Evidence for sub-functionalization of tandemly duplicated XPB nucleotide excision repair genes in Arabidopsis thaliana.

Plants are continuously exposed to agents that can generate DNA lesions. Nucleotide Excision Repair (NER) is one of the repair pathways employed by plants to protect their genome, including from sunlight. The Xeroderma Pigmentosum type B (XPB) protein is a DNA helicase shown to be involved in NER and is also an essential subunitof the Transcription Factor IIH (TFIIH) complex. XPB was found to be a single copy gene in eukaryotes, but found as a tandem duplication in the plant Arabidopsis thaliana, AtXPB1 and AtXPB2. We aimed to investigate whether the XPB in tandem duplication was common within members of the Brassicaceae. We analyzed genomic DNA of species from different tribes of the family and the results indicate that the tandem duplication occurred in Camelineae tribe ancestor, of which A. thaliana belongs, at approximately 8 million years ago. Further experiments were devised to study possible functional roles for the A. thaliana AtXPB paralogs. A non-coincident expression profile of the paralogs was observed in various plant organs, developmental and cell cycle stages. AtXPB2 expression was observed in proliferating cells and clustered with the transcription of other components of the TFIIH such as p44, p52 and XPD/UVH6 along the cell cycle. AtXPB1 gene transcription, on the other hand, was enhanced specifically after UV-B irradiation in leaf trichomes. Altogether, our results reported herein suggest a functional specialization for the AtXPB paralogs: while the AtXPB2 paralog may have a role in cell proliferation and repair as XPB of other eukaryotes, the AtXPB1 paralog is most likely implicated in repair functions in highly specialized A. thaliana cells.

Biotechnology approaches for production of antiulcerogenic dihydro-epideoxyarteannuin B isolated from Artemisia annua L. / Ferramentas biotecnológicas para a produção de dihidro-epideoxiarteanuina B, um antiulcerogênico isolado de Artemisia annua L.

Methodologies were developed for the establishment and cultivation of Artemisia annua L (CPQBA 2/39 x PL5 hybrid) roots submitted to light conditions and genetic transformation performed with Agrobacterium rhizogenes (15834 and 8196 strains). The transgenic and non-transgenic (normal) roots were cultured in Murashige and Skoog (1962) medium, kept under different photoperiodic conditions and analyzed for evaluation of the antiulcerogenic dihydro-epideoxyarteannuin B (compound A) contents. The Dot Blot technique was used to confirm the transgenic nature of the roots. The plants¢s crude extracts were analyzed by Gas Chromatography coupled to Mass Spectrum (CG/MS). The chromatograms of the extracts taken from normal roots revealed the presence of dihydro-epideoxyarteannuin B and other compound (compound B). Photoperiods during cultivation influenced the production of these two compounds: under continuous darkness dihydro-epideoxyarteannuin B was intensely produced and the compound B present in small amounts, while on 16 h photoperiod, the inverse occurred. The quantification of dihydro-epideoxyarteannuin B by Gas Chromatography coupled to Flame Detector Ionization (CG/FID) revealed an approximately fivefold increase in the production of this compound by normal roots kept under continuous darkness compared to roots kept under 16 h light period. The terpene dihydro-epideoxiarteannuin B was not present in transgenic hairy roots.

Foram desenvolvidas metodologias para o estabelecimento e cultivo de raízes de Artemisia annua L. (híbrido CPQBA 2/39 x PL5). Estas raízes foram submetidas a diferentes condições de luz e a transformação genética com Agrobacterium rhizogenes (cepas 8196 e 15834). As raízes transgênicas e não-transgênicas (normais) foram cultivadas em meios de Murashige e Skoog (1962), mantidas sobre diferentes condições de fotoperíodo e analisadas para avaliação do conteúdo do composto antiulcerogênico dehidro-epideoxiarteanuína B (composto A). A confirmação do caráter transgênico das raízes foi obtida por Dot Blot. Os extratos dos materiais vegetais foram analisados por Cromatografia Gasosa acoplada a um Espectômetro de Massas (CG/EM). Os cromatogramas dos extratos das raízes normais revelaram a presença de dehidro-epideoxiarteanuína B e de um outro composto (composto B). As condições fotoperiódicas de cultivo influenciaram na produção destes dois compostos, sendo que sobre condição de escuro contínuo, dehidro-epideoxiarteanuína B foi intensamente produzido e o composto B foi detectado em pequenas proporções, enquanto que sob fotoperíodo de 16 horas, o inverso ocorreu. A quantificação de dehidro-epideoxiarteanuína B por Cromatografia Gasosa acoplada a um Detector de Ionização de Chamas (CG/FID) revelou um aumento de aproximadamente cinco vezes na produção deste composto pelas raízes normais cultivadas sobre escuro contínuo em relação às raízes cultivadas na presença de 16 horas de luz. O terpeno dehidro-epideoxiarteanuína B não estava presente nas raízes transgênicas.

Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis.

The xeroderma pigmentosum complementation group B (XPB) protein is involved in both DNA repair and transcription in human cells. It is a component of the transcription factor IIH (TFIIH) and is responsible for DNA helicase activity during nucleotide (nt) excision repair (NER). Its high evolutionary conservation has allowed identification of homologous proteins in different organisms, including plants. In contrast to other organisms, Arabidopsis thaliana harbors a duplication of the XPB orthologue (AtXPB1 and AtXPB2), and the proteins encoded by the duplicated genes are very similar (95% amino acid identity). Complementation assays in yeast rad25 mutant strains suggest the involvement of AtXPB2 in DNA repair, as already shown for AtXPB1, indicating that these proteins may be functionally redundant in the removal of DNA lesions in A. thaliana. Although both genes are expressed in a constitutive manner during the plant life cycle, Northern blot analyses suggest that light modulates the expression level of both XPB copies, and transcript levels increase during early stages of development. Considering the high similarity between AtXPB1 and AtXPB2 and that both of predicted proteins may act in DNA repair, it is possible that this duplication may confer more flexibility and resistance to DNA damaging agents in thale cress.