Invasive <Emphasis Type="Italic">Spiraea tomentosa</Emphasis>: a new host for monophagous <Emphasis Type="Italic">Earias clorana</Emphasis>?

The introduction of alien species can have a significant impact on the food preferences of native phytophagous insects. The moth Earias clorana L. has previously been considered to be monophagous, ingesting only plants in the genus Salix. In recent years, we have observed larval E. clorana feeding on Spiraea tomentosa L., an invasive shrub species in Central Europe that is native to North America. We hypothesised that this insect can feed on Spiraea tomentosa leaves with no negative effects on its growth and development, and that the leaves of Spiraea tomentosa as a source of food for E. clorana are equally as good as leaves of Salix viminalis L. Our results showed that despite significant differences in the chemical composition of the studied species’ leaves, including a much higher concentration of defence compounds (total soluble phenols and condensed tannins) in Spiraea tomentosa leaves than in those of Salix viminalis, feeding on a new host plant did not significantly affect the survival of larvae. The change in host plant had an unfavourable effect, however, on several parameters of growth and development for the larvae (masses of larvae and pupae, relative growth rates, and efficiency of conversion of ingested food). We conclude that, in comparison to Salix viminalis, Spiraea tomentosa is not a particularly favourable food for larval development. Perhaps, even without direct improvements in adult foraging efficiency, however, the costs of switching hosts may be minimised in larvae that develop on very abundant, invasive species, such as Spiraea tomentosa in Central Europe.     

Deoxyribonucleic Acid Polymerase Activity in a Deoxyribonucleic Acid Polymerase I-Deficient Mutant of Bacillus subtilis Infected with Temperate Bacteriophage SPO2

Increased deoxyribonucleic acid (DNA) polymerase activity is found in soluble extracts from a polymerase I-negative mutant of Bacillus subtilis after infection with temperate phage SPO2, or after induction of SPO2 prophage in lysogenic derivatives of this mutant. No increased enzyme activity is found after SPO2 infection in the presence of chloramphenicol. Infection of the polymerase-negative mutant with the DNA-negative sus mutant SPO2 L244 gives no increased enzyme activity, whereas infection with DNA-negative sus mutant SPO2 J385 gives enzyme activities comparable to those found in wild-type infected cells. These findings suggest that SPO2 determines a DNA polymerase activity essential for synthesis of phage DNA.     

Mapping of Prophage and Mature Deoxyribonuleic Acid from Temperate Bacillus Bacteriophage φ105 by Marker Rescue

By using temperature-sensitive (ts) and suppressor-sensitive (sus) mutants, 11 essential genes have been identified in phage phi105. The order of the genes has been established in two- and three-factor crosses. The genes can be arranged in a linear order; this order is identical in the vegetative phage and in the prophage. One gene essential for phage deoxyribonucleic acid (DNA) synthesis has been found. Marker rescue from prophage and mature DNA, taken up by competent bacteria, was studied by superinfection with phage carrying one sus and one ts mutation. In prophage DNA, all single markers studied are rescued at similar frequencies. The frequency of co-rescue of two markers is proportional to the recombinational distance between the markers. Thus, colinearity between the genetic map and the position on the DNA molecule of those mutations used to establish the map is demonstrated. The results indicate that the recombination frequencies observed in vegetative crosses are a relative measure of the physical distance between markers. All single markers are not rescued at equal frequencies from mature DNA. The frequency of co-rescue of two markers is related to the recombinational distance only over a distance about one-fourth or less of the genetic map. Markers separated by 10% recombination, or more, are co-rescued at 5 to 10% of the frequency of rescue of single markers. Shearing of mature DNA into half-sized molecules reduces the efficiency by which single markers are rescued by a factor of 5 to 10. The results of experiments on co-rescue of two markers from half-sized mature DNA indicate a preferred break-point near the middle of the genetic map; the results are compatible with a nonpermuted sequence in mature DNA. It is pointed out and discussed that mature DNA exhibits several anomalies in marker rescue experiments.     

Induction of Prophage SPO2 in Bacillus subtilis: Prophage Excision in the Absence of Bacterial or Bacteriophage DNA Synthesis

Bacillus subtilis lysogenic for SPO2 wild type was induced under conditions preventing synthesis of both bacterial and phage DNA. The infectivity of phage DNA in transfection is strongly decreased under these conditions, whereas the activity of single phage genes as measured by marker rescue with superinfecting phage is unaffected. DNA from induced cells was sedimented in neutral sucrose gradients. After induction, phage DNA was detected at a position in the gradients, which was different from the bulk of the bacterial DNA, corresponding to linear double-stranded DNA of about 25 x 10(6) daltons. Similar results were obtained with bacteria lysogenic for a SPO2 prophage carrying a DNA-negative mutation. No separation of phage and bacterial DNA activity was detected when chloramphenicol was present during the induction period. These experiments show that prophage SPO2 can excise from the bacterial chromosome without previous replication.     

Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies.

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.     

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls – Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2–4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose–response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

Ants accelerate succession from mountain grassland towards spruce forest

Question: What is the role of mound‐building ants (Lasius flavus) in successional changes of a grassland ecosystem towards a spruce forest? Location: Slovenské Rudohorie Mountains, Slovakia; ca. 950 m a.s.l. near the Obrubovanec point (1020 m a.s.l.; 48°41′N, 19°39′E). Methods: Both chronosequence data along a successional gradient and temporal data from long‐term permanent plots were collected on ants, spruce establishment, and vegetation structure, together with additional data on spruce growth. Results: There are more spruce seedlings on ant mounds (4.72 m^?2) than in the surrounding vegetation (0.81 m^?2). Spruce seedlings grow faster on these mounds compared to surrounding areas. The first colonization wave of seedlings was rapid and probably occurred when grazing prevailed over mowing. Ant colony presence, mound volume, and plant species composition change along the successional gradient. Mounds become bigger when partly shaded but shrink in closed forest, when ant colonies disappear. Shade‐tolerant acidophylic species replace grassland plants both on the mounds and in surrounding areas. Conclusions: The massive occurrence of Lasius flavus anthills contributes to a runaway feedback process that accelerates succession towards forest. The effect of ants as ecosystem engineers is scale‐dependent: although they stabilize the system at the scale of an individual mound, they may destabilize the whole grassland system over a longer time scale if combined with changes in mowing regime.     

The 3��-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3′-extrahelical nucleotide (3′-flap) binding pocket. When presented with 5′-flap substrates having a 3′-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3′-flap on substrates reduced K_m and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3′-flap, and the absence of a 3′-flap from a 5′-flap substrate was more detrimental to hFEN1 activity than removal of the 5′-flap or introduction of a hairpin into the 5′-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3′-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5′-phosphorylated product. Together the results indicate that the presence of a 3′-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA)⁶ two-way junction from which a 5′-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5′-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5′-flap structures are imprecisely cleaved by DNA2 in a replication protein A -dependent manner (7). Subsequent to the DNA2 cleavage, Rad27 (yeast homologue of FEN1) cleaves precisely to generate an intermediate suitable for ligation (2). The recent discovery that human DNA2 is predominantly located in mitochondria in various human cell lines (8, 9) suggests that hFEN1 is the paramount 5′-flap endonuclease in the nuclei of human cells. This observation potentially provides a plausible rationale for why deletion of RAD27 (yeast FEN1 homologue) is tolerated in Saccharomyces cerevisiae (10), whereas deletion of FEN1 in mammals is embryonically lethal (11). Recent models wherein mice carrying a mutation (E160D) in the FEN1 gene, which was shown in vitro to alter enzymatic properties (12), have demonstrated that FEN1 functional deficiency in mice (S129 and Black 6) increases the incidence of cancer, albeit different types presumably due to genetic background (13, 14). Thus, the function of mammalian FEN1 in vivo is vital to the prevention of genomic instability. In addition to its importance in the nucleus, hFEN1 has recently been detected in mitochondrial extracts (15, 16) and implicated in mitochondrial long patch base excision repair (15). Considering the pivotal roles of hFEN1 in DNA replication and repair, it is of interest to understand how hFEN1 and homologues achieve substrate and scissile phosphate selectivity in the absence of sequence information.Since its initial discovery as a nuclease that completes reconstituted Okazaki fragment maturation (17) and subsequent rediscovery as a 5′-flap-specific nuclease (DNaseIV) from bacteria (18), mouse (19), and HeLa cells (20), FEN1 proteins ranging from phage to human have been studied biochemically, computationally, and structurally (5, 6, 21). Biochemical characterizations of FEN1 proteins from various organisms have shown that this family of nucleases can perform phosphodiesterase activity on a wide variety of substrates; however, the efficiency of catalysis on various substrates differs among the species. For instance, phage FEN1s prefer pseudo-Y substrates (22, 23), whereas the archaeal and eukaryotic FEN1s prefer 5′-flap substrates (21, 24, 25), which have two dsDNA domains, one upstream and downstream of the site of cleavage, and a 5′-ssDNA protrusion (Fig. 1A). Primary sequence analysis indicates that FEN1 proteins share characteristic N-terminal (N) and Intermediate (I) “domains,” which harbor the highly conserved carboxylate residues that bind the requisite divalent metal ions (26–28). Structural studies of FEN1 nucleases from phage to humans (22, 29–36), have shown that the N and I domains comprise a single nuclease core domain consisting of a mixed, six- or seven-stranded β-sheet packed against an α-helical structure on both sides. The α-helices on either side of the β-sheet are “bridged” by a helical arch that spans the active site groove (supplemental Fig. S1). On one side of the β-sheet, the α-helical bundle (αb1) creates the floor of the active site and a DNA binding motif (helix-3-turn-helix) (32). Similarly, the opposite α-helical bundle (αb2) has also been observed to interact with DNA (35). Based on site-directed mutagenesis studies with T5 phage FEN1 (T5FEN1) (37) and hFEN1 (38, 39), and crystallographic studies of T4 phage FEN1 (T4FEN1) (22) and Archaeoglobus fulgidus FEN1 (aFEN1) (35) in complex with DNA, a general model for how FEN1 proteins recognize flap DNA has emerged. The helix-3-turn-helix motif is involved in downstream dsDNA binding, whereas the upstream dsDNA domain is bound by αb2. The helical arch is likely involved in 5′-flap binding (22).Open in a separate windowFIGURE 1.Secondary structure schematics of hFEN1 substrates. A, illustration of a general flap substrate created using a bimolecular approach whereby a template strand (T-strand), which partially folds into a hairpin, anneals with the duplex strand (d-strand). The T-strand hairpin creates the upstream dsDNA domain, whereas the d-strand base pairs with the T-strand to create the downstream dsDNA domain. The flap or any other structure is created by addition of nucleotides to the 5′-end of the d-strand. The interface between the upstream and downstream dsDNA domains may be viewed as a derivative of a two-way junction (74). Annealing of either the F(5), E, or G(15) d-strands with the T3F T-strand results in the formation of a (B) double flap substrate (Flap of 5-nt d-strand paired with a Template with a 3′-Flap, F(5)·T3F), C, exonuclease substrate with a 3′-extrahelical nucleotide (EXO d-strand paired with a Template with a 3′-Flap, E·T3F), and a D, fork-GEN substrate with a 3′-extrahelical nucleotide and a 15-nt ssDNA gap capped by a 23-nt hairpin structure (fork-Gap of 15-nt d-strand paired with a Template with a 3′-Flap, G(15)·T3F). E, annealing the F(5) d-strand with the T oligonucleotide creates a single flap (Flap of 5-nt d-strand paired with a Template, F(5)·T).Unlike phage FEN1s, studies of FEN1s from eubacterial (40), archaeal (21), and eukaryotic origins (41) have shown that the addition of a 3′-extrahelical nucleotide (3′-flap) to the upstream duplex of a 5′-flap substrate results in a rate enhancement and an increase in cleavage site specificity. Moreover, substrates possessing a 3′-flap, which mimic physiological “equilibrating flaps,” were cleaved exactly one nucleotide into the downstream duplex, thereby resulting in 5′-phosphorylated dsDNA product that was a suitable substrate for DNA ligase I (21, 41). As postulated by Kaiser et al. (21), the structure of an archaeal FEN1 in complex with dsDNA with a 3′-overhang showed that the protein contains a cleft adjacent to the upstream dsDNA binding site that binds the 3′-flap by means of van der Waals and hydrogen bonding interactions with the sugar moiety (35). Once the residues associated with 3′-flap binding were identified, sequence alignment analyses showed that the amino acid residues in the 3′-flap binding pocket are highly conserved from archaea to human. Furthermore, mutation of the conserved amino acid residues in the 3′-flap binding pocket of hFEN1 resulted in reduced affinity for and cleavage specificity on double flap substrates (42). Although the effects of the addition of a 3′-flap to substrates on hFEN1 catalysis are known qualitatively, a detailed understanding of the relationship between changes in catalytic parameters and rate enhancement by the presence of a 3′-flap is unknown. Here, we describe a detailed kinetic analysis of hFEN1 using four well characterized DNA substrates and show that the presence of a 3′-flap on a substrate not only contributes to substrate binding (42), but also increases multiple and single turnover rates of reaction in the presence of near physiological monovalent salt concentrations. We also demonstrate that, like T5FEN1, hFEN1 is rate-limited by product release, and thus multiple turnover rates at saturating concentrations of substrate are predominantly a reflection of product release and not catalysis as was previously concluded (39). Furthermore, this study provides insight into the mechanism of hFEN1 substrate recognition.