Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.     

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.     

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.     

High diversity in DNA of soil bacteria.

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

High diversity in DNA of soil bacteria

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA reassociation kinetics in diploid and phylogenetically tetraploid cyprinidae.

Four diploid and three phylogenetically tetraploid Cyprinidae (Ostariophysi) have been characterized as for nuclear DNA content, modal chromosome number and DNA reassociation kinetics (hydroxyapatite chromatography). Among the diploid species nuclear DNA content (10(-12) g DNA/2C) was 1.62 for Tinca tinca, 1.87 for Scardinius erythrophthalmus, 2.53 for Leuciscus cephalus and 2.75 for Alburnus alburnus, while the phylogenetically tetraploid species Carassius auratus, Barbus barbus and Cyprinus carpio attained 3.40, 3.66 and 3.80 respectively. Modal chromosome number was 2n = 48-50 for diploid individuals and 2n = 100-104 for phylogenetically tetraploid ones. In all the species 5--8% of the genome is represented by highly repetitive and foldback DNA. In DNA reassociation kinetics of phylogenetically tetraploid Cyprinidae a distinct plateau separates an intermediate reassociating sequence fraction (about 22% of the genome; with average repetition frequencies between 1,000 and 1,400) from a slow reassociating one (unique DNA; about 72% of the genome). These two genome fractions are not clearly distinguishable from each other in Cot curves of the diploid Cyprinidae, where a similar plateau is not evident. Since simple ploidy changes are not expected to affect DNA reassociation kinetics we suggest a different evolution in the genome organization of the two ploidy groups. Some possible hypotheses are discussed.     

Polynucleotide Sequence Relationships among Members of Enterobacteriaceae

Polynucleotide relationships were examined among many representatives of the Enterobacteriaceae by means of agar, membrane filter, and hydroxyapatite procedures. The amount of deoxyribonucleic acid (DNA) that reassociated was dependent, especially in interspecific reactions, on the annealing temperature. In only three cases: Escherichia coli-Shigella flexneri, Salmonella typhimurium-S. typhi, and Proteus mirabilis-P. vulgaris, was relative interspecific duplex formation 80% or higher. In most cases interspecies DNA duplex formation was 40% or less of that obtained from intraspecies DNA reassociation reactions. The stability of E. coli-S. flexneri DNA duplexes formed at either 60 or 75 C was virtually identical to that of homologous E. coli DNA duplexes, and the degree of interspecies duplex formation was minimally affected by the temperature increase (86% at 60 C; 77% at 75 C). The thermal stability of DNA duplexes formed at 60 C between DNA from E. coli and DNA from strains of Aerobacter aerogenes, S. typhimurium, S. typhi, and P. mirabilis was about 12 to 14 C below that of reassociated E. coli DNA. At 75 C, the formation of the interspecific DNA duplexes was markedly decreased, but the stability of the DNA able to reassociate at this temperature approximated that of reassociated E. coli DNA. The degree of reassociation and the thermal stability of E. coli-S. flexneri DNA duplexes suggests relatively little evolutionary divergence in these organisms. The other enterobacteria tested, however, have diverged to a point where less than one-half of their DNA can reanneal with E. coli DNA at 60 C and less than 10% reacts at 75 C. The degree of divergence between various enterobacteria does not appear to be uniform along the DNA molecule. Ribosomal ribonucleic acid (RNA)-specific sequences are conserved among most enterobacteria. An examination of messenger RNA relatively specific for the lactose operon suggests that specific chromosomal genes may diverge more or less than the genome as a whole.     

DNA sequence organization in the genome of Petroselinum sativum (Umbelliferae)

The genome of parsley was studied by DNA/DNA reassociation to reveal its spectrum of DNA reiteration frequencies and sequence organization. The reassociation of 300 nucleotide DNA fragments indicates the presence of four classes of DNA differing in repetition frequency. These classes are: highly repetitive sequences, fast intermediate repetitive sequences, slow intermediate repetitive sequences, and unique sequences. The repeated classes are reiterated on average 136,000, 3000, and 42 times respectively. A minor part of the genome is made up of palindromes. — The organization of DNA sequences in the P. sativum genome was determined by the reassociation kinetics of DNA fragments of varying length. Further information was derived from S1 nuclease resistance and from hyperchromicity measurements on DNA fragments reassociated to defined C₀t values. — The portion of the genome organized in a short period interspersion pattern amounts to 47%, with the unique sequences on an average 1000 nucleotides long, and most of the repetitive sequences about 300 nucleotides in length, whereas the weight average length may be up to 600 nucleotides. — About 5% unique DNA and 11% slow intermediate repetitive DNA consist of sequences from 10³ up to 10⁴ nucleotides long; these are interspersed with repetitive sequences of unknown length. Long repetitive sequences constitute 33% of the genome, 13% are satellite-like organized, and 20% in long stretches of intermediate repetitive DNA in which highly divergent sequences alternate with sequences that show only minimal divergence. — The results presented indicate remarkable similarities with the genomes of most animal species on which information is available. The most intriguing pecularity of the plant genome derives from its high content of repetitive DNA and the presumed organization of the latter.     

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 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.     

Absence of short period interspersion of repetitive and non-repetitive sequences in the DNA of Drosophila melanogaster

A sensitive search has been made in Drosophila melanogaster DNA for short repetitive sequences interspersed with single copy sequences. Five kinds of measurements all yield the conclusion that there are few short repetitive sequences in this genome: 1) Comparison of the kinetics of reassociation of short (360 nucleotide) and long (1,830 nucleotide) fragments of DNA; 2) reassociation kinetics of long fragments (2,200 nucleotide) with an excess of short (390 short nucleotide) fragments; 3) measurement of the size of S1 nuclease resistant reassociated repeated sequences; 4) measurement of the hyperchromicity of reassociated repetitive fragments as a function of length; 5) direct assay by kinetics of reassociation of the amount of single copy sequence present on 1,200 nucleotide long fragments which also contain repetitive sequences.     

Deoxyribonucleic Acid Relatedness Among Species of Erwinia and Between Erwinia Species and Other Enterobacteria

Relatedness in species of Erwinia was assessed by determining the extent of reassociation in heterologous deoxyribonucleic acid preparations. Thermal elution chromatography on hydroxyapatite was used to separate reassociated nucleotide sequences from nonreassociated sequences and to determine the thermal stability of related nucleotide sequences. An apparent 15% core of relatedness is present between fire blight, soft-rot, and "atypical" Erwinia species. All Erwinia species showed low to moderate reaction with representative enteric bacteria.     

Thermal Stability of Escherichia coli–Salmonella typhimurium Deoxyribonucleic Acid Duplexes

Single-stranded, labeled deoxyribonucleic acid (DNA) fragments from Escherichia coli were incubated at 60 and 66 C with a large excess of single-stranded, unlabeled DNA fragments from E. coli and Salmonella typhimurium. The resulting reassociated DNA was adsorbed to hydroxylapatite and eluted in a series of washes at increasing temperatures. The thermal stability of the reassociated DNA was determined by means of this procedure. Neither the extent of reassociation nor stability of the reassociated E. coli DNA was affected by increasing the incubation temperature from 60 to 66 C. The double-stranded molecules resulting from the reassociation of E. coli DNA with S. typhimurium DNA had a markedly lower thermal stability than reassociated E. coli DNA. More reassociation occurred between E. coli and S. typhimurium at 60 C than at 66 C. In addition, the product of interspecies reassociation occurring at 66 C had a higher thermal stability than that occurring at 60 C. Preliminary results indicate that the decreased thermal stability of the interspecies duplex is in part the result of unpaired bases.     

Quantitation of bovine papilloma viral DNA in viral-induced tumors.

Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching.     

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.     

Formation of the genome of the alligator gar Lepisosteus osseus (Ganoidomorpha) genome

Genome structure of the alligator gar was studied by means of a comparison of reassociation kinetics of short and long DNA fragments, an estimation of hyperchromicity of reassociated repetitive DNA as a function of fragments length, and length estimation of S1-resistant duplexes by gel filtration. It was shown that most of the repeated sequences in the alligator gar DNA are no less than 2000 b.p. long and weakly divergent. Little or no interspersion of unique and short repeated sequences were observed in this genome. No highly divergent repeats were found in the alligator gar genome.     

DNA nucleotide sequence heterogeneity between the Towne and AD169 strains of cytomegalovirus.

The results of reciprocal DNA-DNA reassociation kinetics indicated that although the DNAs of human cytomegalovirus (CMV) strains Towne and AD169 shared approximately 90% of their nucleotide sequences, about 10% heterogeneity did exist. The implication was that, with respect to one another, the DNAs of CMV Towne and CMV AD169 contained unique nucleotide sequences. To obtain more direct evidence, 32P-labeled DNA of one virus strain was reassociated in the presence of excess unlabeled DNA of the heterologous virus strain. Those 32P-labeled DNA sequences remaining single stranded were separated from double-stranded DNA on hydroxyapatite columns and incubated with Southern blots containing XbaI restriction enzyme fragments of the homologous virus DNA. This approach not only enriched for nucleotide sequences unique to each strain of virus, but also provided for the identification of the restriction enzyme fragments in which the unique sequences were contained. The CMV Towne unique sequences were found in XbaI fragments A, C, G, L, N, and Q of CMV Towne DNA. The CMV AD169 unique sequences were found in XbaI fragments A, C, G, and J of CMV AD169 DNA. The possible significance of these data with respect to variation among other CMV isolates is discussed.     

Reassociation kinetics of DNA from x-irradiated ascites hepatoma cells of the rat.

The kinetics of the reassociation fo DNA from ascites hepatoma cells has been studied. The curve exhibited three zones corresponding to 'fast', 'intermediate' and 'slow' speeds of DNA reassociation. The difference was observed in the DNA reassociation curves of the control and irradiated (1500 rad) cells which was particularly expressed in the 'slow' zone (10(2) less than C0t less than 10(4). The same dose, however, does not qualitatively effect the secondary DNA structure, which was estimated by the method of thermal elution from the hydroxyapatite column.     

Homology of single copy and repeated sequences in chicken, duck, Japanese quail, and ostrich DNA.

The extent of reassociation of 3H-labeled repetitive or single copy DNA sequences from the chicken with excess unlabeled DNA from the duck, the Japanese quail, and the ostrich, respectively, was measured by hydroxylapatite chromatography. Chicken repetitive DNA reassociated to an equal or greater extent than chicken single copy DNA with the DNA of each of the other birds. Using an isolated subfraction of chicken repetitive DNA representing those DNA sequences common to the chicken and ostrich genomes, we determined that many repetitive DNA sequences that occur at high repetition frequency in the chicken genome have a much lower repetition frequency in ostrich DNA. The data indicate that there has been a striking change in the number of copies of many repetitive DNA sequences during avian evolution.     

Characterization of the genome of the basidiomycete Schizophyllum commune

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.