Reassociation of eukaryotic DNA fragments containing interspersed repeats

This paper offers a criticism of the common approach to the reassociation kinetics of eukaryotic DNA assuming an independent reassociation of nucleotide sequences with different frequencies of reiteration. The reassociation of randomly sheared DNA containing reiterated sequences interspersed with unique ones is described in terms of the model for randomly sheared DNA proposed by Gavrilov & Mazo (1977). Computations performed for different values of the interspersion parameters demonstrate their influence on the reassociation rate of total DNA and its repeat-enriched fraction. The reassociation rate of repeated sequences increases with their length. In the case of short-period interspersion appreciable differences are observed between the reassociation kinetics computed in terms of the random shearing model and the curves obtained for an admittedly independent reassociation of repeated and single-copy sequences.     

Genomic distribution of repeated and single copy DNA sequences in Penicillium funiculosum

Study of the reassociation kinetics of Penicillium funiculosum DNA (fragment size > 20 kb) revealed an interspersion of repeated and single-copy DNA sequences. The fragment length of interspersed single-copy DNA was estimated to be more than 9 kb from a curve relating the fraction of DNA binding to hydroxyapatite as a function of DNA fragment length. The length of interspersed short and long repeated nucleotide sequences was determined to be 0.55 kb and > 20 kb, respectively, by agarose gel electrophoresis. The genomic organization of P. funiculosum is thus significantly different from that in other fungi.     

Capillary electrophoresis as a method to study DNA reassociation

To develop analytical methodology to assess the genetic complexity of a DNA sample, capillary electrophoresis with laser-induced fluorescence detection is used to monitor the annealing process of DNA samples. Coated columns are filled with an entangled polymer solution shown to optimally separate DNA through size-selective capillary electrophoresis. DNA samples are denatured by heating in a boiling water bath for approximately 10 min and then cooled to approximately 25 degrees C below the melting point of the DNA sample to initiate the reassociation process. The DNA is detected by means of the laser-induced fluorescence of intercalated ethidium bromide, which produces a substantially greater signal for double- versus single-stranded DNA. The rate of reassociation is dependent upon the rate at which complimentary strands of DNA encounter each other and the degree of repeating base sequences in the sample (hence, the diversity of the DNA). Experimental parameters also influence the reassociation rate. The effects of salt concentration and incubation temperature are presented. Traditional plots of C(o)t (C(o) = DNA concentration and t = reassociation time) versus % recovery of double-stranded DNA signal are generated for PhiX 174 Hae III digest and 50 bp stepladder DNA, individually and combined, to calculate the reassociation rate constants for these samples. Because reassociation of individual fragments is observed by the CE-LIF method, more information about the samples is available than with less specific and time-consuming traditional methods of investigating DNA reassociation.     

Size and complexity of the nuclear genome of Colletotrichum graminicola.

DNA reassociation was used to estimate GC content, size, and complexity of the nuclear genomes of Colletotrichum from maize and sorghum. Melting-temperature analysis indicated that the GC content of the maize pathotype DNA was 51% and that the GC content of the sorghum pathotype was 52%. DNA reassociation kinetics employing S1 nuclease digestion and an appropriately modified second-order equation indicated that the genome sizes of the maize and sorghum pathotypes were 4.8 x 10(7) bp, and 5.0 x 10(7) bp, respectively. Genomic reconstruction experiments based on Southern blot hybridization between a cloned single-copy gene, PYR1 (orotate phosphoribosyl transferase), and maize-pathotype DNA confirmed the size of the nuclear genome. The single-copy component of the genomes of both pathotypes was estimated at about 90%. For both pathotypes, ca. 7% of the genome represented repetitive DNA, and 2 to 3% was foldback DNA.     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

Molecular characterization of an insect genome: Chironomus thummi.

DNA extracted from Chironomus thummi larvae was studied by isopycnic centrifugation in CsCl, thermal denaturation and DNA-DNA reassociation techniques. The mean G+C content of the C. thummi DNA is 28-29% as indicated both by centrifugation in CsCl and thermal denaturation. According to optical reassociation analysis of total DNA and of isolated DNA fractions the C. thummi genome is composed of at least four components. About 80% of the DNA is classified as unique with a kinetic complexity of nearly 7 X 10(10) daltons. 6-8% intermediate DNA exhibits a kinetic complexity slightly above 10(8) daltons with a mean repetition frequency of 35. 11-13% fast-reassociating DNA has a kinetic complexity slightly above 10(6) daltons with a mean repetition frequency of 6000. 3-5% of the DNA cannot be properly studied by the optical reassociation technique and probably contains inverted repeats. The thermal denaturation behaviour of isolated DNA fractions indicated that most of the repetitive sequences in the C. thummi genome are tightly interspersed.     

The reassociation curve of human DNA amended

The reassociation kinetics of human DNA was studied, utilizing S1 nuclease digestion in aqueous dioxane and hydroxyapatite chromatography for isolating renatured DNA. The percentage of DNA reassociated at C0t = 10(-3) was 5--7% and that at C0t = 18 000 was about 85%, C0t being the product of the molar concentration of DNA and the reassociation period in s. The shape of the amended reassociation curve was roughly that of a rectangular hyperbola. It showed pronounced differences from the curves obtained by direct hydroxyapatite chromatography of reassociated DNA. The S1 nuclease-dioxane procedure offered two advantages over the conventional method. It was applicable to the study of reassociation with high molecular weight DNA, and the reassociated DNA so obtained was devoid of low-melting strands. The analysis of the new data took into account the possible effects of the diploid condition on the reassociation rate of DNA, the source of the DNA used in this study being placental tissue. The new reassociation profile was compared to ideal second-order reassociation curves calculated for the human genome (2.5 . 10(9) nucleotide pairs), and for a genome twice this size, containing various proportions of single-copy sequences. The results showed that approximately 85--90% of th total DNA may consist of unique sequences. This estimate is considerably higher than those reported previously.     

Herpes simplex virus type 1 Angelotti and a defective viral genotype: analysis of genome structures and genetic relatedness by DNA-DNA reassociation kinetics.

DNA-DNA reassociation kinetics of herpes simplex virus type 1 Angelotti DNA and a class of defective viral DNA revealed that the viral standard genome has a total sequence complexity of about 93 X 10(6) daltons and that a portion of 11 X 10(6) daltons occurs twice on the viral genome. These results agree with structural features of herpes simplex virus type 1 DNA derived from electron microscopic studies and restriction enzyme analyses by several investigators. The defective viral DNA (molecular weight, about 97 X 10(6)) displays a sequence complexity of about 11 X 10(6) daltons, suggesting that the molecule is built up by repetitions of standard DNA sequences comprising about 15,000 base pairs. A 2 X 10(6)-dalton portion of these sequences maps in the redundant region and a 9 X 10(6)-dalton portion maps in the unique part of the standard herpes simplex virus type 1 Angelotti DNA, as could be shown by reassociation of viral standard DNA in the presence of defective DNA and vice versa. No cellular DNA sequences could be detected in defective DNA. A 12% molar fraction of the defective DNA consists of highly repetitive sequences of about 350 to 500 base pairs in length.     

Non-repetitive DNA sequence divergence in phylogenetically diploid and tetraploid teleostean species of the family cyprinidae and the order isospondyli

Non-repetitive DNA of anciently tetraploid teleostean species was analysed for the presence of duplicated sequences. Closely related diploid species were investigated in comparison. From the reassociation kinetics of total nuclear DNA, rate constants and fraction sizes of classes of repetitive and non-repetitive sequences were determined. DNA fractions enriched in the slowest renaturing sequence class were prepared and subjected to reassociation. The rate constants of these reactions were compared with the values expected for single-copy DNA from analytical genome size determinations. From reassociated DNA enriched in non-repetitive sequences also the melting temperatures were determined as a measure of internal base sequence heterogeneity. It has been shown that the two ancient tetraploids Cyprinus carpio and Thymallus thymallus are, with regard to the thermal stability of reassociated non-repetitive DNA, and with regard to the correspondence of reaction rates with the values expected for single copy DNA, indistinguishable from diploid controls (Rutilus rutilus, Clupea harengus and Sprattus sprattus). The tetraploid species Salmo irideus, Salvelinus fontinalis and Coregonus lavaretus appear as very recent tetraploids with regard to these criteria. The significance of the results for estimating the time of occurence of polyploidisation events in these taxa is discussed.     

Genome analysis of ground squirrels of the genus Citellus (Rodentia,Sciuridae) II. DNA sequence organization

The organization of the DNA sequences in five specics of Citellus (C. pygmaeus, C. fulvus, C. major, C. parryi and C. undulatus) was determined from the reassociation kineties of DNA fragments of various lengths and the size distribution of SI-nuclease-resistant duplexes of repetitive DNA. Only 15% of the genome of all the species studied consists of short unique and repeated sequences interspersed with a period less than 2 3 kb, whereas the major part of the genome is occupied by much more extensive sequences of two types, moderately long (3–15 kb) and very long (much more than 15 kb). On the basis of the number of moderately long single-copy sequences the species under study are divided into two groups, coinciding with their division into short-tailed and long-tailed ground squirrels: the short-tailed (C. pygmaeus, C. major and C. fulvus) possess far more such sequences (17–24%) than do the long-tailed ones (C. parryi and C. undulatus) (1–7%). The same division is observed in the amount of very long single-copy sequences. The repeated DNA sequences of Citellus vary widely in size, i.e. from 70 up to some thousands of nucleotide pairs, sequences of more than 1200 nucleotide pairs being most common. In addition, part of the repetitions contain between 70 and 150 base pairs. About one-third of C. parryi repeats (10% of the genome) are characterized by such very short sequences whereas their amont is much less in the other Citellus species (1–4% of the genome).     

Evolution of muntjac DNA

The extent of nuclear single-copy DNA divergence between Muntiacus reevesi and Muntiacus muntjak vaginalis (Cervidae), a species pair showing extreme karyotype differences but striking morphological similarity, is 2%, as judged from the thermal stability of interspecific DNA-DNA hybrids. A comparison of the total nuclear DNA reassociation kinetics of the two species indicates a reduction of lowly repetitive sequences in M. m. vaginalis.     

Repetitive DNA of Higher Plants: SEPARATION OF FRACTIONS OF DIFFERENT COMPLEXITY FROM JERUSALEM ARTICHOKE TISSUES BY REASSOCIATION KINETICS

Jerusalem artichoke DNA, extracted from resting rhizomes, wasanalysed by the reassociation kinetics of sheared denaturedfragments. Various fractions were isolated according to theirreassociation rates. The slowest fractions consist of unrepeatedor single-copy DNA which makes up 55 per cent of the total DNA.The total sequence length of the haploid genome is estimatedas corresponding to 0.23?10¹² daltons. Among the fractions ofrepeated DNA isolated, two were relatively homogeneous withan average complexity of 2?10⁸ and 10⁹ daltons respectively.     

DNA reassociation kinetic analysis of the brine shrimp, Artemia salina

DNA reassociation kinetics have been partly elucidated for the brine shrimp $\text{Artemia salina}$, using calf thymus DNA as a standard. The $\text{Artemia}$ single-copy DNA sequences comprise 45% of the genome; sequences having a repetition frequency of about 2–90 are not detectable. The average repetition frequency of the intermediately redundant DNA component is about 5,000 copies. Reassociation kinetic data are consistent with a unit genome size of 1.5 pg.     

Distribution of repetitious sequences in chick nuclear DNA

By an improved method of hydroxylapatite chromatography, the reassociated sequences of chick nuclear DNA were isolated, and their base composition analysed. By increasing the amount of reassociation, the G + C content of the renatured sequences decreased progressively to reach a mean value corresponding to that of the total DNA. In order to study the distribution of the families, or group of families having different amount of reassociation, DNA was fractionated by CsC1 density gradient centrifugation. Fractions having different G + C content were obtained, and their reassociation rates analysed. At high C(o)t value of renaturation (C(o)t=50) the amount of reassociated sequences included in the high or in the low buoyant density DNA fractions was approximately the same, but their G + C content was as expected different. At lower C(o)t values of renaturation (between C(o)t of 0.2 and the C(o)t of 10), the results indicated an heterogeneity of the repeated sequences in the A + T rich DNA fractions, as compared to the G + C rich ones.     

Genome structure and divergence of nucleotide sequences in echinodermata

The arrangement of repetitive and single-copy DNA sequences has been studied in DNA of some species of Echinodermata — sea urchin, starfishes and sea-cucumber. Comparison of the reassociation kinetics of short and long DNA fragments indicates that the pattern of DNA sequence organization of all these species is similar to the so called Xenopus pattern characteristic of the genomes of most animals and plants. However, substantional variations have been found in the amount of repetitive nucleotide sequences in DNA of different species and in the length of DNA regions containing adjacent single-copy and repetitive sequences. Measurements of the size of S₁-nuclease resistant reassociated repetitive DNA sequences show a variability of ratios between long and short repetitive DNA sequences of different species. — The degree of divergence of short and long repetitive DNA sequences and single-copy DNA was studied by molecular hybridization of the sea urchin Strongylocentrotus intermedius ³H-DNA with the DNA of other species and by determination of the thermostability of the hybridized molecules so obtained. All three fractions of S. intermedius DNA contain sequences homologous to DNA of the other echinoderm species studied. The results obtained suggest that short repetitive DNA sequences are those which have been most highly conserved throughout the evolution of Echinodermata. A new hypothesis is proposed to explain the nature of the evolutionary changes in DNA sequence interspersion patterns.     

The Aedes aegypti genome: complexity and organization.

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.     

Relationship between the study of DNA reassociation kinetics of various chromosomal forms of Ellobius and questions concerning the pathways of chromosome rearrangement during evolution

Fifteen chromosome forms of Ellobius talpinus (from 2n = 31 to 2n = 54) were found in the small area in the Pamirs. Low-chromosome karyotypes evolved from 54-chromosomal ancestral form by Robertsonia centric fusions. The DNA reassociation kinetics of 34- and 54-chromosome forms of E. talpinus have been studied. For comparison DNA of E. lutescens (2n = 17) the karyotype of which seems to have arisen from 54-chromosome ancestor by Robertsonian and other types rearrangements was examined. Reassociation profiles of Ellobius DNA suggest the existence of several repeated sequences families with different frequences of repetitions. The reassociation curves of DNA from 34- and 54-chromosome forms were identical. These data indicate absence of changes in DNA molecular organization during the evolution of E. talpinus karyotypes by Robertsonian fusions. Comparative analysis of DNA reassociation kinetics of E. talpinus and E. lutescens showed identical characteristics of highly repeated sequences and of one from the three intermediate fractions, however Cot 1/2, complexity and repetitive frequencies of two intermediate fractions of E. talpinus and E. lutescens were different. It is possible that non-robertsonian rearrangements of E. lutescens karyotype affected only intermediate repetitions. The alternative explanation of these data is a simple divergence of repeated sequences during the evolution of E. lutescens DNA.     

Structural organization of the genome of the cellular slime mold Dictyostelium discoideum: interspersion of repetitive and single-copy DNA sequences.

The length and interspersion of reiterated and single-copy DNA sequences in Dictyostelium have been examined. The results indicate that approximately 50-60% of the single-copy sequences in DNA fragments 1500 nucleotides long and 75% of the single-copy sequences in fragments 3000 nucleotides long are linked to short interspersed repeat DNA sequences. The average length of these single-copy sequences is 1500 nucleotides. The length of the reiterated DNA has also been analyzed and shows a bimodal distribution. One half is present in sequences greater than 2000 nucleotides long, while the remainder is present as short fragments 250-450 nucleotides long. These shorter fragments are interspersed with the bulk of the single-copy DNA.     

The mitochondrial genome is large and variable in a family of plants (cucurbitaceae)

The genome sizes of mitochondrial DNA from darkgrown (etiolated) shoots of several higher plants were determined by reassociation kinetics and restriction analysis. Kinetic complexities obtained from reassociation kinetics measured spectrophotometrically indicate a mitochondrial genome size of 1600 Md for muskmelon, 1000 Md for cucumber, 560 Md for zucchini squash and 220 Md for watermelon (four species in the cucurbit family), as well as 240 Md for pea and 320 Md for corn. The kinetic curves also reveal the presence (except in corn) of sequences of a few magadaltons of complexity, reiterated about 10-50 times and representing 5%- 10% of the DNA in each mitochondrial genome. Molecular weight summation of fragments resulting from digestion with restriction endonucleases Sal I and Kpn I give genome size estimates similar to those obtained from reassociation kinetics, except for muskmelon and cucumber, for which the large number of fragments of similar size limits our estimate to at least 500 Md. The number of mitochondrial genomes per diploid cell is estimated to be about 110 to 140 for muskmelon, zucchini and watermelon. We consider the possible evolutionary mechanisms by which the mitochondrial genome has grown within the cucurbit family and the possible reasons for the existance of a seven to eight-fold range in mitochondrial genome size among such closely related species.