Biomedical subjects

Hauke Holtorf

Publications and source records attributed to Hauke Holtorf.

4 recordsLinked to original sources

PubMed2003

An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.

The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.

Amino Acid Sequence↗

PubMed2002

High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library.

BACKGROUND: The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant. RESULTS: As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics. CONCLUSIONS: The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.

Bryopsida↗

PubMed2002

Plant functional genomics.

Functional genome analysis of plants has entered the high-throughput stage. The complete genome information from key species such as Arabidopsis thaliana and rice is now available and will further boost the application of a range of new technologies to functional plant gene analysis. To broadly assign functions to unknown genes, different fast and multiparallel approaches are currently used and developed. These new technologies are based on known methods but are adapted and improved to accommodate for comprehensive, large-scale gene analysis, i.e. such techniques are novel in the sense that their design allows researchers to analyse many genes at the same time and at an unprecedented pace. Such methods allow analysis of the different constituents of the cell that help to deduce gene function, namely the transcripts, proteins and metabolites. Similarly the phenotypic variations of entire mutant collections can now be analysed in a much faster and more efficient way than before. The different methodologies have developed to form their own fields within the functional genomics technological platform and are termed transcriptomics, proteomics, metabolomics and phenomics. Gene function, however, cannot solely be inferred by using only one such approach. Rather, it is only by bringing together all the information collected by different functional genomic tools that one will be able to unequivocally assign functions to unknown plant genes. This review focuses on current technical developments and their impact on the field of plant functional genomics. The lower plant Physcomitrella is introduced as a new model system for gene function analysis, owing to its high rate of homologous recombination.

Databases, Factual↗

PubMed2002

Cloning and functional characterisation of an enzyme involved in the elongation of Delta6-polyunsaturated fatty acids from the moss Physcomitrella patens.

The moss Physcomitrella patens contains high proportions of polyunsaturated very-long-chain fatty acids with up to 20 carbon atoms. Starting from preformed C18 polyunsaturated fatty acids, their biosynthesis involves a sequence of Delta6-desaturation, Delta6-elongation and Delta5-desaturation. In this report we describe for the first time the characterisation of a cDNA (PSE1) of plant origin with homology to the ELO-genes from Saccharomyces cerevisiae, encoding a component of the Delta6-elongase. Functional expression of PSE1 in S. cerevisiae led to the elongation of exogenously supplied Delta6-polyunsaturated fatty acids. By feeding experiments with different trienoic fatty acids of natural and synthetic origin, both substrate specificity and substrate selectivity of the enzyme were investigated. The activity of Pse1, when expressed in yeast, was not sensitive to the antibiotic cerulenin, which is an effective inhibitor of fatty acid synthesis and elongation. Furthermore, the PSE1 gene was disrupted in the moss by homologous recombination. This led to a complete loss of all C20 polyunsaturated fatty acids providing additional evidence for the function of the cDNA as coding for a component of the Delta6-elongase. The elimination of the elongase was not accompanied by a visible alteration in the phenotype, indicating that C20-PUFAs are not essential for viability of the moss under phytotron conditions.

Amino Acid Sequence↗