A new reliable reference gene UBA52 for quantitative real-time polymerase chain reaction studies in pyloric cecal tissues of the starfish Asterias rubens.

The starfish Asterias rubens is one of the most abundant echinoderm species in the White, Barents, North, and Baltic Seas. This species is an important component of marine ecosystems and a model object for certain biological studies, in particular those requiring quantitative estimation of gene expression. As a rule, expression at the transcriptional level is estimated by real-time qPCR using the ΔΔCt method, which allows the comparison of the copy number of target gene transcripts in samples with unknown mRNA/cDNA concentration. Application of this method requires normalization of the results relative to genes with stable expression levels (reference genes). The identification of reference genes is still a challenging task since data of this kind are missing for certain taxa, whereas the use of "standard" endogenous control genes without additional tests might lead to erroneous conclusions. We performed a preliminary analysis of the expression of many housekeeping genes in the pyloric ceca of A. rubens by high-throughput sequencing under normal and heat shock conditions. For one of them, the ubiquitin gene UBA52, low variation of expression (not greater than 2-fold) was shown using real-time qPCR. Tissues of pyloric ceca of normal adults and underyearlings and of adults after heat shock were used. The data obtained suggest that the UBA52 gene may be used as reference for normalization of gene expression at the mRNA level in the starfish A. rubens and probably in closely related species.

[Dimeric bisberzimidazoles: cytotoxicity and effects on DNA methylation in normal and cancer human cells].

Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a small extent stimulate the proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) display ability to penetrate into the nucleus of HeLa and F-977 cells and accumulate in various parts of the nuclei. DB(11) is not able to penetrate into the nuclei of these cells. The incubation of F-977 cells with 26 microM of DB(1) or DB(3) led to a decrease of the methylation of 18S rRNA gene, which is located in the region of DB(1) and DB(3) accumulation. A similar effect produces the same concentration of DB (3) in the F-977 cells. However, the overall level of genomic DNA methylation was not changed. These data suggest that DB(n) can be directed to act on specific genes demethylation and in the future may selectively inhibit the proliferation of cancer cells.

[Differential expression of genes that encode glycolysis enzymes in kidney and lung cancer in humans].

Glycolysis is a main catabolic pathway of glucose metabolism, accompanied by ATP synthesis. More than 30 enzymes are involved in glycolysis, and genes that encode them can be considered housekeeping genes due to the high conservatism and evolutionary antiquity of the process. We studied the expression of these genes in kidney papillary cancer and planocellular lung cancer via the bioinformatic analysis of transcriptome database and method of quantitative real time PCR. Quantitative analysis of mRNA level demonstrated that only a part ofgenes that encode glycolysis enzymes maintain relatively stable mRNA level, including the HK1, ADPGK, GPI, PGK1, and PKM2 genes in kidney papillary cancer and the ADPGK, ALDOA, GAPDH, PGK1, BPGM, ENO1, and PKM2 genes in planocellular lung cancer. The frequent increase in the mRNA expression of PFKP, ALDOA, and GAPDH genes in kidney cancer, as well as the GPI gene in lung cancer, were detected for the first time by real time PCR. For other genes, their differential expression was demonstrated; the cases of both a decrease and increase in the mRNA level were detected. Thus, several genes that can be used as control genes in transcriptome analysis by real time PCR in kidney and lung cancer, as well as a number of differentially expressed genes that can be potential oncomarkers, were identified.

[Purification and site-directed mutagenesis of DNA methyltransferase SssI].

Prokaryotic DNA methyltransferase SssI (M.SssI) methylates C5 position of cytosine residue in CpG sequences. To obtain functionally active M.SssI and its mutants as His6-tagged proteins, bacterial strains have been produced. To test a possible role of Ser300 in recognition of CpG site by this enzyme, M.SssI mutants containing Ser300 replacements with Gly or Pro have been obtained. These replacements have practically no effect on DNA binding and methylation by M.SssI except small disturbance of DNA binding affinity in the case of S300P mutant. It indicates that there are no interactions of both the side chain and, probably, the main chain of Ser300 with DNA. A replacement of highly conserved Va1188 residue with Ala has been performed. Vall88 may participate in the stabilization of the flipped target cytosine during reaction. The replacement results in a 5-fold decrease of dissociation constant of the enzyme-substrate complex and a 2-fold decrease of initial velocity of DNA methylation. Though there are no noticeable differences in the functioning of the mutant in comparison with the wild-type enzyme, the formation of contact between Val 188 and cytosine could not be excluded. In the case of V 188A mutant the contact may be probably formed between Ala and cytosine residue.