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Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2'-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m(1)A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m(1)A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.
Place based frequency discrimination (tonotopy) is a fundamental property of the coiled mammalian cochlea. Sound vibrations mechanically conducted to the hearing organ manifest themselves into slow moving waves that travel along the length of the organ, also referred to as traveling waves. These traveling waves form the basis of the tonotopic frequency representation in the inner ear of mammals. However, so far, due to the secure housing of the inner ear, these waves only could be measured partially over small accessible regions of the inner ear in a living animal. Here, we demonstrate the existence of tonotopically ordered traveling waves covering most of the length of a miniature hearing organ in the leg of bushcrickets in vivo using laser Doppler vibrometery. The organ is only 1 mm long and its geometry allowed us to investigate almost the entire length with a wide range of stimuli (6 to 60 kHz). The tonotopic location of the traveling wave peak was exponentially related to stimulus frequency. The traveling wave propagated along the hearing organ from the distal (high frequency) to the proximal (low frequency) part of the leg, which is opposite to the propagation direction of incoming sound waves. In addition, we observed a non-linear compression of the velocity response to varying sound pressure levels. The waves are based on the delicate micromechanics of cellular structures different to those of mammals. Hence place based frequency discrimination by traveling waves is a physical phenomenon that presumably evolved in mammals and bushcrickets independently.
Economically feasible production of second-generation biofuels requires efficient co-fermentation of pentose and hexose sugars in lignocellulosic hydrolysates under very harsh conditions. Baker’s yeast is an excellent, traditionally used ethanol producer but is naturally not able to utilize pentoses. This is due to the lack of pentose-specific transporter proteins and enzymatic reactions. Thus, natural yeast strains must be modified by genetic engineering. Although the construction of various recombinant yeast strains able to ferment pentose sugars has been described during the last two decades, their rates of pentose utilization is still significantly lower than D-glucose fermentation. Moreover, pentoses are only fermented after D-glucose is exhausted, resulting in an uneconomical increase in the fermentation time. In this addendum, we discuss novel approaches to improve utilization of pentoses by development of specific transporters and substrate channeling in enzyme cascades. Addendum to: T Subtil, E Boles. Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 2012; 5: 14
PMID: 22424089 DOI: 10.1186/1754-6834-5-14
5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
BACKGROUND:
Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes.
RESULTS:
Plasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates.
CONCLUSIONS:
The analyses showed that extensive ingestion of DNA (100 μg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.
The synthesis of the recently characterized depsipeptide szentiamide (1), which is produced by the entomopathogenic bacterium Xenorhabdus szentirmaii, is described. Whereas no biological activity was previously identified for 1, the material derived from the efficient synthesis enabled additional bioactivity tests leading to the identification of a notable activity against insect cells and Plasmodium falciparum, the causative agent of malaria.
Background: The genome of the carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii, Order: Dasyuromorphia), was sequenced in the hopes of finding a cure for or gaining a better understanding of the contagious devil facial tumor disease that is threatening the species’ survival. To better understand the Tasmanian devil genome, we screened it for transposable elements and investigated the dynamics of short interspersed element (SINE) retroposons.
Results: The temporal history of Tasmanian devil SINEs, elucidated using a transposition in transposition analysis, indicates that WSINE1, a CORE-SINE present in around 200,000 copies, is the most recently active element. Moreover, we discovered a new subtype of WSINE1 (WSINE1b) that comprises at least 90% of all Tasmanian devil WSINE1s. The frequencies of WSINE1 subtypes differ in the genomes of two of the other Australian marsupial orders. A co-segregation analysis indicated that at least 66 subfamilies of WSINE1 evolved during the evolution of Dasyuromorphia. Using a substitution rate derived from WSINE1 insertions, the ages of the subfamilies were estimated and correlated with a newly established phylogeny of Dasyuromorphia. Phylogenetic analyses and divergence time estimates of mitochondrial genome data indicate a rapid radiation of the Tasmanian devil and the closest relative the quolls (Dasyurus) around 14 million years ago.
Conclusions: The radiation and abundance of CORE-SINEs in marsupial genomes indicates that they may be a major player in the evolution of marsupials. It is evident that the early phases of evolution of the carnivorous marsupial order Dasyuromorphia was characterized by a burst of SINE activity. A correlation between a speciation event and a major burst of retroposon activity is for the first time shown in a marsupial genome.
Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.
Background: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds.
Results: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds.
Conclusion: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries.
Organelles are surrounded by membranes with a distinct lipid and protein composition. While it is well established that lipids affect protein functioning and vice versa, it has been only recently suggested that elevated membrane protein concentrations may affect the shape and organization of membranes. We therefore analyzed the effects of high chloroplast envelope protein concentrations on membrane structures using an in vivo approach with protoplasts. Transient expression of outer envelope proteins or protein domains such as CHUP1-TM–GFP, outer envelope protein of 7 kDa–GFP, or outer envelope protein of 24 kDa–GFP at high levels led to the formation of punctate, circular, and tubular membrane protrusions. Expression of inner membrane proteins such as translocase of inner chloroplast membrane 20, isoform II (Tic20-II)–GFP led to membrane protrusions including invaginations. Using increasing amounts of DNA for transfection, we could show that the frequency, size, and intensity of these protrusions increased with protein concentration. The membrane deformations were absent after cycloheximide treatment. Co-expression of CHUP1-TM–Cherry and Tic20-II–GFP led to membrane protrusions of various shapes and sizes including some stromule-like structures, for which several functions have been proposed. Interestingly, some structures seemed to contain both proteins, while others seem to contain one protein exclusively, indicating that outer and inner envelope dynamics might be regulated independently. While it was more difficult to investigate the effects of high expression levels of membrane proteins on mitochondrial membrane shapes using confocal imaging, it was striking that the expression of the outer membrane protein Tom20 led to more elongate mitochondria. We discuss that the effect of protein concentrations on membrane structure is possibly caused by an imbalance in the lipid to protein ratio and may be involved in a signaling pathway regulating membrane biogenesis. Finally, the observed phenomenon provides a valuable experimental approach to investigate the relationship between lipid synthesis and membrane protein expression in future studies.
Cellular models of aging
(2012)
We report on new localities for Anolis gruuo Köhler, Ponce, Sunyer and Batista, 2007 along the Serranía de Tabasará in the Comarca Ngöbe-Buglé and Veraguas province of western Panama. These records extend the known geographic distribution of this lizard about 80 km eastward, and the known vertical distribution approximately 40 m lower and 630 m higher. We provide photos of specimens from different localities and comment on their morphology. Only the easternmost populations of this Panamanian endemic live inside a protected area.
Reporting on the first locality in Bocas del Toro province of extreme western Panama, we extend the known geographic distribution of the lizard Leposoma rugiceps (Cope, 1869) about 275 km westwards from the nearest locality in Panamá province. We provide photos of Panamanian specimens, comment on their morphology, and map the distribution of this binational endemism.
The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.
Crista junctions (CJs) are tubular invaginations of the inner membrane of mitochondria that connect the inner boundary with the cristae membrane. These architectural elements are critical for mitochondrial function. The yeast inner membrane protein Fcj1, called mitofilin in mammals, was reported to be preferentially located at CJs and crucial for their formation. Here we investigate the functional roles of individual domains of Fcj1. The most conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function. In its absence, formation of CJ is strongly impaired and irregular, and stacked cristae are present. This domain interacts with full-length Fcj1, suggesting a role in oligomer formation. It also interacts with Tob55 of the translocase of outer membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex, which is required for the insertion of β-barrel proteins into the outer membrane. The association of the TOB/SAM complex with contact sites depends on the presence of Fcj1. The biogenesis of β-barrel proteins is not significantly affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in the number of CJs. We propose that the C-terminal domain of Fcj1 is critical for the interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in close proximity to the outer membrane. These results assign novel functions to both the C-terminal domain of Fcj1 and the TOB/SAM complex.
Background: Elucidating the genomic basis of adaptation and speciation is a major challenge in natural systems with large quantities of environmental and phenotypic data, mostly because of the scarcity of genomic resources for non-model organisms. The Atlantic molly (Poecilia mexicana, Poeciliidae) is a small livebearing fish that has been extensively studied for evolutionary ecology research, particularly because this species has repeatedly colonized extreme environments in the form of caves and toxic hydrogen sulfide containing springs. In such extreme environments, populations show strong patterns of adaptive trait divergence and the emergence of reproductive isolation. Here, we used RNA-sequencing to assemble and annotate the first transcriptome of P. mexicana to facilitate ecological genomics studies in the future and aid the identification of genes underlying adaptation and speciation in the system.
Description: We provide the first annotated reference transcriptome of P. mexicana. Our transcriptome shows high congruence with other published fish transcriptomes, including that of the guppy, medaka, zebrafish, and stickleback. Transcriptome annotation uncovered the presence of candidate genes relevant in the study of adaptation to extreme environments. We describe general and oxidative stress response genes as well as genes involved in pathways induced by hypoxia or involved in sulfide metabolism. To facilitate future comparative analyses, we also conducted quantitative comparisons between P. mexicana from different river drainages. 106,524 single nucleotide polymorphisms were detected in our dataset, including potential markers that are putatively fixed across drainages. Furthermore, specimens from different drainages exhibited some consistent differences in gene regulation.
Conclusions: Our study provides a valuable genomic resource to study the molecular underpinnings of adaptation to extreme environments in replicated sulfide spring and cave environments. In addition, this study adds to the increasing number of genomic resources in the family Poeciliidae, which are widely used in comparative analyses of behavior, ecology, evolution, and medical genetics.
Analysis of photosystem I (PSI) complexes from Cyclotella meneghiniana cultured under different growth conditions led to the identification of three groups of antenna proteins, having molecular weights of around 19, 18, and 17 kDa. The 19-kDa proteins have earlier been demonstrated to be more peripherally bound to PSI, and their amount in the PSI complexes was significantly reduced when the iron supply in the growth medium was lowered. This polypeptide was almost missing, and thus the total amount of fucoxanthin-chlorophyll proteins (Fcps) bound to PSI was reduced as well. When treating cells with high light in addition, no further changes in antenna polypeptide composition were detected. Xanthophyll cycle pigments were found to be bound to all Fcps of PSI. However, PSI of high light cultures had a significantly higher diatoxanthin to diadinoxanthin ratio, which is assumed to protect against a surplus of excitation energy. PSI complexes from the double-stressed cultures (high light plus reduced iron supply) were slightly more sensitive against destruction by the detergent treatment. This could be seen as a higher 674-nm emission at 77 K in comparison to the PSI complexes isolated from other growth conditions. Two major emission bands of the Fcps bound to PSI at 77 K could be identified, whereby chlorophyll a fluorescing at 697 nm was more strongly coupled to the PSI core than those fluorescing at 685 nm. Thus, the build up of the PSI antenna of several Fcp components enables variable reactions to several stress factors commonly experienced by the diatoms in vivo, in particular diatoxanthin enrichment under high light and reduction of antenna size under reduced iron conditions.
The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.
The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme b(D) ring C (b(D)-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the b(D)-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the b(D)-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme b(D) ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.