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     

Clustered and interspersed repetitive DNA sequences in four amphibian species with different genome size.

We have compared the amount of clustered and interspersed repetitive sequences in the genome of four Amphibia with different DNA contents per haploid nucleus: two Anura (Xenopus laevis, 3 pg and Bufo bufo, 7 pg) and two Urodela (Triturus cristatus, 23 pg and Necturus maculosus, 52 pg). High molecular weight DNA of the four species was denatured and reassociated to the same Cot in order to obtain duplex sequences with a similar reiteration frequency. Single-stranded DNA was digested off with the Aspergillus S1 nuclease. DNA was then fractionated according to the molecular weight through an agarose A-50 column. We found that the amount of long repetitive sequences is roughly proportional to the genome size in the four species, while the number of short (about 300 base pairs) repetitive sequences is increased many-fold in the species with the larger DNA content, both in Anura and in Urodela.     

Structural characteristics of genome organization in amphibians: Differential staining of chromosomes and DNA structure

Summary Differential staining patterns on amphibian chromosomes are in some respects distinct from those on mammalian chromosomes; C-bands are best obtained, whereas G- and Q-bands are either unobtainable (on anuran chromosomes) or coincide with C-bands (chromosomes of urodeles). In amphibians, rRNA genes are located at secondary constrictions, but in urodeles they are also found at other chromosome sites, the positions of these sites being strictly heritable. DNA content in amphibian cells is tens and hundreds times higher than in mammals. DNA contents in anurans and urodeles differ within certain limits: from 2 to 25 pg/N and from 30 to over 160 pg/N respectively. Species characterized by slow morphogenesis have larger genomes. Genome growth is normally due to an increase in the amount of repetitive DNA (mostly intermediate repetitive sequences), the amount of unique sequences being almost constant (11 pg/genome in urodeles, and 1.5 pg/genome in anurans). In anurans in general no satellite DNA was found, whereas such fractions were found in manyUrodela species. Nucleosome chromatin structure in amphibians is identical to that of other eukariotes. It is postulated that differences in chromosome banding between amphibians and mammals are due to differences in chromatin packing which in turn is related to the distinct organization of DNA repetitive sequences. It is likely that fish chromosomes have a similiar structure. A comparison of such properties as the chromosome banding patterns, variations in nuclear DNA content and some genome characteristics enable us to group fishes and amphibians together as regards chromosome structure, as distinct from amniotes - reptiles, birds and mammals. It is probable that in the ancient amphibians - ancestors of reptiles - chromatin packing underwent a radical transformation, following changes in the organization of DNA repetitive sequences.     

Localization of specific repetitive DNA sequences in individual rice chromosomes

This paper describes the characterization and chromosomal distribution of three different rice (Oryza sativa) repetitive DNA sequences. The three sequences were characterized by sequence analysis, which gave 355, 498 and 756 bp for the length of the repeat unit in Os48, OsG3-498 and OsG5-756, respectively. Copy number determination by quantitative DNA slot-blot hybridization analysis showed 4000, 1080 and 920 copies, respectively, per haploid rice genome for the three sequences. In situ DNA hybridization analysis revealed that 95% of the silver grains detected with the Os48 probe were localized to euchromatic ends of seven long arms and one short arm out of the 12 rice chromosomes. For the OsG3-498 repetitive sequence, the majority of silver grains (58%) were also clustered at the same chromosomal ends as that of Os48. The minority (28%) of silver grains were located at heterochromatic short arms and centromeric regions. For the OsG5-756 repetitive sequence, 81% of the silver grains labeled the heterochromatic short arms and regions flanking all of the 12 centromeres. Thus, each of these three repetitive sequences was distributed at specific defined chromosomal locations rather than randomly at many chromosomal locations. The approximate copy number of a given repetitive DNA sequence at any specific chromosomal location was calculated by combining the information from in situ DNA hybridization analysis and the total copy number as determined by DNA slot-blot hybridization.by J. Huberman     

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.     

Length and interspersion of repetitive and non repetitive DNA sequences in four Amphibian species with different genome sizes

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Karyotype analyses and studies on the nuclear DNA content in 30 genotypes of potato (Solanum tuberosum) L

The cytophotometric estimation of 4C DNA content, and karyotypic and somatic chromosome number analyses were carried out in 30 genotypes comprising seven cultivars and 23 advanced breeding lines of Solanum tuberosum. Detailed karyotype analysis revealed genotype specific chromosomal characteristics and structural alterations in chromosomes of the genome, with a rare phenomenon of aneusomatic (2n = 4x + 2 = 50) condition in cv.K. Chandramukhi. The origin of this variation could be attributed to mitotic non-disjunction in the shoots giving rise to aneusomatic roots. Highly significant variations in the genome length, volume and total form percentage were noted at the cultivar level. The total chromosome length varied from 84.56 microm in cv.K. Pukhraj to 127.62 microm in MS/89-60, with an average value of 100.94 microm +/- 1.82. Total chromosome volume varied from 57.22 microm3 in MS/92-1090 to 132.64 microm3 in JW-160. Significant variations in the 4C DNA content (7.28-15.83 pg) were recorded at the cultivar level, with an exceptionally high DNA content (22.24 pg) in cv.K. Chandramukhi. This could be due to the aneusomatic condition of this genotype. Correlation studies revealed interdependence between the chromosomal and nuclear parameters of the genotypes. Structural alterations in the chromosomes, as well as loss or addition of highly repetitive sequences in the genome, caused variations in DNA content at the cultivar level. Variations in genomic structure and nuclear DNA content of the 48-chromosome genotypes suggest a genetic drift during microevolution, leading to the development of new cultivars.     

A new moderately repetitive DNA sequence family of novel organization.

In cloning adenovirus homologous sequences, from a human cosmid library, we identified a moderately repetitive DNA sequence family consisting of tandem arrays of 2.5 kb members. A member was sequenced and several non-adjacent, 15-20 bp G-C rich segments with homology to the left side of adenovirus were discovered. The copy number of 400 members is highly conserved among humans. Southern blots of partial digests of human DNA have verified the tandem array of the sequence family. The chromosomal location was defined by somatic cell genetics and in situ hybridization. Tandem arrays are found only on chromosomes 4 (4q31) and 19 (q13.1-q13.5). Homologous repetitive sequences are found in DNA of other primates but not in cat or mouse. Thus we have identified a new family of moderately repetitive DNA sequences, unique because of its organization in clustered tandem arrays, its length, its chromosomal location, and its lack of homology to other moderately repetitive sequence families.     

Chromosomes,DNA sequences,and evolution in salamanders of the genus Plethodon

Chromosomes and DNA sequence homologies have been studied in 15 species of North American salamander belonging to the genus Plethodon. These include 4 Eastern small species, 5 Eastern large species, 5 Western, and 1 New Mexican species. All species have 14 metacentric or sub-metacentric chromosomes. Their karyotypes are closely similar, but their C values range from 18–69 pg. DNA:DNA molecular hybridization studies showed that salamanders belonging to the same species group had between 60 and 90% of the observed repetitive DNA sequences in common, different groups of Eastern species had between 40 and 60% in common, and Eastern and Western groups had less than 10% in common. The slowly reassociating DNA sequences were also diverse among species, but higher levels of homology were observed than in the case of repetitive sequences. The New Mexican species was exceptional in showing little homology with other species with respect to either repetitive or slowly reassociating sequences.     

Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza

Large variation in genome size as determined by the nuclear DNA content and the mitotic chromosome size among diploid rice species is revealed using flow cytometry and image analyses. Both the total chromosomal length (r_0.939) and the total chromosomal area (r_0.927) correlated well with the nuclear DNA content. Among all the species examined, Oryza australiensis (E genome) and O. brachyantha (F genome), respectively, were the largest and smallest in genome size. O. sativa (A genome) involving all the cultivated species showed the intermediate genome size between them. The distribution patterns of genome-specific repetitive DNA sequences were physically determined using fluorescence in situ hybridization (FISH). O. brachyantha had limited sites of the repetitive DNA sequences specific to the F genome. O. australiensis showed overall amplification of genome-specific DNA sequences throughout the chromosomes. The amplification of the repetitive DNA sequences causes the variation in the chromosome morphology and thus the genome size among diploid species in the genus Oryza.     

Genome evolution in the genus Sorghum (Poaceae)

BACKGROUND AND AIMS: The roles of variation in DNA content in plant evolution and adaptation remain a major biological enigma. Chromosome number and 2C DNA content were determined for 21 of the 25 species of the genus Sorghum and analysed from a phylogenetic perspective. METHODS: DNA content was determined by flow cytometry. A Sorghum phylogeny was constructed based on combined nuclear ITS and chloroplast ndhF DNA sequences. KEY RESULTS: Chromosome counts (2n = 10, 20, 30, 40) were, with few exceptions, concordant with published numbers. New chromosome numbers were obtained for S. amplum (2n = 30) and S. leiocladum (2n = 10). 2C DNA content varies 8.1-fold (1.27-10.30 pg) among the 21 Sorghum species. 2C DNA content varies 3.6-fold from 1.27 pg to 4.60 pg among the 2n = 10 species and 5.8-fold (1.52-8.79 pg) among the 2n = 20 species. The x = 5 genome size varies over an 8.8-fold range from 0.26 pg to 2.30 pg. The mean 2C DNA content of perennial species (6.20 pg) is significantly greater than the mean (2.92 pg) of the annuals. Among the 21 species studied, the mean x = 5 genome size of annuals (1.15 pg) and of perennials (1.29 pg) is not significantly different. Statistical analysis of Australian species showed: (a) mean 2C DNA content of annual (2.89 pg) and perennial (7.73 pg) species is significantly different; (b) mean x = 5 genome size of perennials (1.66 pg) is significantly greater than that of the annuals (1.09 pg); (c) the mean maximum latitude at which perennial species grow (-25.4 degrees) is significantly greater than the mean maximum latitude (-17.6) at which annual species grow. CONCLUSIONS: The DNA sequence phylogeny splits Sorghum into two lineages, one comprising the 2n = 10 species with large genomes and their polyploid relatives, and the other with the 2n = 20, 40 species with relatively small genomes. An apparent phylogenetic reduction in genome size has occurred in the 2n = 10 lineage. Genome size evolution in the genus Sorghum apparently did not involve a 'one way ticket to genomic obesity' as has been proposed for the grasses.     

Molecular characterisation and chromosomal localisation of a telomere-like repetitive DNA sequence highly enriched in the C genome of Brassica

The aim of this work was to find C genome specific repetitive DNA sequences able to differentiate the homeologous A (B. rapa) and C (B. oleracea) genomes of Brassica, in order to assist in the physical identification of B. napus chromosomes. A repetitive sequence (pBo1.6) highly enriched in the C genome of Brassica was cloned from B. oleracea and its chromosomal organisation was investigated through fluorescent in situ hybridisation (FISH) in B. oleracea (2n = 18, CC), B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC) genomes. The sequence was 203 bp long with a GC content of 48.3%. It showed up to 89% sequence identity with telomere-like DNA from many plant species. This repeat was clearly underrepresented in the A genome and the in situ hybridisation showed its B. oleracea specificity at the chromosomal level. Sequence pBo1.6 was localised at interstitial and/or telomeric/subtelomeric regions of all chromosomes from B. oleracea, whereas in B. rapa no signal was detected in most of the cells. In B. napus 18 to 24 chromosomes hybridised with pBo1.6. The discovery of a sequence highly enriched in the C genome of Brassica opens the opportunity for detailed studies regarding the subsequent evolution of DNA sequences in polyploid genomes. Moreover, pBo1.6 may be useful for the determination of the chromosomal location of transgenic DNA in genetically modified oilseed rape.     

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.     

Evolution of gene sequence in response to chromosomal location

Evolutionary forces acting on the repetitive DNA of heterochromatin are not constrained by the same considerations that apply to protein-coding genes. Consequently, such sequences are subject to rapid evolutionary change. By examining the Troponin C gene family of Drosophila melanogaster, which has euchromatic and heterochromatic members, we find that protein-coding genes also evolve in response to their chromosomal location. The heterochromatic members of the family show a reduced CG content and increased variation in DNA sequence. We show that the CG reduction applies broadly to the protein-coding sequences of genes located at the heterochromatin:euchromatin interface, with a very strong correlation between CG content and the distance from centric heterochromatin. We also observe a similar trend in the transition from telomeric heterochromatin to euchromatin. We propose that the methylation of DNA is one of the forces driving this sequence evolution.     

DNA fragments associated with chromosome scaffolds

Following extensive digestion of HeLa metaphase chromosomes with either Hae III endonuclease or micrococcal nuclease, nonhistone protein scaffolds may be isolated. Scaffolds isolated after Hae III digestion have about 1.5% of the chromosomal DNA attached to them. This DNA is heterogeneous in size, ranging from about 0.2 to 20 kbp. It can be cleaved with either Eco RI or Hae III - Eco RI, producing a series of repeated fragments, of which the most abundant is 1.7 kbp in length. The 1.7-kdp fragment is tandemly repeated and is enriched (about 50-fold) in the scaffold-associated DNA. It is located primarily on human chromosome 1 and is probably a component of human satellites II and III. Scaffolds isolated after micrococcal nuclease digestion have about 0.1% of chromosomal DNA attached. This DNA is present in two size classes - fragments larger than 10 kbp and fragments approximately 0.2 kbp long. Restriction enzyme digestion of this DNA gives no prominent repeated fragments. Its reassociation kinetics are similar to those of total DNA, indicating that it is not enriched in either highly repetitive or middle repetitive sequences.     

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluroescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromosome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.     

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.