Retroelements contribute to the excess low-copy-number DNA in pine

Excess DNA in the single-copy component is rarely recognized as a contributor to the C-value paradox yet the single-copy component of the pine genome is reported to comprise over 3000 Mb of DNA, in large excess over the estimated 100 Mb required for gene expression. Two hypotheses regarding the factors that might contribute to the excess low-copy-number DNA were tested. The first hypothesis proposes that the excess low-copy kinetic component is actually overestimated by reassociation data analysis. To test this, a previously published C0t curve for Pinus strobus was reanalyzed using a new estimate of genome size based on laser flow cytometry. Part of the excess low-copy-number DNA in the pine genome could be attributed to the choice of parameters used in the analysis of the reassociation data. The second hypothesis holds that diverged retrotransposons contribute to the excess low-copy DNA. Sequences randomly sampled from single-copy and low-repetitive kinetic components of the P. taeda genome were characterized. Twelve of 46 fragments cloned from these fractions were found to show sequence similarity to retroelements: hence diverged retroelements contribute to the excess low-repetitive kinetic component in the pine genome. Similarity search was shown to be a conservative method for identifying retroelements, and thus the number of retroelements in the low-copy component was actually underestimated. Most of the retroelements in this fraction were nonfunctional. divergent from known retroelement families and previously reported only for flowering plants. Divergent retrotransposons are thus a major factor contributing to the expansion of the low-repetitive DNA component in higher plants.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.     

Sequence heterogeneity in kinetoplast DNA: Reassociation kinetics

Melting and reannealing of purified kinetoplast DNA (kDNA) from Crithidia fasciculata, Trypanosoma mega, and T. brucei have been studied with an automated optical system. The slow reassociation rate of trypanosome kDNA is due neither to the formation of hyperpolymers nor to mispairing of bases and certainly reflects extensive sequence heterogeneity. Simulation of the reassociation kinetics indicates that the kDNA comprises essentially two kinetic components: a fast renaturing component which might be a common sequence present in all the minicircles and a slow renaturing component which is responsible for minicircle heterogeneity. The rapidly renaturing component is more abundant in Crithidia than in trypanosomes.     

Hydrogen peroxide lethality is associated with a decreased ability to maintain positive DNA supercoiling

Hydrogen peroxide is more toxic to mammalian cells at 37 degrees C than 0 degree C at all concentrations studied. Histone-free nuclei (nucleoids) extracted from treated cells have a reduced ability to maintain positive DNA supercoiling, with the maximum effect at the higher temperature. Prior exposure of cells to sodium ascorbate at 0 degree C increased both toxicity and the inhibition of nuclear supercoil rewinding. After exposure at 0 degrees C, normal levels of supercoiling returned with both a fast and a slow component, kinetics characteristic of DNA single-strand break repair; the fast component was eliminated when cells were exposed at 37 degrees C due to in situ rejoining. At least a portion of the lethal lesions induced by hydrogen peroxide are DNA double-strand breaks (dsb) because the dsb repair-deficient mutant, xrs-5, is approximately two to three times more sensitive than wild-type cells over the initial portion of the survival curve. However, the increased toxicity found after exposure at 37 degrees C is observed equally in both cell lines, indicating that temperature-dependent cell killing is not directly linked to DNA dsb. It is suggested that cell killing at 37 degrees C is mediated through two linked processes. First, hydrogen peroxide may disrupt cation-stabilized nuclear supercoiling by direct ion oxidation. Second, as a part of the oxidation process, hydrogen peroxide will produce potentially cytotoxic free radicals close to the DNA-linked metal site, limited in extent only by the presence of chemicals capable of reducing metal ions prior to reoxidation.     

Tension relaxation after stretch in resting mammalian muscle fibers: stretch activation at physiological temperatures.

Tension responses to ramp stretches of 1-3% Lo (fiber length) in amplitude were examined in resting muscle fibers of the rat at temperatures ranging from 10 degrees C to 36 degrees C. Experiments were done using bundles of approximately 10 intact fibers isolated from the extensor digitorum longus (a fast muscle) and the soleus (a slow muscle). At low temperatures (below approximately 20 degrees C), the tension response consisted of an initial rise to a peak during the ramp followed by a complex tension decay to a plateau level; the tension decay occurred at approximately constant sarcomere length. The tension decay after a standard stretch at approximately 3-4.Lo/s contained a fast, an intermediate, and a (small amplitude) slow component, which at 10 degrees C (sarcomere length approximately 2.5 microns) were approximately 2000.s-1, approximately 150.s-1, and approximately 25.s-1 for fast fibers and approximately 2000.s-1, approximately 70.s-1 and approximately 8.s-1 for slow fibers, respectively. The fast component may represent the decay of interfilamentary viscous resistance, and the intermediate component may be due to viscoelasticity in the gap (titin, connectin) filament. The two- to threefold fast-slow muscle difference in the rate of passive tension relaxation (in the intermediate and the slow components) compares with previously reported differences in the speed of their active contractions; this suggests that "passive viscoelasticity" is appropriately matched to contraction speed in different muscle fiber types. At approximately 35 degrees C, the fast and intermediate components of tension relaxation were followed by a delayed tension rise at approximately 10.s-1 (fast fibers) and 2.5.s-1 (slow fibers); the delayed tension rise was accompanied by sarcomere shortening. BDM (5-10 mM) reduced the active twitch and tetanic tension responses and the delayed tension rise at 35 degrees C; the results indicate stretch sensitive activation in mammalian sarcomeres at physiological temperatures.     

Induction of cellular DNA synthesis in G0-specific ts mutant, tsJT60, following Infection with SV40 and adenoviruses

tsJT60 cells, a temperature-sensitive G0 mutant of a Fischer rat cell line, grew normally in an exponential growth phase at both permissive (34 degrees C) and nonpermissive (39.5 degrees C) temperatures, but when stimulated with fetal bovine serum in the growth-arrested state (G0 phase) they entered S phase at 34 degrees C but not at 39.5 degrees C. Infection of G0-arrested tsJT60 cells with SV40, adenovirus (Ad) 5 wild type and its E1B mutant dl313, and Ad12 wild type and its E1B mutants in205B, in205C, dl205, and in206B induced DNA synthesis at both temperatures. The DNA synthesized after virus infection was shown to be cellular by Hirt separation of DNA from SV40-infected cells and by CsCl equilibrium density gradient centrifugation of DNA from Ad5-infected cells.     

Nature of the heterogeneity in mispairing of reannealed middle-repetitive fern DNA

The genome of the homosporous fern Thelypteris normalis contains a large middle-repetitive component, essentially a single second-order kinetic class, which exhibits heterogeneity in the precision of pairing of the reformed duplexes upon remelting. There are two possible models to explain this observed sequence heterogeneity. Either different families of the middle-repetitive class exhibit different degrees of sequence divergence and T_m reduction (inter familial heterogeneity), or else all are equally diverged, and the broad melt is the sum of thousands of equally broad melts for all the families (intra familial heterogeneity). To differentiate between these two hypotheses, iodinated Thelypteris DNA, reannealed through middle-repetitive C₀t, was thermally fractionated on hydroxyapatite into low (65–75° C), medium (75–85° C), and high (85–95° C) thermal stability classes. When reannealed with excess cold DNA, each class remelted over its characteristic temperature range. C₀t curves of these thermal fractions reannealed with cold driver demonstrated that all were from the middle-repetitive class. It was shown that these results were not due to G + C differences nor to artifacts of the labeling technique. Therefore it was concluded that, although all families consisted of approximately the same number of repeats, the families ranged from those with virtually no sequence divergence to those barely able to reanneal at the criterion used, in accordance with the model of inter familial heterogeneity. Though this model may have wide applicability to middle-repetitive DNAs, a different pattern appears to prevail in Selaginella, a heterosporous pteridophyte. Some evolutionary implications are discussed.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting.

We have investigated the kinetics of dissociation of actinomycin D from DNA by a variation of the footprinting technique. Complexes of actinomycin with a radiolabelled DNA fragment (tyrT) were dissociated by addition of a large excess of unlabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37 degrees C, 20 degrees C and 4 degrees C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20 degrees C. In contrast, the dissociation of distamycin was too fast to measure (< 5 s) even at 4 degrees C.     

Characteristics of DNA in vegetative specimens of Bursaria truncatella]

DNA from Bursaria truncatella was isolated and purified by conventional methods. The DNA base content was calculated from both centrifugation in CsCl and melting. The GC-content is 24%. In CsCl density gradient 3H-DNA is banded as a single peak at a range 1.682--1.688 g/cm3 with the maximum at 1.684 g/cm3. The Tm in 0.12 M FB (pH 6.8) was 79 degrees C. About 50% of DNA seems to be represented by highly repetitive sequences, another 50% being made of single-copy sequences (a preliminary data on DNA-DNA reassotiation kinetics). For the estimation of the molecular weight of DNA, the cells were lysed immediately before the centrifugation, at the surface of the alkaline isokinetic sucrose gradient solution. Two components of DNA were detected with molecular weights 10-10(6) and 100-10(6) daltons. It is likely that these two components belong to the macronuclear DNA, because according to cytophotometrical evidence the DNA content in the macronucleus of B. truncatella is 2500 times as much as that in its micronucleus.     

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.     

Cat satellite DNA. Isolation using netropsin with CsCl gradients

The absence of centromeric bands in the karyotype of Felis catus is confirmed. It is also confirmed that no satellite band is visible in CsCl density gradients. However, a satellite is observed both by recentrifuging the fraction of the DNA that bands at high density in CsCl and by using netropsin to enhance the resolution of a CsCl gradient containing total F. catus DNA. The satellite, about 0.5% of total DNA, was isolated by repeated centrifugation in CsCl alone and in CsCl with netropsin. Netropsin was removed and a pure satellite DNA obtained. The reassociation kinetics (C0t1/2 less than 10(-3) M . s) show that the satellite is of the simple sequence type and hence a candidate for centromeric heterochromatin. Its cytological localisation awaits in situ hybridisation experiments.     

The kinetic properties of cruciform extrusion are determined by DNA base-sequence.

The extrusion kinetics of two cruciforms derived from unrelated DNA sequences differ markedly. Kinetic barriers exist for both reactions, necessitating elevated temperatures before extrusion proceeds at measureable speeds, but the dependence upon temperature and ionic strength is quite different for the two sequences. One, the ColE1 inverted repeat, exhibits a remarkably great temperature dependence of reaction rate and is suppressed by moderate amounts of NaCl or MgCl2. In contrast, the other, a synthetic inverted repeat present in pIRbke8, shows more modest temperature dependence and has a requirement for the presence of salt, with optimal concentrations being 50 mM NaCl or 100 microM MgCl2. Under optimal conditions, cruciform extrusion rates are fast (t1/2 less than 60m) at 37 degrees C for both sequences at native superhelix densities. In 50 mM NaCl the pIRbke8 inverted repeat is characterised by an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole -1. The differences in kinetic properties between the two sequences indicate that DNA base sequence is itself an important factor in determining cruciform kinetics, and possibly even in the selection of the mechanistic pathway.     

Evolutionary change in the repetition frequency of sea urchin DNA sequences

The frequency of occurrence of particular repetitive sequence families has been estimated in the DNA of the three sea urchin species Strongylocentrotus purpuratus, Strongylocentrotus franciscanus and Lytechinus pictus using individual cloned S. purpuratus repetitive sequence elements. Cloned repetitive sequence elements as described by Scheller et al. (1977a) were prepared by reassociation of S. purpuratus DNA fragments to repetitive Cot, digestion with single-strand-specific nuclease S1 and ligation of synthetic restriction sites to their ends. The sequences were cloned by insertion at the Eco RI site of plasmid RSF2124, labeled, strand-separated and reassociated with 800–900 nucleotide long unlabeled DNA. Both kinetic (genomic DNA excess) and saturation (cloned DNA excess) estimates of frequencies were made. For nine cloned fragments, the ratio of the repetition frequency in S. purpuratus DNA to that in S. franciscanus DNA ranges from about 20 to about 1. In the four cases examined, only a few copies were detected in the DNA of L. pictus. Estimates have also been made of frequency changes in many repetitive families by measuring the reassociation of labeled repetitive DNA fractions of each species with total DNA from other species. In each reciprocal comparison, the labeled repetitive sequences reassociate more slowly with DNA of other species than with DNA of the species from which they were prepared. Thus it appears that the dominant repetitive sequence families in the DNA of each species are present at lower frequencies in the DNA of closely related species. Measurements of thermal stability have been made of S. purpuratus cloned repetitive sequences reassociated with S. franciscanus DNA or S. purpuratus DNA. Most families have changed both in frequency and sequence, although some have changed little in sequence but show great changes in frequency.     

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families

We have examined high affinity interactions of chick brain microtubule proteins with ³⁵S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1–4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65–75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. ³⁵S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM^–1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three ³⁵S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720–1.740 g/cm³; 1.750–1.765 g/cm³). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.     

The DNAs of the A and B chromosomes of the mealy bug,Pseudococcus obscurus

When the DNA of mealy bugs carrying B chromosomes (+ B:DNA) was compared to the DNA of individuals not possessing Bs (-B:DNA), no significant differences were found using isopycnic centrifugations in CsCl or thermal denaturation analyses. Both DNAs had buoyant densities of 1.693 g/cm³ in neutral CsCl gradients and 1.748 g/cm³ in alkaline CsCl gradients. Satellite DNAs were not detected. The average T_m of +B:DNA was 67.9° C in 0.1 SSC while -B:DNA had an average T_m of 67.4° C in the same solution. However, in situ molecular hybridizations with complementary RNAs (cRNAs) transcribed in vitro from each type of DNA showed considerable differences with regard to the amount of labeling of B chromosomes. Using cRNA to +B:DNA, the average number of silver grains over a B chromosome was 2.1 × the average number of silver grains over individual non-B chromosomes (A chromosomes). In contrast, the ratio (B/A) using cRNA to -B:DNA was less than 0.14. The results are interpreted as meaning that very little DNA is shared in common by both A and B chromosomes.     

Structural characteristics (reassociation and melting kinetics) of kinetoplast DNA from Crithidia oncopelti

A highly purified associate of kinetoplast DNA is isolated from C. oncopelti, and its physico-chemical properties are studied. Both native associate and its ultrasonic fragments are found to have a complex character of melting. 5-6 melting zones (3 of them being the main) are found on the melting curve. Analysis of reassociation kinetics of sonicated associate of kinetoplast DNA has revealed the presence of at least two components: fast reassociating component (65-70% of complex DNA), which reassociation kinetics is equivalent to the unique sequence with molecular weight of 2.3. - 10(6) daltons, and slow reassotiating component (15% of complex DNA), having reassociation kinetics equivalent to unique sequence of 26 - 10(6) daltons. The data obtained suggest that complex associate of kinetoplast DNA is heterogenous for its nucleotide sequence and base composition.     

Temperature dependence of kinetic parameters of single potential dependent K+ channels in mollusc neurons. Similarity in the action of potential and temperature]

Using the patch-voltage-clamp method on excised membrane fragments from molluscan neurones temperature dependences of kinetic parameters of the fast and slow K(+)-channels were investigated in the temperature range 1 to 40 degrees C. Temperature dependences of probability of the channel open state (P0) for the slow and fast K(+)-channels are, generally, opposite, that is P0 increases for the slow channel and decreases for the fast channel with temperature. Similar dependences characterize durations of single channel open intervals (tau 0) and burst durations (t(p)). Durations of interburst and interpulse intervals (respectively, t(i) and tau) decrease for the slow channel and increase, in contrast, for the fast channel with temperature. For the channels of both types temperature dependences of P0 (as for other parameters) are essentially nonmonotonous. There are two local extrema, at least: for the slow K(+)-channel-maximum at 15 degrees C (minimum for the fast channel) and minimum at 20-25 degrees C (maximum for the fast channel). In some cases the number of local extrema may be greater than two. Some similarity in the action of temperature and membrane potential on the kinetic parameters was observed. For the slow K(+)-channel P0, tau 0 and t p increase with temperature and membrane potential. For the fast channel these parameters decrease at the same conditions. Moreover, for the channels of both types temperature dependences of the kinetic parameters are slightly pronounced at the potentials where potential dependences of the parameters are least. As a whole, temperature measurements showed that there are, possibly, several points of structural transitions (similar to phase transitions) in the temperature range 0 to 40 degrees C. Primarily, the kinetic parameters are determined by these transitions.