Refine
Document Type
Institute
Has Fulltext
- yes (11)
Is part of the Bibliography
- yes (11)
Keywords
- genotyping (2)
- microsatellite (2)
- multiplex PCR (2)
- next-generation sequencing (2)
- parentage analysis (2)
- tardigrade (2)
- COLONY software (1)
- Circus pygargus (1)
- Dipetalogaster maxima (1)
- Montagu’s harrier (1)
Genomics technologies, especially transcriptional profiling, allows comparisons of gene expression within and across different organisms. By the use of the model group Tardigrada, also known as water bear, we try to understand the phenomenon of cryptobiosis. The dry organisms can survive for years without water. When re-exposed to water, the animals rehydrate and come back to life. The expression of genes in response to dehydratation and rehydratation is being examined in our laboratory through the generation of expressed sequence tags (ESTs), representational difference analysis (RDA) and subsequent microarray analysis. Molecular dissection of this complex phenomenom, including gene regulation, will allow the development of techniques for preservation and stabilisation of biological materials in a dried state.
Throughout the recent years, 454 pyrosequencing has emerged as an efficient alternative to traditional Sanger sequencing and is widely used in both de novo whole-genome sequencing and metagenomics. Especially the latter application is extremely sensitive to sequencing errors and artificially duplicated reads. Both are common in 454 pyrosequencing and can create a strong bias in the estimation of diversity and composition of a sample. To date, there are several tools that aim to remove both sequencing noise and duplicates. Nevertheless, duplicate removal is often based on nucleotide sequences rather than on the underlying flow values, which contain additional information.
Limno-terrestrial tardigrades are small invertebrates that are subjected to periodic drought of their micro-environment. They have evolved to cope with these unfavorable conditions by anhydrobiosis, an ametabolic state of low cellular water. During drying and rehydration, tardigrades go through drastic changes in cellular water content. By our transcriptome sequencing effort of the limno-terrestrial tardigrade Milnesium tardigradum and by a combination of cloning and targeted sequence assembly, we identified transcripts encoding eleven putative aquaporins. Analysis of these sequences proposed 2 classical aquaporins, 8 aquaglyceroporins and a single potentially intracellular unorthodox aquaporin. Using quantitative real-time PCR we analyzed aquaporin transcript expression in the anhydrobiotic context. We have identified additional unorthodox aquaporins in various insect genomes and have identified a novel common conserved structural feature in these proteins. Analysis of the genomic organization of insect aquaporin genes revealed several conserved gene clusters.
Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.
The phenomenon of desiccation tolerance, also called anhydrobiosis, involves the ability of an organism to survive the loss of almost all cellular water without sustaining irreversible damage. Although there are several physiological, morphological and ecological studies on tardigrades, only limited DNA sequence information is available. Therefore, we explored the transcriptome in the active and anhydrobiotic state of the tardigrade Milnesium tardigradum which has extraordinary tolerance to desiccation and freezing. In this study, we present the first overview of the transcriptome of M. tardigradum and its response to desiccation and discuss potential parallels to stress responses in other organisms.
Die medizinischen Blutegel, Hirudo medicinalis und Hirudo verbana, werden wegen ihrer kurativen Wirkung in der Humanmedizin angewandt. Während des Blutsaugens injizieren sie über ihren Speichel eine Vielzahl bioaktiver, derzeit noch unbekannter Moleküle. Eine vollständige Aufklärung aller Inhaltsstoffe mit Wirkmechanismen ist für die Entwicklung von neuen Pharmaka von großem Interesse. Vor diesem Hintergrund wurden verschiedene Organe beider Arten auf ihre Besiedlung durch symbiontische Bakterien untersucht. Dazu wurden die Bakterien zunächst unter geeigneten Bedingungen kultiviert und mittels biochemischer Methoden charakterisiert. Die Identifizierung der Symbionten erfolgte durch Polymerasekettenreaktion (PCR) und Sequenzierung der 16S rDNA. Die biochemischen Tests ergaben, dass die kultivierbaren Bakterien Amylase positiv, Gram negativ und Ornithin Decarboxylase negativ sind. Mit Hilfe von datenbankgestützten Analysen der 16S rDNA-Sequenzen konnte Aeromonas veronii biovar sobria nachgewiesen werden. Hochdurchsatzsequenzierungen der gesamtgenomischen DNA des Bakteriums aus H. medicinalis zeigten deutliche Abweichungen zum Referenzgenom von Aeromonas veronii B565.
Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum.
To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions.
ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum.
These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant.
Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress.
During the last decades the ground-breeding Montagu’s harrier ( Circus pygargus , Linnaeus, 1758) has changed its breeding habitats in Europe to agricultural areas in which many local populations would be close to extinction without a special nest protection regime. Although Montagu’s harrier is a well-studied species in terms of ecology and breeding biology, its genetic structure and population genetics are almost unknown. As there is a lack of good genetic markers we developed a set of 19 microsatellite markers comprising 16 new STR markers which were identified by next-generation sequencing (NGS) using 454 shot-gun pyrosequencing of genomic DNA. The STR markers were arranged into three multiplex PCR sets for high throughput genotyping and characterised. The marker set provides a powerful tool for kinship analysis. The combined non-exclusion probability for parent pairs was 1.13* 10-11. Only three loci showed PIC values < 0.50. In total, 121 known family relationships were compared with genetically calculated ones to test the markers suitability for parentage analysis. In 97.5% of all cases full-sibships were accurately determined and 97.6% of all mothers were assigned correctly to their chicks. The present multiplex PCR panels can be used to investigate several hypotheses concerning breeding behaviour, kinship, exchange rates between populations and phylogeography.
