Hans Söderlund

A functional genomics approach toward the understanding of secondary metabolism in plant cells

Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonate-mediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general.

Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei.

Hydrophobins are surface‐active proteins produced by filamentous fungi, where they seem to be ubiquitous. They have a variety of roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension. Hydrophobins can be divided into two classes based on the hydropathy profile of their primary sequence. We have studied the adhesion behavior of two Trichoderma reesei class II hydrophobins, HFBI and HFBII, as isolated proteins and as fusion proteins. Both hydrophobins were produced as C‐terminal fusions to the core of the hydrolytic enzyme endoglucanase I from the same organism. It was shown that as a fusion partner, HFBI causes the fusion protein to efficiently immobilize to hydrophobic surfaces, such as silanized glass and Teflon. The properties of the surface‐bound protein were analyzed by the enzymatic activity of the endoglucanase domain, by surface plasmon resonance (Biacore), and by a quartz crystal microbalance. We found that the HFBI fusion forms a tightly bound, rigid surface layer on a hydrophobic support. The HFBI domain also causes the fusion protein to polymerize in solution, possibly to a decamer. Although isolated HFBII binds efficiently to surfaces, it does not cause immobilization as a fusion partner, nor does it cause polymerization of the fusion protein in solution. The findings give new information on how hydrophobins function and how they can be used to immobilize fusion proteins.

SEQAID: a DNA sequence assembling program based on a mathematical model

A program package, called SEQAID, to support DNA sequencing is presented. The program automatically assembles long DNA sequences from short fragments with minimal user interaction. Various tools for controlling the assembling process are also available. The main novel features of the system are that SEQAID implements several new well-behaved algorithms based on a mathematical model of the problem. It also utilizes available information on restriction fragments to detect illegitimate overlaps and to find relationships between separately assembled sequence blocks. Experiences with the system are reported including an extremely pathological real sequence which offers an interesting benchmark for this kind of programs.

Yeast protein translocation complex: Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit

A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion‐defective sec15–1 mutation. This gene encodes a protein homologous to the β subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane‐spanning region at the C‐terminus but no N‐terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61β subunit and 34% identity to the Arabidopsis thaliana Sec61β subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61–2 and sec61–3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N‐terminus exposed to the cytoplasm. The in vitro translated Seb1p was post‐translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature‐sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis. The sequences of SEB1 and SEB2 have been deposited in the EMBL database under Accession Numbers Z47789 and Z50012, respectively. The sequence of the Seb1 protein is identical to that of Sbh1p independently identified by Panzner et al. (Cell 81, 561–570. 1995) using a biochemical approach.

Transient dimers of allergens.

Background Allergen-mediated cross-linking of IgE antibodies bound to the FcεRI receptors on the mast cell surface is the key feature of the type I allergy. If an allergen is a homodimer, its allergenicity is enhanced because it would only need one type of antibody, instead of two, for cross-linking. Methodology/Principal Findings An analysis of 55 crystal structures of allergens showed that 80% of them exist in symmetric dimers or oligomers in crystals. The majority are transient dimers that are formed at high protein concentrations that are reached in cells by colocalization. Native mass spectrometric analysis showed that native allergens do indeed form transient dimers in solution, while hypoallergenic variants of them exist almost solely in the monomeric form. We created a monomeric Bos d 5 allergen and show that it has a reduced capability to induce histamine release. Conclusions/Significance The results suggest that dimerization would be a very common and essential feature for allergens. Thus, the preparation of purely monomeric variants of allergens could open up novel possibilities for specific immunotherapy.

Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries

Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human γ-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 γ-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and β-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 β-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency. (Journal of Biomolecular Screening 2009:282-293)

Multiplexed Quantification of Bacterial 16S rRNA by Solution Hybridization with Oligonucleotide Probes and Affinity Capture

Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin–nucleic acid–probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA–specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 102. The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

IgE antibodies distinctively recognising allergenic epitopes would be ideal reagents in immunodiagnostics to detect and quantify allergens, as well as for the development of allergy diagnostics and therapeutics. We have isolated recombinant human IgE antibodies specific for the major latex allergen, hevein, from antibody phage display libraries using a green fluorescent protein (GFP)-hevein fusion as a selection antigen. Human IgE phage display libraries were constructed by combining the IgE heavy chain genes to kappa and lambda light-chain genes which were isolated from lymphocytes of a latex allergic patient. The screening of antibody libraries resulted in the enrichment of two hevein-binding scFvs designated as 1A4 and 1C2. Both antibodies showed specific binding to the hevein that could be inhibited by both the recombinant GFP-hevein and native hevein isolated from latex examination gloves. The scFvs were prone to aggregate and, thus, for further characterisation, they were converted to Fab fragments with human IgG1 or IgE isotype. Similar hevein-binding properties of the 1A4 and 1C2 Fab fragments and human IgE serum pool, conventionally used in the detection of latex allergens, demonstrate the potential utility of these recombinant antibodies for the analysis of latex allergen.

Algorithms for the search of amino acid patterns in nucleic acid sequences.

Some algorithms are described for the search of regions in a nucleic acid sequence that, when translated into amino acids, are homologous to a given amino acid pattern. All algorithms are modifications of the dynamic programming method for sequence comparison such that the translation of codons is taken into account. One of the algorithms has been implemented as a FORTRAN 77 program. The program operates on files that follow the format of the EMBL Nucleotide Sequence Data Library.

RNA synthesis directed by a temperature-sensitive mutant of Semliki Forest virus.

RAN synthesis induced by a temperature-sensitive RNA negative mutant, ts-4, of Semliki Forest virus was studied both at the permissive (28 degrees C) and restrictive temperature (39 degrees C). At 28 degrees C ts-4 directed RNA synthesis was qualitatively similar to the wild type but overall synthesis remained somewhat lower. Only about 3% of RNA was synthesized compared to the wild type virus when ts-4 was maintained at 39 degrees C throughout the infection. The sedimenting radioactivity was almost exclusively in 42S RNA. When ts-4 infected cultures were shifted from 28 to 39 degrees C at 5 h post-infection, the synthesis of 26S RNA was shut off while 42S RNA synthesis continued. The synthesis of 26S RNA started again even in the presence of cycloheximide if the cultures were back to 28 degrees C. Our results suggest that 26S RNA synthesis is controlled by a virus-specific protein, the function of which is not required for the synthesis of 42S RNA, In ts-4 this protein is denatured at 39 degrees C and becomes non-functional, but renatues and becomes functional upon shift down to 28 degreesC.