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Publications - Cell and Metabolic Biology
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Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one‐pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline.
Publications
A T-DNA insertion mutant of FUSCA3 (fus3-T) in Arabidopsis thaliana exhibits several of the expected deleterious effects on seed development, but not the formation of brown seeds, a colouration which results from the accumulation of large amounts of anthocyanin. A detailed phenotypic comparison between fus3-T and a known splice point mutant (fus3-3) revealed that the seeds from both mutants do not enter dormancy and can be rescued at an immature stage. Without rescue, mature fus3-3 seeds are non-viable, whereas those of fus3-T suffer only a slight loss in their germinability. A series of comparisons between the two mutants uncovered differences with respect to conditional lethality, in histological and sub-cellular features, and in the relative amounts of various storage compounds and metabolites present, leading to a further dissection of developmental processes in seeds and a partial reinterpretation of the complex seed phenotype. FUS3 function is now known to be restricted to the acquisition of embryo-dependent seed dormancy, the determination of cotyledonary cell identity, and the synthesis and accumulation of storage compounds. Based on DNA binding studies, a model is presented which can explain the differences between the mutant alleles. The fus3-T lesion is responsible for loss of function only, while the fus3-3 mutation induces various pleiotropic effects conditioned by a truncation gene product causing severe mis-differentiation.
This page was last modified on 27 Jan 2025 .
