Expression and functional analysis of TaASY1 during meiosis of bread wheat (Triticum aestivum)

Background: Pairing and synapsis of homologous chromosomes is required for normal chromosome segregation and the exchange of genetic material via recombination during meiosis. Synapsis is complete at pachytene following the formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is essential for formation of the SC, and localises along chromosome axes during prophase I. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC. Results: The full length wheat cDNA and genomic clone, TaASY1, has been isolated, sequenced and characterised. TaASY1 is located on chromosome Group 5 and the open reading frame displays significant nucleotide sequence identity to OsPAIR2 (84%) and AtASY1 (63%). Transcript and protein analysis showed that expression is largely restricted to meiotic tissue, with elevated levels during the stages of prophase I when pairing and synapsis of homologous chromosomes occur. Immunolocalisation using transmission electron microscopy showed TaASY1 interacts with chromatin that is associated with both axial elements before SC formation as well as lateral elements of formed SCs. Conclusion: TaASY1 is a homologue of ScHOP1, AtASY1 and OsPAIR2 and is the first gene to be isolated from bread wheat that is involved in pairing and synapsis of homologous chromosomes.Scott A Boden, Nadim Shadiac, Elise J Tucker, Peter Langridge and Jason A Abl

Cell-free protein synthesis of membrane (1,3)-beta-D-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.Agalya Periasamy, Nadim Shadiac, Amritha Amalraj, Soňa Garajová, Yagnesh Nagarajan, Shane Waters, Haydyn D.T. Mertens, Maria Hrmov