Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

Embryonic inheritance of the chromatin organisation of the imprinted H19 domain in mouse spermatozoa

Insulin-like growth factor 2 (Igf 2) and H19 genes are oppositely imprinted and as such have been most extensively studied imprinted genes both genetically and at the molecular level. Imprints of the H19 gene, being established during spermatogenesis, are epigenetically transmitted to the somatic cells of the embryo. Current hypotheses attempting to explain the allele-specific silence of the H19 gene include DNA methylation and chromatin condensation. In order to understand the molecular basis of H19 epigenesis, it is crucial to identify the markings in the chromatin organising the imprinted domain in spermatozoa. Using Micrococcal nuclease (MNase), DNase I and Methidiumpropyl-EDTA. iron II (MPE·Fe(II)) as chromatin probes, we demonstrate that in mouse epididymal spermatozoa, at least 4 kb DNA upstream of the H19 ‘cap’ site, containing the imprinted and differentially methylated domain (DMD), is heterochromatic. The cleavage sites in this domain (−2 to −4 kb) exhibit ~425 bp periodicity. This structure is maintained in the paternal allele of normal embryos and is disrupted at −2.2, −2.65 and at −3.5 kb in embryos maternally disomic for the distal end of chromosome 7 (MatDp 7). The hypersensitive sites in chromatin precisely register the MPE·Fe(II) cleavage sites in chromosomal DNA. Therefore, the DNA sequences in the imprinted domain constrain the chromatin structure in a way similar to that of 1.688 g/cm³ Drosophila satellite chromatin. In addition, we find that condensation of the paternal allele correlates with methylation-dependent alteration in the structure of DNA sequences in DMD. These results suggest that CpG-methylation induces localised changes in DNA conformation and these facilitate consequent remodelling of chromatin thereby allowing the paternal and maternal H19 alleles to be distinguished.     

Role of mutation Y6F on the binding properties of Schistosoma japonicum glutathione S-transferase

The role of the hydroxyl group of tyrosine 6 in the binding of Schistosoma japonicum glutathione S-transferase has been investigated by isothermal titration calorimetry (ITC). A site-specific replacement of this residue with phenylalanine produces the Y6F mutant, which shows negative cooperativity for the binding of reduced glutathione (GSH). Calorimetric measurements indicated that the binding of GSH to Y6F dimer is enthalpically driven over the temperature range investigated. A concomitant net uptake of protons upon binding of GSH to Y6F mutant was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The entropy change is favorable at temperatures below 26 °C for the first site, being entropically favorable at all temperatures studied for the second site. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative ΔC°_p1=−3.45±0.62 kJ K⁻¹ mol⁻¹ for the first site, whereas a small ΔC°_p2=−0.33±0.05 kJ K⁻¹ mol⁻¹ for the second site was obtained. This large heat capacity change is indicative of conformational changes during the binding of substrate.     

DNA damage and repair in gastric cancer--a correlation with the hOGG1 and RAD51 genes polymorphisms

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by ³²P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N⁶-yl]-aristolactam I, 7-[deoxyadenosin-N⁶-yl]-aristolactam II and 7-[deoxyguanosin-N²-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10⁸ nucleotides in liver and 95–4598 adducts/10⁸ nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10⁻⁶ in liver compared with the MFs of 78–1319 × 10⁻⁶ that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.