Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.     

Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance

Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.     

A new, versatile field immunosensor for environmental pollutants: development and proof of principle with TNT, diuron, and atrazine

This paper presents a new, versatile, portable miniaturized flow-injection immunosensor which is designed for field analysis. The temperature-controlled field prototype can run for 6h without external power supply. The bio-recognition element is an analyte-specific antibody immobilized on a gold surface of pyramidal structures inside an exchangeable single-use chip, which hosts also the enzyme-tracer and the sample reservoirs. The competition between the enzyme-tracer and the analyte for the antigen-binding sites of the antibodies yields in the final step a chemiluminescence signal that is inversely proportional to the concentration of analyte in the given range of detection. A proof of principle is shown for nitroaromatics and pesticides. The detection limits (DL; IC20) reached with the field prototype in the laboratory was below 0.1 microg l(-1) for 2,4,6-trinitrotoluene (TNT), and about 0.2 microg l(-1) for diuron and atrazine, respectively. Important aspects in this development were the design of the competition between analyte and enzyme-tracer, the unspecific signal due to unspecific binding and/or luminescence background signal, and the flow pattern inside the chip.     

Face adaptation depends on seeing the face

Retinal input that is suppressed from visual awareness can nevertheless produce measurable aftereffects, revealing neural processes that do not directly result in a conscious percept. We here report that the face identity-specific aftereffect requires a visible face; it is effectively cancelled by binocular suppression or by inattentional blindness of the inducing face. Conversely, the same suppression does not interfere with the orientation-specific aftereffect. Thus, the competition between incompatible or interfering visual inputs to reach awareness is resolved before those aspects of information that are exploited in face identification are processed. We also found that the face aftereffect remained intact when the visual distracters in the inattention experiment were replaced with auditory distracters. Thus, cross-modal or cognitive interference that does not affect the visibility of the face does not interfere with the face aftereffect. We conclude that adaptation to face identity depends on seeing the face.     

Global gene expression profiling and cluster analysis in Xenopus laevis

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.     

Isolation and characterization of Tn-Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1

A new 9.9 kb catabolic transposon, Tn-Dha1, containing the gene responsible for tetrachloroethene (PCE) reductive dechlorination activity, was isolated from Desulfitobacterium hafniense strain TCE1. Two fully identical copies of the insertion sequence ISDha1, a new member of the IS256 family, surround the gene cluster pceABCT, a truncated gene for another transposase and a short open reading frame with homology to a member of the twin-arginine transport system (tatA). Evidence was obtained by Southern blot for an alternative form of the transposon element as a circular molecule containing only one copy of ISDha1. This latter structure most probably represents a dead-end product of the transposition of Tn-Dha1. Strong indications for the transposition activity of ISDha1 were given by polymerase chain reaction (PCR) amplification and sequencing of the intervening sequence located between both inverted repeats (IR) of ISDha1 (IR junction). A stable genomic ISDha1 tandem was excluded by quantitative real-time PCR. Promoter mapping of the pceA gene, encoding the reductive dehalogenase, revealed the presence of a strong promoter partially encoded in the right inverted repeat of ISDha1. A sequence comparison with pce gene clusters from Desulfitobacterium sp. strains PCE-S and Y51 and from Dehalobacter restrictus, all of which show 100% identity for the pceAB genes, indicated that both Desulfitobacterium strains seem to possess the same transposon structure, whereas only the pceABCT gene cluster is conserved in D. restrictus.     

Subproteomic analysis of metal-interacting proteins in human B cells

Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.     

Synthesis of covalent DNA-protein conjugates by expressed protein ligation

Semisynthetic DNA-protein conjugates are versatile tools for many applications in bioanalytics and nanobiotechnology. We here report a method based on expressed protein ligation (EPL) for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins. The latter contain a C-terminal thioester, enabling the mild and highly specific reaction with N-terminal cysteine compounds. To conveniently couple commercially available DNA oligomers with cysteine groups a universal chemical modifier was developed, containing a protected cysteine and an amino-reactive N-hydroxysuccinimide group connected by a hexaethyleneglycol moiety. Using maltose-binding protein (MBP) and green fluorescent protein mutant EYFP as a model systems, we demonstrate the feasibility of this approach, as well as the integrity and functionality of the DNA-protein conjugates synthesized. We anticipate that our concept will enable many applications, such as the generation of large arrays of surface-bound, recombinant proteins assembled by means of DNA-directed immobilization.