are conflicting. Prolonged fixation has been found to change the fluorescence character in certain instances, indicating that other factors than the base sequence of the DNA also play a role in fluorochrome staining of chromosomes. The results are discussed in relation to the taxonomy and phylogeny of the four species.     

Repeated sequence DNA relationships in four cereal genomes

The effect of DNA fragment size on the extent of hybridisation that occurs between repeated sequence DNAs from oats, barley, wheat and rye has been investigated. The extent of hybridisation is very dependent on fragment size, at least over the range of 200 to 1000 nucleotides. This is because only a fraction of each fragment forms duplex DNA during renaturation. From these results estimates of the proportions of repeated sequences of each of the cereal genomes that are homologous with repeated sequences in the other species have been determined and a phylogenetic tree of cereal evolution constructed on the basis of the repeated sequence DNA homologies. It is proposed that wheat and rye diverged after their common ancestor had diverged from the ancestor of barley. This was preceded by the divergence of the common ancestor of wheat, rye and barley and the ancestor of oats. Once introduced in Gramineae evolution most families of repeated sequences appear to have been maintained in all subsequently diverging species. — The repeated sequences of oats, barley, wheat and rye have been divided into Groups based upon their presence or absence in different species. Repeated sequences of related families are more closely related to one another within a species than between species. It is suggested that this is because repeated sequences have been involved in many rounds of amplification or quantitative change via unequal crossing over during species divergence in cereal evolution.     

Elucidation of the B-genome donor to Triticum turgidum by unique- and repeated-sequence DNA hybridizations

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid Triticum species ancestral to the B genome of T. turgidum. Unique and repeated sequences from the various Triticum species were separated by hybridization and thermal elution on hydroxyapatite. Unique- and repeated-sequence fractions of labeled T. turgidum var. durum DNA were hybridized to the corresponding fractions of unlabeled DNAs of T. searsii, T. speltoides, T. longissimum, T. sharonensis, and T. bicorne. Thermal stability profiles were constructed to evaluate base-sequence complementarity between T. turgidum var. durum and the diploid Triticum species. The heteroduplex thermal stabilities indicated that, of the five species examined, T. searsii was the most closely related to the B genome of T. turgidum var. durum. The thermal stability profiles further indicated that the repeated DNA fractions from the Triticum species are more similar than the unique-sequence fractions. This indicates that all of the Triticum species are very closely related and, in all probability, have diverged from a single progenitor species.Published with the approval of the Director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Paper No. 1931.     

Identification of the G-genome donor to Triticum timopheevii by DNA:DNA hybridizations

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid Triticum species ancestral to the G genome of Triticum timopheevii. Total genomic, unique-sequence, and repeated-sequence fractions of ³H-T. timopheevii DNA were hybridized to the corresponding fractions of unlabeled DNAs of T. searsii, T. speltoides, T. sharonensis, T. longissimum, and T. bicorne. The heteroduplex thermal stabilities indicated that, of the five species examined, T. speltoides was the most closely related to the G genome of T. timopheevii. Thus, T. spelotides appears to be the G-genome donor to T. timopheevii. The thermal stability profiles further indicated that the repeated DNA fractions from the five diploid species and the tetraploid T. timopheevii are more similar than the unique DNA fractions. This indicates that all of these species are closely related and that the sequences which comprise the current repeated fractions in the various species have not undergone any significant change since the formation of various species.Published with the approval of the Director of the West Virginia Agriculture and Forestry Experiment Station as Scientific Paper No. 1850.     

Human cellular sequences detectable with adenovirus probes

Previous studies suggesting homology between human cellular DNA and the DNAs from adenovirus types 2 and 5 are extended in the present paper. A clone (ChAd^h), isolated from a human genomic DNA library using an adenovirus probe, hybridized to discrete regions of adenovirus 2 DNA, including part of the transforming genes E1a and E1b, as well as to repeated sequences within human DNA. The E1a and E1b genes both hybridize to the same 300 base pair Sau3AI fragment within ChAd^h although there is no obvious homology between E1a and E1b. The Ad 2 E1a gene was also used as a probe to screen other cellular DNAs to determine whether repeated sequences detectable with Ad 2 DNA probes were conserved over long evolutionary periods. Hybridization was detected to the genomes of man, rat, mouse and fruit fly, but not to those of yeast and bacteria. In addition to a smear hybridization, discrete fragments were detected in both rodent and fruit fly DNAs. The experiments reported suggest the existence of two different types of cellular sequences detected by Ad 2 DNA: (1) repeated sequences conserved in a variety of eukaryote genomes and (2) a possible unique sequence detected with an E1a probe different from that responsible for hybridization to repeated sequences. This unique sequence was detected as an EcoRI fragment in mouse DNA and had a molecular size of about 8.8 kb.     

DNA homologies among homothallic, pseudo-homothallic and heterothallic species of Neurospora.

Summary Highly purified DNAs from three homothallic speciesNeurospora africana, N. dodgei andN. lineolata; three reference strains representing authentic heterothallic species,N. crassa, N. intermedia andN. sitophila; and two strains of pseudo-homothallic speciesN. tetrasperma were characterized by spectrophotometry and DNA reassociation using hydroxyapatite chromatography. All of these known species are closely related on the basis of DNA characteristics such as base composition and thermal denaturation profiles of major DNA components. Minor components of ascospore DNA was, however, only 5–7% of total DNA instead of 15–20% minor component DNA shown by mycelial DNA. Species belonging to same group were not distinguishable morphologically, but all of these species were distinguishable by DNA:DNA homology studies. Greater DNA homology was noticed between DNAs of heterothallic species and DNAs of pseudohomothallic species than DNA of true homothallic species. Difference on DNA-nucleiotide sequences among homothallic species was very little. Pseudo-homothallic speciesN. tetrasperma was found to be distinctly different from homothallic species but closer to heterothallic species based on such studies.Supported in part by a contract No. E(40-1)4182 with the U.S. Energy Research and Development Administration. We are grateful to Departments of Oncology and Radiotherapy, College of Medicine, Howard University, Washington, D.C. for providing us with material assistance     

The relatedness and evolution of repeated nucleotide sequences in the genomes of some gramineae species

Reassociation kinetics of sheared denatured DNAs from wheat, barley, rye, and oats at 60 C in 0.18 cm Na⁺ indicate that approximately 80% of these genomes consist of repeated nucleotide sequences, using hydroxylapatite chromatography to detect reannealed DNA. The remaining DNA appears to consist of sequences present in only one or a few copies per haploid genome. Studies on heterologous duplexes formed in vitro between the repeated sequence DNA fractions from each of the species in turn indicate that many of the families of repeated sequences in these cereals evolved from common ancestral sequences. The extent of heteroduplex formation and duplex thermal stabilities suggest a scheme for the evolution of these species which agrees with taxonomic and genetic evidence. Further analyses of these parameters indicate that many quantitative changes in the chromosomal complement of repeated sequences have occurred during divergence of these species.     

Evolution of Satellite DNAs in a Radiation of Endemic Hawaiian Spiders: Does Concerted Evolution of Highly Repetitive Sequences Reflect Evolutionary History?

Satellite DNAs are known for an unusual and nonuniform evolution characterized by rapid evolutionary change between species and concerted evolution leading to molecular homogeneity within species. In this paper we use satellite DNAs for phylogenetic analysis of a rapidly evolving lineage of spiders and compare the phylogeny with a hypothesis previously generated based on mitochondrial DNA and allozymes. The spiders examined include almost all species within a monophyletic clade of endemic Hawaiian Tetragnatha species, the spiny-leg clade. The phylogeny based on satellite sequences is largely congruent to those produced by mtDNA and allozymes, except that the satellite DNA yields much longer branches, with higher levels of support for any given node. Closely related species that have differentiated ecologically within an island are well resolved with satellite DNA but much less so with mtDNA. These results suggest that Tetragnatha stDNA repeats seem to be evolving gradually and cohesively during the diversification of these endemic Hawaiian spiders. The study also reveals gain–loss of satellite DNA copies during species diversification. We conclude that satellite DNA sequences may potentially be very useful for resolving relationships between rapidly evolving taxa within an adaptive radiation. In addition, satellite DNA as a nuclear marker suggests that hybridization or peripatry could play a possible role in species formation that cannot be revealed by mitochondrial markers due to its maternal inheritance.This article contains online supplementary data.Reviewing Editor: Dr. Rafael Zardoya