<p> The Notch signalling pathway is a lateral inhibition pathway that serves to
limit the number of cells in a proneural cluster (a group of equipotent cells) that
will adopt a neural cell fate during neurogenesis in Drosophila. The proper
segregation of neural and epidermal progenitor cells during neurogenesis requires
the expression of both the proneural genes and the neurogenic genes. Expression
of proneural genes, such as achaete, gives cells the potential to commit to a neural
cell fate. The neurogenic genes encode proteins that act in the Notch signalling
cascade and are required for cell fate determination during Drosophila
neurogenests. Notch and Delta are neurogenic genes that encode large
transmembrane proteins. Interaction between the extracellular domains of Notch
and Delta is thought to transmit a signal to the nucleus by way of the DNAbinding
Suppressor of Hairless protein. In response to Notch activation
Suppressor of Hairless is translocated to the nucleus where it activates the
transcription ofthe neurogenic genes ofthe Enhancer of split complex (E(spl)-C).
The products of the E(spl)-C are bHLH transcription factors. They possess a Cterminal
tryptophan-arginine-proline-tryptophan (WRPW) motif that interacts
with the product of another neurogenic gene, groucho. The groucho gene
product encodes a protein containing a WD40 repeat element. When bound to
Groucho, E(spl) bHLH proteins are able to repress transcription of proneural
genes, such as achaete, thereby directing the cell to adopt a non-neural cell fate.</p> <p> A number of murine groucho homologues have been identified and named
Grg's (Groucho related genes). Three full length Grg proteins have been identified which contain all five domains found in the Drosophila Groucho protein. Two short Grg proteins have also been identified which only contain one
of the domains found in the full-length Grg proteins. A number of murine
homologues of the Drosophila E(spl)-C have also been identified and named
Hes (Hairy/Enhancer of split) proteins. Like the gene products of the Drosophila
E(spl)-C, the Hes proteins are bHLH proteins containing a C-terminal WRPW
motif. One of the Hes proteins, Hes3, is lacking a basic domain and therefore
lacks the DNA-binding activity possessed by the other Hes proteins. </p> <p> Attempts were made to detect interactions between Grg and Hes proteins using co-immunoprecipitation techniques. The anti-WD40 antibody, which recognizes the long WD40-containing Grg proteins, was able to specifically
immunoprecipitate 35S-labelled Grgl . This antibody was also able to recognize
WD40-containing Grg proteins present in Pl9 cell extracts. However, attempts to
co-immunoprecipitate radiolabelled Hesl and AMLlb proteins with Grg proteins
present in P19 cell extract were unsuccessful due to the low affinity of the antiWD40
antibody and the background caused by the binding of the test proteins to
Sepharose. A second method of co-immunoprecipitation was attempted using an
HA-tagged Grgl fusion protein and a commercially available anti-HA antibody.
The attempt to co-immunoprecipitate 35S-labelled Hesl with radiolabelled HAtagged
Grg 1 was unsuccessful due to a high degree of background caused by
Hesl binding to protein G Agarose. Using the Yeast Two-Hybrid interaction
assay, the WD40-containing Grg proteins, Grgl and Grg4, were found to interact
with Hesl. However, using the same assay WD40-containing Grg proteins were
found not to interact with Hes3, which lacks DNA-binding activity. A Western
blot was performed to determine if the Hes3 fusion proteins were being expressed in transformed yeast but none were detected. This may have been due to the poor affinity of the anti-GAL4 activation domain antibody. A similar Western blot demonstrated that the Grg proteins, fused to the GAL4 DNA binding domain,
were being expressed in transformed yeast extract. The WD40-containing Grg
proteins, Grgl and Grg4, were also found not to interact with AMLlb, a protein
which contains a C-terminal VWRPY domain which is reminiscent of the Cterminal
WRPW interaction domain found in Hes proteins and Drosophila E(spl) proteins. However, WD40-containing Grg proteins were able to interact with an AML 1 b mutant in which the VWRPY motif was mutated to VWRPW in the Yeast Two Hybrid assay. </p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22638 |
| Date | 06 1900 |
| Creators | Taylor, Catherine |
| Contributors | Lobe, Corinne, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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