Expression of the SOD gene from Trichoderma harzianum in Saccharomyces cerevisiae

Superoxide dismutases are metalloproteins which play a major role in defense against oxygen radicalmediated toxicity in aerobic organisms. Such proteins are important endogeneity cytoprotection factor involving defence. A 751-bp full-length cDNA sequence of an SOD gene was isolated from the Trichoderma harzianum. The full-length cDNA of the SOD gene consists of one 465-bp open reading frame nucleotide, which encodes a 15.7-kDa polypeptide consisting of 154 amino acid residues. Sequence analysis revealed that SOD gene has more than 72%-86% amino acid sequence homology with those of other fungi. The SOD gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2-SOD. SOD expressed by pYES2-SOD was induced by galactose. We test whether SOD could offer abiotic stress resistance when it was introduced into yeast ceils. A transgenic yeast harboring T. harzianum SOD was generated under the control of a constitutively expressed GAL promoter. The results indicated that SOD yeast transformants had significantly higher resistance to salt and drought stress.

Induced pluripotent stem cell-derived motor neurons from amyotrophic lateral sclerosis(ALS)patients carrying different superoxide dismutase 1 mutations recapitulate pathological features of ALS

Background:Investigations of the pathogenic mechanisms in motor neurons(MNs)derived from amyotrophic lateral sclerosis(ALS)disease-specific induced pluripotent stem(iPS)cell lines could improve understanding of the issues affecting MNs.Therefore,in this study we explored mutant superoxide dismutase 1(SOD1)protein expression in MNs derived from the iPS cell lines of ALS patients carrying different SOD1 mutations.Methods:We generated induced pluripotent stem cell(iPSC)lines from two familial ALS(FALS)patients withSOD1-V14M andSOD1-C111Y mutations,and then differentiated them into MNs.We investigated levels of the SOD1 protein in iPSCs and MNs,the intracellular Ca2+levels in MNs,and the lactate dehydrogenase(LDH)activity in the process of differentiation into the MNs derived from the controls and ALS patients’iPSCs.Results:The iPSCs from the two FALS patients were capable of differentiation into MNs carrying different SOD1 mutations and differentially expressed MN markers.We detected high SOD1 protein expression and high intracellular calcium levels in both the MN and iPSCs that were derived from the twoSOD1 mutant patients.However,at no time did we observe stronger LDH activity in the patient lines compared with the control lines.Conclusions:MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis,providing a cell-based disease model to further elucidate disease pathogenesis and explore gene repair coupled with cell-replacement therapy.Incremental mutant expressions of SOD1 in MNs may have disrupted MN function,either causing or contributing to the intracellular calcium disturbances,which could lead to the occurrence and development of the disease.