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El pdf del artículo es la versión pre-print. ; A study of the in vitro reconstitution of sugar beet cytochrome b 559 of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b 559 form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b 559. Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b 559 subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b 559-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b 559-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b 559. A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme. ; This work was supported by Grants AGL2008-00377, MAT2008-03461, and BFU2007-68107-C02-01 from the Spanish Ministry of Science and Innovation (MICINN), PADI CVI-261 from the Andalusia Regional Government, and DGA-GC E33 and DGA-GE B18 from Aragon Regional Government. All these Grants were partially financed by the EU FEDER Program. M. A. Luján would like to thank the FPI Fellowship Program of the MICINN for financial support. ; Peer reviewed

8 páginas, 3 figuras, 1 tabla ; Conventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU law ; This work was funded by the European Union INTERREG Atlantic Area Program ("LabelFish", project 2011-1/163) ; Peer reviewed

Non-invasive methods have the potential to circumvent problems associated with using more traditional techniques when surveying for rare and elusive species. In this study, non-invasive molecular-based methods have been used to analyse the scats of several species of marsupials. DNA was successfully extracted from scats of the quokka, Setonix brachyurus, and three other macropods (Macropus fuliginosus, M. irma and M. eugenii) sympatric with the quokka and with similar-appearing scats. Partial sequence from the mitochondrial cytochrome b gene from these four species and seven other macropods was used to measure genetic differentiation among them to determine whether the quokka could be unambiguously identified from the scats alone. The results confirm that molecular approaches can be used for macropod species identification using scats as the source material. The approach will have potential survey and management applications, and, more specifically, may lead to more accurate assessment of the quokka's geographic range, leading to implementation of more appropriate management strategies for its conservation.

The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre. ; This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO2012-35271, BIO2015-64169-P, MAT2011-23861 and CTQ2015-64486-R) the Andalusian Government (PAIDI BIO-022) and the Aragón Government (Grupo consolidado B-18). All these grants were partially financed by the EU FEDER Program. ; Peer reviewed

The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre. ; This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO2012-35271, BIO2015-64169-P, MAT2011-23861 and CTQ2015-64486-R) the Andalusian Government (PAIDI BIO-022) and the Aragón Government (Grupo consolidado B-18). All these grants were partially financed by the EU FEDER Program ; Peer reviewed

Twenty-five chipmunk species occur in the world, of which only the Siberian chipmunk, Tamias sibiricus, inhabits Asia. To investigate mitochondrial cytochrome b sequence variations and population structure of the Siberian chipmunk in northeastern Asia, we examined mitochondrial cytochrome b sequences (1140 bp) from 3 countries. Analyses of 41 individuals from South Korea and 33 individuals from Russia and northeast China resulted in 37 haplotypes and 27 haplotypes, respectively. There were no shared haplotypes between South Korea and Russia - northeast China. Phylogenetic trees and network analysis showed 2 major maternal lineages for haplotypes, referred to as the S and R lineages. Haplotype grouping in each cluster was nearly coincident with its geographic affinity. In particular, 3 distinct groups were found that mostly clustered in the northern, central and southern parts of South Korea. Nucleotide diversity of the S lineage was twice that of lineage R. The divergence between S and R lineages was estimated to be 2.98-0.98 Myr. During the ice age, there may have been at least 2 refuges in South Korea and Russia - northeast China. The sequence variation between the S and R lineages was 11.3% (K2P), which is indicative of specific recognition in rodents. These results suggest that T. sibiricus from South Korea could be considered a separate species. However, additional information, such as details of distribution, nuclear genes data or morphology, is required to strengthen this hypothesis. ; This study was supported, in part, by a Korea Research Foundation Grant funded by the Korean Government (Ministry of Education and Human Resources Development; KRF-2007-C00193-I00755), Brain Korea 21 Program for Veterinary Science and Research Institute of Veterinary Science, Seoul National University.

During oxygenic photosynthesis, cytochromec6 shuttles electrons between the membrane-bound complexes cytochrome bf and photosystem I. Complex formation between Phormidium laminosum cytochromef and cytochrome c6 from bothAnabaena sp. PCC 7119 and Synechococcus elongatus has been investigated by nuclear magnetic resonance spectroscopy. Chemical-shift perturbation analysis reveals a binding site on Anabaena cytochrome c6, which consists of a predominantly hydrophobic patch surrounding the heme substituent, methyl 5. This region of the protein was implicated previously in the formation of the reactive complex with photosytem I. In contrast to the results obtained for Anabaena cytochromec6, there is no evidence for specific complex formation with the acidic cytochrome c6 fromSynechococcus. This remarkable variability between analogous cytochromes c6 supports the idea that different organisms utilize distinct mechanisms of photosynthetic intermolecular electron transfer. ; European Commission HPRN-CT-1999-00095 ; Spanish Ministry of Science and Technology BMC2000-0444 ; Andalusian Government CVI-0198