Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae.

BACKGROUND: Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host. RESULTS: As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5-7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology. CONCLUSION: The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds.

SNP mining porcine ESTs with MAVIANT, a novel tool for SNP evaluation and annotation.

MOTIVATION: Single nucleotide polymorphisms (SNPs) analysis is an important means to study genetic variation. A fast and cost-efficient approach to identify large numbers of novel candidates is the SNP mining of large scale sequencing projects. The increasing availability of sequence trace data in public repositories makes it feasible to evaluate SNP predictions on the DNA chromatogram level. MAVIANT, a platform-independent Multipurpose Alignment VIewing and Annotation Tool, provides DNA chromatogram and alignment views and facilitates evaluation of predictions. In addition, it supports direct manual annotation, which is immediately accessible and can be easily shared with external collaborators. RESULTS: Large-scale SNP mining of polymorphisms bases on porcine EST sequences yielded more than 7900 candidate SNPs in coding regions (cSNPs), which were annotated relative to the human genome. Non-synonymous SNPs were analyzed for their potential effect on the protein structure/function using the PolyPhen and SIFT prediction programs. Predicted SNPs and annotations are stored in a web-based database. Using MAVIANT SNPs can visually be verified based on the DNA sequencing traces. A subset of candidate SNPs was selected for experimental validation by resequencing and genotyping. This study provides a web-based DNA chromatogram and contig browser that facilitates the evaluation and selection of candidate SNPs, which can be applied as genetic markers for genome wide genetic studies. AVAILABILITY: The stand-alone version of MAVIANT program for local use is freely available under GPL license terms at http://snp.agrsci.dk/maviant. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

In vitro assessment of recombinant, mutant immunoglobulin G anti-D devoid of hemolytic activity for treatment of ongoing hemolytic disease of the fetus and newborn.

BACKGROUND: A specific treatment for ongoing hemolytic disease of the fetus and newborn (HDFN) due to anti-D would be very attractive. One approach could be administration to the mother of nonhemolytic anti-D, which by crossing the placenta can block the binding of hemolytic maternal anti-D. STUDY DESIGN AND METHODS: Two anti-D immunoglobulin G3 (IgG3) heavy-chain mutants were expressed in Chinese hamster ovary cells. To investigate whether these anti-D IgG3 mutants could inhibit the red blood cell-destructive activity of recombinant human (rHu)IgG1 with identical antigen-binding region as well as polyclonal anti-D having multiple D epitope specificities, two assays were used, antibody-dependent cell-mediated cytotoxicity (ADCC) and a chemiluminescence (CL)-based method for detection of respiratory burst in peripheral blood monocytes. RESULTS: The two IgG3 anti-D heavy-chain mutants inhibited the ADCC and CL responses mediated by a rHuIgG1 anti-D with identical antigen-binding region as the mutant antibodies, as well as the destructive activity mediated by a polyclonal anti-D. CONCLUSION: The use of nonhemolytic anti-D may be an effective countermeasure against hemolysis in HDFN due to anti-D.

Functional in vitro studies of recombinant human immunoglobulin G and immunoglobulin A anti-D.

BACKGROUND: The use of anti-D purified from human serum to prevent hemolytic disease of the fetus and newborn due to D is well established. Owing to supply and safety reasons, however, an unlimited and non-plasma-derived source of antibodies for Rhesus prophylaxis is needed. STUDY DESIGN AND METHODS: Recombinant human immunoglobulin G (IgG)1, IgG2, IgG3, IgG4, IgA1, and IgA2 anti-D with the same variable region were expressed in Chinese hamster ovary cells. The effector functions of these antibodies were assessed by an antibody-dependent cell-mediated cytotoxicity (ADCC) assay and a chemiluminescence (CL) method for detection of respiratory burst. RESULTS: In the ADCC assay, IgG1, IgG3, and IgA1 did the best and were as active as a currently used prophylactic polyclonal anti-D. IgG4 and IgA2 were moderately active, whereas IgG2 was not active. In the CL assay, IgG1 and IgG3 were active but much less so than a currently used prophylactic polyclonal anti-D. For some effector cell preparations, IgG4 was active in the CL assay, whereas IgG2, IgA1, and IgA2 were not. A mixture of IgG1 and IgG3 showed a synergistic effect in the CL assay and did as well as the prophylactic polyclonal anti-D in ADCC and CL. Mixtures of IgA1 and either IgG1 or IgG3 showed no synergistic effect. CONCLUSION: A mixture of recombinant human IgG1 and IgG3 anti-D could be of value in future Rhesus prophylaxis.

Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags.

BACKGROUND: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages. RESULTS: Using the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories. CONCLUSION: This EST collection, the largest to date in pig, represents an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcription studies.

Human anti-rhesus D IgG1 antibody produced in transgenic plants.

Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D antigen, which is responsible for alloimmunization of RhD- mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs.