Podcasts about chlamydomonas

In this episode, Assistant Editor for Functional Ecology, Frank Harris, sits down with Hannah Meier who has been nominated for the 2022 Haldane Prize. This prize is awarded to the best research from an early career researcher. Hannah has been nominated for her paper: Temperature-mediated transgenerational plasticity influences movement behaviour in the green algae Chlamydomonas reinhardtii Paper: https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/1365-2435.14214 Plain language Summary: https://fesummaries.wordpress.com/2022/11/07/the-effects-of-temperature-can-influence-the-movement-of-green-algae-individuals-across-multiple-generations/ English Blogpost: https://functionalecologists.com/2023/01/24/hannah-meier-transgenerational-behavioral-plasticity-in-chlamydomonas-reinhardtii/ German Blogpost: https://functionalecologists.com/2023/01/24/hannah-meier-transgenerational-behavioral-plasticity-in-chlamydomonas-reinhardtii-german-translation/

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.07.536038v1?rss=1 Authors: Dougherty, L. L., Avasthi, P. Abstract: At the core of cilia are microtubules which are important for establishing length and assisting ciliary assembly and disassembly; however, another role for microtubule regulation on ciliogenesis lies outside of the cilium. The microtubule cytoskeleton is a highly dynamic structure which reorganizes rapidly to assist in cellular processes. Cytoplasmic microtubule dynamics have previously been thought to be necessary to free up tubulin and proteins in the ciliary precursor pool for ciliogenesis. However, we previously found that low concentrations of taxol can stabilize cytoplasmic microtubules during deciliation while allowing normal cilium regrowth. Here we look at the relationship between ciliogenesis and cytoplasmic microtubule dynamics in Chlamydomonas reinhardtii using chemical and mechanical perturbations. We find that not only can stabilized cytoplasmic microtubules allow for normal ciliary assembly, but high calcium concentrations and low pH-induced deciliation cause microtubules to depolymerize separately from ciliary shedding. In addition, we find that through mechanical shearing, cilia regenerate more quickly despite intact cytoplasmic microtubules. Our data suggests that cytoplasmic microtubules are not a sink for a limiting pool of cytoplasmic tubulin, reorganization that occurs following deciliation is a consequence rather than a requirement for ciliogenesis, and intact microtubules in the cytoplasm and the proximal cilium support more efficient ciliary assembly. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.14.528553v1?rss=1 Authors: Kubo, T., Tani, Y., Yanagisawa, H., Kikkawa, M., Oda, T. Abstract: - and {beta}-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTT). The function of this region in cilia/flagella is still unclear, except that glutamates in CTT act as the sites for posttranslational modifications that affect ciliary motility. A unicellular alga Chlamydomonas possesses only two -tubulin genes and two {beta}-tubulin genes, each pair encoding an identical protein. This simple gene organization may enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutants expressing tubulins with modified CTTs. We found that the mutant whose four glutamate residues in the -tubulin CTT have been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant lacking the glutamate-rich region of the {beta}-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to the mutants harboring a mutation in a subunit of katanin, whose function has been shown to depend on the {beta}-tubulin CTT. Therefore, our study reveals distinct and important roles of - and {beta}-tubulin CTT in the formation and function of cilia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.12.515357v1?rss=1 Authors: Kafri, M., Patena, W., Martin, L., Wang, L., Gomer, G., Sirkejyan, A. K., Goh, A., Wilson, A. T., Gavrilenko, S. E., Breker, M., Roichman, A., McWhite, C. D., Rabinowitz, J. D., Cross, F. R., Wuhr, M., Jonikas, M. C. Abstract: Photosynthesis is central to food production and the Earth's biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (FDR less than 0.11) 70 previously-uncharacterized genes required for photosynthesis. We then provide a resource of mutant proteomes that enables functional characterization of these novel genes by revealing their relationship to known genes. The data allow assignment of 34 novel genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Additional analysis uncovers at least seven novel critical regulatory proteins, including five Photosystem I mRNA maturation factors and two master regulators: MTF1, which impacts chloroplast gene expression directly; and PMR1, which impacts expression via nuclear-expressed factors. Our work provides a rich resource identifying novel regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.02.514835v1?rss=1 Authors: Liu, D., Lopez-Paz, C., Li, Y., Zhuang, X., Umen, J. Abstract: Coordination of growth and division in eukaryotic cells is essential for populations of proliferating cells to maintain size homeostasis, but the underlying mechanisms that govern cell size have only been investigated in a few taxa. The green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle that involves a long G1 phase followed by a rapid series of successive S and M phases (S/M) that produces 2n daughter cells. Two control points show cell-size dependence: Commitment in mid-G1 phase requires attainment of a minimum size to enable at least one mitotic division during S/M, and the S/M control point where mother cell size governs cell division number (n), ensuring that daughter distributions are uniform. tny1 mutants pass Commitment at a smaller size than wild type and undergo extra divisions during S/M phase to produce small daughters, indicating that TNY1 functions to inhibit size-dependent cell cycle progression. TNY1 encodes a cytosolic hnRNP A-related RNA binding protein and is produced once per cell cycle during S/M phase where it is apportioned to daughter cells, and then remains at constant absolute abundance as cells grow, a property known as subscaling. Altering the dosage of TNY1 in heterozygous diploids or through overexpression increased Commitment cell size and daughter cell size, indicating that TNY1 is a limiting factor for both size control checkpoints. Epistasis placed TNY1 function upstream of the retinoblastoma tumor suppressor complex (RBC) and one of its regulators, Cyclin-Dependent Kinase G1 (CDKG1). Moreover, CDKG1 protein and mRNA were found to over-accumulate in tny1 cells suggesting that CDKG1 may be a direct target of repression by TNY1. Our data expand the potential roles of subscaling proteins outside the nucleus and imply a control mechanism that ties TNY1 accumulation to pre-division mother cell size. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.512090v1?rss=1 Authors: Brewer, K. M., Engle, S. E., Bansal, R., Brewer, K. K., Jasso, K. R., McIntyre, J. C., Vaisse, C., Reiter, J. F., Berbari, N. F. Abstract: Primary cilia are small immotile cellular appendages which mediate diverse types of singling and are found on most mammalian cell types including throughout the central nervous system. Cilia are known to localize certain G protein-coupled receptors (GPCRs) and are critical for mediating the signaling of these receptors. Several of these neuronal GPCRs have recognized roles in feeding behavior and energy homeostasis. Heterologous cell line and model systems like C. elegans and Chlamydomonas have implicated both dynamic GPCR cilia localization and cilia length and shape changes as key for signaling. However, it is unclear if mammalian ciliary GPCRs utilize similar mechanisms in vivo and under what physiological conditions these processes may occur. Here, we use the ciliary GPCRs, melanin concentrating hormone receptor 1 (MCHR1) and neuropeptide-Y receptor 2 (NPY2R) as model ciliary receptors to determine if dynamic localization to cilia occurs. We tested physiological conditions in which these GPCRs have been implicated such as feeding behavior, obesity, and circadian rhythm. Cilia were imaged using confocal microscopy and analyzed with a computer assisted approach allowing for unbiased and high throughput analysis of cilia. We analyzed GPCR positive cilia, cilia frequency as well as cilia length and receptor occupancy. Interestingly we observed changes in ciliary length, receptor occupancy, and cilia frequency under different conditions, but no consistent theme across GPCRs or brain nuclei was observed. A better understanding of the subcellular localization dynamics of ciliary GPCRs could reveal unrecognized molecular mechanisms regulating behaviors like feeding. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

This shorter episode is about a tiny, single-celled alga – Chlamydomonas reinhardtii – that's managed to have a big impact. UC Berkeley plant biologist Sabeeha Merchant explains why she works on this alga, how researchers managed to sequence its genome, and what it has to teach us about other organisms – like plants. Links from this episodeEpisode TranscriptJGI@25: The Little Alga That CouldChlamydomonas reinhardtii on Phytozome and PhycoCosm JGI Blog Post: Green Algae Reveal One mRNA Encodes Many ProteinsJGI News Release: Green Alga Genome Project Catalogs Carbon Capture Machinery and Reveals Identity as Ancient Cousin of Land Plants and AnimalsThe original sequence: ScienceOur contact info:Twitter: @JGIEmail: jgi-comms at lbl dot govGenome Insider is a production of the Joint Genome Institute. 

This week we discuss cilia length, ciliopathies and Chlamydomonas reinhardtii (also known as ‘Chlamy') with Brae Briggs (@BiggeBrae), a graduate student from at Dartmouth (@dartmouth),Geisel School of Medicine (@GeiselMed). We find out about a range of ciliopathies, the main composition of cilia as well as using ‘Chlamy' as a model to study cilia length. We also discuss the difficulties of moving in the middle of a PhD and pandemic, open science principles including preprints as well as how we try to have healthy work life balance. Read the full preprint: https://www.biorxiv.org/content/10.1101/2022.04.18.488674v1.full This episode was produced by Emma Wilson and edited by John D Howard. If you enjoyed this show then hit that subscribe button and leave a review (on Apple Podcasts or Spotify). If you love what we are trying to do then buy us a coffee https://www.buymeacoffee.com/preprints! Any contribution is greatly appreciated. For the latest podcast news and updates follow us on Twitter @MotionPod or visit our website; www.preprintsinmotion.com. Produced by JEmJ Productions (find us on Twitter: Jonny @JACoates, Emma @ELWilson92, John @JohnDHoward8) and generously supported by ASAPbio (https://asapbio.org | @asapbio_).

Article: Alternative photosynthesis pathways drive the algal CO2-concentrating mechanism Journal: Nature Year: 2022 Guest: Adrien Burlacot Host: Arif Ashraf Abstract Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption. The high efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, which enhances CO2 fixation. Although many cellular components involved in the transport and sequestration of inorganic carbon have been identified how microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains unknown. Here we show that in the green alga Chlamydomonas reinhardtii, the combined action of cyclic electron flow and O2 photoreduction—which depend on PGRL1 and flavodiiron proteins, respectively—generate a low luminal pH that is essential for CCM function. We suggest that luminal protons are used downstream of thylakoid bestrophin-like transporters, probably for the conversion of bicarbonate to CO2. We further establish that an electron flow from chloroplast to mitochondria contributes to energizing non-thylakoid inorganic carbon transporters, probably by supplying ATP. We propose an integrated view of the network supplying energy to the CCM, and describe how algal cells distribute energy from photosynthesis to power different CCM processes. These results suggest a route for the transfer of a functional algal CCM to plants to improve crop productivity. Art credit: Solène Moulin Cover art design and audio editing: Ragib Anjum --- Send in a voice message: https://anchor.fm/no-time-to-read-podcast/message

En el episodio de hoy echaremos una ojeada a vista de pájaro a las algas verdes y las carófitas, que son respectivamente primas lejanas y primas hermanas de las plantas terrestres. Hoy hemos hablado de: -Archeplastidia: las viejas plastudas, compuestas por algas rojas, algas verdes y glaucófitas. -Plasmodium: el parásito de la malaria. -Viridiplantae: Las que antes eran algas verdes pero que ahora comprenden algas verdes + plantas terrestres. En cuanto a algas verdes en general, vamos a ver unos ejemplos: -Chlamydomonas, un alga verde unicelular muy típica: 03aea4820e90259ec7b3cbd97bee6867.jpg (452×360) (pinimg.com)-Ulva, la lechuga de mar: Meersalat-Ulva-lactuca.jpg (3000×2126) (wikimedia.org)-Caulerpa, el alga macroscópica que consiste en una única célula: 3-Caulerpa-racemosa-1.jpg (960×640) (monaconatureencyclopedia.com)-Volvox, la simpática alga colonial con desarrollo embrionario: colonies-Volvox-aureus-daughter-movements-environment-flagella.jpg (800×450) (britannica.com)Aquí algunos ejemplos de carófitas (Charophyta). Si las juntamos con las plantas terrestres el grupo se llama Streptophyta: -Micrasterias, unas bonitas algas unicelulares: Micrasterias_radiata.jpg (640×480) (wikimedia.org)-Spirogyra, la masa viscosa verde que se forma en la superficie de los estanques pero que al microscopio es muy bonita: Light-microscopic-images-of-Spirogyra-morphotypes-from-the-Lake-Baikal-region-A.jpg (850×1193) (researchgate.net)-Chara, una carófita con una organización corporal bastante compleja: 1200px-CharaFragilis.jpg (1200×1753) (wikimedia.org)

In this episode, I converse with Prof. Prachee Avasthi, an Associate Professor of Biochemistry and Cell Biology at the Geisel School of Medicine at Dartmouth College. She completed a PhD in neuroscience under the supervision of Wolfgang Baehr at the University of Utah and a Postdoc with Wallace Marshall at the University of California San Francisco (UCSF). Prachee started her own research group at the University of Kansas Medical Center before moving to Dartmouth in 2020 and she also serves on the Board of Directors of eLife and is the incumbent President of ASAPbio (Accelerating Science and Publication in biology), a non-profit initiative promoting innovation and transparency via preprints and open peer review. Prachee's group uses the unicellular green alga Chlamydomonas reinhardtii to study the formation of the cellular antenna, the cilium, and how its assembly is coordinated with other cellular processes. We indulge in a terrific conversation on her phenomenal journey through science and life; being exposed to research as an undergrad and graduate school in neuroscience; wonderful mentors who have inspired her and making mistakes by the plenty; the fun and collaborative aspects of science; the importance of fundamental research; actively reforming science and scientific publication through preprints and fixing the broken pipeline; communicating science through Twitter and stitching unexpectedly remarkable collaborations; and many more things!!

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.05.369801v1?rss=1 Authors: Stehnach, M. R., Waisbord, N., Walkama, D. M., Guasto, J. S. Abstract: Gradients in fluid viscosity characterize microbiomes ranging from mucus layers on marine organisms and human viscera to biofilms. While such environments are widely recognized for their protective effects against pathogens and their ability to influence cell motility, the physical mechanisms controlling cell transport in viscosity gradients remain elusive, primarily due to a lack of quantitative observations. Through microfluidic experiments with a model biflagellated microalga (Chlamydomonas reinhardtii), we show that cells accumulate in high viscosity regions of weak gradients as expected, stemming from their locally reduced swimming speed. However, this expectation is subverted in strong viscosity gradients, where a novel viscophobic turning motility - consistent with a flagellar thrust imbalance - reorients the swimmers down the gradient and causes striking accumulation in low viscosity zones. Corroborated by Langevin simulations and a three-point force model of cell propulsion, our results illustrate how the competition between viscophobic turning and viscous slowdown ultimately dictates the fate of population scale microbial transport in viscosity gradients. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.21.349399v1?rss=1 Authors: Schulze, S., Oltmanns, A., Fufezan, C., Kragenbring, J., Mormann, M., Pohlschroder, M., Hippler, M. Abstract: Motivation: Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Results: Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae. Availability: The source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.27.315606v1?rss=1 Authors: Cortese, D., Wan, K. Y. Abstract: Helical swimming is a ubiquitous strategy for motile cells to generate self-gradients for environmental sensing. The model biflagellate Chlamydomonas reinhardtii rotates at a constant 1 - 2 Hz as it swims, but the mechanism is unclear. Here, we show unequivocally that the rolling motion derives from a persistent, non-planar flagellar beat pattern. This is revealed by high-speed imaging and micromanipulation of live cells. We construct a fully-3D model to relate flagellar beating directly to the free-swimming trajectories. For realistic geometries, the model reproduces both the sense and magnitude of the axial rotation of live cells. We show that helical swimming requires further symmetry-breaking between the two flagella. These functional differences underlie all tactic responses, particularly phototaxis. We propose a control strategy by which cells steer towards or away from light by modulating the sign of biflagellar dominance. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.31.276428v1?rss=1 Authors: Bauer, D., Ishikawa, H., Wemmer, K., Marshall, W. F. Abstract: Stochastic variations (noise) in gene expression have been extensively characterized, but the ramifications of this gene-level variation for cellular structure and function remain unclear. To what extent are cellular structures subject to noise? We show that flagellar length in Chlamydomonas exhibits significant variation that results from a combination of intrinsic fluctuations within the flagella and extrinsic cell to cell variation. We analyzed a series of candidate genes affecting flagella and found that flagellar length variation is increased in mutations which increase the average flagellar length, an effect that can be explained using a theoretical model for flagellar length regulation. Cells with greater differences in their flagellar lengths show impaired swimming but improved gliding motility, raising the possibility that cells have evolved mechanisms to tune intrinsic noise in length. Taken together our results show that biological noise exists at the level of subcellular structures, with a corresponding effect on cell function. Copy rights belong to original authors. Visit the link for more info

Don’t you dare disrespect our favourite model organism, Arabidopsis. No wait, we love tobacco and hate Arabidopsis. No! Both are the WORST, we love Chlamydomonas above everything else! Or were we team sunflower?

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.20.212159v1?rss=1 Authors: Mojiri, S., Isbaner, S., Muehle, S., Jang, H., Bae, A. J., Gregor, I., Gholami, A., Enderlein, J. Abstract: Axonemes are the basic structure of motile cilia and flagella, and the investigation of how they function and move requires rapid three-dimensional imaging. We built a multi-plane phase-contrast microscope for imaging the three-dimensional motion of unlabeled flagella of the model organism Chlamydomonas reinhardtii with sub-m spatial and 4 ms temporal resolution. This allows us to observe not only bending but also the three-dimensional torsional dynamics of these small structures. We observe that flagella swim counter-clockwise close to a surface, with negatively-valued torsion at their basal and positively-valued torsion at their distal tips. To explain the torsional dynamics and signature, we suggest the existence of an intrinsic negative twist at the basal end that is untwisted by active positive-twist-inducing dynein motor proteins. Moreover, dyneins walking towards the basal induce an opposite twist at the distal tip. Bending of the whole axoneme structure then translates this twist into an observable torsion. This interconnection between chiral structure, twist, curvature, and torsion is fundamental for understanding flagellar mechanics. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.20.211904v1?rss=1 Authors: Goli Pozveh, S., Bae, A., Gholami, A. Abstract: Cilia-driven motility and fluid transport is ubiquitous in nature and essential for many biological processes, including swimming of eukaryotic unicellular organisms, mucus transport in airway apparatus or fluid flow in brain. The-biflagellated micro-swimmer Chlamydomonas reinhardtii is a model organism to study dynamics of flagellar synchronization. Hydrodynamic interactions, intracellular mechanical coupling or cell body rocking are believed to play crucial role in synchronization of flagellar beating in green algae. Here, we use freely swimming intact flagellar apparatus isolated from wall-less strain of Chlamydomonas to investigate wave dynamics. Our analysis in phase coordinates show that, when the frequency difference between the flagella is high, neither mechanical coupling via basal body nor hydrodynamics interactions are strong enough to synchronize two flagella, indicating that beating frequency is controlled internally by the cell. We also examined the validity of resistive force theory for a flagellar apparatus swimming freely in the vicinity of a substrate and found a quantitative agreement between experimental data and simulations with drag anisotropy of ratio 2. Finally, using a simplified wave form, we investigated the influence of phase and frequency differences, intrinsic curvature and wave amplitude on the swimming trajectory of flagellar apparatus. Our analysis shows that by controlling phase or frequency differences between two flagella, steering can occur. Copy rights belong to original authors. Visit the link for more info

This episode: Producing both biodiesel and bioethanol fuels from cold-loving Arctic algae! Download Episode (8.7 MB, 12.6 minutes) Show notes: Microbe of the episode: Royal Farm virus Takeaways Renewable fuels such as biofuels can allow existing infrastructure and vehicles to continue to operate in a more sustainable manner, which could reduce the cost and impact of switching to new/different systems of transportation like electricity. Economically competitive methods of producing biofuels are still being explored and developed.   In this study, Arctic algae are grown in cold temperatures using only light, carbon dioxide, and a few minerals, and then broken down to produce biodiesel and bioethanol, which can be used as fuel in many different internal combustion engines. The amounts produced are comparable to other algae-based systems being researched, and use of the cold-loving organisms could reduce the cost of production in colder latitudes and seasons. Journal Paper: Kim EJ, Kim S, Choi H-G, Han SJ. 2020. Co-production of biodiesel and bioethanol using psychrophilic microalga Chlamydomonas sp. KNM0029C isolated from Arctic sea ice. Biotechnol Biofuel 13:20. Other interesting stories: Certain foods could activate or inhibit bacteriophages and modulate the gut microbiota Some antibiotic-producing bacterial colonies have specialized members that do all the antibiotic production   Email questions or comments to bacteriofiles at gmail dot com. Thanks for listening! Subscribe: Apple Podcasts, Google Podcasts, Android, or RSS. Support the show at Patreon, or check out the show at Twitter or Facebook.

Människans tillvaro hänger antagligen samman med en två miljarder år gammal passion mellan en bakterie och en arké. Mikrobiologen Farshid Jalalvand funderar över uppkomsten av livet som vi känner det. ESSÄ: Detta är en text där skribenten reflekterar över ett ämne eller ett verk. Åsikter som uttrycks är skribentens egna. Är hon en Capulet?! O Gud, mitt liv är då en oväns rätt! Det utbrister Shakespeares Romeo när han får reda på vem tjejen han kärat ner sig i är. Julia, är av huset Capulet, arvfiender till familjen Montague, huset Romeo tillhör. Paret är oförenliga av börd, men ändå uppslukade av varandra, som genom en naturkraft. Det som inte kunde vara, blir. För cirka 2 miljarder år sedan, långt innan människor eller ens multicellulärt liv existerade, inträffade en liknande händelse en händelse som kom att drastiskt förändra jordens historia. Två individer, från livets två olika, oförenliga grenar, träffades någonstans ute i en de stora oceanerna. Efter en kyss, bjöd den mer sofistikerade den enklare och ivrigare parten in till sig. Och så inleddes ett äktenskap som varat i 2 miljarder år och som evolutionärt är en av de mest lyckade sammankomsterna i livets historia. De två individerna som gifte sig, där ute i havet, för eoner sen, lever kvar än idag, bland annat genom dig. Du är en direkt ättling till den där eukaryota cellen. Livet på jorden är vetenskapligt indelat i tre huvudgrenar. Och ingen av dem är djur eller växter, som många skulle kunna tro. Faktum är att om man granskar släktskapet mellan alla organismer på jorden är djur och växter bara två små intilliggande kvistar på en gren av livets stora träd. Det betyder att en katt och en gran är relativt närbesläktade, jämfört med allt annat som finns där ute. Huvudgrenen som den kvisten sitter på, och som samlar såväl människor som hårlösa marsvin, olivträd, flugsvampar och amöbor, är de eukaryota cellernas rike. Dessa celler har en DNA-skyddande cellkärna, en mer komplicerad cellarkitektur och mer avancerade förmågor än cellerna på den andra grenen på livets träd den bakteriella. Bakterier är så kallade prokaryoter och saknar flera av den eukaryota cellens finesser, som till exempel cellkärna. Den tredje återstående huvudgrenen är desto mer okänd för allmänheten, vilket är konstigt. Dels för den utgör en tredjedel av livets träd, och dels för den är en systergren till den eukaryota gren vi sitter på. Det är den arkéella grenen. Kärlek kan ju fråga bara Romeo och Julia vara alldeles förtärande. Hur det nu än gick till, hamnade den mindre bakterien inne i den större arkén. Men innan den ena hann förgöra den andra, hände något. Arkéer är, liksom bakterier, prokaryoter och de är fullständigt fascinerande. Fram till sent 70-tal visste vi inte ens att de fanns, eller snarare, att de var vad de var. För det som forskare trodde var bakterier som levde i extrema miljöer som kokande svavelkällor, frätande syra eller exceptionella saltkoncentrationer visade sig vara en helt egen livsform, och utgjorde alltså en helt egen huvudgren på livets träd. En tredjedel av livet på jorden hade gömt sig mitt framför näsan på oss. Och nu pekar allt fler bevis på att den eukaryota cellen den som vi är uppbyggda av ursprungligen var en arké. Allt går tillbaka till den där kritiska dagen för 2 miljarder år sen. Då existerade endast encelligt liv. Enligt den så kallade endosymbiontteorin, som det råder stor konsensus om bland biologer, skedde då följande: två encelliga organismer möttes. Den ena var en bakterie och som nämnts tyder mycket på att den andra var en arké. Det verkar sedan som att arkén försökte äta sin nya bekantskap, eller om bakterien försökte parasitera på arkén. Kärlek kan ju  fråga bara Romeo och Julia vara alldeles förtärande. Hur det nu än gick till, hamnade den mindre bakterien inne i den större arkén. Men innan den ena hann förgöra den andra, hände något. De två prokaryota cellerna började av någon anledning att samarbeta. Bakterien, som nu bodde inne i arkén, fokuserade på att alstra energi. Arkén kunde då släppa den mödosamma processen, och dirigera om sina resurser till att göra allt det andra som behövs för att föröka sig. Varje gång arkén delade sig, tog varje dottercell några inneboende bakterier med sig, som alltså hade vuxit till sig inne i arkén under tiden. Och på så vis fortgick samarbetet. Allt eftersom generationer kom och gick, började bakterien flytta mer och mer av sitt DNA till arkéns genom. Och arkén fortsatte förse bakterien med allt den behövde i utbyte mot energi. Till slut blev bakterien en del av arkécellen, en cellorganell, det vi idag kallar mitokondrien, cellens energikraftverk. Och vips så hade den moderna eukaryota cellen uppstått. En supercell med superkrafter. Men utvecklingssagan slutade inte där. Kärlekshistorier brukar ju inte sällan involvera en tredje part. Efter ett par 100 miljoner år träffade en av den ursprungliga eukaryota cellens ättlingar på en fotosyntetiserande cyanobakterie. Utan att kunna motstå denna gröna skönhet, svalde den helt sonika bakterien. Symbiogenes, det vill säga sammansmältningen av två samarbetande organismer, skedde därefter framgångsrikt för andra gången i världshistorien: cyanobakterien degenererade nämligen med tiden till den fotosyntetiserande cellorganellen kloroplast, och den pimpade cellen blev alla växters anfader. Men vad är då den stora konsekvensen av dessa passionerade möten? Jo, av anledningar som inte är helt klarlagda, har komplexa multicellulära organismer, som växter och djur, endast uppstått bland eukarya. Multicellularitet innebär är att ett stort antal genetiskt identiska celler tillsammans bildar en större organism. Du själv var från början en enda cell, ett embryo, innan den delade sig i miljarder kopior. Alla celler i din kropp, vare sig om det är muskelceller, njurceller eller vita blodkroppar, är genetiskt identiska dubbletter. Men de har gått ihop och skapat ett nätverk. Och trots att de alla ursprungligen har identiska kapaciteter utför cellerna endast specialistfunktioner. Ett gäng har specialiserats till musklerceller, ett gäng till njurceller, och ett gäng till immunceller. Genom att alla celler slipper göra allt själva kan de avsätta större resurser till sina spetskompetenser och skapa avancerade vävnader. Komplexitet uppstår ur denna specialisering. Romeo hade i sin tur begått självmord då han felaktigt trodde att Julia hade dött. Hängivenheten mellan det mikrobiella paret i vår historia är lika häftig. Den här sortens avancerad samarbete har prokaryoter aldrig klarat av att åstadkomma under de miljarder år de funnits. Anledningen till att tulpanerna på ditt bord, katten i ditt knä, granen i skogen, och människorna på din gata kan finnas, är att en arké och en bakterie för 2 miljarder år sen började samarbeta istället för att äta upp varandra. Vi komplexa organismer har endosymbiogenesen att tacka för vår existens. Ett möte mot alla odds, som skapade den värld vi känner. När Shakespeares Julia i slutet av pjäsen upptäcker att Romeo dött tar hon sitt liv, för hon inte förmår att leva utan honom. Romeo hade i sin tur begått självmord då han felaktigt trodde att Julia hade dött. Hängivenheten mellan det mikrobiella paret i vår historia är lika häftig. Cellens energikraftverk mitokondrien kan efter alla dessa år inte längre leva utanför cellen, och cellen inte överleva utan mitokondrien. Det verkar som de starkaste band skapas när oförenliga par förenas. Farshid Jalalvand, skribent och forskare i klinisk mikrobiologi Vidare läsning Buttery, S. 2017. Rediscovering symbiogenesis: The latest research in understanding the origins of eukaryotic cells. Crosstalk, Cellpress Sällström, S. 2015. Mikroorganism från havet ger ledtrådar om cellers ursprung. Vetenskapsradion, Sveriges Radio. Miller, S. M. 2010. Volvox, Chlamydomonas, and the Evolution of Multicellularity. Nature Education Morell, V. 1997. Microbiologys Scarred Revolutionary. Science.

Människans tillvaro hänger antagligen samman med en två miljarder år gammal passion mellan en bakterie och en arké. Mikrobiologen Farshid Jalalvand funderar över uppkomsten av livet som vi känner det. ESSÄ: Detta är en text där skribenten reflekterar över ett ämne eller ett verk. Åsikter som uttrycks är skribentens egna. Är hon en Capulet?! O Gud, mitt liv är då en oväns rätt! Det utbrister Shakespeares Romeo när han får reda på vem tjejen han kärat ner sig i är. Julia, är av huset Capulet, arvfiender till familjen Montague, huset Romeo tillhör. Paret är oförenliga av börd, men ändå uppslukade av varandra, som genom en naturkraft. Det som inte kunde vara, blir. För cirka 2 miljarder år sedan, långt innan människor eller ens multicellulärt liv existerade, inträffade en liknande händelse en händelse som kom att drastiskt förändra jordens historia. Två individer, från livets två olika, oförenliga grenar, träffades någonstans ute i en de stora oceanerna. Efter en kyss, bjöd den mer sofistikerade den enklare och ivrigare parten in till sig. Och så inleddes ett äktenskap som varat i 2 miljarder år och som evolutionärt är en av de mest lyckade sammankomsterna i livets historia. De två individerna som gifte sig, där ute i havet, för eoner sen, lever kvar än idag, bland annat genom dig. Du är en direkt ättling till den där eukaryota cellen. Livet på jorden är vetenskapligt indelat i tre huvudgrenar. Och ingen av dem är djur eller växter, som många skulle kunna tro. Faktum är att om man granskar släktskapet mellan alla organismer på jorden är djur och växter bara två små intilliggande kvistar på en gren av livets stora träd. Det betyder att en katt och en gran är relativt närbesläktade, jämfört med allt annat som finns där ute. Huvudgrenen som den kvisten sitter på, och som samlar såväl människor som hårlösa marsvin, olivträd, flugsvampar och amöbor, är de eukaryota cellernas rike. Dessa celler har en DNA-skyddande cellkärna, en mer komplicerad cellarkitektur och mer avancerade förmågor än cellerna på den andra grenen på livets träd den bakteriella. Bakterier är så kallade prokaryoter och saknar flera av den eukaryota cellens finesser, som till exempel cellkärna. Den tredje återstående huvudgrenen är desto mer okänd för allmänheten, vilket är konstigt. Dels för den utgör en tredjedel av livets träd, och dels för den är en systergren till den eukaryota gren vi sitter på. Det är den arkéella grenen. Kärlek kan ju fråga bara Romeo och Julia vara alldeles förtärande. Hur det nu än gick till, hamnade den mindre bakterien inne i den större arkén. Men innan den ena hann förgöra den andra, hände något.  Arkéer är, liksom bakterier, prokaryoter och de är fullständigt fascinerande. Fram till sent 70-tal visste vi inte ens att de fanns, eller snarare, att de var vad de var. För det som forskare trodde var bakterier som levde i extrema miljöer som kokande svavelkällor, frätande syra eller exceptionella saltkoncentrationer visade sig vara en helt egen livsform, och utgjorde alltså en helt egen huvudgren på livets träd. En tredjedel av livet på jorden hade gömt sig mitt framför näsan på oss. Och nu pekar allt fler bevis på att den eukaryota cellen den som vi är uppbyggda av ursprungligen var en arké. Allt går tillbaka till den där kritiska dagen för 2 miljarder år sen. Då existerade endast encelligt liv. Enligt den så kallade endosymbiontteorin, som det råder stor konsensus om bland biologer, skedde då följande: två encelliga organismer möttes. Den ena var en bakterie och som nämnts tyder mycket på att den andra var en arké. Det verkar sedan som att arkén försökte äta sin nya bekantskap, eller om bakterien försökte parasitera på arkén. Kärlek kan ju  fråga bara Romeo och Julia vara alldeles förtärande. Hur det nu än gick till, hamnade den mindre bakterien inne i den större arkén. Men innan den ena hann förgöra den andra, hände något. De två prokaryota cellerna började av någon anledning att samarbeta. Bakterien, som nu bodde inne i arkén, fokuserade på att alstra energi. Arkén kunde då släppa den mödosamma processen, och dirigera om sina resurser till att göra allt det andra som behövs för att föröka sig. Varje gång arkén delade sig, tog varje dottercell några inneboende bakterier med sig, som alltså hade vuxit till sig inne i arkén under tiden. Och på så vis fortgick samarbetet. Allt eftersom generationer kom och gick, började bakterien flytta mer och mer av sitt DNA till arkéns genom. Och arkén fortsatte förse bakterien med allt den behövde i utbyte mot energi. Till slut blev bakterien en del av arkécellen, en cellorganell, det vi idag kallar mitokondrien, cellens energikraftverk. Och vips så hade den moderna eukaryota cellen uppstått. En supercell med superkrafter. Men utvecklingssagan slutade inte där. Kärlekshistorier brukar ju inte sällan involvera en tredje part. Efter ett par 100 miljoner år träffade en av den ursprungliga eukaryota cellens ättlingar på en fotosyntetiserande cyanobakterie. Utan att kunna motstå denna gröna skönhet, svalde den helt sonika bakterien. Symbiogenes, det vill säga sammansmältningen av två samarbetande organismer, skedde därefter framgångsrikt för andra gången i världshistorien: cyanobakterien degenererade nämligen med tiden till den fotosyntetiserande cellorganellen kloroplast, och den pimpade cellen blev alla växters anfader. Men vad är då den stora konsekvensen av dessa passionerade möten? Jo, av anledningar som inte är helt klarlagda, har komplexa multicellulära organismer, som växter och djur, endast uppstått bland eukarya. Multicellularitet innebär är att ett stort antal genetiskt identiska celler tillsammans bildar en större organism. Du själv var från början en enda cell, ett embryo, innan den delade sig i miljarder kopior. Alla celler i din kropp, vare sig om det är muskelceller, njurceller eller vita blodkroppar, är genetiskt identiska dubbletter. Men de har gått ihop och skapat ett nätverk. Och trots att de alla ursprungligen har identiska kapaciteter utför cellerna endast specialistfunktioner. Ett gäng har specialiserats till musklerceller, ett gäng till njurceller, och ett gäng till immunceller. Genom att alla celler slipper göra allt själva kan de avsätta större resurser till sina spetskompetenser och skapa avancerade vävnader. Komplexitet uppstår ur denna specialisering. Romeo hade i sin tur begått självmord då han felaktigt trodde att Julia hade dött. Hängivenheten mellan det mikrobiella paret i vår historia är lika häftig.  Den här sortens avancerad samarbete har prokaryoter aldrig klarat av att åstadkomma under de miljarder år de funnits. Anledningen till att tulpanerna på ditt bord, katten i ditt knä, granen i skogen, och människorna på din gata kan finnas, är att en arké och en bakterie för 2 miljarder år sen började samarbeta istället för att äta upp varandra. Vi komplexa organismer har endosymbiogenesen att tacka för vår existens. Ett möte mot alla odds, som skapade den värld vi känner. När Shakespeares Julia i slutet av pjäsen upptäcker att Romeo dött tar hon sitt liv, för hon inte förmår att leva utan honom. Romeo hade i sin tur begått självmord då han felaktigt trodde att Julia hade dött. Hängivenheten mellan det mikrobiella paret i vår historia är lika häftig. Cellens energikraftverk mitokondrien kan efter alla dessa år inte längre leva utanför cellen, och cellen inte överleva utan mitokondrien. Det verkar som de starkaste band skapas när oförenliga par förenas. Farshid Jalalvand, skribent och forskare i klinisk mikrobiologi   Vidare läsning Buttery, S. 2017. Rediscovering symbiogenesis: The latest research in understanding the origins of eukaryotic cells. Crosstalk, Cellpress Sällström, S. 2015. Mikroorganism från havet ger ledtrådar om cellers ursprung. Vetenskapsradion, Sveriges Radio. Miller, S. M. 2010. Volvox, Chlamydomonas, and the Evolution of Multicellularity. Nature Education Morell, V. 1997. Microbiologys Scarred Revolutionary. Science.

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler The TWiVites discuss Zika virus seroprevalence in wild monkeys, Zika virus mRNA vaccines, and a gamete fusion protein inherited from viruses. Become a patron of TWiV! Links for this episode Nido2017 Meeting ASMCUE Seroprevalence of Zika virus in wild monkeys and baboons (mSphere) Zika virus mRNA vaccines (paper one, paper two) Gamete fusion protein is type II viral fusogen (paper one, paper two) Image credit Letters read on TWiV 432 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free with your first purchase – WITH FREE SHIPPING – by going to blueapron.com/twiv Weekly Science Picks Alan - Raspberry pi Kathy - The Worst F&#%ing Words Ever Dickson - NASA images of climate changeBrianne - How herd immunity works Vincent - Radioactive boars in Fukushima Listener Pick Margaret - DNA socks and gloves Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

In the present study, the structure and mechanism of two assembly chaperones of Rubisco, Raf1 and RbcX, were investigated. The role of Raf1 in Rubisco assembly was elucidated by analyzing cyanobacterial and plant Raf1 with a vast array of biochemical and biophysical techniques. Raf1 is a dimeric protein. The subunits have a two-domain structure. The crystal structures of two separate domains of Arabidopsis thaliana (At) Raf1 were solved at resolutions of 1.95 Å and 2.6–2.8 Å, respectively. The oligomeric state of Raf1 proteins was investigated by size exclusion chromatography connected to multi angle light scattering (SEC-MALS) and native mass spectrometry (MS). Both cyanobacterial and plant Raf1 are dimeric with an N-terminal domain that is connected via a flexible linker to the C-terminal dimerization domain. Both Raf1 poteins were able to promote assembly of cyanobacterial Rubisco in an in vitro reconstitution system. The homologous cyanobacterial system resulted in very high yields of active Rubisco (>90%), showing the great efficiency of Raf1 mediated Rubisco assembly. Two distinct oligomeric complex assemblies in the assembly reaction could be identified via native PAGE immunoblot analyses as well as SEC-MALS and native MS. Furthermore, a structure-guided mutational analysis of Raf1 conserved residues in both domains was performed and residues crucial for Raf1 function were identified. A new model of Raf1 mediated Rubisco-assembly could be proposed by analyzing the Raf1-Rubisco oligomeric complex with negative stain electron microscopy. The final model was validated by determining Raf1-Rubisco interaction sites using chemical crosslinking in combination with mass spectrometry. Taken together, Raf1 acts downstream of chaperonin-assisted Rubisco large subunit (RbcL) folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by Rubisco small subunit (RbcS) results in holoenzyme formation. In the second part of this thesis, the role of eukaryotic RbcX proteins in Rubisco assembly was investigated. Eukaryots have two distinct homologs of RbcX, RbcX-I and RbcX-II. Both, plant and algal RbcX proteins were found to promote cyanobacterial Rubisco assembly in an in vitro reconstitution system. Mutation of a conserved residue important for Rubisco assembly in cyanobacterial RbcX also abolished assembly by eukaryotic RbcX, underlining functional similarities among RbcX proteins from different species. The crystal structure of Chlamydomonas reinhardtii (Cr) RbcX was solved at a resolution of 2.0 Å. RbcX forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Structural analysis of a fusion protein of CrRbcX and the C-terminal peptide of RbcL suggests that the peptide binding mode of CrRbcX may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution. Preliminary analysis of RbcX in Chlamydomonas indicated that the protein functions as a Rubisco assembly chaperone in vivo. Therefore, RbcX was silenced using RNAi in Chlamydomonas which resulted in a photosynthetic growth defect in several transformants when grown under light. RbcX mRNA levels were highly decreased in these transformants which resulted in a concomitant decrease of Rubisco large subunit levels. Biochemical and structural analysis from both independent studies in this thesis show that Raf1 and RbcX fulfill similar roles in Rubisco assembly, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

Cells often face low-oxygen conditions at night. When this happens, some organisms such as the single-cell alga Chlamydomonas are able to generate cellular energy from the breakdown of sugars without taking up oxygen.Although critical to the survival of common aquatic and terrestrial organisms that are found all over the planet, many of the details regarding this low-oxygen energy creation process are poorly understood.

This episode: Green algae's hydrogen production is analyzed and improved! Download Episode (3.8 MB, 4.1 minutes)Show notes:News item/Journal Paper Other interesting stories: Scientists take protein from flesh-eating bacteria and make useful adhesive Fungus from horse gut could be good for breaking down plant material for biofuels Dogs are an important influence on our microbiota Bacterial fossils could contain evidence of an ancient supernova E. coli modified to produce imitation petroleum Post questions or comments here or email to bacteriofiles at gmail dot com. Thanks for listening! Subscribe at iTunes, check out the show at Twitter or Facebook

Mon, 23 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17329/ https://edoc.ub.uni-muenchen.de/17329/1/Wang_Fei.pdf Wang, Fei

Thu, 10 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15080/ https://edoc.ub.uni-muenchen.de/15080/1/JALAL_ABDULLAH.pdf Jalal, Abdullah ddc:570, ddc:500, Fakultät für Biologie

Fri, 13 Jan 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13881/ https://edoc.ub.uni-muenchen.de/13881/1/Schwarz_Christian.pdf Schwarz, Christian d

Ursula Goodenough, PhD The Sacred Depths of Nature Join Michael Lerner in conversation with professor and author Ursula Goodenough about her work and book, The Sacred Depths of Nature. As well as her biology courses, Ursula co-teaches The Epic of Evolution, with a physicist and a geologist, for non-science students. Her research has focused on the cell biology and (molecular) genetics of the sexual phase of the life cycle of the unicellular eukaryotic green alga Chlamydomonas reinhardtii and, more recently, on the evolution of the genes governing mating-related traits. Ursula Goodenough Ursula is professor of biology at Washington University in St. Louis, Missouri. She is the author of The Sacred Depths of Nature (Oxford University Press, 1998), which offers religious perspectives on our scientific understandings of nature, particularly biology at a molecular level. Ursula was educated at Radcliffe and Barnard Colleges, Columbia University, and Harvard University. She did two years of postdoctoral work at Harvard, and was assistant and associate professor of biology at Harvard from 1971-1978 before moving to Washington University. Find out more about The New School at tns.commonweal.org.

Phylloquinone is a compound present in all plants serving as cofactor for photosystem I mediated electron transport during photosynthesis. This work reports on the identification and analysis of several Arabidopsis thaliana phylloquinone absence (pha) and isochorismate synthase (ics) mutants impaired in the biosynthesis of PhQ (vitamin K1). Besides the complete lack of PhQ, these plants show a typical phenotype characterized by seedling lethality, photosynthetic defects specifically related to impaired photosystem I accumulation/activity to 5-15% of wild-type levels and partial recovery of 15% PhQ content and 50-70% PSI accumulation/activity after feeding with the metabolic precursor of vitamin K1, 1,4-dihydroxy-2-naphthoate. Map-based localization of the mutated allele in the pha plants identified a new gene, called PHYLLO. It consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of the photosynthetic phylloquinone in cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes still reside as polycistronic units in plastomes of red algae and in eubacterial chromosomes strongly suggests that PHYLLO derived from an operon present in the proto-organelle precursor of all plastids. The principle architecture of the PHYLLO locus is conserved in the nuclear genomes of plants and the green alga Chlamydomonas reinhardtii, indicating that selective forces have been acting to maintain the cluster structure in the form of a gene fusion, presumably as an adaptation of an multifunctional association of four enzymatic activities already pre-existing in the chloroplast. In line with this finding, the data present in this work suggest that the PHYLLO composite product is part of a metabolon for the biosynthesis of phylloquinone. The menF module of PHYLLO in Chlamydomonas, encoding the isochorismate synthase activity, is full-length, whereas in higher plants this module surprisingly lacks the functional 3’ part, uncovering a recent gene splitting event during evolution. Such a gene fission event, which resulted in inactivation of the encoded ICS enzymatic activity from PHYLLO, must have been preceded by establishment of a second functional copy of the menF gene. Accordingly, double-knockouts of the ICS1 and ICS2 genes in Arabidopsis analysed during this work, were unable to synthesize PhQ, demonstrating that the activity of the menF module of PHYLLO has been replaced after the splitting of the 3’-region by at least one more ICS gene present in genomes of higher plants. The fact that ICS1 is also required for salicylic acid biosynthesis in Arabidopsis, establishes a metabolic link between photosynthesis and systemic acquired resistance. Therefore, gene fusion, duplication and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants. Despite the essential function of PhQ for PSI stability and plant viability, analyses of ics heterozygous knockout plants, as well as complementation of the pha mutants by NA feeding and transgenic forms of PHYLLO demonstrate that the bulk of cellular phylloquinone is not associated with photosystem I, opening the possibility for additional functions of vitamin K1 in plant cell membranes.

Die plastidäre DNA höherer Pflanzen wird allgemein als zirkuläres Molekül von der Größe eines Monomers beschrieben. Die DNA-Replikation soll von einem Paar Replikationsursprünge ausgehen. Mittels theta- (displacement loop) und sigma-Replikation (rolling circle) würden aus zirkulären Ausgansprodukten erneut zirkuläre Produkte entstehen. In Nicotiana tabacum sollen diese Mechanismen auf zwei beschriebenen Replikationsursprüngen beruhen: oriA und oriB. In früheren Arbeiten wurde bereits gezeigt, dass oriA nicht essentiell ist, aber vermutet, dass eine Kopie des oriB unverzichtbar sei. Mittels Plastidentransformation wurde jetzt auch gezeigt, dass plastidäre DNA-Replikation auch erfolgt, wenn beide Kopien des oriB inaktiviert sind. In weiteren Experimenten konnten in einer Linie drei der vier Ori deletiert werden. Untersuchungen mittels Pulsfeldgelelektrophorese und Southern-Analysen zum Replika-tionsmechanismus wiesen auf lineare ptDNA-Moleküle mit definierten Enden hin. Eine mögliche Erklärung für diese Enden wäre, dass diese an der Position von Replikationsursprüngen liegen. Tatsächlich wurde eine entsprechende Korrelation mit oriA – und weniger deutlich – mit oriB gefunden. Andere Enden liegen auf Positionen, auf denen in Chlamydomonas reinhardtii, Glycine max, Oenothera elata ssp. hookeri, Oryza sativa und Zea mays Replikationsursprünge beschrieben wurden. Dazu kommen noch weitere mögliche Replikationsursprünge. Die Mechanismen der plastidärer DNA-Replikation werden basierend auf diesen neuen Ergebnissen und neuen Erkenntnissen in der Literatur diskutiert.

Plants respond to changes in illumination conditions by modifying the thylakoid proteins post-translationally and by reorganizing the photosynthetic machinery. However, the mechanisms that characterize the short-term and the long-term responses are different. In the first case, the organism reacts to rapid illumination changes via phosphorylation of photosystem II (PSII) and light-harvesting (LHCII) proteins. Phosphorylation of PSII is thought to be relevant for PSII turnover, whereas LHCII phosphorylation is required for state transitions, which ensure the redistribution of the excitation energy between the two photosystems. Long-term imbalances in the energy distribution elicit changes in the composition and stoichiometry of the photosynthetic apparatus (photosynthetic acclimation). Two types of thylakoid protein kinases have been previously associated with LHCII phosphorylation, the TAK (thylakoid-associated kinase) proteins in Arabidopsis thaliana and Stt7 in Chlamydomonas reinhardtii. This work shows that the TAK proteins (TAK1, TAK2, and TAK3) are neither involved in LHCII phosphorylation nor in state transitions. In addition, evidences are provided that exclude any role of TAK2 and TAK3 in the photosynthetic electron flow. In Arabidopsis, two Stt7-like proteins exist, STN7 and STN8. Loss of STN7 blocks both LHCII phosphorylation and state transitions, indicating that this protein is a genuine Stt7 homolog. In contrast, STN8 is required for the quantitative phosphorylation of PSII core proteins. PSII activity under high-intensity light is affected only slightly in stn8 mutants, and D1 turnover is indistinguishable from the wild-type (WT), implying that reversible protein phosphorylation is not essential for PSII repair. Functional characterization of stn7 mutants showed that STN7 is not only associated with the short term response, but it is also required for the adaptation to long-term illumination changes including light-quality-induced changes in the mRNA expression of nuclear and plastid genes for photosynthetic proteins. This indicates that short-term and long-term photosynthetic adaptations are coupled and that phosphorylation of LHCII, or of an unknown substrate of STN7, is crucial for the control of photosynthetic gene expression and readjustment of photosystem stoichiometry.

Fri, 20 Dec 2002 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/802/ https://edoc.ub.uni-muenchen.de/802/1/Waltenberger_Harald.pdf Waltenberger, Harald

Die regulatorischen Fähigkeiten des RNS-Bindeproteins CHLAMY 1 aus C. reinhardtii wurden unter Verwendung eines chimären Reporterkonstruktes überprüft. Hierdurch konnte gezeigt werden, dass ein in CHLAMY 1- angereichertes Proteingemisch die Translation dieses Reporterkonstruktes in einem zellfreien System um durchschnittlich 14,56 % reprimieren konnte. Durch eine Molekularmassenbestimmung unter nativen und denaturierenden Bedingungen wurde der Aufbau von CHLAMY 1 aus mehreren Untereinheiten gezeigt. Der native Komplex hat eine Größe von ca. 160 kDa und besteht, ausgehend von den in dieser Arbeit durchgeführten Experimenten unter denaturierenden Bedingungen, aus den drei Untereinheiten C1 bis C3. Die Aufreinigung von CHLAMY 1 wurde in drei Schritten ausgeführt: (a) Herstellung eines Rohextraktes, (b) Durchführung einer 0,5-1 M AS-Fällung und (c) Ausführung einer spezifischen RNS-Affinitätschromatographie. Es konnten dadurch drei für CHLAMY 1 spezifische Proteine mit den Molekularmassen 60, 50 und 44 kDa aufgereinigt werden. Durch eine Peptid-Sequenzierung wurden insgesamt neun Peptide mit einer Länge von bis zu 24 Aminosäuren sequenziert. Hierbei wurde ein Peptid identifiziert, das Bestandteil der zwei Untereinheiten C1 und C2 ist. In Western Blot-Analysen mit C. reinhardtii-Rohextrakten und Peptid- Antikörpern wurde die Abundanz der CHLAMY 1-Untereinheiten über einen Tag-Nacht-Verlauf bestimmt. Die Untereinheit C1 (60 kDa) scheint konstitutiv exprimiert zu werden, wohingegen die Expression von C3 (44 kDa) von der inneren Uhr kontrolliert zu sein scheint. Durch die Sichtung einer Expressions-Genbank aus C. reinhardtii mit Peptid- Antikörpern wurden zwei für CHLAMY 1 kodierende cDNS-Fragmente isoliert und durch EST-Klone aus dem Sequenzierungsprojekt von C. reinhardtii Richtung 5’-Ende verlängert. Alle Peptide der Untereinheiten C1 und C2 konnten in einer cDNS identifiziert werden, deren ORF für ein Protein von 51,73 kDa kodiert. Für die Untereinheit C3 wurde ein ORF identifiziert, der für ein Protein der Molekularmasse 45,00 kDa kodiert. In allen Proteinen wurden Regionen für mögliche posttranslationale Modifikationen identifiziert. Im Rahmen dieser Arbeit konnte gezeigt werden, dass „UG“-Bindeproteine auch in anderen Organismen vorkommen. CHLAMY 1 kann spezifisch an die 16 „UG“-Wiederholungen in der 3’-NTR der regA-mRNS aus V. carteri binden. In dieser Kugelalge wurde außerdem ein RNS-Bindeprotein (VOLVO 1) identifiziert, das spezifisch an diese „UG“-haltige Region bindet. Im Pilz N. crassa konnte ein CHLAMY 1 analoges Protein identifiziert werden (CRASSA 1), das an die sieben „UG“-Wiederholungen in der 3’-NTR von gs2wt aus C. reinhardtii binden kann. Die Bindeaktivität in einem Tag-Nacht-Verlauf scheint von der circadianen Uhr gesteuert zu werden.

Plastid chromosomes from the variety of plant species contain several conserved open reading frames of unknown function, which most probably represent functional genes. The primary aim of this thesis was the analysis of the role of two such ORFs, designated ycfs or hypothetical chloroplast reading frames, namely ycf9 (ORF62) and ycf10 (ORF229, cemA). Both were analyzed in Nicotiana tabacum (tobacco) via their inactivation using biolistic plastid transformation. A new experimental protocol, based on pulsed-field gel electrophoresis (PFGE), was established to reliably assess the homoplastomic state of transformed plants. 1. Functional analysis of the ycf9 gene product: The inactivation of ycf9 in N. tabacum as well as in Chlamydomonas reinhardtii yielded a homoplastomic mutant phenotype after several rounds of regeneration under selective pressure. The mutant plants grew photoautotrophically, but displayed two clear phenotypes, a light-sensitive one, increasing with the light intensity, and a dwarf phenotype under low-light combined with temperatures below 20°C. The ycf9 gene product was exclusively located in PSII core complexes. This localization was based on the isolation of protein complexes released from thylakoids by controlled, partial lysis, followed by sucrose density gradient centrifugation or 2D gel electrophoresis. This finding revised data of the literature. Biochemical analysis indicated an involvement of the protein in the interaction of the light harvesting antenna II complex (LHCII) with PSII cores. In particular, PSII-LHCII supercomplexes could no longer be isolated from transplastomic tobacco plants. Furthermore, the minor chlorophyll a/b-binding proteins CP26, and to a lesser extent CP29, were substantially reduced under most growth conditions analyzed, in both, tobacco and photoautotrophically grown Chlamydomonas mutants (Swiatek et al. 2001). The gene was therefore renamed psbZ. The ∆psbZ-related alterations in the supramolecular organization of PSII complexes were accompanied by considerable modification in (i) the phosphorylation pattern of PSII subunits, (ii) the rate of deepoxydation of xanthophylls, and (iii) the kinetics and amplitude of non-photochemical quenching. The proposed position of PsbZ in close proximity to CP43 enables the protein to interact with PSII cores to elicit an adaptation process in response to excess light excitation. The molecular mechanism underlying this energy dissipation process remains to be investigated. 2. Functional analysis of the ycf10 gene product: Biolistic plastid transformation was also used to inactivate the ycf10 reading frame in tobacco. After several rounds of regeneration under selective pressure, homoplastomic plants were obtained. Northern analysis uncovered co-transcription of ycf10 within the psaI-ycf4-ycf10-petA gene cluster, with at least two promotor regions upstream of the psaI gene. The mutant plants grew photoautotrophically and developed dark green leaves with numerous pale green to white regions, the latter devoid of photosynthetic activity. The loss of ycf10 did not affect photosynthetic activity, as indicated by unaltered chlorophyll fluorescence. The tobacco ycf10 gene product was localized in the chloroplast inner envelope membrane. Neither protein composition of stroma or thylakoid fractions, nor the stability of the photosynthetic protein complexes were affected in the mutant plants. In contrast, CO2- dependent oxygen evolution was strongly reduced, with a maximum rate of Ci-dependent photosynthesis being approximately 50% lower than in wild-type plants. Two explanations can account for the observed phenomenon: (i) de-regulation of carbonconcentrating mechanisms in transformed cells, or (ii) an indirect effect on CO2-uptake in ∆ycf10 plants. 3. Pulsed-field gel electrophoresis is an ideal tool to verify the homoplastomic state of transformed plants: To enhance the sensitivity of detection of heteroplastomic states, and to distinguish between plastome-located wild-type segments in transplastomic material and promiscuous DNA, a new approach was developed. Customary Southern and PCR techniques are not sensitive enough or not discriminating the latter alternatives, respectively. Pulsed-field gel electrophoresis allows to isolate virtually contamination-free plastid DNA. Plastid DNA isolated this way lacked traces of nuclear and mitochondrial DNA at a detection level of 50 DNA molecules. This excludes that gene-specific PCR amplification products originate from promiscuous nuclear or mitochondrial gene copies. Therefore, PFGE appears to be an ideal tool to investigate the homoplastomic state of transformed plants, especially when combined with radiolabeled probes and Southern techniques.

Chloroplast gene expression is predominantly regulated at the posttranscriptional levels of mRNA stability and translation efficiency. The expression of psbA, an important photosynthesis-related chloroplast gene, has been revealed to be regulated via its 5’- untranslated region (UTR). Some cis-acting elements within this 5’UTR and the correlated trans-acting factors have been defined in Chlamydomonas. However, no in vivo evidence with respect to the cis-acting elements of the psbA 5’UTR has been so far achieved in higher plants such as tobacco. To attempt this, we generated a series of mutants of the tobacco psbA 5’UTR by base alterations and sequence deletions, with special regard to the stem-loop structure and the putative target sites for ribosome association and binding of nuclear regulatory factors. In addition, a versatile plastid transformation vector pKCZ with an insertion site in the inverted repeat region of the plastid genome was constructed. In all constructs, the psbA 5’UTR (Wt or modified) was used as the 5’ leader of the reporter gene uidA under control of the same promoter, Prrn, the promoter of the rRNA operon. Through biolistic DNA delivery to tobacco chloroplasts, transplastomic plants were obtained. DNA and RNA analyses of these transplastomic plants demonstrated that the transgenes aadA and uidA had been correctly integrated into the plastome at the insertion site, and transcribed in discrete sizes. Quantitative assays were also done to determine the proportion of intact transplastome, the uidA mRNA level, Gus activity, and uidA translation efficiency. The main results are the following: 1) The insertion site at the unique MunI between two tRNA genes (trnR-ACG and trnNGUU) is functional. Vector pKCZ has a large flexibility for further DNA manipulations and hence is useful for future applications. 2) The stem-loop of the psbA 5’UTR is required for mRNA stabilisation and translation. All mutants related to this region showed a 2~3 fold decrease in mRNA stability and a 1.5~6 fold reduction in translation efficiency. The function of this stem-loop depends on its correct sequence and secondary conformation. 3) the AU-box of the psbA 5’UTR is a crucial translation determinant. Mutations of this element almost abolished translation efficacy (up to 175-fold decrease), but did not significantly affect mRNA accumulation. The regulatory role of the AU-Box is sequencedependent and might be affected by its inner secondary structure. 4) The internal AUG codon of the psbA 5’UTR is unable to initiate translation. An introduction of mRNA translatability from this codon failed to direct the translation of reporter uidA gene, overriding the mutation of the AU-Box. 5) The 5’end poly(A) sequence does not confer a distinct regulatory signal. The deletion of this element only insignificantly affected mRNA abundance and translation. However, this mutation might slightly disturb the conformation of the stem-loop, resulting in a moderate decrease in translation efficiency (~1.5 fold). 6) The SD(Shine-Dalgarno)-like RBS (ribosome binding site) of the psbA 5’UTR appears to be an indispensable element for translation initiation. Mutation of this element led to a dramatically low expression of the uidA gene as seen by Gus staining. 7) The 5’end structural sequence of the rbcL 5’UTR does not convey a high mRNA stabilising effect to the psbA 5’UTR in a cycling condition of the light and the dark. Their distinct roles appear to be involved in darkness adaptation. Furthermore, with respect to the overall regulatory function of the psbA 5’UTR, two models are proposed, i.e. dual RBS-mediated translation initiation, and cpRBPs-mediated mRNA stability and translation. The mechanisms for mRNA stabilisation entailed by the rbcL 5’UTR are also discussed. Direct repeat-mediated transgene loss after chloroplast transformation and other aspects related to the choice of insertion site and plastid promoter are also analysed.