Excision and repair of mismatched base pairs in transformation of Streptococcus pneumoniae

Summary The use of heteroduplex DNA molecules as donors in pneumococcal transformation makes it possible to follow the fate of each DNA strand. The integration efficiency of each strand depends strongly upon the single base changes it carries. The function (hex) which reduces drastically the transformation yield of markers referred to as low efficiency (LE) tends to remove either donor strand without respect to which one is introduced. In the case of high efficiency (HE) markers the reduction in the transformation yield involves the elimination of only one donor strand. For a given locus it can be either one depending upon the mutation. The reduction in transformation yield can be less drastic for HE markers than for both strands of the LE markers. These data are discussed in terms of differences in the affinity for mismatched base pairs.We have studied the transfer of information from each donor DNA strand to the recipient genome, on the basis of differences in the rates of phenotypic expression of a given marker introduced on opposite strands. Results show that, as in the case of LE markers, the information from HE markers, when introduced on the strand recognized by the hex function, is transmitted to both strands of the recipient molecule. Correction of the recipient strand to homozygosis probably accounts for this information transfer. These results, together with earlier investigations, strongly suggest that the hex function is an excision-repair system acting on donor-recipient base pair mismatches.     

Effect of mismatched base pairs on the fate of donor DNA in transformation of Streptococcus pneumoniae

Summary Investigation of the mechanism that discriminates against mismatched base pairs in transformation of Streptococcus pneumoniae of genotype hex ⁺ was based on the use of a radioactively labeled cloned fragment of pneumococcal DNA as donor in transformation. The fate of the donor label was followed by lysis of the transformed cells and separation by agarose gel electrophoresis of DNA fragments generated by restriction endonucleases. As a result of Hex action, most of the donor DNA fragment, which was a few kilobases in length, was lost when a mismatched base pair occurred between donor and recipient DNA. This was not observed in hex ^- recipient cells. Kinetic studies of mismatch-induced donor DNA loss showed that the process is faster in strain 800, an R6 derivative, than in DP 1601, a strain of different origin. In the latter strain, the amount of donor label that becomes double stranded rises substantially, indicating extensive formation of donorrecipient heteroduplex structures, before falling to the expected level. At 30°C the process is essentially completed 15 min after entry.     

The use of unilateral PCR to identify prominent heteroduplexes formed during PCR of the mouse microsatellite locus D17Mit23

Microsatellite markers are useful tools for understanding the evolutionary history of discrete segments of the mammalian genome. We used the microsatellite marker D17Mit23 to study the portion of the mouse genome known as the t complex, a naturally occurring variant of Chromosome 17. We identified an allelic variant of D17Mit23, which is shared by two forms of the t complex, the t haplotypes t ^w2 and t ^Lub2 . Polymerase chain reaction (PCR) analysis of DNA samples from mice that were heterozygous for either haplotype resulted in gel patterns with prominent bands of higher molecular weight in addition to the bona-fide D17Mit23 alleles. The appearance of these higher molecular weight bands, although consistent with heteroduplex formation, was not diminished through the use of reconditioning PCR. We used a modified form of asymmetric PCR, called “unilateral PCR”, to show that the higher molecular weight bands are heterodu-plexes and to identify their constituent strands. Certain microsatellite motifs may be especially prone to the production of prominent heteroduplex products, and this may lead to the erroneous genotyping of DNA samples.     

An integrated genetic map of Populus deltoides based on amplified fragment length polymorphisms

Amplified fragment length polymorphism (AFLP) is an efficient molecular technique for generating a large number of DNA-based genetic markers in Populus. We have constructed an integrated genetic map for a Populus backcross population derived from two selected P. deltoides clones using AFLP markers. A traditional strategy for genetic mapping in outcrossing species, such as forest trees, is based on two-way pseudo-testcross configurations of the markers (testcross markers) heterozygous in one parent and null in the other. By using the markers segregating in both parents (intercross markers) as bridges, the two parent-specific genetic maps can be aligned. In this study, we detected a number of non-parental heteroduplex markers resulting from the PCR amplification of two DNA segments that have a high degree of homology to one another but differ in their nucleotide sequences. These heteroduplex markers detected have served as bridges to generate an integrated map which includes 19 major linkage groups equal to the Populus haploid chromosome number and 24 minor groups. The 19 major linkage groups cover a total of 2,927 cM, with an average spacing between two markers of 23. 3 cM. The map developed in this study provides a first step in producing a highly saturated linkage map of the Populus deltoides genome. Received: 10 September 1999 / Accepted: 3 November 1999     

One way to do experiments on gene conversion?

Summary Competent cells of B. subtilis were transfected with heteroduplex SPP1 DNA, made by annealing complementary strands of wild type and 21 plaque type mutant DNAs. The frequencies of cells yielding mutant and wild type, only wild type, and only mutant phages were determined by single burst analyses of transfected cells. The data obtained reveal that an effective mechanism is operating in B. subtilis, which converts heterozygous to homozygous molecules prior to their replication. This correction mechanism is asymmetric with regard to the strand which is preferentially corrected in a given heteroduplex pair. The direction of asymmetry thus defined depends on the marker introduced into a particular heteroduplex. The efficiency of correction varies with the markers used and is correlated to the position of markers in the genetic map. From this correlation, the direction of replication of the SPP1 genome is deduced. The frequency distribution of wild type and mutant phages in cells yielding both genotypes indicates that both strands of the input DNA contribute equally to the production of progeny, i.e. DNA replication is symmetric.     

Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers

Ten codominant RAPD markers, ranging in size from about 300 to about 1350 bp, were identified in mapping populations of chickpea (Cicer arietinum L.) and diploid strawberry (Fragaria vesca L.). A distinguishing feature of all ten markers, and perhaps of codominant RAPD markers in general, was the presence in heterozygous individuals of a non-parental, heteroduplex band migrating more slowly than either of the respective parental bands. This non-parental band could also be generated by mixing parental DNAs before PCR (template mixing). As a means of identifying primers likely to detect codominant RAPD markers, parental and mixed-template (parent-parent) PCR-product gel lanes were compared for 20 previously untested RAPD primers (10-base oligomers). Four primers that produced a total of five non-parental, heteroduplex bands in mixed-template reactions were selected, and then used to detect a total of five segregating, codominant markers and nine dominant markers in the respective F₂ mapping population, a codominant marker frequency of 35.7%. When closely migrating fast and slow bands of codominant RAPDs were difficult to differentiate, parent-progeny template mixing was used to deliberately generate heteroduplex bands in fast- or slow-band F₂ homozygotes, respectively, allowing confirmation of marker phenotype.     

The roles of RecBCD, Ssb and RecA proteins in the formation of heteroduplexes from linear-duplex DNA in vitro

Summary The formation of heteroduplexes from linear duplex DNA, where one molecule possesses a DNA doublestrand break, was assayed by agarose gel electrophoresis. Using unlabeled whole-length linear duplex DNA and ³H-labeled half-length linear duplex DNA (obtained from plasmid pACYC184), the appearance of ³H-labeled DNA that migrated as whole-length linear DNA was taken as evidence for formation of heteroduplex DNA. When the DNA mixtures were incubated with RecA, RecBCD, or Ssb proteins, or any double or triple combination of these proteins under a variety of reaction conditions, no heteroduplex DNA was detected. However, heteroduplex DNA was detected when the DNA mixtures were first incubated briefly with the RecBCD and Ssb proteins under reaction conditions that allow unwinding to proceed, and then the MgCl₂ concentration was raised such that renaturation could proceed. The inclusion of the RecBCD and Ssb proteins was sufficient to catalyze the slow formation of heteroduplex DNA, but the presence of RecA protein greatly increased the kinetics. The roles of the RecBCD, Ssb and RecA proteins in heteroduplex formation in vitro are discussed.     

Influence of genetic markers and of the fusing agent polyethylene glycol on chromosomal gene transfer inSpiroplasma citri

Gene transfer-recombination involving three different chromosomal markers (Ars^R, Van^R, and Xyl^R) and influence of the fusing agent PEG on transfer efficiency were investigated in matings between single-resistant or single and double-resistant strains ofSpiroplasma citri. Efficiency and kinetics vary, inS. citri, with the markers involved in the crosses and are not enhanced by the membrane fusion effect of PEG. These features allow one to distinguish the chromosomal gene transfer inS. citri from the closely related transfer by protoplast fusion in Gram-positive bacteria. However, the results may be explained by a spontaneous high frequency of the membrane fusion events ofS. citri cells and the existence of variations in the recombination frequency of the markers. Indeed, inS. citri, the formation of the heterozygotic polyploid structure formed after membrane fusion may be considered as a nonlimiting step of the mechanism and allows one, accordingly, to point out differences in recombination frequencies of the markers.     

Evaluation of the calcium mobilizing action of acetaminophen and bromobenzene in rat hepatocyte cultures

Acetaminophen (APAP) and bromobenzene (BrB) are reported to selectively inhibit plasma membrane (PM) but not endoplasmic reticulum (ER) Ca²⁺ transport in rat liver (1). The ability of these hepatotoxicants to increase cytoplasmic Ca²⁺ levels as a result of disrupted Ca²⁺ pumping was determined in cultured rat hepatocytes by monitoring the activity of glycogen phosphorylase a, a Ca²⁺ -sensitive (via phosphorylase kinase) enzyme. Following exposure to 2.5 to 10 mM APAP for five minutes, dose-dependent increases in phosphorylase a activity were observed (58 to 190 U/g protein). Endoplasmic reticulum Ca²⁺ pump activity was not inhibited after any dose of APAP (56 nmol Ca²⁺ per milligram protein per 30 minutes). Phosphorylase a activity remained elevated for 60 minutes after exposure to APAP (124 μl/g protein). Following exposure to 0.5 to 2 mM BrB for five minutes, phosphorylase a activity also increased (58 to 229 U/g protein) in a dose-related manner. Endoplasmic reticulum Ca²⁺ pump activity was inhibited after BrB exposure (from 58 to 16 nmol Ca²⁺ per milligram protein per 30 minutes). Phosphorylase a activity remained elevated for 60 minutes after exposure to BrB (147 U/g protein). Evidence of elevated cytoplasmic Ca²⁺ is consistent with the inhibition of Ca²⁺ -extruding/sequestering mechanisms at hepatocyte PM and/or ER. Prolonged elevation of cytosolic Ca²⁺ levels could overstimulate Ca²⁺ -sensitive processes within liver cells and thus initiate or contribute to hepatotoxic injury.     

Role of the Plasmalemma in Controlling the Gravitropic Response of Wheat Coleoptiles

The changes in the specific binding of ³H-IAA to the plasmalemma from segments of wheat (Triticum aestivumL.) coleoptiles and the physiological activity of the IAA–protein complexes thus formed in dependence on the duration of gravitational stimulation (GS) (1 g) were studied. The overall inhibition of the formation of IAA–protein complexes was accompanied by a transverse polarization of their functional activity occurring as early as within two minutes after the onset of GS. The pretreatment of plasmalemmal vesicles with 0.1 M CaCl₂prevented the in vitroIAA–protein complex formation in the plasmalemma. It is suggested that the GS results in an increase in the plasmalemma permeability for Ca²⁺, which reduces the capacity of the plasmalemma to bind IAA at the early stages of the gravitropic response.     

Mapping and validation of chromosome regions conferring a new boron-efficient locus in <Emphasis Type="Italic">Brassica napus</Emphasis>

Considerable genotypic variation exists in the response of different cultivars of rapeseed (Brassica napus) to B deficiency. This raises the possibility of genetic improvement of a B nutrition trait that will make the plant more tolerant to low B stress. The results of our study showed that B-efficient backcross plants had lower B concentration and more dry matter when grown at low levels of B when compared with the recurrent parent. Accordingly, we proposed that the improved B efficiency was attributed to either a high B utilization efficiency or less demand for B. The results of the genetic analysis showed that B efficiency is a dominant trait that is controlled by a single locus, namely BnBE2. By using bulked segregant analysis (BSA) in combination with amplified fragment length polymorphism (AFLP) and sequence related amplified polymorphism (SRAP) techniques, five SRAP markers and one converted single strand conformation polymorphism (SSCP) marker were identified to be linked to BnBE2 after screening 1,800 primer combinations. The six markers together with BnBE2 were mapped in a region that covered a genetic distance of 6.9 cM on a linkage group using a BC₆ population. This region was located on linkage group N14 after mapping these markers in two doubled haploid (DH) populations (TNDH and BQDH). The SRAP and AFLP markers were sequenced and found to be homologous to a BAC sequence from Brassica oleracea (CC). This finding suggested that the segment containing BnBE2 locus originated from the C genome of Brassica oleracea. Three SSR markers were identified to be linked to BnBE2 through comparative mapping. All these markers might have potential value for facilitating the pyramiding of the BnBE2 gene with other B efficient genes in order to improve the B efficiency trait and for further fine mapping of the BnBE2 gene in Brassica napus.     

Combination of photodynamic therapy with aspirin in human-derived lung adenocarcinoma cells affects proteasome activity and induces apoptosis

Objectives: Photodynamic treatment (PDT) of human lung carcinoma cells A549 (p53^+/+) and H1299 (p53^?/?) induces fast but transient stalling of proteasome activity. We have explored the possibility of prolonging this effect by combining PDT with drugs capable of sustaining the stall, and promote apoptosis of surviving cells. We show that aspirin can be used to accomplish this. Materials and methods: Cells were irradiated at doses ranging from 0.54 to 1.10 J cm^?2, and subsequently were incubated with aspirin at either high (10 and 5 mm ) or low concentration (2.5 and 1.5 mm ). Photofrin concentration and incubation time were constant (2.5 μg/ml and 16 h). Under these conditions, we analysed cell viability, colony‐forming efficiency, cycle profile, expression patterns of specific proteins and ubiquitination state, after individual or combined administration. Results: Treatment with either PDT or aspirin, rapidly induced proteasome malfunction and accumulation of cells in G₂M, but did not induce apoptosis. However, when aspirin was added to cells (even at low concentrations) after PDT, the proteasome block was sustained. Moreover, significant cytotoxic effects, including apoptosis, were observed along with cytostatic effects (G₂M accumulation/decreased colony formation). Conclusions: Combination of PDT and low‐toxicity drugs (such as aspirin) resulted in protracted inhibition of proteasome activity and induced apoptosis even in apoptosis‐resistant cancer cells.     

An in vitro method for increasing UV-tolerance in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) nucleopolyhedrovirus

A method for increasing tolerance to ultraviolet (UV) radiation in a strain of nucleopolyhedrovirus of cotton bollworm, Helicoverpa armigera (Hübner) (HearNPV) using a solar simulator is described. The Coimbatore isolate (CBE I) of HearNPV was subjected to a five-step sequence of selection to increase its UV tolerance. Each step consisted of irradiation of wet deposits of the virus to near UV (at energy level of 300W/m²), bioassay against second instar H. armigera larvae and propagation in early fifth instar larvae. Selection steps carried out at 15, 30, 60 and 90 minutes of exposure revealed that the continuous exposure of HearNPV-CBE I at low doses of UV irradiation (270–540 KJ/m²) did not significantly affect the virus activity as measured by its biological activity against second instar larvae. Selection at higher doses (1620 KJ/m²) led to loss of viral activity in the first two exposure cycles; however, there was retention of virulence coupled with increased tolerance to UV doses from third cycle onwards. Further, studies on the persistence of UV tolerant strain of HearNPV-CBE I in comparison with original strain showed that the tolerant strain had more persistence even after 7 days of weathering both under exposed (18% original activity remaining) and shaded (26% original activity remaining) condition on potted cotton plant.     

Hybridization of Antisense Oligonucleotides with α‐Sarcin Loop Region of Escherichia coli 23S rRNA

Binding of complementary oligonucleotides (ONs) with α‐sarcin loop region (2638–2682) of Escherichia coli 23S rRNA was investigated. Four of the tested pentadecanucleotides efficiently bound to target sequences with association rate and equilibrium constants ～ 10³ M^? 1s^? 1 and 10⁷ M^? 1, respectively. ON S5 (CGAGAGGACCGGAGU) complementary to the sequence 2658–2672 displayed the highest affinity to the target. Activation energy for binding of ON S5 was measured to be 11 kcal/mol; this value corresponds to ～ 10% of the calculated enthalpy of the local RNA structure unfolding in the presence of this oligonucleotide. The activation energy value is evidence for the heteroduplex formation to occur via strand displacement pathway; the initiation of heteroduplex formation requires disruption of 1–2 base pairs in RNA hairpin.     

Microbial flocculation, a potentially low-cost harvesting technique for marine microalgae for the production of biodiesel

Microbial flocculation is investigated as a separation technique for harvesting marine microalgae for the production of biodiesel. Organic carbon (acetate, glucose or glycerine) was used as substrate for the growth of flocculating microbes in situ. Under stress, due to nutrient depletion, these microbes produced extracellular polymeric substances that promote flocculation of the coccolithophorid alga, Pleurochrysis carterae. Maximum recovery efficiency was achieved at low concentration of organic substrate (0.1 g L⁻¹) and with a long mixing time (24 h); an average recovery efficiency of over 90% and a concentration factor of 226 were achieved. The recovery efficiency is positively correlated with mixing time (R ² = 0.90). The concentration factor is negatively correlated to the product of substrate concentration and mixing time (R ² = 0.73). The microalgae cells were not under stress and remained viable, thus potentially allowing media to be reused in large-scale processes without further treatment. Other advantages of the process are that no metallic flocculants were required and the organic substrates are readily available, e.g. glycerine is a by-product of biodiesel production and acetic acid may be produced by anaerobic digestion of the biomass residue after lipid extraction. Further research is required to optimise the process.     

Reduction of heteroduplex formation in PCR amplification

Heteroduplex formation is known to occur during mixed-template polymerase chain reaction (PCR) using universal primers, and may represent a serious problem in several PCR-based analyses. A common way to eliminate heteroduplex formation is to use reconditioning PCR. Because we detected that reconditioning PCR was not always sufficient to prevent heteroduplex formation, we focused on developing methods for the elimination of heteroduplexes during PCR. We detected that the heteroduplex to homoduplex ratio can be decreased by the addition of Taq polymerase and by a decrease in the number of PCR cycles. An appropriate combination of both of these approaches can be a method generally applicable to decrease the formation of heteroduplexes.     

Activity and feeding of Dotilla fenestrata (Brachyura: Ocypodidae) in a warm,temperate South African estuary

The activity of and consumption of organic material by the sand-bubbler crab Dotilla fenestrata was studied over neap and spring tides on a sheltered sand bank close to the mouth of the warm, temperate Kowie Estuary, South Africa. Crabs emerged from their burrows only after the tide receded, and it was light. Time to emergence therefore varied from about 30 minutes to three hours after exposure, depending on the time of low water in the early morning vs at midday. General activity of the crab population was longer on a spring tide (about five hours) than on a neap tide (about three hours). Maximum densities of active crabs were 140 m^–2 and 41 m^–2 on spring and neap tides, respectively. After emergence, crabs spent 60% to 80% of their time feeding. In the Kowie Estuary, D. fenestrata produced between 7 and 12 pseudofaecal pellets, average weight 0.0358 g per pellet, per minute. These pellets had a significantly lower organic and chlorophyll a content than the substratum, and it was estimated that crabs removed about 25% of the organic content from the sediment.     

Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism<Emphasis Type="Italic"> Deinococcus radiodurans</Emphasis>

We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous Rif^R mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration—suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to -rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Baldacci     

Biostoning of denims by Penicillium occitanis (Pol6) cellulases

The Pol6 mutant of Penicillium occitanis, secreting a large quantity of cellulases, was cultivated in fermentor using a local paper pulp as an inducer substrate. A high titer of extracellular cellulase activity was reached after a fed batch process: 23 IU ml⁻¹ filter paper activity, 21 IU ml⁻¹ CMCases activity (endoglucanase units) and 25 mg ml⁻¹ of proteins. Various tests were done to compare the action of the P. occitanis cellulases with those commercially available and with the traditional stonewashing process. This cellulase preparation was successfully applied in a biostoning process at an industrial scale. The abrasive effect of the P. occitanis cellulases was very uniform and with an efficiency comparable to that obtained by the commercial ones.