Characterization of the genome of the free-living nematode Panagrellus silusiae: absence of short period interspersion.

The complexity of the DNA of the free-living nematode Panagrellus silusiae has been examined. Reassociation kinetics of pressure-sheared fragments (approximately 290 nucleotides) in 0.18 M Na+ at 60 degrees C showed the presence of foldback, repetitive, and unique DNA sequence elements. The three classes comprise 9.3%, 26.1%, and 61.3% of the total DNA, respectively. The mean length of the foldback duplex DNA after digestion with S1 nuclease is about 185 nucleotides. There are about 1.8 x10(4) inverted repeats per genome. Sequence arrangement was deduced from (1) renaturation kinetic profiles of long and short fragments on hydroxylapatite; (2) the pattern of renaturation of tracer DNA, labeled in vitro with 125I, of various sizes after incubation with excess short fragments; and (3) thermal denaturation behavior of DNA that had been reassociated to various C0t values. It was found that DNA fragments of the repetitive fraction that are, at least, 2000 nucleotides in length are virtually free of unique sequences. Moreover, it is estimated that the repeated segments in this species could extend for 10,000 nucleotide pairs. Thus, Panagrellus DNA lacks the pattern of extensive short period interspersion that is typified by the DNA of Xenopus.     

DNA-sequence organization in the genome of the domestic chicken (Gallus domesticus).

The sequence organization in the DNA of chicken (Gallus domesticus) was studied using hydroxyapatite-monitored reassociation kinetics. DNA 320-nucleotides long reassociates as though it is composed of three components, i.e., a very rapidly reacting fold-back fraction, a component composed of sequences repeated an average of 640 times in the genome, and a large unique fraction representing about 80% of the genome. The sizes of the fold back and repeated components increase only moderately with large increases in fragment size, indicating that these sequences are not extensively interspersed in the genome. Even at a fragment size of 4500 nucleotides, the unique component represents 68% of the DNA. Thus, the chicken genome is not organized in the short-period (Xenopus) interspersion pattern described for a large number of other organisms; rather, the DNA-sequence organization of this vertebrate bears more resemblance to the long-period interspersion pattern of Drosophila.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

Electron microscopy of Achlya deoxyribonucleic acid sequence organization.

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.     

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.     

A comparative study of genomes in angiosperms

Angiosperms investigated by DNA/DNA reassociation studies were classified and tested for a taxonomic class- and subclass-specifity in a biometrical fashion. Monocotyledons and Dicotyledons differ significantly from each other with respect to a genomic parameter (U/R-ratio;U single copy DNA fraction;R = 1-U fraction of repetitive DNA). This difference is discussed from an evolutionary and molecular point of view.—Intercorrelations between the fraction of fast repeats, slow repeats, and single copy DNA can be detected. The amount of DNA organized in a short period pattern of interspersion is found to depend on the fraction of repetitive and single copy DNA. The number of DNA segments tandemly arranged in a short period pattern is linearly correlated withR/U-values. This correlation allows for a formula suitable for the estimation of the number of active genes in angiosperms. The analytical complexities of repetitive and single copy DNA are linearly correlated with the genome size of higher plants. The ratioU/R depends on the genome size of angiosperms in a hyperbolic fashion.     

Contrasting DNA sequence organisation patterns in sauropsidian genomes

The genomic DNA organisation patterns of four sauropsidian species, namely Python reticularis, Caiman crocodilus, Terrapene carolina triungius and Columba livia domestica were investigated by reassociation of short and long DNA fragments, by hyperchromicity measurements of reannealed fragments and by length estimations of S₁-nuclease resistant repetitive duplexes. While the genomic DNA of the three reptilian species shows a short period interspersion pattern, the genome of the avian species is organised in a long period interspersion pattern apparently typical for birds. These findings are discussed in view of the close phylogenetic relationships of birds and reptiles, and also with regard to a possible relationship between the extent of sequence interspersion and genome size.     

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome.Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$pure⁵ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 4.6 × 10}{}^{\mathrm{?4}}$) and intermediately repetitive sequences constitute the remaining 27% of the genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 1.46}$). Hybridization of ¹²⁵I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome.The interspersion of repetitive sequences that reassociate between C₀t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component.Highly repetitive sequence elements that are reassociated by a C₀t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C₀t = 10^?4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.     

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.     

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.     

Genome analysis of Amphioxus and speculation as to the origin of contrasting vertebrate genome organization patterns.

1. The genome of Amphioxus was investigated by DNA reassociation techniques for the amount of repetitive and non-repetitive sequences and its pattern of organization. 2. A comparison of the amount of non-repetitive DNA between Amphioxus and the tunicate Ciona intestinalis does not support the hypothesis that the Cephalochordates have arisen from the Tunicates by polyploidy. 3. In the Amphioxus genome repetitive and non-repetitive elements are predominantly arranged in a short period interspersion pattern. Conclusions are presented as to the evolution of contrasting genome organization patterns among vertebrates.     

DNA sequence organization in finger millet (Eleusine coracana)

Approximately 39 to 49% of the genome of finger millet consists of repetitive DNA sequences which intersperse with 18% of single copy DNA sequences of 1900 nucleotide pairs. Agarose gel filtration and electrophoresis experiments have yielded the sizes of interspersed repeated sequences as 4000–4200 nucleotide pairs and 150–200 nucleotide pairs. Approximately 20% of the repeated DNA sequences (4000–4200 nucleotide pairs) are involved in long range interspersion pattern, while 60% of the repeated DNA sequences (150–200 nucleotide pairs) are involved in short period interspersion pattern. Based on the data available in literature and the results described here on DNA sequence organization in plants, it is proposed that plants with haploid DNA content of more than 2.5 pg exhibit mostly the short period interspersion pattern, while those with haploid DNA content of less than 2.5 pg show diverse patterns of genome organization. NCL Communication No.: 2708     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

Active DNA transcription sites released from the genome of normal embryonic chicken cells.

Single-stranded DNA (ssDNA) isolated from (and amounting to 1.5-2% of) native nuclear DNA of cultured embryonic chicken cells labelled 1-2 days with 3H-thymidine was analyzed by self-hybridization, hydroxyapatite chromatography (HAC) partial digestion with S1 nuclease, isopycnic centrifugation. Two main fractions were rehybridized to excess amounts of bulk nuclear DNA or total cytoplasmic RNAs. The major fraction, equivalent to 75% of total ssDNA, consists of unique DNA sequences, apparently derived from multiple coding regions of the cell genome, since they are not self-reassociating but are hybridizable to the non repetitious portion of bulk nuclear DNA and 40-45% of them are complementary to cell RNAs. About half of these ssDNA sequences hybridizable to cell RNAs seem to be closely connected with molecules belonging to the minor ssDNA fraction. The latter fraction consists of self-reassociating, moderately repeated DNA sequences, mainly derived from non coding regions of the cell genome. These findings are discussed in the light of others, showing interspersion of coding and non coding DNA sequences and susceptibility of active genes to certain nucleasic attacks.     

The genome of Zea mays,its organization and homology to related grasses

The pattern of genome organization of Zea mays has been analyzed, and the relationship of maize to possible progenitor species assessed by DNADNA hybridization. Reassociation of 470 and 1,350 bp fragments of maize DNA to various C₀t values demonstrates that the genome is composed of 3 major kinetic classes: highly repetitive, mid-repetitive, and unique. Mini-C₀t curves of the repetitive sequences at short fragment length indicate that the highly repetitive sequence class is 20% of the genome and is present at an average reiteration frequency of 800,000 copies; the mid-repetitive sequence class is 40% of the genome and is present at an average reiteration frequency of 1,000 copies. Thermal denaturation studies show that the highly repetitive sequences are 12% divergent and mid-repetitive sequences are 6% divergent. Most of the genome is organized in two interspersion patterns. One, approximately one-third of the genome, is composed of unique sequences of average length 2,100 bp interspersed with mid-repetitive sequences; the other, also one-third of the genome, is mid-repetitive sequences interspersed with highly repetitive sequences. The repetitive sequences are 500 to 1,000 bp by electron microscopic measurement. The remaining third of the genome is unique sequences farther than 5,000 bp from a palindromic or repetitive sequence. Hybridization of maize DNA from Midwestern Dent to popcorn and related grasses indicates that both the unique and repetitive sequence elements have diverged. Teosinte and popcorn are approximately equally divergent from Midwestern Dent whereas Tripsacum is much more divergent. The divergence times calculated from the depression of T_m in heterologous duplexes indicate that the divergence within Zea mays and between maize and near relatives is at least an order of magnitude greater than expected. This high degree of divergence may reflect the pressures of domestication of maize.     

Organization of the micronuclear genome of oxytricha nova

In the hypotrichous ciliated protozoan Oxytricha nova, approximately 95% of the micronuclear genome, including all of the repetitive DNA and most of the unique sequence DNA, is eliminated during the formation of the macronuclear genome. We have examined the interspersion patterns of repetitive and unique and eliminated and retained sequences in the micronuclear genome by characterizing randomly selected clones of micronuclear DNA. Three major classes of clones have been defined: (1) those containing primarily unique, retained sequences; (2) those containing only unique, eliminated sequences; and (3) those containing only repetitive, eliminated sequences. Clones of type one and three document two aspects of organization observed previously: clustering of macronuclear destined sequences and the presence of a prevalent repetitive element. Clones of the second type demonstrate for the first time that eliminated unique sequence DNA occurs in long stretches uninterrupted by repetitive sequences. To further examine repetitive sequence interspersion, we characterized the repetitive sequence family that is present in 50% of the clones (class three above). A consensus map of this element was obtained by mapping approximately 80 phage clones and by hybridization to digests of micronuclear DNA. The repeat element is extremely large (approximately 24 kb) and is interspersed with both macronuclear destined sequences and eliminated unique sequences.     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.     

Sequence organization of the soybean genome

The total complexity of one constituent soybean (Glycine max) genome is estimated to be 1.29 . 10(9) nucleotide pairs, as determined by analysis of the reassociation kinetics of sheared (0.47 kilobase) DNA. Single copy sequences are estimated to represent from 53 to 64% of the genome by analysis of hydroxyapatite binding of repetitive DNA as a function of fragment length. From 65 to 70% of these single copy sequences have a short period interspersion with 1.11--1.36 kilobase lengths alternating with 0.3--0.4 kilobase repetitive sequence elements. The repetitive sequences of soybean DNA are interspersed both among themselves and among single copy regions of the genome.     

DNA sequence organization in the soybean plant

The arrangement of repetitive and nonrepetitive DNA sequences in the soybean genome was ascertained by a comparison of the reassociation kinetics of short (250 nucleotides) and long (2700 nucleotides) DNA fragments, the size distribution of S-1 nuclease resistant repetitive duplexes, and a direct assay of the spectrum of DNA sequences present on long DNA fragments enriched in repetitive DNA. These measurements reveal the following: (1) The 1N genome size of the soybean plant is 1.97 pg. (2) Approximately 40% of the soybean genome consists of nonrepetitive or single-copy DNA sequences, while 60% is repetitive DNA. (3) The repetitive DNA is partitioned into three discrete classes termed very fast, fast, and slow, containing DNA sequences repeated an average of 290,000, 2800, and 19 times each. (4) Approximately 35–50% of the soybean genome is arranged in a short-period interspersion pattern of 250 nucleotide slow sequences and single-copy DNA averaging up to 2700 nucleotides in length. (5) From 30% to 45% of the soybean genome is organized into long stretches of repetitive DNA at least 1500 nucleotides in length. (6) Minimal interspersion of repetitive sequence classes occurs in soybean DNA.These experiments were supported by NSF Grants BMS74-21461 and PCM76-24593 and were conducted while the author was in the Department of Biology, Wayne State University, Detroit, Michigan.     

Genome complexity and organization in the red imported fire ant Solenopsis invicta Buren

The red imported fire ant Solenopsis invicta is the most destructive invading arthropod in the southern United States, yet little is known about its genome complexity and organization. Here we report the size, organization and GC content of S. invicta genome. DNA reassociation kinetics using S1 nuclease assay and a modified second-order kinetics model indicated that the S. invicta genome is approximately 0.62 picograms or 5.91 x 10(8) base pairs, composed of 36% unique, 41% moderately repetitive and 23% highly repetitive/foldback sequences. Comparison of the reassociation kinetics of short and long DNA fragments revealed that the sequence arrangement follows a pattern of short period interspersion, as in most organisms with relatively large genomes. Melting-temperature analysis showed that the GC content of the fire ant genomic DNA is 34.8%, similar to that of most eukaryotic organisms. The results reveal that the fire ant genome is much larger and more complex than those of a number of hymenopteran insects studied to date. Our study provides a foundation for further analysis and genetic manipulation of the S. invicta genome.