Construction of DNA damage response gene pprI functiondeficient and function-complementary mutants in Deinococcus radiodurans

PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprl locus in the genome of the wild-type strain R1. The resultingpprl-deficient strain, designated YR1,was very sensitive to ionizing radiation. Meanwhile, the recombinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. radiodurans. The fragments containing complete pprl gene and 3‘-terminal deletion pprIΔ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprIΔ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. raiodurans R1, whereas YR1 pRADKpprIΔ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. radiodurans is not only useful to elucidating the relationship between domains and functions of PprI protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.     

Engineering <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> into biosensor to monitor radioactivity and genotoxicity in environment

Based on a genetically modified radioresistant bacteria Deinococcus radiodurans, we constructed a real time whole cell biosensor to monitor radioactivity and genotoxicity in highly radioactive environment. The enhanced green fluorescence protein （eGFP） was fused to the promoter of the crucial DNA damage-inducible recA gene from D. radiodurans, and the consequent DNA fragment （PrecA-egfp） carried by plasmid was introduced into D. radiodurans R1 strain to obtain the biosensor strain DRG300. This engineered strain can express eGFP protein and generate fluorescence in induction of the recA gene promoter. Based on the correlation between fluorescence intensity and protein expression level in live D. radiodurans cells, we discovered that the fluorescence induction of strain DRG300 responds in a remarkable dose-dependent manner when treated with DNA damage sources such as gamma radiation and mitomycin C. It is encouraging to find the widely detective range and high sensitivity of this reconstructed strain comparing with other whole cell biosensors in former reports. These results suggest that the strain DRG300 is a potential whole cell biosensor to construct a detective system to monitor the biological hazards of radioactive and toxic pollutants in environment in real time.     

Engineering Deinococcus radiodurans into biosensor to monitor radioactivity and genotoxicity in environment

Based on a genetically modified radioresistant bacteria Deinococcus radiodurans, we constructed a real time whole cell biosensor to monitor radioactivity and genotoxicity in highly radioactive environ-ment. The enhanced green fluorescence protein (eGFP) was fused to the promoter of the crucial DNA damage-inducible recA gene from D. radiodurans, and the consequent DNA fragment (PrecA-egfp) car-ried by plasmid was introduced into D. radiodurans R1 strain to obtain the biosensor strain DRG300. This engineered strain can express eGFP protein and generate fluorescence in induction of the recA gene promoter. Based on the correlation between fluorescence intensity and protein expression level in live D. radiodurans cells, we discovered that the fluorescence induction of strain DRG300 responds in a remarkable dose-dependent manner when treated with DNA damage sources such as gamma radiation and mitomycin C. It is encouraging to find the widely detective range and high sensitivity of this re-constructed strain comparing with other whole cell biosensors in former reports. These results suggest that the strain DRG300 is a potential whole cell biosensor to construct a detective system to monitor the biological hazards of radioactive and toxic pollutants in environment in real time.     

RadA： A protein involved in DNA damage repair processes of Deinococcus radiodurans R1

RadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein su-perfamily found in bacteria,archaea and eukarya. In Archaea,it plays a critical role in homologous re-combination process due to its RecA-like function. In Escherichia coli,it takes part in conjugational recom-bination and DNA repair but is not as important as that of archaea. Using PSI-BLAST searches,we found that Deinococcus radiodurans RadA had a higher similarity to that of bacteria than archaea and eukarya. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet,but had no effect on the resistance to hydrogen peroxide. Complementa-tion of the radA disruptant by both E. coli radA and D. radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype to gamma and UV radiation than that of the radA mutant,while the absence of the Lon protease domain exhib-ited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.     

Functional analysis of the sbcD （dr1921 ） gene of the extremely radioresistant bacterium Deinococcus radiodurans

In eukaryotes, the Mre11-Rad50-Nbs1 (MRN) complex, which resides at the crossroads of DNA repair and checkpoint signaling, rapidly forms prominent foci at damage sites following double-strand break (DSB) induction. This complex carries out the initial processing of the DSB ends. Mutations in the genes that encode components of this complex result in DNA-damage hypersensitivity, genomic in- stability, telomere shortening, and aberrant meiosis. Therefore, the MR proteins are highly conserved during evolution. The bacterial orthologs of Mre11 and Rad50 are the SbcD and SbcC proteins, respec- tively. Deinococcus radiodurans, an extremely radioresistant bacterium, is able to mend hundreds of radiation-induced DSBs. The SbcD and SbcC proteins were identified as the products of the Dr1921 and Dr1922 genes. Disruption of the sbcD gene, by direct reverse-orientation insertional mutagenesis tech- nology, remarkably increases the cells’ sensitivity to various types of DNA damaging agents, such as ionizing radiation, ultraviolet irradiation, hydrogen peroxide, and mitomycin C. We also provide evidence that the drSbcD protein plays an important role in both growth and DNA repair in this organ- ism, especially in repair of DSBs generated after cellular exposure to 6000 Gy of IR. These results demonstrate that the drSbcD protein plays an important role in DSBs repair in D. radiodurans.     

Characterization of a PDR type ABC transporter gene from wheat （Triticum aestivum L.）

DON, as a virulence factor, plays an important role in the infection of Fusarium graminearum in wheat. The infection ability of F. graminearum depends on its capacity of producing DON. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. In this study, GeneChip analysis indicated that an EST encoding an ATP-binding cassette （ABC） transporter was up-regulated by 45 times in a wheat landrace Wangshuibai, which is resistant to DON accumulation. A pair of EST-derived primers were designed based on the EST sequence, and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp, consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kD. BLAST analysis indicated that the gene may belong to pleiotropic drug resistance （PDR） sub-family, and hence designated as TaPDR1 （Triticum aestivum pleiotropic drug resistance）. TaPDR1 was located on chromosome 5A of wheat using nullisomic-tetrasomic lines of Chinese Spring. TaPDR1 was up-regulated by induction of both DON and F. graminearum. Expression patterns of TaPDR1 were different in wild-type Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON accumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress, such as JA and SA, nor by abiotic stresses, such as heat, cold, wounding and NaCI. However, TaPDR1 expression was regulated by Al^3＋ and [Ca^2＋], indicating that [Ca^2＋]1 might mediate the signal of TaPDR1 expression.     

Mechanism of oxidative damage to DNA by Fe-loaded MCM-41 irradiated with visible light

The mechanism of oxidative damage to deoxyribonucleic acid (DNA) by iron-containing mesoporous molecular sieves (MCM-41) irradiated with visible light was elucidated. Fe-loaded MCM-41 (Fe/MCM-41) was used as a photocatalyst and the damage to calf thymus DNA caused by hydrogen peroxide (H2O2) was studied. The damage and extent of oxidation of DNA were measured by high-performance liquid chromatography (HPLC) and intermediate products were detected by HPLC/electrospray ionization tandem mass spectrometry. Electron spin resonance was used to detect changes in reactive oxygen species and peroxidase catalytic spectrophotometry was used to determine the concentration of H2O2. The results indicated that Fe/MCM-41 efficiently activated H2O2 in solution at pH 4.0-8.0 under irradiation with visible light. The photocatalytic system degraded DNA most effectively at pH 5.0-6.0 but also operated at pH 8.0. At pH 4.2, the degree of DNA damage reached 25.65% after 5 h and the kinetic constant was 5.89×10 2 min 1. Damage to DNA was predominantly caused by hydroxyl radicals generated in the system. The mechanism of DNA damage is of potential concern to human health because it can occur in neutral solutions irradiated by visible light.     

Clustered DNA damage induced by protons radiation in plasmid DNA

Clustered DNA damage is considered as a critical type of lesions induced by ionizing radiation, which can be converted into the fatal or strong mutagenic complex double strand breaks (DSBs) during damage processing in the cells. The new data show that high energy protons produce more potentially lethal DSBs than low LET radiation. In this study, plasmid DNA were used to in-vestigate and re-evaluate the biological effects induced by the protons with the LET of ～3.6 keV/μm at the molecular level in vitro, including single strand breaks (SSBs), DSBs, isolated and clustered base damages. The results of complex DNA damage detections indicated that protons at the given LET value induce about 1.6 fold more non-DSB clustered DNA damages than the prompt DSB. The DNA damage yields by protons were greater than that by γ-rays, specifically by 6 fold for the isolated type of DNA damage and 14 fold for the clustered damage. Furthermore, the spectrum of damages was also demonstrated to be depended on the radiation quality, with protons producing more DSBs relative to clusters than do γ-rays.     

Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleurotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBlue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P. nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a ＂reporter＂ gene, green fluorescent protein gene can be successfully stably exoressed in this Pleurotus species.     

The R- and AR-indices： Complementing the h-index

Based on the foundation laid by the h-index we introduce and study the R- and AR-indices. These new indices eliminate some of the disadvantages of the h-index, especially when they are used in combina-tion with the h-index. The R-index measures the h-core’s citation intensity, while AR goes one step further and takes the age of publications into account. This allows for an index that can actually in-crease and decrease over time. We propose the pair (h, AR) as a meaningful indicator for research evaluation. We further prove a relation characterizing the h-index in the power law model.     

Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

Mutagenesis of Ser24 of Cytochrome b559 α Subunit Affects PSII Acitivies in Chlamydomonas reinhardtii

In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSII) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in α subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSII activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSII (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore, SDS-PAGE and Western-blotting analysis indicated that the expression levels of α subunit of Cyt b559, LHCII and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSII complex activity of oxygen evolution although it is not directly bound to heme group.     

Composition dependence of viscosity for Al（1-x）Mgx（0≤x≤0. 10） alloys

Viscosities of molten Al（1-x）Mgx（0≤x≤0. 10 alloys have been measured in the temperature range of 973 K--1173 K by a torsional oscillation cup method. The viscosity dependence on temperature for Al（1-x）Mgx（0≤x≤0. 10 melts obeys Arrhenius equation. The viscosity increases with increasing magnesium concentration in the investigated system. There is an important relationship between viscosity and its glass-forming ability for metallic melt.     

Formation of ζ phase in Cu-Ge peritectic alloys

Rapid growth behavior of ζ phase has been investigated in the undercooling experiments of Cu-14%Ge, Cu-15%Ge, Cu-18.5%Ge and Cu-22%Ge alloys. Alloys of the four compositions obtain the maximum undercoolings of 202 K（0.17TL）, 245 K（0.20TL）, 223 K（0.20TL） and 176 K（0.17TL）, respectively. As the content of Ge increases, the microstructural transition of ＂a（Cu） dendrite ＋ ζ＂ peritectic phase → ζ＂ peritectic phase →, ζ dendrite ＋ （ε＋ζ） eutectic＂ takes place in the alloy at small undercooling, while the microstructural transition of ＂fragmented α （Cu）dendrite ＋ ζ peritectic phase →, ζ peritectic phase →ζ dendrite ＋ ε phase＂ happens in the alloy at large undercooling. EDS analysis of the Ge content in peritectic phase indicates that undercooling enlarges the solid solubility of ζ rdendrite, which leads to a decrease in the Ge content in ζ phase as undercooling increases. In the Cu-18.5%Ge alloy composed of ζ peritectic phase, the Ge content in ζ phase increases when undercooling increases, which is due to the restraint of the Ge enrichment on the grain boundaries by high undercooling effect.     

Icariin suppresses bone resorption activity of rabbit osteoclasts in vitro

The effect of icariin on the bone resorption activity of rabbit osteoclasts is assessed in vitro. Osteoclasts were isolated from Japanese white rabbits and cultured on plates with a sterilized bone slice in each well. After treatment with icariin at various concentrations, the bone resorption activity of osteoclasts was evaluated by examining pit areas, superoxide anion (·O2-) generation, size and number of actin rings and intracellular calcium concentration [Ca2 ]i. As revealed by these data, icariin elicited continuous decline of [Ca2 ]i, making actin ring constricted and ·O2- generation decreased. These events resulted in smaller and fewer pits which indicate suppressed bone resorption activity of rabbit osteoclasts by icariin.     

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using 3＇-RACE technology and named AtKP1 （for Arabidopsis kinesin protein 1）. This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coil and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.     

Molecular identification and phylosenetic position of Cuora yunnanensis

The Yunnan box turtle （Cuora yunnanensis, Boulenger, 1906）, which has drawn much attention in conservation biology, was regarded as extinct since it was previously known only from 12 specimens collected in Yunnan, China, before 1908. Recently, live specimens have been discovered which are suggested to be C. yunnanensis. To determine whether the newly discovered specimens are really C. yunnanensis, we have established a molecular phylogeny, with a 1725-bp fragment of mitochondrial DNA, using samples from three live individuals of C. yunnanensis, together with sequence data from a museum specimen of C. yunnanensis （MNHN 1907.10） and other members of the genus Cuora. We found that the three newly discovered individuals and the old museum specimen of C. yunnanensis are very similar both in morphology and in mitochondrial DNA sequence, suggesting that the three new individuals are the very C. yunnanensis, and thus the species is not extinct. Our phylogenetic analysis also demonstrates that C. yunnanensis is not of recent hybrid origin, but rather represents a distinct evolutionary lineage.