The rainbow trout metallothionein-B gene promoter: contributions of distal promoter elements to metal and oxidant regulation.

In this report, the contributions of the distal 5'-regulatory sequences of the rainbow trout (Oncorhynchus mykiss) metallothionein (tMT)-B gene promoter (-738 to +5) were studied. Transfection of the -738 promoter fragment in a rainbow trout hepatoma cell line (RTH-149) resulted in 4- to 5-fold greater activity compared to the proximal -137 promoter region. Mutation of the proximal MREa abolishes the basal activity of the -738 fragment indicating that the distal regulatory elements require a cooperative interaction with MREa. However, the fragments containing both distal MREs, c and d (positioning -570 and -680, respectively), or MREc alone could confer basal and metal-induced activity when fused to the TATA box. This suggests that these distal elements are functional and therefore may play a role as basal elements in their natural state. The trout MT genes are also induced by oxidants including H2O2, tBHP and tBHQ. The larger promoter fragment -738 responds to H2O2, while the -137 fragment does not. However, fusion of the isolated MREc fragment (-648 to -533) in its native orientation, upstream of the -137 promoter elicits a response to H2O2, although no response is seen with MREc in reverse. These data suggest that this distal fragment contains functional oxidant responsive elements which have resemblance to the mammalian antioxidant responsive element (AREs).

Isolation, characterization and disruption of the casein kinase II alpha subunit gene of Leishmania chagasi.

To elucidate the role played by casein kinase II in Leishmania survival, we have isolated and characterized the Leishmania chagasi casein kinase II alpha subunit cDNA, (L.c CKIIalpha). The 1083 bp coding region is flanked by 148 bp of 5' UTR and 1155 bp of 3' UTR. L.c CKIIalpha shows a remarkable degree of similarity with other isolated casein kinase II alpha subunit sequences. L.c CKIIalpha protein is encoded by a single copy gene that transcribes a mRNA of 2.4 kb. The 41.2 kDa L.c CKIIalpha protein expressed in vitro has been shown to be catalytically active. A single allele disruption of the L.c CKIIalpha gene that removes 94 bp from the coding region which contains one of the 15 conserved amino acids closest to the carboxy-terminus of the protein has been generated. This mutant is viable and results in a reduction of L.c CKIIalpha transcript levels over 14-fold and that of an iron superoxide dismutase mRNA by 5-fold. As well, the kinase activity of the single allele disrupted cells showed a 3-fold reduction as compared to the wild type cells suggesting a decrease in activity of the L.c CkIIalpha enzyme.

Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi.

Two cDNAs (FeSODA and FeSODB cDNAs) corresponding to superoxide dismutase (1.15.1.1., SOD) were isolated from a Trypanosoma cruzi cDNA library. Comparison of the deduced amino acid sequences with previously reported SOD protein sequences revealed that the T. cruzi open reading frames had considerable homology with FeSODs. The coding region of the T. cruzi FeSODB cDNA has been expressed in fusion with glutathione-S-transferase using an Escherichia coli mutant QC779, lacking both MnSOD and FeSOD genes (sodA sodB). Staining of native polyacrylamide gels for SOD activity of T cruzi crude lysate and the recombinant SOD suggests that this protein is an FeSOD. The recombinant enzyme also protected the E. coli mutant QC779 from paraquat toxicity. Northern blot analysis showed that FeSODB is differentially expressed, showing a higher level at the epimastigote stage of T. cruzi development; whereas, FeSODA is constitutively expressed at a lower level in all developmental stages. Furthermore, Southern hybridization shows that both FeSODA and FeSODB genes appear to be present in the T. cruzi genome as multiple repeating units (multi-copy gene family).

Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis.

We have isolated and characterized two superoxide dismutase (SOD) cDNAs from a Leishmania chagasi promastigote cDNA library using degenerate primers derived from conserved amino acid residues of previously isolated manganese and iron SODs. Comparison of these two L. chagasi SOD deduced amino acid sequences with previously isolated MnSOD and FeSOD amino acid sequences revealed that they have higher homology to, and complete conservation of, invariant residues found in iron-containing SODs. Southern blot analysis showed that one gene, L.c.FeSODA, is a single copy gene, whereas the other gene, L.c.FeSODB, belongs to a multi-gene family. Transcript levels and enzyme activities of L.c.FeSODA and L.c.FeSODB show differential stage expression, with higher levels present in the amastigote stage of the parasite compared to the promastigote stage. Expression of the L.c.FeSODs in an E. coli SOD null strain protected the bacteria against free radical generating agents. Overexpression of these FeSODs in L. chagasi parasites also showed enhanced protection against the free radical generating agents, paraquat and nitroprusside. The cloning, characterization and overexpression of the L.c.FeSODA and L.c.FeSODB genes and proteins demonstrates the possible role of SODs in Leishmania pathogenesis.

Functional analyses of the human metallothionein-IG gene. In vitro and in vivo studies.

We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region (-174 to +5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa. Transient transfection of HepG2 cells was complemented by in vitro transcription with rat liver nuclear extracts. In both systems, mutations of the TATA box and conserved core of metal responsive element (MRE)a were detrimental to hMT-IG promoter activity suggesting that both elements make significant contributions to hMT-IG transcription. Although MRE binding factors were active in vitro, further metal activation of MT promoter activity was accomplished only by in vivo metal treatment rather than addition of zinc in vitro. Southwestern blotting identified nuclear proteins in rat liver and HepG2 cells which physically interact with MREa in a zinc-dependent manner and could be responsible for MREa function in each system. In addition, the functional effects of the TATCA mutation correlate with altered physical interaction with TATA box-binding protein as observed using DNase I protection.