ORCID Profile
0000-0002-7405-8518
Current Organisation
University of Adelaide
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Publisher: Springer Science and Business Media LLC
Date: 18-02-2012
Abstract: The synaptonemal complex (SC) is a proteinaceous tripartite structure used to hold homologous chromosomes together during the early stages of meiosis. The yeast ZIP1 and its homologues in other species have previously been characterised as the transverse filament protein of the synaptonemal complex. Proper installation of ZYP1 along chromosomes has been shown to be dependent on the axial element-associated protein, ASY1 in Arabidopsis. Here we report the isolation of the wheat ( Triticum aestivum ) ZYP1 ( TaZYP1 ) and its expression profile (during and post-meiosis) in wild-type, the ph1b deletion mutant as well as in Taasy1 RNAi knock-down mutants. Ta ZYP1 has a putative DNA-binding S/TPXX motif in its C-terminal region and we provide evidence that Ta ZYP1 interacts non-preferentially with both single- and double-stranded DNA in vitro . 3-dimensional dual immunofluorescence localisation assays conducted with an antibody raised against Ta ZYP1 show that Ta ZYP1 interacts with chromatin during meiosis but does not co-localise to regions of chromatin where Ta ASY1 is present. The Ta ZYP1 signal lengthens into regions of chromatin where Ta ASY1 has been removed in wild-type but this appears delayed in the ph1b mutant. The localisation profile of Ta ZYP1 in four Taasy1 knock-down mutants is similar to wild-type but Ta ZYP1 signal intensity appears weaker and more diffused. In contrast to previous studies performed on plant species where ZYP1 signal is sandwiched by ASY1 signal located on both axial elements of the SC, data from the 3-dimensional dual immunofluorescence localisation assays conducted in this study show that Ta ZYP1 signal only lengthens into regions of chromatin after Ta ASY1 signal is being unloaded. However, the observation that Ta ZYP1 loading appears delayed in both the ph1b and Taasy1 mutants suggests that Ta ASY1 may still be essential for Ta ZYP1 to play a role in SC formation during meiosis. These data further suggest that the temporal installation of ZYP1 onto pairing homologous chromosomes in wheat is different to that of other plant species and highlights the need to study this synaptonemal complex protein on a species to species basis.
Publisher: CSIRO Publishing
Date: 2012
DOI: 10.1071/FP11253
Abstract: Various genetic-based approaches including mutant population screens, microarray analyses, cloning and transgenesis have broadened our knowledge of gene function during meiosis in plants. Nonetheless, these genetic tools are not without inherent limitations. One alternative approach to studying plant meiosis, especially in polyploids such as Triticum aestivum L. (bread wheat), is proteomics. However, protein-based approaches using proteomics have seldom been described, with only two attempts at studying early plant meiosis reported. Here, we report the investigation of early bread wheat meiosis using proteomics. Five differentially expressed protein spots were identified using 2D gel electrophoresis (2DGE) on protein extracts from four pooled stages of meiosis and three genotypes (Chinese Spring wild-type, ph1b and ph2a wheat mutant lines). Tandem mass spectrometry (MS/MS) identification of peptides from these protein spots led to the isolation and characterisation of the full-length clones of a wheat Speckle-type POZ protein, an SF21-like protein and HSP70, and a partial coding sequence of a hexose transporter. Significantly, the putative functions of the Speckle-type POZ protein and HSP70 were confirmed using in vitro DNA binding assays. Through the use of a 2DGE proteomics approach, we show that proteomics is a viable alternative to genetic-based approaches when studying meiosis in wheat. More significantly, we report a potential role for a Speckle-type POZ protein and a HSP70 in chromosome pairing during the early stages of meiosis in bread wheat.
Publisher: Springer Science and Business Media LLC
Date: 28-07-2018
Publisher: Springer Science and Business Media LLC
Date: 18-01-2019
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-0446-4_3
Abstract: Temperature Switch PCR (TSP) is a robust single-marker single nucleotide polymorphism (SNP) genotyping technique with broad applications in genetic studies of various organisms. The technique consists of a biphasic PCR with two sets of primers, a locus-specific set and a nested locus-specific set. The PCR products can be easily assessed for polymorphism based on different band sizes using agarose gel electrophoresis.
Publisher: CSIRO Publishing
Date: 2008
DOI: 10.1071/FP08203
Abstract: The RADiation sensitive protein 51 (RAD51) recombinase is a eukaryotic homologue of the bacterial Recombinase A (RecA). It is required for homologous recombination of DNA during meiosis where it plays a role in processes such as homology searching and strand invasion. RAD51 is well conserved in eukaryotes with as many as four paralogues identified in vertebrates and some higher plants. Here we report the isolation and preliminary characterisation of four RAD51 gene family members in hexaploid (bread) wheat (Triticum aestivum L.). RAD51A1, RAD51A2 and RAD51D were located on chromosome group 7, and RAD51C was on chromosome group 2. Q-PCR gene expression profiling revealed that RAD51A1 was upregulated during meiosis with lower expression levels seen in mitotic tissue, and bioinformatics analysis demonstrated the evolutionary linkages of this gene family to other eukaryotic RAD51 sequences. Western blot analysis of heterologously expressed RAD51 from bread wheat has shown that it is detectable using anti-human RAD51 antibodies and that molecular modelling of the same protein revealed structural conservation when compared with yeast, human, Arabidopsis and maize RAD51A orthologues. This report has widened the knowledge base of this important protein family in plants, and highlighted the high level of structural conservation among RAD51 proteins from various species.
Publisher: Hindawi Limited
Date: 06-02-2012
DOI: 10.1155/2012/514398
Abstract: Chromosome pairing, synapsis, and DNA recombination are three key processes that occur during early meiosis. A previous study of Poor Homologous Synapsis 1 ( PHS1 ) in maize suggested that PHS1 has a role in coordinating these three processes. Here we report the isolation of wheat ( Triticum aestivum ) PHS1 ( TaPHS1 ), and its expression profile during and after meiosis. While the Ta PHS1 protein has sequence similarity to other plant PHS1/PHS1-like proteins, it also possesses a unique region of oligopeptide repeat units. We show that Ta PHS1 interacts with both single- and double-stranded DNA in vitro and provide evidence of the protein region that imparts the DNA-binding ability. Immunolocalisation data from assays conducted using antisera raised against Ta PHS1 show that Ta PHS1 associates with chromatin during early meiosis, with the signal persisting beyond chromosome synapsis. Furthermore, Ta PHS1 does not appear to colocalise with the asynapsis protein ( Ta ASY1) suggesting that these proteins are probably independently coordinated. Significantly, the data from the DNA-binding assays and 3-dimensional immunolocalisation of Ta PHS1 during early meiosis indicates that Ta PHS1 interacts with DNA, a function not previously observed in either the Arabidopsis or maize PHS1 homologues. As such, these results provide new insight into the function of PHS1 during early meiosis in bread wheat.
No related grants have been discovered for Kelvin Khoo.