The B-chromosome system of Allium schoenoprasum

Nine morphologically distinct euchromatic B-chromosomes have been identified in Allium schoenoprasum from the River Wye, South Wales. The most common type (89%) is telocentric (B^t–1) and it is likely that the non-standard Bs are derivatives of B^t–1 by deletion, centric shift and/or centric misdivision. New B-types have also been produced from standard Bs in controlled crosses. In general, the Bs are mitotically extremely stable, although occasional plants, particularly those carrying non-standard Bs, are conspicuously variable in their B-constitution between root-tip cells. In addition, B-chromosome number is enhanced in some anthers of about one third of plants. Behaviour of B-chromosomes during meiosis is described. Although there is little bivalent formation, less than 5% of the Bs are lost during meiosis in anthers. There is, however, no evidence of B-chromosome accumulation in the offspring of controlled crosses, usually a slight loss, and Bs have deleterious effects on aspects of vigour and fertility. Thus, no satisfactory explanation for populations with up to 65% B-containing individuals has yet been found.     

The B-chromosome system of Allium schoenoprasum

Populations of Allium schoenoprasum, 2n=16, from six of the eight British localities have been examined. A B-chromosome polymorphism has been found which is limited to the River Wye, South Wales, where A. schoenoprasum grows along a 15 kilometre stretch. Four interrelated types of small, euchromatic, mitotically-stable B-chromosomes occur. Twelve different B-karyotypes, including an individual with 18 Bs, have been recorded. Populations contain up to 39% of plants with Bs. The B-chromosomes are not uniformly distributed along the river but are limited to populations in the downstream part of the range. An abrupt change from 34% to 0% B-contaiuing plants takes place over only 1.2 kilometres. The unique combination of riverside populations in linear order and the sudden discontinuity in the cytological structure of these populations may enable the factors influencing B-chromosome distribution to be established.     

Chromosomal localizations by in situ hybridization of the repetitious human DNA families and evidence of their satellite DNA equivalents

Four of the five major repetitious human DNA families, have been mapped by the in situ hybridization technique at their T_OPT values. Two of the lighter density DNA families have autoradiographic grain patterns over heterochromatic chromosomal regions that resemble those of known satellite DNAs. The two heaviest density DNA families have autoradiographic grain patterns of middle repetitious DNAs, with all chromosomes showing labelling. Some evidence suggests that one of these DNA families is concentrated in certain chromosomal regions. Both DNA families exhibit biphasic T_OPT curves. The presence of two thermal stability classes of hybrids suggests sequence interspersion. By co-enrichment studies in Ag⁺-Cs₂SO₄ gradients, evidence suggests the origin of the three lightest density renaturated human DNA families to be satellites I, II and III.     

Influence of a rare unstable B-chromosome on chiasma frequency and nonhaploid sperm production in Dichroplus pratensis (Melanoplinae,Acrididae)

Three B-containing individuals of Dichroplus pratensis were found among 300 males collected in several Argentine localities. The B-chromosome is a partially euchromatic short telocentric and it is mitotically unstable. The B-chromosome number of testis cells ranged from 0 to 4 in two males and from 0 to 3 in the remaining one; this variation was inter- as well as intrafollicular. Meiotic behaviour of the Bs was very regular, association of Bs was usually chiasmate and no lagging univalents were observed during first division. The presence of Bs increased the production rate of abnormal spermatids and this effect was more pronounced in follicles with odd numbers of Bs. Chiasma frequency and between-cell variance were also increased in B-containing cells. A possible interaction between the Bs and the polymorphic centric fusions present in the species is discussed in relation to chiasma frequency determination.     

Characterisation of DNA from condensed and dispersed human chromatin

Condensed and dispersed chromatin fractions were isolated from human placental nuclei. The DNA of each fraction was purified and characterised by isopycnic centrifugation, thermal fractionation on hydroxylapatite (HAP) and sequence complexity studies. The DNAs had identical buoyant densities in neutral CsCl (1.698 g/cm³) and similar melting profiles on HAP. Analytical ultracentrifugation in Ag⁺-Cs₂SO₄, however, showed that satellite DNAs were present in the condensed fraction DNA (DNA_C) but were not visible in the dispersed fraction DNA (DNA_D). In addition, DNA_C was found to be enriched in highly reiterated sequences (20% reassociated by C₀t 10^?3) which can be correlated with the presence of satellite DNAs, whereas DNA_D contained only 3% of these fast reassociating sequences. In contrast DNA_D contained 30% intermediate sequences (reassociating between C₀t 10^?3 and C₀t 100) which represent only 10% of DNA_C. The reassociated highly repeated sequences of DNA_C showed the presence of two components in both CsCl density gradients and HAP thermal elution studies. This suggests that either there are sequence relationships resulting in partial mismatching between the different highly repeated DNA sequences in this fraction, or that highly repeated sequences are associated with less repetitious DNA. The results are discussed in terms of possible differences in genetic activity between the chromatin fractions.     

Effect of tetramethylammonium ions on conformational changes of DNA in the premelting temperature range

The reversible conformational change of DNAs and polydeoxyribonucleotides occurring before melting was followed by circular dichroism. Δθ/δT, the rate of change of ellipticity θ with temperature, was used mainly as a measure of this premelting phenomenon. If sodium ions were replaced by tetramethylammonium ions Δθ/δT decreased for poly (dA) poly (dT) and poly (dA.dT) poly (dT.dA), but increased for poly (dG.dC) poly (dC.dG). DNAs of different base composition showed no more premelting (Δθ/ΔT ~ 0) even at low molarities of TMACl provided the Na/TMA ratio was very small. For all cases studied the θ values at 0°C and at a given ionic strength were smaller in NaCl than in TMACl. When studying the series of ammonium ions from NH⁺₄ to (C₂H₅)₄,N⁺ the Δθ/ΔT values first decreased, going through zero with TMA⁺ io and then increased again. A tentative and qualitative explanation of our results can be given: (a) Hydration of the polymers increases in presence of TMA ions and their average stability decreases; locally, however, (AT) pairs are preferentially stabilized by TMA ions owing to a specific interaction at the level of O₂ of thymine. (b) In order to explain the different behaviour of (AT) polymers and DNA, it is assumed that only the B structure is able to accommodate TMA ions in the small groove of the double stranded helix.     

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

The B-chromosome system of Hypochoeris maculata

The meiotic process in PMCs of Hypochoeris maculata is progressively disrupted in the presence of two or more B-chromosomes. Bivalent formation and chiasma conditions are unaffected by up to 3Bs although some univalence occurs with higher numbers. Spindle behaviour, however, is inefficient at both first and second division in the presence of two or more Bs. At metaphase I, regular equatorial alignment breaks down and A-bivalents sometimes show amphitelic or monosyntelic orientation. Anaphase I is characterised by irregular segregation, equatorial laggards and centric division products. The proportion of normal anaphase segregations declines by 20% for every B more than one. A-chromosome laggards, but not Bs, can induce nuclear restitution at telophase I. Following centric division at anaphase II poleward movement can fail leading to further nuclear restitution. Telophase II nuclei thus can be approximately haploid, diploid or tetraploid with aneuploid variation around the haploid and diploid levels. The frequency of numerical mutants in the offspring indicates that EMC meiosis is much less susceptible to the presence of Bs than PMC meiosis: only 4- and 5B^t plants have an enhanced frequency of numerically-aberrant offspring. The deleterious effects of Bs on meiotic efficiency will contribute to setting an upper limit on B-numbers in natural populations of this species.     

The B-chromosome system of Hypochoeris maculata

Populations of Hypochoeris maculata (Compositae) over a wide area of Europe contain a small proportion of individuals with mitoticallystable B-chromosomes. The telocentric Bs are about one sixth the length of the smallest A-chromosome and are heterochromatic. Offspring grown from seeds of natural populations have one or at most two B-chromosomes although plants with up to five Bs have been produced in experiment. The Bs rarely pair during meiotic prophase and the univalents usually undergo centric division at anaphase I. Despite lagging, the Bs are rarely lost during male meiosis. The inheritance of the B-chromosomes differs between the two sides with accumulation in the egg and random inheritance or loss on the pollen side. Although meiotic drive may play some part in the maintenance of the B-polymorphism there is a strict upper limit on the number of Bs in natural populations.     

The effect of B chromosomes on meiotic and pre-meiotic spindles and chromosome pairing in Triticum/Aegilops hybrids

Diploid populations of Aegilops mutica and Aegilops speltoides containing B chromosomes have been used as male parents in crosses with aneuploid genotypes of Triticum aestivum to investigate the effect of B chromosomes on meiotic homologous and homoeologous chromosome pairing. F₁ hybrids of T. aestivum/Ae. mutica and T. aestivum/Ae. speltoides segregated into four classes with regard to the degree of meiotic chromosome pairing, irrespective of the presence of B chromosomes. The B chromosomes do not introduce factors altering the level of pairing other than that due to the natural allelic and gene variation occurring in the diploids. Similarly no reduction in pairing of homologous chromosomes was observed in genotypes in which pairs of homologues co-existed with B chromosomes. However, a significant drop in chiasma frequency was observed in F₁ hybrids of T. aestivum × Ae. mutica with B chromosomes and T. aestivum × Ae. mutica nullisomic for wheat chromosome 5D with B chromosomes, in temperature regimes of 12° C. No asynapsis occurred in similar hybrids in the absence of Mutica B chromosomes at low temperatures. The low-temperature sensitive phase lies early in the pre-meiotic interphase. In this instance the Mutica B chromosomes are interacting with specific gene loci of the A chromosomes. Synaptic pairing has been observed between A and B chromosomes in Ae. mutica. A high frequency of pollen mother cells with twice the number of chromosomes was observed in hybrids in the presence of Mutica B chromosomes due to failure of spindle formation at the last pre-meiotic mitosis. Meiotic spindle irregularities occurred in hybrids containing Speltoides B chromosomes. Hybrids of Ae. speltoides + B's X Ae. mutica + B's displayed the mitotic and meiotic spindle abnormalities introduced by the presence of the B chromosomes of each parent.     

High Temperature Stabilization of DNA in Complexes with Cationic Lipids

The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100°C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T_m(labile), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T_m(stable), at 105 to 115°C. The parameter T_m(stable) did not depend on the charge ratio, R(±). Instead, the amount of thermostable DNA and the enthalpy ratio ΔH_(stable)/ΔH_(labile) depended upon R(±) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(±) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(±) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130°C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization.     

DNA with damage in both strands as affinity probes and nucleotide excision repair substrates

Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC^FAB and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC^FAB/dG (probe I), dC^FAB/nFlu₊₄ (probe II), and dC^FAB/nFlu_?3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC—HR23B (Kd_um > Kd_I > Kd_II ≈ Kd_III) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC^FAB results in (i) decreased melting temperature (ΔT_m = ?3°C) and (ii) 12° DNA bending. The extended dC^FAB/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC—HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC^FAB/nFlu₊₄, dC^FAB/nFlu_?3) or irreparable (nFlu/nFlu₊₄, nFlu/nFlu_?3, nAnt/nFlu₊₄, nAnt/nFlu_?3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis.     

Studies of DNA dumbbells. V. A DNA triplex formed between a 28 base-pair DNA dumbbell substrate and a 16 base linear single strand

CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T₄ single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ～20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (～ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of f_T(T), the fraction of triplex as a function of temperature, was obtained. Analysis of f_T vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔH_T = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔS_T = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from f_T = 0 to 0.55 and a higher, sharper transition above f_T = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′_T = 44.8 ± 9.6 kcal/mol and ΔS′_T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′_T = 128.0 ± 2.3 kcal/mol and ΔS′_T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.     

B-chromosome systems in the pocket mouse,Perognathus baileyi: Meiosis and C-band studies

The heterochromatin characteristics and meiotic behavior of the B-chromosome system of the pocket mouse, Perognathus baileyi, are described. B-chromosomes are associated both with a meiotic accumulation mechanism and with an increase in average chiasma frequency in the A-chromosome set in males. Three morphological classes of B-chromosomes are recognizable, and the mechanisms of origin of each are discussed.     

Thermal melting and circular dichroism of DNA complexes with (Lys-Ala-Ala)10 and (Lys-Ala-Ala)n: effect of polypeptide chain length

DNA complexes with polypeptides (Lys-Ala-Ala)₁)] and (Lys-Ala-Ala)₃₄ have been studied using the methods of thermal melting and circular dichroism. Derivative melting curves of (Lys-Ala-Ala)₁₀ DNA differed substantially from those of (Lys-Ala-Ala)₃₄ prepared either by salt gradient dialysis or by direct mixing. Melting curves of the former complex were unimodal or bimodal with T_m increasing continuously withn input lysin-to-DNA phosphate ratio (r); those of the latter complex consisted of three separate transitions with T_m values almost independent of r. Complete reversibility of binding in the (Lys-Ala-Ala)₁₀-DNA system but a slow redistribution of (Lys-Ala-Ala)₃₄ on DNA at low temperature were found in the redistribution experiments Much faster redistribution from denatured to native DNA occurs at the temperature of melting, contributing to the unusual trimodal melting pattern. Circular dichroism curves are very similar for both complexes and indicate little change of DNA conformation upon polypeptide binding.     

Trisomics inArabidopsis thaliana and the location of linkage groups

Different populations of the grasshopper Arcyptera fusca located through a small valley of the Pyrenees present an unstable B-chromosome system. Frequencies of individuals carrying Bs ranged from 11% to 50%. In the testes of these males the number of Bs varied among the different follicles ranging from 0 to 4 with 2 being the number most commonly found. The variation in the number of supernumeraries probably resulted from their preferential non-disjunction in the carly mitosis prior to the differentiation of the follicles. The meiotic behaviour of Bs depends on their number within cach follicle. When two or more Bs are present they usually pair and segregate regularly; B univalents divide in anaphase I and segregate without further division in anaphase II in 75% of the cells observed. The presence of Bs does not affect the chiasma frequency, however, the males with Bs had fewer follicles in their testes; this event could be related with the non-existence of follicles with more than 4 Bs.     

Exploration of DNA processing features unravels novel properties of ICE conjugation in Gram-positive bacteria

Integrative and conjugative elements (ICEs) are important drivers of horizontal gene transfer in prokaryotes. They are responsible for antimicrobial resistance spread, a major current health concern. ICEs are initially processed by relaxases that recognize the binding site of oriT sequence and nick at a conserved nic site. The ICESt3/Tn916/ICEBs1 superfamily, which is widespread among Firmicutes, encodes uncanonical relaxases belonging to a recently identified family called MOB_T. This family is related to the rolling circle replication initiators of the Rep_trans family. The nic site of these MOB_T relaxases is conserved but their DNA binding site is still unknown. Here, we identified the bind site of RelSt3, the MOB_T relaxase from ICESt3. Unexpectedly, we found this bind site distantly located from the nic site. We revealed that the binding of the RelSt3 N-terminal HTH domain is required for efficient nicking activity. We also deciphered the role of RelSt3 in the initial and final stages of DNA processing during conjugation. Especially, we demonstrated a strand transfer activity, and the formation of covalent DNA-relaxase intermediate for a MOB_T relaxase.     

Thermodynamics of DNA Containing Very Stable Chemically Modified Base Pairs

Abstract

DNA chemical modifications caused by the binding of some antitumor drugs give rise to a very strong local stabilization of the double helix. These sites melt at a temperature that is well above the melting temperatures of ordinary AT and GC base pairs. In this work we have examined the melting behavior of DNA containing very stable sites. Analytical expressions were derived and used to evaluate the thermodynamic properties of homopolymers DNA with several different distributions of stable sites. The results were extended to DNA with a heterogeneous sequence of AT and GC base pairs. The results were compared to the melting properties of DNA with ordinary covalent interstrand cross-links. It was found that, as with an ordinary interstrand cross-link, a single strongly stabilized site makes a DNA's melting temperature (T_m ) independent of strand concentration. However in contrast to a DNA with an interstrand cross-link, a strongly stabilized site makes the DNA's T_m independent of DNA length and equal to T _∞, the melting temperature of an infinite length DNA with the same GC-content and without a stabilized site. Moreover, at a temperature where more than 80% of base pairs are melted, the number of ordinary (non-modified) helical base pairs (n) is independent of both the DNA length and the location of the stabilized sites. For this condition, n(T) = (2ω-a) S (1- S ) and S = exp[ΔS(T∞-T)/(RT)] where ω is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and ΔS, T, and R are the base pair entropy change, the temperature, and the universal gas constant per mole. The above expression is valid for a temperature interval that corresponds to n<0.2N for ω=1, and n<0.1N for ω>1, where N is the number of ordinary base pairs in the DNA chain.     

Evolution of a complex B-chromosome polymorphism in the grasshopper Eyprepocnemis plorans

B-chromosome systems in several Spanish natural populations of Eyprepocnemis plorans are reported. The geographical distributions of the fourteen types of B chromosomes, which were classified according to size, C-banding pattern, and meiotic behaviour, are described. The results indicate a common origin for most types and a possible independent origin for a few secondary B variants. The origin and mechanisms of evolution of this B chromosome polymorphism are discussed.