Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta . Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo . Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.     

Comparison of acceptor activity of rabbit liver tRNA in ontogenesis

The rabbit liver tRNA was studied in such functional states when there occurred essential qualitative and quantitative changes in the protein synthesis. On the basis of the maximum level of aminoacylation it is established that the contents of corresponding biologically active individual tRNA in the total preparation for adult animals is 1.5-3 times as high as that for 3-5 day rabbits. Exception is alanine tRNA whose number decreases sharply in the total preparation of the liver tRNA of one- and two-year animals. No correlation was found for any age group between the quantitative ratio of active individual tRNA and the amino acid composition of albumins whose synthesis in the liver is intensive. Thus, the synthesis and, respectively, the content of certain types of tRNA in the liver characterized by a wide spectrum of proteins with different functions are governed by other regularities, whose search is one of the most difficult and at the same time high-promising problems.     

Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable and sequence-specific complexes with duplex DNA in a very salt-dependent manner. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with supercoiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate DNA duplexes in a virtually sequence-unrestricted manner.     

Duplex DNA capture

This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques.     

Infection with Wolbachia does not influence crossing-over in Drosophila melanogaster

The influence of endosymbiotic bacterium Wolbachia on the recombination processes in the sex chromosome of Drosophila melanogaster on the region between white and cut genes has been studied. The analysis of the crossing-over frequency in various variants of strain crossing, infected and noninfected, with bacteria has been carried out. The results have shown the absence of the influence of infection with Wolbachia on the frequency of crossing-over in the studied region of the X chromosome of D. melanogaster.     

The temporal and spatial pattern of histone H3 phosphorylation at serine 28 and serine 10 is similar in plants but differs between mono- and polycentric chromosomes

Immunolabeling using site-specific antibodies against phosphorylated histone H3 at serine 10 or serine 28 revealed in plants an almost similar temporal and spatial pattern of both post-translational modification sites at mitosis and meiosis. During the first meiotic division the entire chromosomes are highly H3 phosphorylated. In the second meiotic division, like in mitosis, the chromosomes contain high phosphorylation levels in the pericentromeric region and very little H3 phosphorylation along the arms of monocentric species. In the polycentric plant Luzula luzuloides phosphorylation at both serine positions occurs along the whole chromosomes, whereas in monocentric species, only the pericentromeric regions showed strong signals from mitotic prophase to telophase. No phosphorylated serine 10 or serine 28 was detectable on single chromatids at anaphase II resulting from equational segregation of rye B chromosome univalents during the preceding anaphase I. In addition, we found a high level of serine 28 as well as of serine 10 phosphorylation along the entire mitotic monocentric chromosomes after treatment of mitotic cells using the phosphatase inhibitor cantharidin. These observations suggest that histone H3 phosphorylation at serine 10 and 28 is an evolutionarily conserved event and both sites are likely to be involved in the same process, such as sister chromatid cohesion.