INHERITANCE OF NUCLEAR 2C DNA CONTENT VARIATION IN INTRASPECIFIC AND INTERSPECIFIC HYBRIDS OF MICROSERIS (ASTERACEAE)

Nuclear DNA content varies over 20% within the diploid (2n = 18) species M. douglasii and M. bigelovii. Two different intraspecific crosses were made between M. douglasii biotypes which differed by about 10% in 2C nuclear DNA content. The F₂ progeny of one intraspecific cross showed no striking evidence of segregation for DNA content. The mean DNA contents of F₂ progeny from two sister hybrids from the second intraspecific cross were significantly different at the 1% level. An interspecific cross was made between biotypes of M. douglasii and M. bigelovii that differed by approximately 10% in DNA amount. The 12 F₁ progeny did not cluster around the parental midpoint, but instead encompassed nearly the entire range between the parental means. The five families of F₂ progeny studied each had a mean DNA content corresponding to that of the particular F₁ from which they were derived, indicating that the F₁ plants were not of identical DNA content. The results of this study suggest that DNA sequences which account for the DNA content differences among the plants are unstable and can undergo deletion or amplification in a hybrid. The altered DNA content may be heritably stable and show little or no segregation in the F₂ progeny.     

Dna Damage by Chemically Generated Singlet Oxygen

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (¹O₂) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10⁸ daltons) was exposed to 120 mol m^?31O₂ (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for ¹O₂-induced interstrand crosslinking was obtained. The capacity of ¹O₂ to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage øX174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of ¹O₂ -exposure were assessed by examining the plaque-forming capacity of ss and ds øX 174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,²⁰) we infer that ¹O₂-induced inactivation of øX174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.     

Copper(II)-DNA denaturation. II. The model of DNA denaturation

In a continuing study of the denaturation of DNA as brought abought about by Cu(II) ions, results are presented for the dependence of T_m and τ (the terminal relaxation time) on ionic strength, pH, reactant concentrations, and temperature. Maximum stability of the double helix, as reflected by the longest relaxation times and highest T_m values, was observed between pH 5.3 and 6.2. Outside this range both T_m and τ decreased sharply. A second, faster relaxation time was deduced from the kinetic cureves. The apparent activation energies of the rapid and slow (“terminal”) relaxations were found to be 12 and 55 kcal/mole, respectively. Several lines of evidence led to the conclusions that (1) the rate-determining step in DNA denaturation, when occurring in the transition region, is determined by chemical events and (2) the interactions which are disrupted kinetically in the rate-determining step are those which account for the major portion of the thermal (T_m) stability of helical DNA.     

Induction of theEscherichia coli UVM response by oxidative stress

UVM (ultravioletmodulation of mutagenesis) is a recently describedrecA-independent, inducible mutagenic phenomenon in which prior UV irradiation ofEscherichia coli cells strongly enhances mutation fixation at a site-specific 3-N⁴-ethenocytosine (C) lesion borne on a transfected single-stranded M13 DNA vector. Subsequent studies demonstrated that UVM is also induced by alkylating agents, and is distinct from both the SOS response and the adaptive response to alkylation damage. Because of the increasing significance being attributed to oxidative DNA damage, it is interesting to ask whether this class of DNA damage can also induce UVM. By transfecting M13 vector DNA bearing a site-specificC lesion into cells pretreated with inducing agents, we show here that the oxidative agent H₂O₂ is a potent inducer of UVM, and that the induction of UVM by H₂O₂ does not requireoxyR-regulated gene expression. UVM induction by H₂O₂ appears to be mediated by DNA damage, as indicated by the observation of a concomitant reduction in cellular toxicity and UVM response in OxyR^c cells. Available evidence suggests that UVM represents a generalized cellular response to a broad range of chemical and physical genotoxicants, and that DNA damage constitutes the most likely signal for its induction.     

Postglacial expansion and genome subdivision in the European grasshopper Chorthippus parallelus

A noncoding nuclear DNA marker sequence (Cpnl-1) was used to investigate subdivision in the grasshopper Chorthippus parallelus and deduce postglacial expansion patterns across its species range in Europe. Investigation of the spatial distribution of 71 Cpnl-1 haplotypes and estimation of levels of genetic differentiation (K_ST values) between populations and geographic regions provided evidence for subdivision of C. parallelus into at least five major geographic regions and indicated that the French form of C. parallelus originated after range expansion from a Balkan refugium, Further evidence for subdivision of C. parallelus between Italy and northern Europe suggests that the Alps may have formed a significant barrier to gene flow in this grasshopper.     

Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

Accurately predicting binding affinity constant (K_A) is highly required to determine the binding energetics of the driving forces in drug–DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining K_A for PD153035, where K_A is determined from the changes in B-form contour length (L) of PD153035–DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that K_A = 1.18(±0.09) × 10⁴ (1/M) at 23 ± 0.5 °C and the minimum distance between adjacent bound PD153035 ≈ 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.     

Production and characterization of interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra and their progenies for the selection of advanced pre-breeding materials

Somatic hybridization is a potential method for gene transfer from wild relatives to cultivated crops that can overcome sexual incompatibilities of two distantly related species. In this study, interspecific asymmetric somatic hybrids of Brassica oleracea var. botrytis (cauliflower) and Brassica nigra (black mustard) were obtained by protoplast fusion and their backcrossed (BC₃) and selfed (S₃) offspring were analyzed. Cytological analysis showed that the B. nigra chromosomes were successively eliminated in the backcrosses with cauliflower. The fertility of the hybrid progenies was quite different due to the asynchronous and abnormal chromosome behavior of pollen mother cells (PMC) during meiosis. Analysis of sequence-related amplified polymorphism (SRAP) showed that all of these hybrids mainly had the DNA banding pattern from the two parents with some alterations. Genetically, the selfed generations were closer to B. nigra, while the backcrossed generations were closer to the cauliflower parent. Analysis of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) showed that all somatic hybrids in this study contained chloroplast (cp) DNA of the donor parent black mustard, while mitochondrial (mt) DNA showed evidence of recombination and variations in the regions analyzed. Furthermore, three BC₃ plants (originated from somatic hybrids 3, 4, 10) with 2–8 B. nigra-derived chromosomes shown by genomic in situ hybridization (GISH) displayed a more cauliflower-like morphology and high resistance to black-rot. These plants were obtained as bridge materials for further analysis and breeding.     

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)₂], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)₂] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100 mA beam current of 0.5-μW power varied from 0.6 h⁻¹ for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)₂] and high salt poly[(dA-dT)₂]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.     

Seed-storage-protein loci in RFLP maps of diploid, tetraploid, and hexaploid wheat

 Linkages between high- and low-molecular-weight (M_r) glutenin, gliadin and triticin loci in diploid, tetraploid and hexaploid wheats were studied by hybridization of restriction fragments with DNA clones and by SDS-PAGE. In tetraploid and hexaploid wheat, DNA fragments hybridizing with a low-M_r glutenin clone were mapped at the XGlu-3 locus in the distal region of the maps of chromosome arms 1AS, 1BS, and 1DS. A second locus, designated XGlu-B2, was detected in the middle of the map of chromosome arm 1BS completely linked to the XGli-B3 gliadin locus. The restriction fragments mapped at this locus were shown to co-segregate with B subunits of low-M_r glutenins in SDS-PAGE in tetraploid wheat, indicating that XGlu-B2 is an active low-M_r glutenin locus. A new locus hybridizing with the low-M_r clone was mapped on the long arm of chromosome 7A^m in diploid wheat. No glutenin protein was found to co-segregate with this new locus. Triticin loci were mapped on chromosome arms 1AS, 1BS, and 1DS. A failure to detect triticin proteins co-segregating with DNA fragments mapped at XTri-B1 locus suggests that this locus is not active. No evidence was found for the existence of Gli-A4, and it is concluded that this locus is probably synonymous with Gli-A3. Recombination was observed within the multigene gliadin family mapped at XGli-A11 (1.2 cM).1 Although these closely linked loci may correspond to the previously named Gli-A1 and Gli-A5 loci, they were temporarily designated XGli-A1.1 and XGli-A1.2 until orthology with Gli-A1 and Gli-A5 is established. Received: 25 March 1997 / Accepted: 23 June 1997     

A reappraisal of the role of Zn2+ in TGF-β1-induced apoptosis in primary hepatocytes

There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn²⁺ on transforming growth factor-β₁ (TGF-β₁)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-β₁ (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 μmol/L Zn²⁺ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-β₁-treated hepatocytes. Surprisingly, Zn²⁺ did not inhibit the formation of high-molecular-weight DNA fragments (30–50 kbp to 250–300 kbp). These data provide evidence that Zn²⁺ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-β₁-induced apoptosis in hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA damage signaling triggers the cytoplasm-to-vacuole pathway of autophagy to regulate cell cycle progression

Budding yeast cells suffering a single unrepaired DNA double-strand break (DSB) trigger the ATR (Mec1)-dependent DNA damage checkpoint and arrest prior to anaphase for 12–15 h, following which they adapt and resume cell division. When the DNA lesion can be repaired, the checkpoint is extinguished and cells “recover” and resume mitosis. In this autophagic punctum, we report that hyperactivation of autophagy—specifically via the cytoplasm-to-vacuole targeting (Cvt) pathway—prevents both adaptation to, and recovery from, DNA damage, resulting in the permanent arrest of cells in G₂/M. We show that Saccharomyces cerevisiae deleted for genes encoding the Golgi-associated retrograde protein transport (GARP) complex are both adaptation- and recovery-defective. GARP mutants such as vps51Δ exhibit mislocalization of the key mitotic regulator, securin (Pds1), and its degradation by the vacuolar protease Prb1. In addition, separase (Esp1), is excluded from the nucleus, accounting for pre-anaphase arrest. Pds1 is degraded via the Cvt pathway. Many of the same defects seen by deleting GARP genes can be mimicked by hyperactivation of the Cvt pathway by overexpressing an unphosphorylatable form of ATG13 or by adding the TORC1 inhibitor rapamycin. These results suggest that nuclear events such as DNA damage can have profound effects on cytoplasmic processes and further expand the burgeoning connections between DNA damage and autophagy.     

Sequence Specific Molecular Recognition and Binding of a Monocationic Bis-Imidazole Lexitropsin to the Decadeoxyribonucleotide d-[(GATCCGTATG) (CATACGGATC)]: Structural and Dynamic Aspects of Intermolecular Exchange Studied by 1H-NMR

Abstract

The non-exchangeable and imino proton NMR resonances of the non self-complementary decadeoxyribonucleotide d-[(GATCCGTATG) · (GATACGGATC)] as well as those of the 1:1 complex of the monocatonic bis-imidazole lexitropsin 1 to this sequence have been assigned by using a combination of NOE difference, COSY and NOESY techniques. Confirmation of complete annealing of the two non self-complementary decamer strands to give the duplex decadeoxyribonucleotide is obtained by the detection of ten imino protons. It is established that the sugar-base orientations of all the bases in the duplex decamer are anti. From NOE studies, it is concluded that the duplex oligomer is right-handed and adopts a conformation in solution that belongs to the B family. A population analysis reveals that the sugar moieties exist predominantly in the S-form (2′-endo-3′-exo). Addition of 1 to the DNA solution leads to doubling of the resonances for CH6(4,5), GH8(6), TH6(7) and T-CH₃(7). The base, anomeric H1′ and imino proton signals for the base sequence 5′-CCGT undergo the most marked drug-induced chemical shift changes. These results provide evidence that the lexitropsin is bound to the sequence 5′-CCGT in the minor groove of the DNA NOE measurements between the amide protons (NH1 and NH4) and the imino proton (IV and V) signals confirmed the location and orientation of 1 in the 1:1 complex, with the amino terminus oriented to C(4). The specific binding of 1 to the sequence 5′-CCGT-3′ deduced in this study is in agreement with the footprinting data obtained using the Hind III/Nci I fragment from pBR322 DNA [Kissinger et al. 1987 (13)]. Intramolecular NOEs observed between H4 and H9 of the lexitropsin suggest that the molecule is not planar, but subjected to propeller twisting, in both the free and bound forms. Furthermore, NOE measurements permit assignment of the DNA duplex in the 1:1 complex to the B-form, which is similar to that of the free DNA The [(T₇A₈T₉)· (A₁₂T₁₃A₁₄)] segment of the DNA shows better stacking, by propeller twisting, compared to the rest of the molecule in the free as well as the complex forms. The intermolecular rate of exchange of 1 between the equivalent 5′-CCGT sites, at a concentration of 12 mM, is estimated to be ～88s^?1 at 308°K with ΔG≠ of 63±5 K.J mol^?1.     

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl₄) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl₄-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl₄ with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl₄ exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl₄ and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl₄ is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl₄.     

Crystallographic evidence for two‐metal‐ion catalysis in human pol η

Extensive evidence exists that DNA polymerases use two metal ions to catalyze the phosphoryl transfer reaction. Recently, competing evidence emerged, suggesting that a third metal ion, known as MnC, may be involved in catalysis. The binding of MnC was observed in crystal structures of the replication complexes of human polymerase (pol) η, pol β, and pol μ. Its occupancy (q_MnC) in the pol η replication complexes exhibited a strong correlation with the occupancy of the formed product pyrophosphate (q_PPi), i.e., q_MnC ∝ q_PPi. However, a key piece of information was missing that is needed to distinguish between two possible sequences of events: (i) the chemical reaction occurs first with only two meal ions, followed by the binding of MnC in a “catch‐the‐product” mode; and (ii) MnC binds first, followed by the chemical reaction with all three metal ions in a “push‐the‐reaction‐forward” mode. Both mechanisms can lead to a strong correlation between q_MnC and q_PPi. However, q_MnC ≤ q_PPi in the first scenario, whereas q_MnC ≥ q_PPi in the second. In this study, an analysis of crystallographic data published recently for pol η complexes shows that the formation of the product pyrophosphate definitely precedes the binding of MnC. Therefore, just like all other DNA polymerases, human pol η employs a two‐metal‐ion catalytic mechanism. Rather than help to catalyze the reaction, MnC stabilizes the formed product, which remains trapped inside the crystals, before it slowly diffuses out.     

The enhancement of oxidative DNA damage by anti-diabetic metformin,buformin, and phenformin,via nitrogen-centered radicals

Metformin (N,N-dimethylbiguanide), buformin (1-butylbiguanide), and phenformin (1-phenethylbiguanide) are anti-diabetic biguanide drugs, expected to having anti-cancer effect. The mechanism of anti-cancer effect by these drugs is not completely understood. In this study, we demonstrated that these drugs dramatically enhanced oxidative DNA damage under oxidative condition. Metformin, buformin, and phenformin enhanced generation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) in isolated DNA reacted with hydrogen peroxide (H₂O₂) and Cu(II), although these drugs did not form 8-oxodG in the absence of H₂O₂ or Cu(II). An electron paramagnetic resonance (EPR) study, utilizing alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide as spin trapping agents, showed that nitrogen-centered radicals were generated from biguanides in the presence of Cu(II) and H₂O₂, and that these radicals were decreased by the addition of DNA. These results suggest that biguanides enhance Cu(II)/H₂O₂-mediated 8-oxodG generation via nitrogen-centered radical formation. The enhancing effect on oxidative DNA damage may play a role on anti-cancer activity.     

Mitotic crisis: The unmasking of a novel role for RPA

Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (PNAS 2008; 105:12903–8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can stimulate the ability of cells to exit mitosis into a 2N G₁ phase. Along with providing additional discussion of this study, we review evidence suggesting that DNA replication and repair factors can modulate mitotic transit by acting through Polo-like kinase-1 (Plk1) and the centrosome. “A crisis unmasks everyone.”—Mason Cooley, US aphorist.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

DNA orientation in shear flow

The linear dichroism (LD) has been measured for DNA molecules 239–164,000 base pairs long oriented in shear flow over a large range of velocity gradients (30–3,000 s ^?1) and ionic strengths (2–250 mM). At very low gradients, the degree of DNA orientation increases quadratically with the applied shear as predicted by the Zimm theory [J. Zimm, (1956) Chemical Physics, Vol. 24, p. 269]. At higher gradients, the orientation of fragments ≥ 7 kilobase pairs (kbp) increases linearly with increasing shear, whereas the orientation of fragments ≥ 15 kbp shows a more complicated dependence. In general, the orientation decreases with increasing ionic strength throughout the studied ionic strength interval, owing to a decrease in the persistence length of the DNA. The effect is most dramatic at ionic strengths below 10 mM, and is more pronounced for longer DNA fragments. For fragments ≥ 15 kbp and velocity gradients ≥ 100 s^?1, the orientation can be adequately described by the empirical relation: LD^r= –(k₁-G)/(k₂ + G), where k₁is a linear function of the square root of the ionic strength and k₂ depends on the DNA contour length. Since the DNA persistence length can be represented as a linear function of the reciprocal square root of the ionic strength [D. Porschke, (1991) Biophysical Chemistry, Vol. 40, p. 169], extrapolation of the empirical relation provides information about the stiffness of the DNA fibers. © 1993 John Wiley & Sons, Inc.     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.