The column was washed several times with TBS and 0.1 x TBS to remove any unbound molecules. Finally the column was eluted with 0.1M glycine-Cl pH2.5. 6ml of this 0.1M glycine-Cl pH2.5 was added to the column, and each sequential 800μl was collected separately. There were two guinea pigs murdered for this experiment designated 13 and 14. The 2nd elution for GP13 and the 3rd elution for GP14 had the highest concentrations of protein as measured by nanodrop. Purified antibody GP14-3 was then used for immunoblots on a transverse section of normal immature tassel, cross section of normal immature tassel and on normal SAM tissue. Tissue was sectioned from wax using a microtome. Sections were laid on a slide, and the wax dissolved with Histoclear. Histoclear was then washed off with 100% EtOH and then the tissue was slowly rehydrated step-wise with lower concentrations of EtOH until finally pure water. The slides were then boiled in citrate buffer and left to stand for 10min. Slides were then incubated in PBS and then blocking solution . Next, the slides were incubated in 2ml of blocking solution and 2μl of antibody overnight, before incubating with the secondary antibody GP-AP, washing, and finally NETN and NBT/BCIP. Slides were examined under a light microscope and signal was observed at the base of the pedicel in the longitudinal section , and in the L2 in the transverse section , of the tassel. A similar localisation pattern was observed by in situ for TS1 RNA35. No signal was observed in the longitudinal section of the SAM, though it is possible that there is signal at the base of the axillary meristem . As a control,vertical grow products a longtitudinal section of a fun tassel was also used for an immunoblot . A similar localisation pattern was observed in this mutant control as was seen in the normal tassel casting doubt on the reliability of the wild-type localisation patterns since the fun mutation causes a truncation of the protein before the region that the antibody was designed to .
This apparent non-specificity prompted a western blot using antibody GP14-3. No band was observed at the predicted size of 90kDa, and a 30kDa band was observed in both mutant and normal samples . Due to this lack of specificity, it was decided to make another antibody, using the entire third exon of FUN. The pENTR clone containing the 53xF/R insert was amplified by Phusion with primers AV53 and AV54 to create a DNA fragment containing the third exon of FUN flanked by appropriate restriction sites. The fragment was then cut with XhoI and BamHI, purified by phenol:choloroform extraction and ligated into the His-tagged expression vector pet21. After verification by sequencing, this plasmid was transformed into Rosetta cells, grown in a 1L culture and induced with IPTG and purified with Ni++ beads as described above. This time, most protein was eluted at the 3rd pH3 elution. As before, this was dialysed into 6M urea and sent to Cocalico for injection into Guinea Pigs 15 and 16. In a dot plot, the test bleeds from GP15 and 16 showed strong reactivity against the antigens sent to Cocalico, no reactivity with control purified Tru1 protein, and a little reactivity with purified Bd1 protein, possibly due to the His tag on this protein. The secondary bleeds showed similar results. Recombination of pENTR-53xF/R and pDEST15 was unsuccessful, so pgex5x-2, a GST-fusion expression plasmid, was used instead. pENTR-53xF/R was amplified by Phusion with primers AV63 and AV64 for blunt end cloning. This PCR fragment was incubated with Polynucleotide Kinase to remove phosphoryl groups at the 3’ end and phosphorylate the 5’ end. pgex5x- 2 was cut with SmaI and incubated with Shrimp Alkaline Phosphotase to remove both 5’ and 3’ phosphate groups. The cut plasmid was then ligated to the PCR product with T4 ligase at 16°C overnight. Sequencing showed successful recombination, so the pgex5x-2-53xF/R plasmid was transformed into Rosetta cells and grown in a 1litre culture. This culture was induced with IPTG and made into a column, as described above.
The pellet and supernatant of this Rosetta-pgex5x-2-53xF/R were used in a dot plot against an anti-GST antibody to test if the protein made was soluble, which it was .The pgex5x-2-53xF/R column was then used to purify sera from GP15 and GP16 . The purified serum was used in a western blot where it reacted strongly with recombinant FUN protein. This western also found a >100kDa band in WT crude extract which was not present in fun crude extract, and a ~34kDa band that was present in both WT and fun crude extract . Crude extract of protein was carried out by grinding 2g of tissue in liquid nitrogen and adding 1ml of loading buffer , which was centrifuged before loading into gel. Nuclei and cytoplasmic extractions were carried out as follows. 2g of tissue was ground in liquid nitrogen and mixed with 1xNIB buffer with DTT; the mixture was filtered through micro-cloth and the filtrate centrifuged at 1260g at 4°C for 10 minutes.The pellet was resuspended in 2ml 1xNIB buffer with 10ul of 10% triton x-100 and protease inhibitors before centrifuging at 1200g at 4°C for 10 minutes. The supernatant was discarded and the pellet resuspended in 0.5ml of JAJ-Extraction Buffer . This suspension was sonicated and centrifuged at 3000g at 4°C for 10 minutes – the resulting supernatant was reserved as the nuclear fraction. Nuclear and cytoplasmic fractions were then run on an acrylamide gel and a western blot was carried out using the purified GP16 serum. A ~90kDa band was observed in the WT cytoplasmic fraction which is the correct size for full length FUN protein, and a ~37kDa band was observed in the fun cytoplasmic fraction. Both WT and fun nuclear fractions displayed a ~70kDa and ~37kDa band . Western blots were then attempted using crude, nuclear and cytoplasmic fractions of protein extracted from fun-2 plants, but none of these gels showed any bands despite confirmation of protein presence in sample by probing with KN1 antibody .
Other extraction procedures were carried out using triton, SDS, dodecyl maltosidase and IGEPAL detergents, but none of these westerns showed any bands . In order to further purify the serum, primers AV242 and AV243 were designed to a highly antigenic region of the protein predicted by antigen prediction software . This 96bp product was cloned into pENTR and recombined into pDEST15 using C3040 cells, which greatly improved transformation efficiency. pDEST15-3500 was cloned into Rosetta cells to purify protein and create a column as described above. This column was used to purify GP15 serum; the purified serum was found to react weakly to FUN-recombinant protein in a dot plot, but none of the controls . 15 western blots were attempted using this purified serum, but none showed bands, or had high background such that any bands present would be obscured . Solubilising the 3500-GST construct before creating a column may help – a dot plot based on extractions solubilised with DDT showed that solubilisation greatly improved 3500-GST construct recovery from culture .In summary, we can say that FUN is a conserved, universally expressed, disordered protein that localises to the nucleus. Cytoskeletal binding has been implied by both bio-informatic prediction programs and Y2H, as has involvement in nucleotide binding, especially RNA and GTPases. The highly conserved nature of FUN across the grasses, and significant conservation across the Plant Kingdom, along with the mutant study presented in Chapter 1, make a compelling case for the importance of this protein in general plant growth and development. The nuclear localisation of FUN, along with its synergistic interaction with the transcription factor WAB1 ,and its interactions with various hormone mutants suggest that FUN is involved at some stage in a signal transduction pathway between the hormones and downstream gene expression. The universal expression of FUN makes it unlikely that FUN can be considered a signalling molecule per se; instead it strengthens the hypothesis that FUN is involved in the transduction of these signals. On the other hand,vertical grow racks for cannabis there is evidence of variance in transcript levels so there could be some merit to the idea that FUN is a real signal. Using Gene Ontology term analysis, it has been shown that disordered proteins are heavily biased toward signalling, regulation and control110, strengthening the signalling network hypothesis. The disorder of FUN may also have contributed to the difficulty in creating a specific antibody. Many disordered proteins are thought to undergo conformational changes in order to transduce signals110 and the high concentration of serines that could be phosphorylated , as well as a serine repeat chain in FUN , could allow these conformational change. Despite the fact that the antibody was shown to be non-specific, and the same immunoblot pattern was produced in both normal and fun tassels , the pattern of expression in the normal tassel fits the feminised tassel phenotype. Additionally, the expression pattern agrees with the nuclear localisation shown by YFP-fusion and bioinformatic prediction . Thus the expression pattern could indeed be real. Since both GO term analysis by FFPred agrees with the Y2H that tubulin interacts with FUN, and that nucleotide binding is likely for FUN, these avenues should not be ignored. While it is difficult to imagine how cytoskeletal binding could be important to the FUN protein’s function in a signal transduction pathway, the importance of nucleotide binding in signalling needs no explanation. Intriguingly, one of the Y2H predictions is ricin, a protein that binds to ribosomes, which are of course rich in RNA. Though ricin is a deadly poison to animals, its presence in low levels in plant cells could indicate that it works as a regulator of ribosome function in plants, and has been co-opted by the infamous Ricinus communis that accumulates it in high concentrations in its beans, presumably as a defence mechanism.
The expression of cannabinoid receptors by human leukocytes suggests that both endogenous ligands and inhaled marijuana smoke might exert immunoregulatory properties that are distinct from their effects on the brain . Furthermore, while brain cells exclusively express cannabinoid receptor type 1 , leukocytes express both CB1 and CB2, with CB2 reported as the predominant sub-type . Both CB1 and CB2 are transmembrane G-protein coupled receptors that inhibit the generation of cyclic adenosine monophosphate and can signal through a variety of pathways including PI3-kinase, MAP kinase, NF-κB, AP-1, and NF-AT . The resulting effects on host immunity have primarily been studied in animal models and suggest a coordinated down-regulation of cellular responses that can occur through altered trafficking, selective apoptosis, or functional skewing of antigen presenting cells and T cells away from T helper type 1 or Th17 response patterns . Similar results have been observed when purified human T cells are stimulated in vitro in the presence of Δ9-tetrahydrocannabinol . However, the extent to which the effects are observed in humans in vivo is unclear. Daily administration of marijuana or oral THC to research subjects in a prospective and randomized study had no obvious effect on T cell proliferation or cytokine production when blood cells were subsequently isolated and stimulated in vitro . Sipe et al. examined the distribution and function of a common polymorphism in the human CB2 gene associated with the replacement of a glutamine by an arginine at amino acid position. Functionally, lymphocytes from subjects with either of these genotypes proliferated normally when stimulated with anti-CD3 antibody. However, when stimulated in the presence of an endocannabinoid, lymphocytes expressing the glutamine residue at position 63 were markedly inhibited while those expressing the arginine were only modestly suppressed. The arginine substitution also correlated with the prevalence of autoimmune disease in the subjects tested. Collectively, this body of work suggests that cannabinoids are biologically active immune regulators in humans. Expanding upon this hypothesis, we examined the expression of cannabinoid receptors by human monocytes and the impact of THC on their differentiation into monocyte-derived dendritic cells . Exposing monocytes to THC blocked many of the features normallyassociated with their differentiation into functional DC and impaired their capacity for T cell activation. Furthermore, the T cell activation that did occur was associated with a change in T cell phenotype and cytokine secretion. However, the impact of THC was partially overcome when DC and T cells were exposed to a combination of activation signals and exogenous cytokines.