Joanna Trzcinska-Danielewicz

Yeast transcription factor Oaf1 forms homodimer and induces some oleate-responsive genes in absence of Pip2

Genes encoding peroxisomal proteins in the yeast Saccharomyces cereviasiae are induced in the presence of oleate in growth medium. This induction is known to be mediated by the binding of a heterodimer of transcription factors Oaf1 and Pip2 to an upstream activating sequence called ORE (oleate response element). By analyzing expression of nine ORE-containing genes we show that the presence of an ORE sequence is not sufficient to confer oleate inducibility, as three such genes were in fact expressed constitutively. Moreover, some of the oleate-inducible genes undergo activation even in the absence of Pip2. Using coimmunoprecipitation we show that, when Pip2 is missing, Oaf1 may form homodimers which apparently substitute for the Oaf1-Pip2 heterodimer.

Controlled delivery of BID protein fused with TAT peptide sensitizes cancer cells to apoptosis

BackgroundLow cellular level of BID is critical for viability of numerous cancer cells. Sensitization of cells to anticancer agents by BID overexpression from adenovirus or pcDNA vectors is a proposed strategy for cancer therapy; however it does not provide any stringent control of cellular level of BID. The aim of this work was to examine whether a fusion of BID with TAT cell penetrating peptide (TAT-BID) may be used for controlled sensitization of cancer cells to anticancer agents acting through death receptors (TRAIL) or DNA damage (camptothecin). Prostate cancer PC3 and LNCaP, non-small human lung cancer A549, and cervix carcinoma HeLa cells were used in the study.MethodsUptake of TAT-BID protein by cells was studied by quantitative Western blot analysis of cells extracts. Cells viability was monitored by MTT test. Apoptosis was detected by flow cytometry and cytochrome c release assay.ResultsTAT-BID was delivered to all cancer cells in amounts depending on time, dose and the cell line. Recombinant BID sensitized PC3 cells to TRAIL or, to lesser extent, to camptothecin. Out of remaining cells, TAT-BID sensitized A549, and only slightly HeLa cells to TRAIL. None of the latter cell lines were sensitized to camptothecin. In all cases the mutant not phosphorylable by CK2 (TAT-BIDT59AS76A) was similarly efficient in sensitization as the wild type TAT-BID.ConclusionsTAT-BID may be delivered to cancer cells in controlled manner and efficiently sensitizes PC3 and A549 cells to TRAIL. Therefore, it may be considered as a potential therapeutic agent that enhances the efficacy of TRAIL for the treatment of prostate and non-small human lung cancer.

Rhythmic Diel Pattern of Gene Expression in Juvenile Maize Leaf

Background Numerous biochemical and physiological parameters of living organisms follow a circadian rhythm. Although such rhythmic behavior is particularly pronounced in plants, which are strictly dependent on the daily photoperiod, data on the molecular aspects of the diurnal cycle in plants is scarce and mostly concerns the model species Arabidopsis thaliana. Here we studied the leaf transcriptome in seedlings of maize, an important C4 crop only distantly related to A. thaliana, throughout a cycle of 10 h darkness and 14 h light to look for rhythmic patterns of gene expression. Results Using DNA microarrays comprising ca. 43,000 maize-specific probes we found that ca. 12% of all genes showed clear-cut diel rhythms of expression. Cluster analysis identified 35 groups containing from four to ca. 1,000 genes, each comprising genes of similar expression patterns. Perhaps unexpectedly, the most pronounced and most common (concerning the highest number of genes) expression maxima were observed towards and during the dark phase. Using Gene Ontology classification several meaningful functional associations were found among genes showing similar diel expression patterns, including massive induction of expression of genes related to gene expression, translation, protein modification and folding at dusk and night. Additionally, we found a clear-cut tendency among genes belonging to individual clusters to share defined transcription factor-binding sequences. Conclusions Co-expressed genes belonging to individual clusters are likely to be regulated by common mechanisms. The nocturnal phase of the diurnal cycle involves gross induction of fundamental biochemical processes and should be studied more thoroughly than was appreciated in most earlier physiological studies. Although some general mechanisms responsible for the diel regulation of gene expression might be shared among plants, details of the diurnal regulation of gene expression seem to differ between taxa.

Time schedule-dependent effect of the CK2 inhibitor TBB on PC-3 human prostate cancer cell viability

Inhibitors of CK2 kinase inhibit cell proliferation and induce apoptosis in numerous cancer cell lines. Due to these properties, they are considered potentially useful in anticancer therapy. In this study, we show that the exact effect of the specific CK2 inhibitor TBB on PC-3 human prostate cancer cell viability depends on the time schedule of administration: it was not observed when the treatment was directly followed by the viability assay but it appeared when the treatment and the assay were separated by a 24-h incubation without the inhibitor. Such a pattern was maintained when the TBB treatment was combined with either camptothecin or TRAIL. The time schedule-dependence of cell viability was not reflected by a similar dependence of induction of apoptosis. Despite this, the schedule in which a treatment with the CK2 inhibitor precedes that with an anticancer drug seems to be a good choice for a potential therapy against androgen-refractory prostate cancer.

Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces cerevisiae

Murine DNA methyltransferases Dnmt1 and Dnmt3a were expressed in the yeast Saccharomyces cerevisiae. Adjustment to yeast preferences of the nucleotide sequences upstream and downstream of the translation initiation sites of both cDNAs was needed to obtain significant levels of the methyltransferases. Both proteins were correctly localized to the nucleus and their presence had no measurable influence on the functioning of yeast cells. Both Dnmt1 and Dnmt3a expressed in yeast cells were enzymatically active in vitro, and in vivo in the genomic DNA of the transgenic S. cerevisiae ca. 0.06% and 0.4%, respectively, of cytosines became methylated. This level of DNA methylation is about 100‐ to 10‐fold less than that observed in mammalian cells. The constructed system may be used to investigate the in vivo specificity of individual mammalian DNA methyltransferases and to search for additional factors needed to allow more efficient in vivo methylation of chromatin‐contained DNA and to study their mechanism of action. Copyright

Synergy of BID with doxorubicin in the killing of cancer cells

Overexpression of the BH3-interacting domain death agonist (BID) protein sensitizes certain cancer cell lines to apoptosis induced by anticancer agents, particularly by those acting through death receptors (e.g. TRAIL). Previously, we showed that recombinant BID fused with TAT cell penetrating peptide (TAT-BID) allowed for controlled delivery of BID to different cancer cell lines and moderately sensitized some of them to TRAIL or slightly to camptothecin. In the present study, we showed that TAT-BID delivered to HeLa cells strongly sensitized them to doxorubicin, as identified by cell viability and apoptosis assays. Another cell line sensitized to doxorubicin was PC3, whereas A549 and LNCaP cells were sensitized moderately or not at all, respectively. Sensitization was more pronounced at 1 μM doxorubicin administered for 48 h than for lower doses and shorter treatments. TAT-BID and doxorubicin may thus be considered as a potential therapeutic combination for cervical carcinoma and advanced prostate cancer treatment.

Global analysis of gene expression in maize leaves treated with low temperature. II. Combined effect of severe cold (8 °C) and circadian rhythm

Key messageIn maize seedlings, severe cold results in dysregulation of circadian pattern of gene expression causing profound modulation of transcription of genes related to photosynthesis and other key biological processes.AbstractPlants live highly cyclic life and their response to environmental stresses must allow for underlying biological rhythms. To study the interplay of a stress and a rhythmic cue we investigated transcriptomic response of maize seedlings to low temperature in the context of diurnal gene expression. Severe cold stress had pronounced effect on the circadian rhythm of a substantial proportion of genes. Their response was strikingly dual, comprising either flattening (partial or complete) of the diel amplitude or delay of expression maximum/minimum by several hours. Genes encoding central oscillator components behaved in the same dual manner, unlike their Arabidopsis counterparts reported earlier to cease cycling altogether upon cold treatment. Also numerous genes lacking circadian rhythm responded to the cold by undergoing up- or down-regulation. Notably, the transcriptome changes preceded major physiological manifestations of cold stress. In silico analysis of metabolic processes likely affected by observed gene expression changes indicated major down-regulation of photosynthesis, profound and multifarious modulation of plant hormone levels, and of chromatin structure, transcription, and translation. A role of trehalose and stachyose in cold stress signaling was also suggested. Meta-analysis of published transcriptomic data allowed discrimination between general stress response of maize and that unique to severe cold. Several cis- and trans-factors likely involved in the latter were predicted, albeit none of them seemed to have a major role. These results underscore a key role of modulation of diel gene expression in maize response to severe cold and the unique character of the cold-response of the maize circadian clock.

Interspecific hybridization in sympatric species of Daphnia inhabiting lakes in northeastern Poland

Populations of three sympatric species (Daphnia cucullata, Daphnia galeata, and Daphnia hyalina (Daphnia longispina complex)) inhabiting three lakes in northeastern Poland (Roś, Mikołajskie, Wigry) were checked for interspecific hybrids. Hybrid Daphnia coexisting with parental individuals were detected in each of the investigated lakes. Several classes of hybrids (F1, F2, backcrosses), including D. cucullata×galeata×hyalina individuals, have been detected. Hybrids constituted a substantial (13%–38%) part of the total abundance of individuals of the D. longispina complex. Deficiency of heterozygotes (hybrids) detected may indicate the existence of barriers for gene flow between the sympatric Daphnia taxa.

Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene ☆

We have cloned the genomic copy of the Ppras1 gene, a homologue of the ras proto-oncogene, from the true slime mold Physarum polycephalum. Ppras1 contains five small introns, four of which have a high content of pyrimidines. The (dC)-homopolymers present in introns 4 and 5 may be responsible for the observed recA-independent deletion in Ppras1 upon amplification of the Ppras1-bearing plasmid by choramphenicol. Although Ppras1 exhibits amino acid and nucleotide homologies with the DdrasG gene, a homologue of ras from another slime mold, Distyostelium discoideum, locations and sequences of their introns are quite different. This discordance suggests that introns of the ras genes in these species were acquired independently.

PPRAS1PPRAS2ANDPPRAP1GENES, MEMBERS OF ARASGENE FAMILY FROM THE TRUE SLIME MOLDPHYSARUM POLYCEPHALUMARE DEVELOPMENTALLY REGULATED☆

The expression patterns of two truerasgenes,Ppras1andPpras2and onerapgene,Pprap1were examined in fourPhysarum polycephalumdevelopmental stages: uninucleate amoebae, plasmodia (multinucleate syncytia), spherules (a vegetative, dormant stage) and fruiting bodies.Ppras1andPprap1are expressed in all stages examined with the maximum levels of their transcripts in amoebae and fruiting bodies, respectively, and the minimum levels in plasmodia, whereas thePpras2transcript is only detectable in amoebae and fruiting bodies. The results obtained indicate thatP. polycephalumis an organism possessing a developmentally regulatedrasgene family and presents a convenient system to study the role ofras/rapgenes in control of growth and differentiation of lower eukaryotic organisms.