Kinetic complexity of chloroplastal deoxyribonucleic acid and mitochondrial deoxyribonucleic acid from higher plants

1. Chloroplasts and mitochondria were isolated by aqueous and non-aqueous cell-fractionation techniques. In a variety of higher plants the mitochondrial DNA bands in a caesium chloride gradient at 1.706g.cm.(-3), whereas chloroplastal DNA has a buoyant density of 1.697g.cm.(-3). 2. In total cellular DNA of moderate molecular weight, the chloroplastal DNA is found within the Gaussian distribution of the nuclear DNA and is not resolved as a satellite. 3. Both chloroplastal DNA and mitochondrial DNA from lettuce renature rapidly. 4. The kinetic complexity of mitochondrial DNA is > 10(8) daltons. 5. Chloroplastal DNA is made up from fast and slow renaturing sequences with kinetic complexities of 3x10(6) and 1.2x10(8) daltons respectively. 6. From the discrepancy between analytical and kinetic complexity it is concluded that chloroplastal DNA is extensively reiterated.     

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.     

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.     

Isolation and characterization of mitochondrial deoxyribonucleic acid of Acanthamoeba castellanii

DNA was prepared from isolated mitochondria of Acanthamoeba castellanii and was shown to behave as a single component in density gradients, on ;melting' and on renaturation. From measurements of renaturation kinetics, sedimentation coefficient and electron micrographs the genome size of the mitochondrial DNA was calculated to be about 3.4x10(7) daltons. A small proportion of the preparations could be isolated as relaxed circular molecules of mean contour length 16.2mum.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

Physico-chemical determination of the ploidy of the unicellular alga, Chlorella pyrenoidosa (strain 211/8b) (author's transl)]

The ploidy of the unicellular green alga Chlorella pyrenoidosa (strain 211/8b) has been determined by means of renaturation kinetics. The nuclear DNA is made up from fast, intermediate and slow renaturing sequences, which represent respectively about 5, 15 and 80% of the DNA. These observations are consistent with the findings in other eukaryotic nuclear DNAs. Nevertheless, the relative importance of the repeated sequences is much lower than that observed in Chlamydomonas reinhardi [16] and in higher plants [18-20], but slightly higher than that obtained in Chlorella vulgaris [17]. The kinetic complexity of the main fraction of the Cl. pyrenoidosa nuclear DNA is found to be 2.94 - 10-10 daltons (mean value of five independant experiments) assuming value of 2.1 - 10-8 daltons for Cl. pyrenoidosa chloroplastic DNA. When compared with the analytical complexity of this fraction (80% of the nuclear DNA analytical complexity, that is 2.02 - 10-10 daltons), one can assume that the slow renaturing fraction of the nuclear DNA is constituted by a unique nucleotide sequence. This result thus suggests that Cl. pyrenoidosa (strain 211/8b) is an haploid organism. The possible existence of an haploid genome in the nuclei of the algae from Chlorella genus and the apparent absence of sexuality might explain the high discrepancy observed in the G + C content of the Chlorella nuclear DNAs.     

Molecular structure of adeno-associated virus variant DNA

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.     

Two repeated DNA sequences from the heterochromatic regions of rye (Secale cereale) chromosomes

Rye DNA sequences renaturing with a C₀t <0.02 mol·sec/l, are largely undigested by the restriction enzyme HindIII. These HindIII-spared sequences are mostly located in telomeric heterochromatin. When digested with EcoRI* and cloned into the EcoRI site of pBR 325, these sequences yielded clones of two classes when hybridized to a probe of rapidly renaturing DNA. One class contains a DNA sequence which is a major constituent of the telomeric heterochromatic blocks, while the other is a minor component of the highly repeated DNA of the genome. The major component was sequenced, its chromosomal distribution mapped using wheat-rye addition lines and its distribution in meiotic prophase nuclei determined. The minor component is present in significant amounts in wheat as well as in rye and is localized at the terminal heterochromatic regions of three rye chromosomes but not in the major blocks of heterochromatin.     

Properties of satellite DNA of Phaseolus vulgaris]

Nuclear DNA components of Ph. vulgaris were preparatively separated by equilibrium ultracentrifugation in Hg++-Cs2SO4 density gradient (buoyant density of the major component in CsCl density gradient 1.694 g/cm3., satellite component--1.703 g/cm3). The properties of individual DNA fractions were investigated. The melting curve of satellite DNA of Ph. vulgaris has biphasic character. The observed heterogeneity of satellite DNA component is of intermolecular nature. This is illustrated by the splitting of unsheared satellite DNA into two components during renaturation, as well as by its behaviour in Hg++-Cs2SO4 density gradient at high rf value. The width of satellite DNA reassociation curve covers three decades of Cot. The length of the major repeating sequences of the satellite component is close to the length of phage T2 DNA. During chromatography on MAK column satellite DNA elutes earlier than the major component due to its higher GC-content. It is suggested that one of the satellite DNA fractions of Ph. vulgaris contains rRNA genes.     

The DNA of Acanthamoeba spp.; A Method for Extraction and Its Characterization

SYNOPSIS. A method is described which permits the recovery of a high yield of undegraded DNA from Acanthamoeba castellanii. The DNA of A. castellanii is comprised of 2 species: the buoyant density of the major component is 1.720 and of the minor component, 1.693. The renaturation characteristics of the minor component suggest that it is of mitochondrial origin. Two components are also present in the DNA of A. palestinensis, A. polyphaga and A. astronyxis. The buoyant densities of major and minor components differ between species.     

Nuclear and plastid DNAs from the binucleate dinoflagellates Glenodinium (Peridinium) foliaceum and Peridinium balticum

The binucleate dinoflagellates Glenodinium (Peridinium) foliaceum Stein and Peridinium balticum (Levander) Lemmermann were found to contain two major buoyant density classes of DNA. The heavier peak (1.730 g/cm3) was derived from the "dinokaryotic" nucleus and the lighter peak (1.706 g/cm3) from the "endosymbiont" nucleus and this allowed for the fractionation of G. foliaceum DNA in CsCl/EtBr density gradients. An initial CsCl/Hoechst Dye gradient removed a minor A-T rich satellite species which was identified as plastid DNA with a size of about 100-106 kb. Analysis of the nuclear DNA by agarose gel electrophoresis and renaturation studies showed that the endosymbiont nucleus lacked amplified gene-sized DNA molecules, however, this nucleus did have a comparatively high level of DNA. The total amount of DNA per cell and the relative contributions of the two nuclei appeared to vary between two strains of G. foliaceum (75 pg/cell in CCAP strain and 58 pg in UTEX strain). The only strain of P. balticum examined contained 73 pg cell. These results are discussed in relation to the status and possible functioning of the endosymbiont nucleus and the idea that these dinoflagellates provide model systems with which to study the evolution of plastids.     

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.     

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.     

Characterization of DNA components from some colorless algae

The DNA components of five colorless algae were characterized by their buoyant densities in cesium chloride. Two DNA components were detected in Polytoma obtusum and Polytoma uvella. Upon renaturation of the thermally denatured DNA the minor and approx. 15% of the major DNA component returned to their native densities. The buoyant densities of the major and minor DNA of P. obtusum and P. uvella are different from that of the morphologically and biochemically similar green alga Chlamydomonas reinhardtii. A major and a minor DNA component with the same buoyant densities as that of the green alga Euglena were also found in Astasia longa, which is morphologically similar to Euglena. The renaturation of the minor but not the major component was readily detectable by the change in buoyant density. Only one DNA component was detected in Polytomella agilis and Polytomella caeca. After thermal denaturation approx. 5% of each of these DNA components were renatured readily. Based on these data, the possible evolutionary origin of these colorless algae is discussed.     

The chloroplastic DNA of Chlorella pyrenoidosa (Emerson Strain): Heterogeneity and complexity

The heterogeneity and the complexity of Emerson strain Chlorella pyrenoidosa chloroplastic DNA have been investigated by means of thermal denaturation and renaturation kinetics, and the results have been compared with those of the strain 211/8b of the same alga. The thermal denaturation properties are very close to those of the other strain: the Tm of 65 degrees C in 0.1 standard saline citrate, the maximal hyperchromicity of 41%, and the dispersion coefficent delta 2/3 of 6.65 degrees C. The first derivated curves of the melting profiles show also five components. Denatured chloroplastic DNA renatures rapidly. Two fractions are found; their kinetic complexities have been estimated: 1.5 times 10(7) daltons for the fast renaturing fraction; 2x 10(8) daltons for the low d (G + C) content of the chloroplastic DNA: 1.24 times 10(8) daltons). The unique nucleotide sequence is present in about 19 copies per chloroplastic genome. This report confirms the homogeneity of the chloroplastic genome of algae.     

The complexity of satellite deoxyribonucleic acid in a higher plant.

Purified satellite DNA from melon (Cucumis melo) was shown to contain at least two components from thermal-denaturation and renaturation studies. Two components were separated after partial renaturation, a fast-renaturing fraction similar in complexity to mouse satellite DNA, and one with 6000 times greater complexity. Both components renatured very accurately, indicating a minimum of sequence divergence. Centrifugation of the purified satellite DNA in Ag+/Cs2SO4 gradients resolved two major and several minor fractions. The two major fractions were only slightly enriched for fast- or slow-renaturing sequences.     

Molecular aspects of chromatin elimination in Ascaris lumbricoides

DNA from spermatids, 4-cell stages, and larvae of Ascaris lumbricoides was isolated, and the genome size before and after chromatin elimination was determined by isotope dilution. According to these determinations, 27% of the DNA is lost during the process of chromatin elimination. This value is based on the assumption that larval nuclei are diploid. The genomes were then characterized by CsCl equilibrium density gradient centrifugation and renaturation kinetics. The eliminated DNA does not differ from the retained DNA in base composition. About 26% of the DNA of 4-cell stage embryos sediments as a light satellite and was shown to be mitochondrial DNA by electron microscopy. Renaturation kinetics revealed that 10% of the retained somatic DNA is repetitious with an average family size of 5500 to 7000 copies, whereas 90% of the retained DNA is presumably composed of unique sequences. By contrast, germ-line DNA contains 23% fast renaturing DNA with a family size of 7000 to 10,000 copies. Thus, eliminated DNA consists of repetitious and unique sequences in a ratio of about 1:1.     

Studies on the involvement of lysosomes in estrogen action, II. Seasonal variation in the sedimentation patterns of endometrial lysosomes from prepuberal pigs

The lysosomal population in endometrial cells of prepuberal pigs varies with the seasons. In summertime, lysosomes equilibrate in sucrose density gradients over a broad, domeshaped area from p = 1.21 g/cm3 to p = 1.15 g/cm3. The winter pattern is characterized by a major sharp peak of aggregated lysosomes at p = 1.20 g/cm3, visible as a "snowflurry"-like band. The two extremes are linked by transition patterns in spring and fall. The possible correlation of this phenomenon to the seasonal fluctuations in the level of soluble cytoplasmic estrogen receptor is discussed.     

Heterogeneity of the kinetoplast DNA molecules of Trypanosoma cruzi.

Kinetoplast DNA (kDNA) of the culture form of Trypanosoma cruzi is cleaved by restriction endonucleases (HpaII, HindII, EcoRI, and HaeIII). The analysis of the cleavage patterns proves that the minicircles (free circulargenome units) are heterogeneous in base sequences. The same results are obtained with the complex kDNA network which is composed of the association of minicircles and linear molecules. Kinetic studies of the renaturation of kDNA previously cleaved by HpaII into fragments of the genome unit size show at least two populations of molecules. About 75% of these molecules correspond to the fast renaturing population having the molecular complexity of the minicircles. The molecules of the slow renaturing population have a much higher molecular complexity than the minicircles and do not seem to be related to the majority of the long linear molecules.