How Do Difficult Features Evolve? Test of a Sperm Tail Tubulin Synergism in the Fly D. melanogaster

Date of Award


Degree Name

M.S. in Biology


Department of Biology


Mark Nielsen


How does a protein evolve while maintaining its function? Structure/function tests of the sperm tail protein β2-tubulin show that even small changes in the protein render it unable to generate a motile sperm tail, raising the question about how it evolved in the first place. In fact, it has not evolved in 60 million years in Drosophila melanogaster and its relatives. In previous work, additive and synergistic amino acid specializations of the β2 protein were identified. Synergism is of particular interest because it makes evolution path-dependent, slowing its progress and potentially contributing to the 60-million-year stasis of the β2 protein. Two amino acids, threonine 55 (Thr55) and alanine 57 (Ala57), were identified as potentially participating in a β2-specific synergism. Their function depends on amino acid contacts that are only present in β2, and their amino acid identities are unique to β2 in comparison to other β-tubulins. Here, the proposed hypothesis states that a third amino acid that is in contact with Thr55 and Ala57 in the folded protein, and is also unique to β2, cysteine 29 (Cys29), completes the synergism. The hypothesis will be tested by generating transgenic flies (called "CTA") expressing a modified major β1 tubulin containing β2 identity Cys29, Thr55 and Ala57. Spermatogenesis and fertility were assessed in CTA/CTA β2 null/β2 null flies. CTA is able to support all post-mitotic tubulin function in the testis (cell division and shaping), except spermatogenesis. CTA flies generate short, immotile sperm and are sterile. No additional sperm-generating function was provided by cysteine 29 compared to the β1-β2 construct containing only threonine 55 and alanine 57. There are 13 remaining amino acid differences between β2-tubulin and the experimental CTA flies spread across the protein. Two differences, sites 37 and 80, are of particular interest due to their unique amino acid Department of Chemistry characteristics and can be tested in a β1-β2 chimeric protein. However, the lack of rescue by CTA, given it is 97% identical to β2, raises the possibility that the β1 backbone in testing for β2 amino acid specializations is not neutral, but contains 9+0 specializations that actively generate immotile axonemes. This hypothesis will be tested in β1-β2 chimeric tubulins using β2 as the tubulin backbone.


Biology, Evolution and Development, Molecular Biology, Organismal Biology, Genetics, Beta 2, Sperm tail, Evolution, Epistasis, Drosophila melanogaster

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