Efficient gene targeting in Drosophila with zinc-finger nucleases

This report describes high-frequency germline gene targeting at two genomic loci in Drosophila melanogaster, y and ry. In the best case, nearly all induced parents produced mutant progeny; 25% of their offspring were new mutants and most of these were targeted gene replacements resulting from homologous recombination (HR) with a marked donor DNA. The procedure that generates these high frequencies relies on cleavage of the target by designed zinc-finger nucleases (ZFNs) and production of a linear donor in situ. Increased induction of ZFN expression led to higher frequencies of gene targeting, demonstrating the beneficial effect of activating the target. In the absence of a homologous donor DNA, ZFN cleavage led to the recovery of new mutants at three loci-y, ry and bw-through nonhomologous end joining (NHEJ) after cleavage. Because zinc fingers can be directed to a broad range of DNA sequences and targeting is very efficient, this approach promises to allow genetic manipulation of many different genes, even in cases where the mutant phenotype cannot be predicted.     

Influence of natural complex compounds with hipolipidemic activity on expression of bacteria virulence factors

Natural complex compounds with hipolipidemic activity, having considerable inhibitory effect on expression of bacteria virulence factors were isolated. Inhibitory properties of the compounds with respect to pyocyanine and protease formation, as well as their influence on the quorum sensing mechanism in Chromobacterium violacium were shown.     

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.KEY WORDS: Ribosomal protein deficiency, Rps19, Rpl11, p53, ATR, RNR, Chk1, ATP, AMPK, Exogenous nucleosides