[The role of ZF5 and CGGBP-20 transcription factors in expression regulation of human FMR1 gene responsible for X-fragile syndrome].

The human FMR1 gene encodes an RNA-binding protein taking part in translation regulation. The 5'-untranslated region of FMR1 gene contains a large number of tandem repeats of GCC triplets (5-50) which increasing (more then 200) is responsible for X-fragile syndrome (human congenital anomaly). As it has been shown earlier, al least two transcription factors (ZF5 and CGGBP-20) are capable of interacting specifically with GCC-repeats in regulatory regions of some genes. In this work, their roles in FMR1 gene expression regulation were studied. It was demonstrated by electrophoretic mobility shift assay that ZF5 recombinant protein specifically bound with GCC-triplet repeats (GCC9). Tissue-specific distributions of ZF5 and FMR1 proteins are very overlapped in mammalian. Inhibition of ZF5 expression in HepG2 cells (by RNA interference) leads to at least 1.5 times stimulations of FMR1 gene expression in these cells. To estimate the contribution of GCC-triplet repeats in FMR1 gene expression regulation we used two alternative variants of genetic construction: containing luciferase reporter gene under 5'-regulatory region fragment devoid of GCC-triplet repeats or including the GCC9 nucleotide sequence. HepG2 cells were co-transfected by these constructions and expressions vectors of ZF5 or (and) CGGBP-20 respectively. It was found that ZF5 downregulated the activity of 5'-regulatory region of FMR1 gene in both cases (acting probably through canonic 5'-GCGCGC3' sites). The presence of GCC-triplet repeats in the construction weakens this ZF5 effect. CGGBP-20 downregulates the activity of 5'-region of FMR1 gene in the presence of GCC-triplets only. The data obtained evidently indicate differently directed ZF5 effects on FMR1 gene expression and suggest the mechanism to explain the earlier demonstrated phenomenon about increasing of mRNA level in permutation FMR1 allele carries.

[Transcription factor ZF5 regulates expression of mammalian gene containing GCC-triplet repeats in 5'-regulatory region in human hepatoma HepG2 cells].

Some nuclear proteins of human HeLa and HepG2 cells are capable of binding to GCC-triplet repeats--(GCC)n > 3 in 5'-regulatory regions of a number of mammalian genes--G-C-elements. According to our previous data, nucleotide sequence (GCC)4 in promoter of mouse ribosomal protein L32 gene (rpL32) between 17 and 6 bp upstream of transcription start site interacts to nuclear proteins from HepG2 cells, and may be considered as a GCC-element. We suggest that one of those proteins, with molecular weight about 52 kDa, which may interact with rpL32 GCC-element, is a known conservative mammalian transcription factor ZF5. DNA-binding domain of ZF5 contains a few Kruppel-like Zn-fingers (Cys2His2-type) interacting with the GC-rich nucleotide sequences in 5'-regulatory regions of a number of mammalian genes. Our results (obtained by EMSA) showed that recombinant GST-ZF5 fused protein containing ZF5 DNA-binding domain specifically binds a few GS-rich sequences: (GCC)g-9riplet repeats, 5'-GCGCGC-3' (known ZF5 consensus binding site) and (more preferable) the fragment (-24...+1 bp) of rpL32 promoter. The high affinity of ZF5 DNA-domain binding with the latter may be explained by the presence in this fragment of two overlapped subsequences, each being capable of binding to ZF5: (GCC)4 and 5'-GCGCGC- 3'. Zf5 cDNA was cloned from HepG2 cells by RT-PCR method, and then used for construction of the gene expression vector. It has been shown that Zf5 cDNA expression vector specifically down-regulates (in luciferase assays) the activity of rpL32 promoter (-155...+159) including the above mentioned GC-rich subsequences by cotransfection of HepG2 cells. Therefore, our results enable us to consider GCC-elements as a novel class of ZF5 targets in 5'-regulatory regions of mammalian genes.

[Proteomic analysis of two isogenic Vibrio cholerae of the classical biovar with the alternative expression of virulence genes].

At carrying out the proteomic analysis of two isogenic Vibrio cholerae Dakka35 of the classical biovar itwas revealed, that toxigenic (1 type) and nontoxigenic (2 type) clones differ from each other not only the expression ofgenes of exopolysaccharide, motility, and soluble haemagglutinin/protease, but also change of activity about other 60 genes. Among 11 identified proteins 5 are the enzymes participating in a metabolism cells. Besides it is revealed, that clones 2 types of Dakka35 strain synthesize in a more level of OmpU and TolC proteins, which provide their more significant stability to action of bile in comparison with clones of 1 type. It was shown, that bile serves as a signal from an environment for switching of gene expression of the genes, coding production of factors as virulence, and carrying out protective function of bacterial cell.