Elisabete José Vicente

Further characterization of the yeast pso4-1 mutant: interaction with rad51 and rad52 mutants after photoinduced psoralen lesions

Thepso4-1 mutant was characterized as deficient in some types of recombination, including gene conversion, crossing over, and intrachromosomal recombination. The mode of interaction betweenpso4-1 andrad51 and betweenpso4-1 andrad52 mutants indicated that thePSO4 gene belongs to theRAD52 epistasis group for strand-break repair. Moreover, the presence of thepso4-1 mutation decreased 8-MOP-photoinduced mutagenesis of therad51 andrad52 mutants. Complementation tests using heterozygous diploid strains showed that thePso4 protein might interact with theRad52 protein during repair of 8-MOP photolesions. Thepso4-1 mutant, even though defective in inter- and intea-chromosomal recombination, conserves the ability for plasmid integration of circular and linear plasmid DNA. On the other hand, similar to therad51 mutant,pso4-1 was able to incise but did not restore high-molecular-weight DNA during the repair of cross links induced by 8-MOP plus UVA. These results, together with those of previous reports, indicate that thePSO4 gene belongs to theRAD52 DNA repair group and its product participates in the DNA rejoining step of the repair of cross-link lesions, which are crucial for induced mutagenesis and recombinogenesis.

Endostatin‐ and interleukin‐2‐expressing retroviral bicistronic vector for gene therapy of metastatic renal cell carcinoma

Metastatic renal cell carcinoma (mRCC) is one of the most treatment‐resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)‐2 using an orthotopic metastatic RCC mouse model.

A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal‐regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram‐positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram‐negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20‐ to 53‐fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram‐negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010;107: 469–477.