The mouse Splotch phenotype is characterized by specific defects in neural tube closure, neural crest cell migration, and skeletal myogenesis, and results from loss-of-function mutations in Pax-3, a transcription factor expressed within a subset of neural and mesodermal lineages. A hypomorphic allele termed Splotch-delayed produces milder neural tube defects and neural crest cell deficiencies than other Splotch locus mutations. We found that this unique phenotype is associated with a glycine to arginine mutation of the ninth amino acid of the Pax-3 paired domain. We also identified and characterized two alternatively spliced Pax-3 isoforms termed Pax-3/Q+ and Pax-3/Q--. These isoforms differ solely by the presence or absence of a single glutamine residue in the linker which joins the two DNA-binding subdomains of the Pax-3 paired domain, and are co-expressed throughout the early stages of mouse embryogenesis. Preliminary biochemical studies suggested that the novel Pax-3/Q-- isoform possessed enhanced C-terminal subdomain-mediated DNA-binding activity. In follow-up studies, we generated derivatives of a classical paired domain binding site (CD19-2/A) in order to further characterize the distinct DNA-binding properties of the two naturally occurring Pax-3 isoforms. We found that the presence of a 5 '-TT-3' dinucleotide at positions 15 and 16 of the paired domain recognition sequence promoted high affinity binding by both Pax-3 isoforms. However, with a guanine at position 15, only the Q-- isoform retained high affinity binding to the derivatives tested, suggesting that the presence of the additional glutamine residue in the Q+ isoform restricts the sequence specificity of the Pax-3 paired domain. Altogether, these studies demonstrate that the C-terminal subdomain makes an important contribution to the DNA-binding activity of the Pax-3 paired domain, and highlight the importance of the paired domain linker region in modulating the interaction of Pax proteins with t
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.35643 |
| Date | January 1998 |
| Creators | Vogan, Kyle J. |
| Contributors | Gros, Philippe (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001605491, proquestno: NQ44620, Theses scanned by UMI/ProQuest. |
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