Honors Capstone Project
Date of Submission
Dr. John Belote
Dr. Eleanor Maine
Arts and Science
Capstone Prize Winner
Won Capstone Funding
Sciences and Engineering
Biology | Ecology and Evolutionary Biology
The genome of eukaryotes is associated with chromosomal proteins that fold the DNA into ordered arrangements. The most abundant of these are the histones. Four of these (H2A, H2B, H3 and H4) form the nucleosomal core around which DNA is wrapped. Other proteins can associate with chromatin and fold it into higher order structures. During spermatogenesis in many organisms the standard chromatin configuration undergoes a transition to a highly condensed form in which small, highly basic proteins called protamines replace the histones. It is thought that protamine-based chromatin is more resistant to mutational damage.
Previous work by the Renkawitz-Pohl group identified and cloned the protamine genes from Drosophila melanogaster. In this species there are two tandemly arranged genes encoding similar small, highly basic proteins, ProtamineA and ProtamineB, that are expressed in late spermatogenesis and that accumulate in the sperm head as the histones disappear. Interestingly, these protamines are not essential for sperm function, although sperm lacking the protamines were more susceptible to mutation.
In the present study I sought to extend these studies to another insect species, the red flour beetle Tribolium castaneum. Here I describe the identification of three clustered genes (called Protamine-1, Protamine-2, and Protamine-3) encoding small highly basic proteins that show similarity to the Drosophila protamines. To see if these are indeed protamines, I have used gene splicing methods to tag each of them with a fluorescent marker (GFP, a green fluorescent protein) and have subcloned the tagged constructs into a germline transformation vector, pB3xP3-EGFP, that will be used to create transgenic beetles that express the fluorescent proteins. To date, Dr. Belote has generated transformed lines of fluorescently tagged Protamine-1 and has confirmed that this gene is expressed during spermatogenesis and that the protein accumulates in the sperm head as expected for a protamine. I have successfully GFP-tagged the Protamine-2 gene and subcloned it into the transformation vector. The next step will be to generate transgenic beetles and examine the sperm-specific expression of this candidate protamine gene. Generation of the Protamine-3 GFP-tagged construct is in progress.
It is anticipated that these fluorescent-tagged protamine lines will be an invaluable genetic tool for a variety of studies in which direct visualization of live sperm is desired.
Martimucci, Kristina, "Identification, cloning and fluorescent-tagging of protamines of the flour beetle, Tribolium castaneum" (2011). Syracuse University Honors Program Capstone Projects. 266.
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