Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • Evaluation of plant growth Transgenic Arabidopsis plants wer

    2023-04-17

    Evaluation of plant growth. Transgenic Arabidopsis plants were examined by microscope for alterations in cell size and shape. Overall plant architecture was also compared with control plants.
    Results and discussion
    Acknowledgments
    Introduction Mammalian ACK1 is a kinase effector for Cdc42 that also interacts with clathrin and can influence receptor endocytosis [1], [2]. SNX9 (sorting nexin 9, also referred to as SH3PX1) was reported to be a binding partner for the non-receptor kinase, activated Cdc42-associated kinase (ACK) in Drosophila[3], and similar interactions were later reported for the mammalian counterparts [4]. This interaction occurs between a proline-rich domain of ACK2 and the Src homology 3 domain (SH3) of SNX9: co-immunoprecipitation studies indicate that ACK2, clathrin, and SNX9 can form a complex in Azimilide [4]. Other SH3 containing proteins including Grb2 have been identified as partners for ACK although the sites of interaction have not been mapped [5]. In common with a number of protein kinases ACK is apparently regulated via heatshock protein (HSP90) chaperones, that are required for the in vivo kinase activity of ACK2 and its association with Cdc42 [6]. The SH2 domain of the Drosophila Nck (Dock) adaptor co-purifies with five proteins from S2 cells including DACK [7]. The largest protein in the complex was identified as an orthologue of Dscam (Down syndrome cell adhesion molecule), which plays a role in directing neurons of the fly embryo to correct positions within the nervous system [8]. The smallest protein in this complex (p63) was DSH3PX1 (DSNX9), that is similar to mammalian SNX9 comprising of an NH2-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. SNX9 is a member of a family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking [9]. Both Dock SH3 and SH2 domains appear to mediate association with DSNX9 but the exact determinants of interaction have not been uncovered. A number of methods have been developed to assess direct protein interaction by protein overlays onto proteins arrayed on membranes. We have previously described the use of a glutathione-S-transferase Ras fusion protein system to generate probes fused to domains of choice which can be rapidly labelled with [γ-32P] GTP with similar sensitivity to direct 32P labelling of proteins [10]. Because of the hazards associated with radiolabelling techniques and requirement for specialized disposal of waste, non-radioactive methods are in ascendancy. Indirect labelling methods include antibodies directed against the protein ‘probe’ or the use of chemical labels that can then be detected via enzymic or chemiluminescent methods. The modification of proteins by covalent attachment of biotin occurs to very few proteins, one in Escherichia coli, three in budding yeast and four in mammalian cells. Biotin attachment is a 2-step reaction that results in the formation of an amide linkage between the carboxyl group of biotin and the ϵ-amino group of the modified lysine. The unique E. coli biotin acceptor is biotin carboxyl carrier protein (BCCP) which is recognized by the BirA enzyme. Specific biotinylation of small peptides (14–23 residues) by BirA in vivo was demonstrated by a phage-based selection protocol: the resultant consensus sequence for biotinylation by BirA has little similarity to the primary sequence of BCCP [11]. Such peptides have formed the basis for the development of hybrid vectors expressing affinity fusion proteins such as maltose binding protein [12]. Here, we describe a vector in which an optimal acceptor peptide of only 10 residues is placed in the pGEX expression vector also encoding a polycistonic mRNA that includes the BirA enzyme. This vector allows for the in vivo attachment of biotin to proteins or domain of choice that can be purified using one step affinity chromatography on glutathione sepharose. Subsequently these proteins can be used as probes, which we have previously shown to be much more effective when arrayed as GST dimers [10] particularly when dealing with SH3 affinities in the 0.1–10μM range. Such modified proteins can also be immobilized via streptavidin to surfaces such as ELISA plates or plasmid resonance chips for solid phase assays.