Enhanced Ascorbate Regeneration Via Dehydroascorbate Reductase Confers Tolerance to Photo-Oxidative Stress in Chlamydomonas reinhardtii.

The role of ascorbate (AsA) recycling via dehydroascorbate reductase (DHAR) in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress was examined. The activity of DHAR and the abundance of the CrDHAR1 (Cre10.g456750) transcript increased after moderate light (ML; 750 µmol m-2 s-1) or high light (HL; 1,800 µmol m-2 s-1) illumination, accompanied by dehydroascorbate (DHA) accumulation, decreased AsA redox state, photo-inhibition, lipid peroxidation, H2O2 overaccumulation, growth inhibition and cell death. It suggests that DHAR and AsA recycling is limiting under high-intensity light stress. The CrDHAR1 gene was cloned and its recombinant CrDHAR1 protein was a monomer (25 kDa) detected by Western blot that exhibits an enzymatic activity of 965 µmol min-1 mg-1 protein. CrDHAR1 was overexpressed driven by a HSP70A:RBCS2 fusion promoter or down-regulated by artificial microRNA (amiRNA) to examine whether DHAR-mediated AsA recycling is critical for the tolerance of C. reinahartii cells to photo-oxidative stress. The overexpression of CrDHAR1 increased DHAR protein abundance and enzyme activity, AsA pool size, AsA:DHA ratio and the tolerance to ML-, HL-, methyl viologen- or H2O2-induced oxidative stress. The CrDHAR1-knockdown amiRNA lines that have lower DHAR expression and AsA recycling ability were sensitive to high-intensity illumination and oxidative stress. The glutathione pool size, glutathione:oxidized glutathione ratio and glutathione reductase and ascorbate peroxidase activities were increased in CrDHAR1-overexpressing cells and showed a further increase after high-intensity illumination but decreased in wild-type cells after light stress. The present results suggest that increasing AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity in C. reinhardtii against photo-oxidative stress.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.

The tomato UV-damaged DNA-binding protein-1 (DDB1) is implicated in pathogenesis-related (PR) gene expression and resistance to Agrobacterium tumefaciens.

Plants defend themselves against potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs). However, the molecular mechanisms underlying this PAMP-triggered immunity (PTI) are largely unknown. In this study, we show that tomato HP1/DDB1, coding for a key component of the CUL4-based ubiquitin E3 ligase complex, is required for resistance to Agrobacterium tumefaciens. We found that the DDB1-deficient mutant (high pigment-1, hp1) is susceptible to nontumorigenic A. tumefaciens. The efficiency of callus generation from the hp1 cotyledons was extremely low as a result of the necrosis caused by Agrobacterium infection. On infiltration of nontumorigenic A. tumefaciens into leaves, the hp1 mutant moderately supported Agrobacterium growth and developed disease symptoms, but the expression of the pathogenesis-related gene SlPR1a1 and several PTI marker genes was compromised at different levels. Moreover, exogenous application of salicylic acid (SA) triggered SlPR1a1 gene expression and enhanced resistance to A. tumefaciens in wild-type tomato plants, whereas these SA-regulated defence responses were abolished in hp1 mutant plants. Thus, HP1/DDB1 may function through interaction with the SA-regulated PTI pathway in resistance against Agrobacterium infection.

The presence of traces of iron and copper ions during gamma-irradiation does not result in clear mutational hot spots in the lacI gene.

Oxidative radicals, which are produced during ionizing irradiation of DNA in water, damage the DNA and may result in mutations, which are in general randomly distributed. Alternatively, the addition of transition metal ions, like iron or copper, to DNA in combination with H(2)O(2) and a reducing agent also results in the production of oxidative radicals. Due to binding of the transition metal ions to DNA, the production of these radicals is very local, and results in a mutational spectrum in which the mutations are not randomly distributed. If transition metal ions are complexed to the DNA during irradiation, and react with radiation-induced species such as hydrogen peroxide, site-specific formation of.OH radicals on these sites may occur, leading to the formation of mutational hot spots. This study examines the influence of the presence of traces of iron or copper ions during gamma-irradiation of plasmid DNA in water, on the possible formation of mutational hot spots in the lacI gene. Comparison of the mutational spectra, after irradiation in the presence or in the absence of transition metal ions, shows that there are indeed relatively more positions in the lacI gene where more than one mutation occurs, suggesting formation of mutational hot spots in the presence of transition metal ions. However, the appearance of these hot spots is rather weak. Although in all three mutational spectra G:C to A:T mutations are predominant, there are also some differences between the types of mutations in these spectra. These differences in mutational spectra might reflect the different preferences of iron and copper ions to bind specific sites in the DNA. Indeed, there appears to be a high association of mutations at CC or GG sites in the mutational spectrum in the presence of copper ions, confirming the observation that copper binds preferably at two adjacent guanines in the DNA. It can be concluded from this study that the presence of small amounts of transition metal ions during gamma-irradiation influences the types and distribution of gamma-radiation-induced mutations, although no major mutational hot spots can be observed.

Radiation-induced carcinogenesis: studies using human epithelial cell lines.

It has proved difficult to develop suitable models to study radiation-induced carcinogenesis by using human epithelial cells. However, immortalised human epithelial cell lines have proved useful. Unirradiated cells from the human keratinocyte cell line (HPV-G) and the human embryonic lung cell line (L132) were found to be tumourigenic in T-cell-deficient mice; thus, they are not suitable for transformation studies. Human urothelial cell lines (SV-HUC-1, NT11, BC16) and the human thyroid epithelial cell line (HTori-3) were nontumourigenic. The urothelial cell lines were refractory to radiation-induced carcinogenesis, and only one small tumour was observed in 57 mice that received irradiated cells. Whereas tumours were not produced following irradiation of these urothelial cells, changes in anchorage-independent growth were observed after a single dose of 8 Gy gamma-irradiation but not after 2 or 4 Gy. Irradiation of the human thyroid epithelial cell line (HTori-3) in vitro resulted in tumour formation. Passaging of the cells in vitro before injection did not seem to be critical. Some of the cell lines derived from the primary thyroid tumours exhibited p53 mutations in exons 5, 6, 7, and 8, as detected by single-stranded conformational polymorphism (SSCP) analysis. Thus, the human thyroid epithelial cell line (HTori-3) looks promising as a model for investigating the molecular events in radiation-induced carcinogenesis.

Correlation of (6-4)photoproduct formation with transforming mutations in UV-irradiated Ha-ras.

Sites and types of mutations in relation to the amount of cyclobutane pyrimidine dimers (CPDs) and (6-4)-photoproducts in UV-irradiated normal Ha-ras sequences were investigated. Mouse BALB/C 3T3 cells were transfected with UV-irradiated pYN-mHras plasmids containing mouse normal Ha-ras sequences, and transformed foci developed. Direct DNA sequencing of the Ha-ras retrieved from the foci revealed that most mutations (23/24, 96%) took place at dipyrimidine sequences, and the C-->T transition at the 3'-cytosine in 5'-TC or 5'-CC sequences was predominant (17/24, 71%) in codons 12, 13 and 60. In codon 61, where 5'-TC or 5'-CC is absent, two mutations were found at the 5'-TT sequence. More (6-4)photoproducts were produced than CPDs in codons 12, 13 and 60, and more CPDs were produced than (6-4)photoproducts in codon 61. These results suggest that (6-4)photoproducts are the major lesion leading to the mutations in the mouse Ha-ras sequence and subsequent transformation of BALB/C 3T3 cells.

Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration, its inheritance and factors affecting these processes in Nicotiana plumbaginifolia.

Freshly isolated haploid mesophyll protoplasts of Nicotiana plumbaginifolia were transformed for kanamycin resistance. In 38% of the 224 transformants analysed, transmission of the NPTII gene occurred as a homozygous trait, while 62% of the transformants were heterozygous for the trait. In the first case, the foreign DNA integration predominantly (95%) resulted in monogenic inheritance. The second group was characterized by a significant (46%) proportion of multiple insertions. However, there was no clear-cut difference in the integration pattern between the two groups. Furthermore, transformation rates were increased by 4- to 10-fold when transformed diploid protoplasts were treated with UV light or with 3-aminobenzamide. The number of insertion sites was also increased by these treatments. These results shed further light on the fate of the foreign DNA in transformed plants and on means to control or manipulate the integration event(s).

Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid.

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.

Quantitative and molecular analyses of radiation-induced mutation in AS52 cells.

pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.

DNA-mediated gene transfer efficiency is enhanced by ionizing and ultraviolet irradiation of rodent cells in vitro. I. Kinetics of enhancement.

The enhancement effects of ionizing and ultraviolet radiation on the efficiency of DNA-mediated gene transfer were studied. The established cell line, Rat-2, consists of cells that are density-dependent contact-inhibited and produce flat monolayers in vitro. When these cells are infected with SV40 virus, a small fraction of cells becomes morphologically "transformed" due to the stable expression of the viral A-gene. Rat-2 cells are competent for DNA-mediated gene transfer, deficient in thymidine kinase activity (TK-), and will die in HAT selective media. Confluent Rat-2 cells were transfected with purified SV40 viral DNA (via calcium phosphate precipitation), irradiated with either X rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were nonlinear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X rays or 330 MeV/amu argon particles at the Berkeley BEVALAC showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. In both cases the enhancement contained a linear and a higher order component in dose, but the argon ions were at least twice more efficient than X rays in producing enhancement per unit dose. Rat-2 cells transfected with pOT-TK5, X-irradiated, and assayed for either TK transformation or A-gene transformation showed the same dose dependence for radiation enhancement. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK transformation by both X rays and ultraviolet radiation. SV40 A-gene products are not necessary for the radiation enhancement of the efficiency of gene transfer. This in vitro system will be used to study the lesions produced by ionizing radiation on mammalian cell DNA that may act as substrates for integration of exogenously introduced plasmid DNA.

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.

Enhanced transformation of human cells by UV-irradiated pSV2 plasmids.

Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation. UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.

Cell variants showing differential susceptibility to ultraviolet light--induced transformation.

Six variant clones isolated from a subclone of BALB/3T3-A31 clone were classified into three groups according to their different susceptibilities to cell transformation by ultraviolet light irradiation: highly susceptible, intermediately susceptible, and resistant. All variant clones showed similar susceptibility to cytotoxic effects induced by ultraviolet light.