DNA of ciliated protozoa: DNA sequence diminution during macronuclear development of oxytricha

We have measured the reassociation kinetics of DNA from the micronucleus and from the macronucleus of the hypotrichous cillate Oxytricha. The micronuclear DNA reassociates with at least a two-component reaction, indicating the presence of both repeated and non-repeated sequences. The kinetic complexity of micronuclear non-repeated DNA is in the range of 2 to 15 × 10¹¹ daltons; the haploid DNA content of the micronucleus is 4 × 10¹¹ daltons (0.66 pg), measured microspectrophotometrically. The DNA of the macronucleus reassociates as a single second-order reaction, with a kinetic complexity of 3.6 × 10¹⁰ daltons. A comparison of the kinetic complexities of micronuclear and macronuclear DNAs suggest a 5 to 30 fold reduction in DNA sequence complexity during the formation of a macronucleus from a micronucleus. Macronuclear DNA is in pleces with an average molecular weight of 2.1 × 10⁶ daltons. Since the kinetic complexity of macronuclear DNA is 3.6 × 10¹⁰ daltons, the macronucleus must contain about 17,000 different kinds of DNA pieces.Each macronucleus contains 3.5 × 10¹³ daltons (58 pg) of DNA, indicating that each sequence must be present about 1000 times per macronucleus or 2000 times per cell.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma

DNA isolated from purified nuclei of Polytoma obtusum has a buoyant density of 1.711 g/ml in CsCl, a Tm of 91.3° C in SSC, and a G + C content of 52.5% as determined by base composition analysis. Thermal dissociation and reassociation studies indicated that this nuclear DNA contains a considerable amount of heterogeneity. Under appropriate reannealing conditions for denatured DNA, about 15% of the DNA reannealed to form a satellite peak at a density of 1.711 g/ml within one hour. Native DNA fractions of different average buoyant densities, ranging from 1.723 to 1.708 g/ml were also obtained in a preparative CsCl gradient, indicating the presence of intermolecular heterogeneity at a molecular size of 8.5×10⁶ daltons. The nuclear DNA reassociated as three distinct classes. The very fast species constituted about 20 % of the total hyperchromicity, the class of intermediate rate comprised roughly 10% of the nuclear DNA, while the remaining 70% consisted of unique sequences. The haploid genome set was estimated by renaturation kinetics studies to contain 5.0×10¹⁰ daltons of DNA or 7.5×10⁷ nucleotide pairs. The analytical complexity of the total nuclear genome was found to be 9.35×10¹⁰ daltons, thus indicating that vegetative cells of P. obtusum are diploid.     

The Kinetic Complexity of Paramecium Macronuclear Deoxyribonucleic Acid

DNA extracted from macronuclei of axenically cultured Paramecium aurelia has been characterized with regard to its kinetic complexity. Renaturation of macronuclear DNA from this protozoon appeared to follow 2nd order kinetics and revealed the presence of 2 components: a main component comprising ～96% of the genome which renatured slowly and a minor component comprising ～4% of the genome which renatured at a rate ～3000 faster than the main component. The value of the kinetic complexity of the main component has been estimated at 3.8 × 10¹⁰ daltons and that of the minor component at 1.45 × 10⁷ daltons. It is suggested that the macronucleus contains ～840 diploid copies of the slowly renaturing component; for each copy of the latter there are ～100 copies of the fast renaturing component.     

Molecular complexity of Paramecium symbiont lambda deoxyribonucleic acid: evidence for the presence of a multicopy genome

DNA was isolated from highly purified symbiont lambda particles of Paramecium aurelia. Renaturation kinetic data revealed a molecular size of 0.71 × 10⁹ daltons. Analytical complexity estimated from chemical data was 7.5 × 10⁹ daltons. These values correspond to about ten copies of the genome per lambda particle. The relationship of symbiont lambda particles to bacteria and cellular organelles, i.e. chloroplasts, mitochondria and kinetosomes, is discussed.     

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.     

Inter- and intraspecific variation of nuclear DNA reassociation kinetics in the Gracilariales (Rhodophyta)

DNA reassociation kinetics were used to determine inter- and intraspecific variation in genome organization and complexities in species ofGracilaria andGracilariopsis. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repeated sequences varied from 13–95%. Three geographic populations ofGracilaria tikvahiae were similar with 13–27% repeated sequences.Gracilaria sp. cultivars G-1 and G-6 with 35% and 95% repeated sequences, respectively, were distinct from each other andG. tikvahiae. No pattern of genome organization and complexity was found which permitted a distinction betweenGracilaria andGracilariopsis. Comparison of the percent of unique and repetitive sequences (U/R) indicated a wide range of ratios, withGracilaria tikvahiae populations having the highest values (2.7–7.3) andGracilaria sp. cultivar G-6,G. blodgettii andGracilariopsis lemanieformis the lowest (0.05–1.80). Unique component complexities varied one order of magnitude, from 10⁸ forGracilaria takvahiae to 10⁷ forGracilaria sp. cultivar G-6,G. blodgettii andGracilariopsis lemanieformis. Information for genome size, organization and complexity is used to develop a nuclear genome profile forGracilaria blodgettii andGracilariopsis lemanieformis which are characterized by commercial grade agars having high gel strengths (> 700 g cm^?2) and elevated melting temperatures (99 °C).     

The Bombyx mori genome: analysis by DNA reassociation kinetics

The size and nucleotide sequence complexity of the Bombyx mori genome has been determined from the kinetics of reassociation of its DNA. Nonrepeated DNA comprises 55% of the genome, and the remainder is divided equally between sequences repeated roughly 500 and 50000 times. Non-repeated sequence DNA virtually free of repeated sequences was prepared by partial reassociation and subsequent fractionation on hydroxyapatite. The nucleotide sequence complexity of this component was determined relative to DNA from B. subtilis and E. coli. After correction for the size of the repeated sequence fraction, the DNA content of the Bombyx mori genome was calculated to be 0.53±0.02×10^?12 g. This value compares favorably with the DNA content of haploid B. mori spermatids and mature sperm determined cytophotometrically by Rasch (1973).     

Analysis of DNA of male and femaleSphaerocarpos donnellii Aust. (Liverwort)

Summary The karyotypes of females and males ofSphaerocarpos donnellii differ in that there is a large essentially heterochromatic X chromosome in the female (approx. 25 volume-% of the autosomes) and a small Y chromosome in the male (0.1–3 volume-% of the autosomes). DNA from females and males differ in buoyant densities in cesium chloride equilibrium gradients (1.702₅ and 1.703₅g cm^-3, respectively) and in melting points (87.5 and 88.5°C in SSC). The differences are statistically significant. Base analyses revealed 2.5 Mol-% of the rare base 5-methyl cytosine. Upon reassociationSphaerocarpos DNA behaves kinetically in a heterogeneous manner. About 22% of the DNA is repetitive with an average kinetic complexity of 1.6×10⁸ daltons. The kinetic complexity of the slow reassociating DNA fraction, considered to be of the single copy type, is 3.2×10¹⁰ daltons. No difference in the renaturation behavior between DNA of males and females could be detected with the techniques used. Our data thus indicate that X chromosomal DNA cannot contain large amounts of highly repeated nucleotide sequences and that it is slightly enriched in AT content compared to the autosomes.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. IV. The F sequences in F14

The structure of F14, in particular the arrangement of the F sequences on this plasmid, has been studied by the electron microscope heteroduplex method. F14 has a molecular size of 311 ± 10 kilobase pairs (M = (206 ± 8) × 10⁶daltons). It contains all of F (94.5 kilobases). A sequence of length 5.7 kilobases, which occurs once in F (with co-ordinates 2.8 to 8.5F), is directly repeated in F14. It occurs at the two junctions of F DNA with chromosomal DNA. Thus, F14 contains about 211 ± 10 kilobases of chromosomal DNA. A previously unidentified direct repeat has also been discovered on F itself; the sequence with co-ordinates 93.2 to 94.5F is directly repeated at 13.7 to 15.0F. Physical observations indicate that the population of closed circular plasmid molecules extracted from F14-containing strains is heterogeneous. In addition to F14 itself, molecules the size of F and 2.3 times the size of F were found. The latter molecules contain all the chromosomal sequences of F14 and one copy of the 2.8 to 8.5F segment. Such heterogeneity was observed in both recA^? and recA⁺ backgrounds. It is proposed that this heterogeneity is due to intramolecular recombination events occurring within F14 between the duplicated 2.8 to 8.5F sequences. Such recombination can account for the previously observed genetic instability of F14. Another F prime plasmid, F186, independently isolated from the Hfr parent of AB313, was found to be identical to F14.     

Characterization of Allomyces genome

Allomyces arbuscula DNA isolated from whole cells (bulk DNA) is composed of a major (alpha) and two minor components (beta & gamma) with buoyant densities in neutral CsCl corresponding to 1.721, 1.710 and 1.702 g/cm3, respectively. The DNA obtained from purified nuclei contains alpha component only. The beta component corresponds to mitochondrial DNA. The gamma component is also extra-nuclear but has not been characterized. The reassociation kinetics of sheared, bulk and nuclear DNA show that (i) 25 % bulk and 10% of nuclear DNA reanneal very rapidly and contain highly repeated sequences; (ii) moderately repeated sequences, accounting for 15% of both bulk and nuclear DNA, have a sequence complexity of approximately 7.2-10(6) daltons and are repeated about 320 times; (iii) the slow reannealing fraction accounts for about 60% of the genome and has kinetic properties similar to single copy sequences. The sequence complexity of this fraction was determined in relation to that of Escherichia coli. After a correction for the size of the repeated sequences the genome size of A. arbuscula was calculated to be 1.7-10(10) daltons.     

Genome size and complexity of the obligate fungal pathogen,Bremia lactucae

The size and organization of the genome of Bremia lactucae, a highly specialized fungal pathogen of lettuce, has been characterized using dot blot genomic reconstructions, reverse genomic blots, and genomic DNA reassociation kinetics. The haploid genome contains 5 × 10⁷ bp of DNA and 65% of the nuclear DNA is repeated. Low copy sequences are interspersed with repeated sequences in a short-period interspersion pattern. This pattern of genome organization is different to that described for other fungi. Although most fungi have been shown to contain some form of repetitive DNA other than the ribosomal repeat, the high percentage of repetitive DNA and the interspersion of low copy and repeated sequences are atypical of fungi characterized previously.     

Herpes Simplex Virus: Genome Size and Redundancy Studied by Renaturation Kinetics

Herpes simplex virus subtype 1 deoxyribonucleic acid (DNA) was sheared in a French press to uniform fragments, denatured by heating, then allowed to reassociate. The renaturation reaction followed second-order kinetics with a single rate constant indicating that at least 95% of the genome was unique and that repetitive sequences, if present, were not detectable by this technique. The kinetic complexity of the herpes simplex genome was determined by DNA renaturation kinetics to be (95 ± 1) × 10⁶ daltons. Since this value is in excellent agreement with the molecular weight of viral DNA [(99 ± 5) × 10⁶ daltons] obtained from velocity sedimentation studies, it is concluded that virions contain only one species of double-stranded DNA molecules 95 × 10⁶ to 99 × 10⁶ daltons in molecular weight.     

Structural organization of the genome of Dictyostelium discoideum: Analysis by EcoR1 restriction endonuclease

The genome of the cellular slime mold Dictyostelium discoideum has been analyzed by limit digestion with EcoR1 restriction endonuclease. Approximately 15% of the nuclear genome is cleaved into nine discrete fragments as analyzed by agarose gel electrophoresis. These fragments appear to be derived from two nuclear buoyant density satellites, one of which contains sequences coding for ribosomal RNA. The bulk of the nuclear DNA is digested into approximately 7000 fragments with a mean molecular weight of 4 × 10⁶ to 5 × 10⁶. The mitochondrial DNA is digested into four fragments. One of the nuclear bands has been cloned in Escherichia coli using plasmid pSC101 carrying tetracyline resistance. Analysis by renaturation kinetics indicates that it is repeated approximately 200 times per haploid genome and that it is not internally repeated.     

Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes.

The DNA content of the nucleus of the yeast Saccharomyces cerevisiae has been determined by both renaturation kinetics and DNA per cell measurements. Renaturation kinetics experiments were performed by following the decrease of optical hyperchromicity at 260 nm and by hydroxyapatite chromatography. DNA per cell measurements were made by the diaminobenzoic acid method and by the ethidium bromide method of Klotz &; Zimm (1972b). The conclusion from the above experiments is that the S. cerevisiae nucleus contains 9 × 10⁹ ± 2 × 10⁹ daltons of DNA. Previously we (Lauer &; Klotz, 1975) had measured the molecular weight of the largest piece of DNA in the yeast nucleus to be 2 × 10⁹ ± 0.2 × 10⁹. Here we extend this work by using a more highly protein-denaturing buffer system and conclude that the largest piece of DNA in the S. cerevisiae nucleus contains 1.5 × 10⁹ to 2.2 × 10⁹ daltons of DNA in both haploid and diploid cell lysates. From genetics, the largest yeast chromosome should contain 13% of the genome, or 0.9 × 10⁹ to 1.5 × 10⁹ daltons of DNA (using our DNA per cell range). Thus, the large DNA we measure contains from one to two times the amount of the DNA predicted from genetics to be in the largest chromosome. In light of these new data, viscoelastic measurements on yeast DNA are now consistent with the idea that each chromosome contains one piece of DNA.     

Characterization of the DNA of Hyalophora cecropia

DNA was isolated from the pupae and various tissues of pharate adults of the silkmoth Hyalophora cecropia. CsCl equilibrium density analysis showed the presence of one major band at 1.693 and two minor satellites, 1.705 and 1.709, comprising 3 and 6% respectively of the total DNA. We could detect no difference in the renaturation kinetics of DNA prepared from pupal or pharate adult tissues. The genome appears to be composed of 30% redundant and 70% unique sequences. The haploid information content of the unique sequences is 11.4 × 10¹⁰ daltons.     

Differentiation between strains ofFlavobacterium breve and allied bacteria by comparisons of deoxyribonucleic acids

Chromosomal deoxyribonucleic acid was isolated and purified from 10 strains ofFlavobacterium breve, originating from human or other animal sources. The mean and standard deviation for the species in base content was 32.4±0.6% G+C, and in genome size was 3.21±0.37×10⁹ daltons. In vitro DNA reassociation showed that sevenF. breve strains (mainly from human sources) had high levels of intraspecific base sequence similarity (>70%) as derived from reassociations done at the optimum temperature of reassociation (TOR) or TOR—10°C (nonstringent conditions). The three otherF. breve strains contained a high degree of base sequence divergence. All 10 strains ofF. breve were readily distinguishable in their DNA characteristics fromF. meningosepticum, F. odoratum, and allied Gram-negative bacteria.     

Estimation of the genome size of blue-green algae from DNA renaturation rates

The molecular weight of the genomes of the blue-green algaeAnacystis nidulans andAnabaena cylindrica have been estimated as 2.27×10⁹ and 2.47×10⁹ daltons respectively from the renaturation kinetics of DNA. Thus the genomes of these organisms are similar in size to that ofEscherichia coli K-12, (2.40×10⁹ daltons) measured by the same technique. No evidence was obtained of repeated sequences in the DNA of the two blue-green algae.