Impaired function of the phage-type RNA polymerase RpoTp in transcription of chloroplast genes is compensated by a second phage-type RNA polymerase

Although chloroplast genomes are small, the transcriptional machinery is very complex in plastids of higher plants. Plastidial genes of higher plants are transcribed by plastid-encoded (PEP) and nuclear-encoded RNA polymerases (NEP). The nuclear genome of Arabidopsis contains two candidate genes for NEP, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. We have analyzed the use of PEP and NEP promoters in transgenic Arabidopsis lines with altered RpoTp activities and in Arabidopsis RpoTp insertion mutants lacking functional RpoTp. Low or lacking RpoTp activity resulted in an albino phenotype of the seedlings, which normalized later in development. Differences in promoter usage between wild type and plants with altered RpoTp activity were also most obvious early in development. Nearly all NEP promoters were used in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene, however, was not used in mutant seedlings lacking RpoTp activity. Our data provide evidence for NEP being represented by two phage-type RNA polymerases (RpoTp and RpoTmp) that have overlapping as well as gene-specific functions in the transcription of plastidial genes

Phagenähnliche RNA-Polymerasen

Chloroplasten höherer Pflanzen haben kleine Genome. Trotzdem ist ihre Transkriptionsmaschinerie sehr komplex. Plastidäre Gene werden von plastidenkodierten (PEP) und kernkodierten RNA-Polymerasen (NEP) transkribiert. In der vorliegenden Arbeit wurden Promotoren plastidärer Gene und Operons von Arabidopsis thaliana charakterisiert. Zur Unterscheidung zwischen NEP- und PEP-Promotoren wurden erstmals spectinomycinbehandelte, chlorophylldefiziente Arabidopsis-Pflanzen mit fehlender PEP-Aktivität verwendet. Obwohl für einige Gene auch einzelne Promotoren lokalisiert wurden, wird die Transkription der meisten plastidären Gene und Operons an multiplen Promotoren initiiert. Der Vergleich plastidärer Promotoren von Tabak und Arabidopsis zeigte eine hohe Vielfältigkeit der Promotornutzung, die möglicherweise auch in anderen höheren Pflanzen vorkommt. Dabei stellt die individuelle Promotornutzung eine speziesspezifische Kontrollmöglichkeit der plastidären Genexpression dar. Das Kerngenom von Arabidopsis beinhaltet zwei Kandidatengene der NEP, RpoTp und RpoTmp, welche Phagentyp-RNA-Polymerasen kodieren. In der vorliegenden Arbeit wurde die Wirkung veränderter RpoTp-Aktivität auf die Nutzung von NEP- und PEP-Promotoren in transgenen Arabidopsis-Pflanzen mit verminderter und fehlender RpoTp-Aktivität untersucht. Im Keimlingsstadium konnten Unterschiede in der Promotornutzung zwischen Wildtyp und Mutanten beobachtet werden. Fast alle NEP-Promotoren wurden in Pflanzen mit verringerter oder fehlender RpoTp-Aktivität genutzt. Dabei zeigten nur einige von ihnen eine geringere Aktivität, andere wiederum waren sogar verstärkt aktiv. Der starke NEP-Promotor des essentiellen ycf1 Gens wurde in jungen Keimlingen ohne funktionelle RpoTp nicht genutzt. Die Ergebnisse zeigen, dass NEP gemeinsam von beiden Phagentyp-RNA-Polymerasen RpoTp und RpoTmp repräsentiert wird und dass beide sowohl eine überlappende, als auch eine spezifische Rolle in der Transkription plastidärer Gene innehaben.Although chloroplasts of higher plants have small genomes, their transcription machinery is very complex. Plastid genes of higher plants are transcribed by the plastid-encoded plastid RNA polymerase PEP and the nuclear-encoded plastid RNA polymerases NEP. Here, promoters of plastid genes and operons have been characterized in Arabidopsis thaliana. For the first time spectinomycin-treated, chlorophyll-deficient Arabidopsis plants lacking PEP activity have been used to discriminate between NEP and PEP promoters. Although there are plastid genes that are transcribed from a single promoter, the transcription of plastid genes and operons by multiple promoters seems to be a common feature. Comparison of plastid promoters from tobacco and Arabidopsis revealed a high diversity, which my also apply to other plants. The diversity in individual promoter usage in different plants suggests that there are species-specific solutions for attaining control over gene expression in plastids. The nuclear genome of Arabidopsis contains two candidate genes for NEP transcription activity, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. In this study the usage of NEP and PEP promoters has been analysed in transgenic Arabidopsis plants with reduced and lacking RpoTp activity. Differences in promoter usage between wild type and mutant plants were most obvious early in development. Nearly all NEP promoters were active in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene was not transcribed in young seedlings without functional RpoTp. These results provide evidence for NEP being represented by two phage-type RNA polymerases RpoTp and RpoTmp that have overlapping as well as specific functions in the transcription of plastid genes

Microcystins as new therapeutic agents for OATP1B3 expressing tumors _ Poster at ICNPR2012.pdf

<p> </p> <p>Microcystins are cyclic heptapeptides produced by cyanobacteria. They potently inhibit the eukaryotic protein phosphatase families PP1 and PP2A. They are able to display toxicity only after transporter-mediated uptake by the cell. This uptake is mediated by the organic anion transporting polypeptides OATP1B1, OATP1B3, and OATP1A2. Liver cells express OATP1B1 and OATP1B3 transporters, which results in a pronounced liver toxicity of microcystins. However, OATP1B3 is also expressed in a significant percentage of hepatocellular carcinoma cells as well as gastrointestinal, lung, breast and colon tumors. Interestingly, compared to OATP1B1, OATP1B3 is found only in low abundance in liver cells. Thus selectivity that favors OATP1B3 over OATP1B1 should lead to a decreased hepatic clearance and toxicity, and an increased uptake in OATP1B3-expressing tumors, thus creating a therapeutic window for the respective compound.</p> <p> </p> <p>Since many naturally occurring microcystin variants are known and many more have not been descri­bed yet, there is high potential for isolating variants with unique properties. In fact, in our initial screening of 20 microcystins, we have found four microcystin structural variants with an IC₅₀ in the low nanomolecular range and with transporter selectivity that favors OATP1B3 over OATP1B1 by a factor of more than ten. Some of these microcystin variants have not yet been described in the literature.</p> <p> </p

Selectivity and Potency of Microcystin Congeners against OATP1B1 and OATP1B3 Expressing Cancer Cells

<div><p>Microcystins are potent phosphatase inhibitors and cellular toxins. They require active transport by OATP1B1 and OATP1B3 transporters for uptake into human cells, and the high expression of these transporters in the liver accounts for their selective hepatic toxicity. Several human tumors have been shown to have high levels of expression of OATP1B3 but not OATP1B1, the main transporter in liver cells. We hypothesized that microcystin variants could be isolated that are transported preferentially by OATP1B3 relative to OATP1B1 to advance as anticancer agents with clinically tolerable hepatic toxicity. Microcystin variants have been isolated and tested for cytotoxicity in cancer cells stably transfected with OATP1B1 and OATP1B3 transporters. Microcystin variants with cytotoxic OATP1B1/OATP1B3 IC₅₀ ratios that ranged between 0.2 and 32 were found, representing a 150-fold range in transporter selectivity. As microcystin structure has a significant impact on transporter selectivity, it is potentially possible to develop analogs with even more pronounced OATP1B3 selectivity and thus enable their development as anticancer drugs.</p></div

General structures of microcystins and nodularins.

<p>Prevalence of residues found within microcystins (left) and nodularins (right) is proportional to the font size of the respective residue. Data used to generate this figure is deposited at <a href="http://dx.doi.org/10.6084/m9.figshare.880756" target="_blank">http://dx.doi.org/10.6084/m9.figshare.880756</a>.</p

Structures of the tested MC congeners originating from ^a<i>Microcystis aeruginosa</i>, ^b<i>Planktothrix rubescens</i>, and ^c<i>Nodularia</i> sp.

<p><b>Hil</b> homoisoleucine, <b>Hty</b> homotyrosine, <b>MeAsp</b> β-methylaspartic acid, <b>Har</b> homoarginine, <b>Mdha</b><i>N</i>-methyl dehydroalanine, <b>Dhb</b> dehydrobutyric acid, <b>Mser</b><i>N</i>-methyl serine, <b>Mdhb</b><i>N</i>-methyl dehydrobutyric acid.</p