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Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
Biotic interchange after the connection of previously independently evolving floras and faunas is thought to be one of the key factors that shaped global biodiversity as we see it today. However, it was not known how biotic interchange develops over longer time periods of several million years following the secondary contact of different biotas. Here we present a novel method to investigate the temporal dynamics of biotic interchange based on a phylogeographical meta-analysis by calculating the maximal number of observed dispersal events per million years given the temporal uncertainty of the underlying time-calibrated phylogenies. We show that biotic influx from mainland Asia onto the Indian subcontinent after Eocene continental collision was not a uniform process, but was subject to periods of acceleration, stagnancy and decrease. We discuss potential palaeoenvironmental causes for this fluctuation.
Photosystem II (PSII) catalyzes the unique reaction of light-dependent water oxidation and subsequent reduction of plastoquinone at the beginning of the photosynthetic electron transport chain. The mature complex consists of at least 20 protein-subunits and over 80 cofactors. Further proteins are required for biogenesis and repair of PSII. Most of these proteins interact specifically with assembly intermediates during defined steps in PSII assembly. This review shall emphasize the function of the two factors Psb27 and Psb28 during the biogenesis and repair of PSII in cyanobacteria and give an impression of their potential biochemical, structural and physiological properties in plants considering the fact that they both have homologues in all oxygenic photosynthetic organisms. We hypothesize that Psb28 may have retained its function in higher plants while the two Psb27 forms bind differently to PSII intermediates depending on PSII core phosphorylation state.
The chloroplast phosphorylation network is important for posttranslational regulation of photosynthetic complexes, gene expression and metabolic pathways. In mass-spectrometric analyses a lot of putative phosphorylation targets have been found but these data need to be confirmed and brought into a physiological context. Here, we present a current protocol to quantify the phosphorylation state of thylakoid proteins and an in situ method to verify putative substrates for thylakoid associated kinases.
The genome of S. cerevisae encodes at least twenty hexose transporter-like proteins. Despite extensive research, the functions of Hxt8-Hxt17 have remained poorly defined. Here, we show that Hxt13, Hxt15, Hxt16 and Hxt17 transport two major hexitols in nature, mannitol and sorbitol, with moderate affinities, by a facilitative mechanism. Moreover, Hxt11 and Hxt15 are capable of transporting xylitol, a five-carbon polyol derived from xylose, the most abundant pentose in lignocellulosic biomass. Hxt11, Hxt13, Hxt15, Hxt16 and Hxt17 are phylogenetically and functionally distinct from known polyol transporters. Based on docking of polyols to homology models of transporters, we propose the architecture of their active site. In addition, we determined the kinetic parameters of mannitol and sorbitol dehydrogenases encoded in the yeast genome, showing that they discriminate between mannitol and sorbitol to a much higher degree than the transporters.
Anthropogenic changes in climate and land use are driving changes in migration patterns of birds worldwide. Spatial changes in migration have been related to long-term temperature trends, but the intrinsic mechanisms by which migratory species adapt to environmental change remain largely unexplored. We show that, for a long-lived social species, older birds with more experience are critical for innovating new migration behaviours. Groups containing older, more experienced individuals establish new overwintering sites closer to the breeding grounds, leading to a rapid population-level shift in migration patterns. Furthermore, these new overwintering sites are in areas where changes in climate have increased temperatures and where food availability from agriculture is high, creating favourable conditions for overwintering. Our results reveal that the age structure of populations is critical for the behavioural mechanisms that allow species to adapt to global change, particularly for long-lived animals, where changes in behaviour can occur faster than evolution.
Importance of latrine communication in European rabbits shifts along a rural–to–urban gradient
(2016)
Background: Information transfer in mammalian communication networks is often based on the deposition of excreta in latrines. Depending on the intended receiver(s), latrines are either formed at territorial boundaries (between-group communication) or in core areas of home ranges (within-group communication). The relative importance of both types of marking behavior should depend, amongst other factors, on population densities and social group sizes, which tend to differ between urban and rural wildlife populations. Our study is the first to assess (direct and indirect) anthropogenic influences on mammalian latrine-based communication networks along a rural-to-urban gradient in European rabbits (Oryctolagus cuniculus) living in urban, suburban and rural areas in and around Frankfurt am Main (Germany).
Results: The proportion of latrines located in close proximity to the burrow was higher at rural study sites compared to urban and suburban ones. At rural sites, we found the largest latrines and highest latrine densities close to the burrow, suggesting that core marking prevailed. By contrast, latrine dimensions and densities increased with increasing distance from the burrow in urban and suburban populations, suggesting a higher importance of peripheral marking.
Conclusions: Increased population densities, but smaller social group sizes in urban rabbit populations may lead to an increased importance of between-group communication and thus, favor peripheral over core marking. Our study provides novel insights into the manifold ways by which man-made habitat alterations along a rural-to-urban gradient directly and indirectly affect wildlife populations, including latrine-based communication networks.
Background: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii.
Results: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs).
Conclusion: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.
Background: Butanol isomers are regarded as more suitable fuel substitutes than bioethanol. n-Butanol is naturally produced by some Clostridia species, but due to inherent problems with clostridial fermentations, industrially more relevant organisms have been genetically engineered for n-butanol production. Although the yeast Saccharomyces cerevisiae holds significant advantages in terms of scalable industrial fermentation, n-butanol yields and titers obtained so far are only low.
Results: Here we report a thorough analysis and significant improvements of n-butanol production from glucose with yeast via the acetoacetyl-CoA-derived pathway. First, we established an improved n-butanol pathway by testing various isoenzymes of different pathway reactions. This resulted in n-butanol titers around 15 mg/L in synthetic medium after 74 h. As the initial substrate of the n-butanol pathway is acetyl-coenzyme A (acetyl-CoA) and most intermediates are bound to coenzyme A (CoA), we increased CoA synthesis by overexpression of the pantothenate kinase coaA gene from Escherichia coli. Supplementation with pantothenate increased n-butanol production up to 34 mg/L. Additional reduction of ethanol formation by deletion of alcohol dehydrogenase genes ADH1-5 led to n-butanol titers of 71 mg/L. Further expression of a mutant form of an ATP independent acetylating acetaldehyde dehydrogenase, adhEA267T/E568K, converting acetaldehyde into acetyl-CoA, resulted in 95 mg/L n-butanol. In the final strain, the n-butanol pathway genes, coaA and adhE A267T/E568K, were stably integrated into the yeast genome, thereby deleting another alcohol dehydrogenase gene, ADH6, and GPD2-encoding glycerol-3-phosphate dehydrogenase. This led to a further decrease in ethanol and glycerol by-product formation and elevated redox power in the form of NADH. With the addition of pantothenate, this strain produced n-butanol up to a titer of 130 ± 20 mg/L and a yield of 0.012 g/g glucose. These are the highest values reported so far for S. cerevisiae in synthetic medium via an acetoacetyl-CoA-derived n-butanol pathway.
Conclusions: By gradually increasing substrate supply and redox power in the form of CoA, acetyl-CoA, and NADH, and decreasing ethanol and glycerol formation, we could stepwise increase n-butanol production in S. cerevisiae. However, still further bottlenecks in the n-butanol pathway must be deciphered and improved for industrially relevant n-butanol production levels.
Background: n-Butanol can serve as an excellent gasoline substitute. Naturally, it is produced by some Clostridia species which, however, exhibit only limited suitability for industrial n-butanol production. The yeast Saccharomyces cerevisiae would be an ideal host due to its high robustness in fermentation processes. Nevertheless, n-butanol yields and titers obtained so far with genetically engineered yeast strains are only low.
Results: In our recent work, we showed that n-butanol production via a clostridial acetoacetyl-CoA-derived pathway in engineered yeast was limited by the availability of coenzyme A (CoA) and cytosolic acetyl-CoA. Increasing their levels resulted in a strain producing up to 130 mg/L n-butanol under anaerobic conditions. Here, we show that under aerobic conditions. this strain can even produce up to 235 mg/L n-butanol probably due to a more efficient NADH re-oxidation. Nevertheless, expression of a bacterial water-forming NADH oxidase (nox) significantly reduced n-butanol production although it showed a positive effect on growth and glucose consumption. Screening for an improved version of an acetyl-CoA forming NAD+-dependent acetylating acetaldehyde dehydrogenase, adhEA267T/E568K/R577S, and its integration into n-butanol-producing strain further improved n-butanol production. Moreover, deletion of the competing NADP+-dependent acetaldehyde dehydrogenase Ald6 had a superior effect on n-butanol formation. To increase the endogenous supply of CoA, amine oxidase Fms1 was overexpressed together with pantothenate kinase coaA from Escherichia coli, and could completely compensate the beneficial effect on n-butanol synthesis of addition of pantothenate to the medium. By overexpression of each of the enzymes of n-butanol pathway in the n-butanol-producing yeast strain, it turned out that trans-2-enoyl-CoA reductase (ter) was limiting n-butanol production. Additional overexpression of ter finally resulted in a yeast strain producing n-butanol up to a titer of 0.86 g/L and a yield of 0.071 g/g glucose.
Conclusions: By further optimizing substrate supply and redox power in the form of coenzyme A, acetyl-CoA and NADH, n-butanol production with engineered yeast cells could be improved to levels never reached before with S. cerevisiae via an acetoacetyl-CoA-derived pathway in synthetic medium. Moreover, our results indicate that the NAD+/NADH redox balance and the trans-2-enoyl-CoA reductase reaction seem to be bottlenecks for n-butanol production with yeast.