Hussam Hassan Nour-Eldin

Bio: Hussam Hassan Nour-Eldin is an academic researcher from University of Copenhagen. The author has contributed to research in topics: Arabidopsis & Glucosinolate. The author has an hindex of 21, co-authored 47 publications receiving 2197 citations. Previous affiliations of Hussam Hassan Nour-Eldin include National Research Foundation of South Africa & University of California, San Diego.

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

Abstract: The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.

NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds

Abstract: Two high-affinity proton-dependent transporters of glucosinolates have been identified in Arabidopsis and termed GTR1 and GTR2; these transporters are essential for transporting glucosinolates to seeds, offering a means to control the allocation of defence compounds in a tissue-specific manner, which may have agricultural biotechnology implications. Glucosinolates are important plant defence compounds. They are synthesized in various tissues and then translocated to the seeds, where they accumulate. In this study, Barbara Halkier and colleagues examine the molecular basis of this long-distance transport process. They identify two high-affinity, proton-dependent glucosinolate-specific transporters in Arabidopsis, termed GTR1 and GTR2. These transporters control the loading of glucosinolates from the apoplast into the phloem. The authors' specific and complete elimination of glucosinolates from Arabidopsis seeds, combined with the compounds' retention in vegetative tissues, establishes transport engineering as a potential approach for eliminating anti-nutritional natural products in high-value crops. In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value1, as demonstrated in the plant model Arabidopsis, in which the major defence compounds, glucosinolates2, are translocated to seeds on maturation3. The molecular basis for long-distance transport of glucosinolates and other defence compounds, however, remains unknown. Here we identify and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters. The gtr1 gtr2 double mutant did not accumulate glucosinolates in seeds and had more than tenfold over-accumulation in source tissues such as leaves and silique walls, indicating that both plasma membrane-localized transporters are essential for long-distance transport of glucosinolates. We propose that GTR1 and GTR2 control the loading of glucosinolates from the apoplasm into the phloem. Identification of the glucosinolate transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

Abstract: We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.

USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.

Abstract: The explosive development of the field of molecular biology has led to the need for simpler and more efficient cloning techniques. These requirements are elegantly met by the ligation-free cloning technique called USER cloning. USER cloning is suitable not only for everyday and high-throughput cloning but also for the one-step construction of complex DNA constructs, which can be achieved in a variant called USER fusion. In this chapter, we present a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.

The Arabidopsis NPF3 protein is a GA transporter

Abstract: Gibberellins (GAs) are plant hormones that promote a wide range of developmental processes. While GA signalling is well understood, little is known about how GA is transported or how GA distribution is regulated. Here we utilize fluorescently labelled GAs (GA-Fl) to screen for Arabidopsis mutants deficient in GA transport. We show that the NPF3 transporter efficiently transports GA across cell membranes in vitro and GA-Fl in vivo. NPF3 is expressed in root endodermis and repressed by GA. NPF3 is targeted to the plasma membrane and subject to rapid BFA-dependent recycling. We show that abscisic acid (ABA), an antagonist of GA, is also transported by NPF3 in vitro. ABA promotes NPF3 expression and GA-Fl uptake in plants. On the basis of these results, we propose that GA distribution and activity in Arabidopsis is partly regulated by NPF3 acting as an influx carrier and that GA-ABA interaction may occur at the level of transport.

Enzymatic assembly of DNA molecules up to several hundred kilobases

Abstract: We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome

Abstract: Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.

Abstract: In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

Methods for the directed evolution of proteins

Abstract: Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties. This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins. We highlight recent cases in which directed evolution generated enzymatic activities and substrate specificities not known to exist in nature.