Nuclear Photosynthetic Mutants of Chlamydomonas reinhardtii

In a previous study (Gumpel et al., 1995) a collection of nuclear photosynthetic mutants of Chlamydomonas reinhardtii were generated using an insertional mutagenesis technique. Subsequent biophysical and fluorescence analysis identified mutants that were affected in either photosystem II, photosystem I or the cytochrome b6f complex. A detailed genetic analysis of the latter mutants identified three (MΦ11, MΦ3O and MΦ37) in which the mutant phenotype co-segregates with the insertional mutagen. Protein and RNA analysis revealed that MΦ11 fails to accumulate transcript for the chloroplast gene (petA) encoding the apoprotein of cytochrome f, whereas MΦ37 shows a severely reduced accumulation of the transcript for the chloroplast gene (petB) encoding the apoprotein of cytochrome b6. In this study, the collection of mutants have been further characterised: i) The photosystem I mutants have been further characterised by fluorescence and EPR spectroscopy and by Southern and western blot analysis. The results indicate that a functional PSI complex fails to assemble but that none of the nuclear genes encoding known PSI subunits are affected, suggesting that nuclear genes other than those encoding structural components are required for PSI biogenesis. ii) Using DNA probes isolated from the genomic region flanking the insertional mutagen in mutant MΦll, a cosmid wildtype genomic library was screened. A 42kb cosmid (termed B5) was shown to rescue the mutant phenotype when transformed into the nuclear genome. B5 transformants show restored photosynthetic function and were able to grow photoautotrophically on minimal medium. Protein analysis of the transformants showed the presence of the cytochrome f protein. Restriction analysis of B5 combined with complementation studies identified a 18kb genomic fragment that retained the ability to complement the mutant. This fragment was used to screen a wildtype C. reinhardtii cDNA library. Two independent cDNA clones were obtained, named ACV4 and ACV5. Southern analysis showed that the gene encoding ACV4 showed restriction fragment length polymorphisms when comparing the genomes of wildtype cells and MΦ11. In the case of ACV5, the genomic sequence corresponding to this cDNA was completely lacking from the mutant, indicating that insertion of the mutagen has lead to the deletion of genomic DNA. Northern analysis revealed that the ACV5 transcript is also absent in MΦ11, whereas the ACV4 transcript was undetectable in both wildtype and mutant cells. Sequence analysis was initiated on both cDNAs and indicates that both appear not to be full-length. In the case of ACV5, the deduced protein sequence has revealed some homology to known nucleic acid binding protein sequences in the database. iii) Southern analysis was carried out on mutant MΦ37 in order to generate a restriction map covering the site of integration of the mutagen. This enabled the cloning of a flanking genomic fragment and the screening of the wildtype cosmid library. A number of hybridising clones were isolated, however none were able to complement the mutant. Simultaneously, a plasmid rescue strategy was also employed to attempt to isolate flanking genomic DNA. However, this too failed to isolate a complementing cosmid clone. Possible reasons for this failure are discussed

Few-photon transport in quasi-one-dimensional geometries

An analysis of some aspects of photon transport through cavities and emitters embedded in a one-dimensional geometries is presented. The concept of photon blockade is defined for few-photon states interacting with a single two-level atom and the strength of achievable blockade is calculated in this setting. A brief review of some promising schemes for achieving photon blockade from the literature is also provided. The conflict between linear and nonlinear optical processes is studied for a novel version of the famous Hong-Ou-Mandel effect in a photonic waveguide with a side-coupled two-level emitter

Medical Practice, Urban Politics and Patronage: The London 'Commonalty' of Physicians and Surgeons of the 1420s.

Medical practice in fifteenth-century England is often seen as suffering from the low status and unregulated practice of which Thomas Linacre later complained. Unlike in many European cities, the provision of physic was uncontrolled, and while urban guilds oversaw surgery as a manual art, no comprehensive system of medical organisation or regulation existed. However, in a remarkable episode of the 1420s, a group of university-trained physicians and elite surgeons associated with Humphrey, Duke of Gloucester, briefly established just such a system. While their efforts initially secured approval for a national scheme, it was only in the City of London that they succeeded in implementing their plans. The detailed ordinances of the collegiate 'commonalty' they founded provide a unique insight into their attitudes. Drawing on continental models, they attempted to control all medicine within the city by establishing a hierarchy of practitioners, preventing illicit and incompetent practice, and offering treatment to even the poorest Londoners. Yet they failed to appreciate the vested interests of civic politics: achieving these aims meant curtailing the rights of the powerful Grocers and the Barbers, a fact made clear by their adjudication of a case involving two members of the Barbers' Company, and the Barbers' subsequent riposte-a mayoral petition that heralded the commonalty's end. Its founder surgeons went on to revitalise their Surgeons' Fellowship, which continued independently of the Barbers until a merger in 1540; in contrast, the physicians withdrew from civic affairs, and physic remained entirely unregulated until episcopal licensing was instituted in 1511.This work was supported by the Wellcome Trust awards 097782/Z/11/Z and 077548.This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/ehr/cev26

Cognitive neuroscience, metaphor and pictures: part 2

This paper provides a sense of the value of a ‘cognitive’ perspective to therapists when thinking about minds and situations that they encounter. As well as helping to establish knowledge, the science also offers a framework for critical assessment. The paper also aims to prepare the ground for future progress in our understanding of the value of metaphor or picture-based communications. This is part 2 of 2

Genetic modification of tomato with the tobacco lycopene β-cyclase gene produces high β-carotene and lycopene fruit

Transgenic Solanum lycopersicum plants expressing an additional copy of the lycopene beta-cyclase gene (LCYB) from Nicotiana tabacum, under the control of the Arabidopsis polyubiquitin promoter (UBQ3), have been generated. Expression of LCYB was increased some 10-fold in ripening fruit compared to vegetative tissues. The ripe fruit showed an orange pigmentation, due to increased levels (up to 5-fold) of beta-carotene, with negligible changes to other carotenoids, including lycopene. Phenotypic changes in carotenoids were found in vegetative tissues, but levels of biosynthetically related isoprenoids such as tocopherols, ubiquinone and plastoquinone were barely altered. Transformants showed tolerance to the bleaching herbicide beta-cyclase inhibitor, 2-(4-chlorophenylthio) triethylamine. The phenotype was inherited for at least three generations

Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis

Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial β-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (∼38%), and low (∼1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g−1 dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g−1 dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin