Date of Award

5-2017

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

John J. Gaynor

Committee Member

Vladislav Snitsarev

Committee Member

Paul A. X. Bologna

Subject(s)

Sea nettle, Nucleotide sequence

Abstract

Jellyfish cnidocysts contain many different venom proteins, each with unique functions that combine synergistically to achieve a more robust and toxic fluid. One of these proteins is hyaluronidase (EC 3.2.1.35), an enzyme that breaks down hyaluronic acid, which is commonly found in the extracellular matrix of many multicellular organisms. Due to its wide application, this enzyme is also found in the venom of many arthropod and reptile families. Putatively, hyaluronidase aids in the dispersion of the other venom proteins as it perforates the matrix in the target tissue. This activity has been detected in Chrysaora quinquecirrha cnidocysts, but the gene encoding hyaluronidase has yet to be identified or sequenced in full. Preliminary analysis of this protein shows high homology (67% homologous) to similar venom proteins found in another Cnidarian, Hydra vulgaris, which both conserve the active and secondary sites found in SPAM1, a protein found on the surface of mammalian sperm as well. Chrysaora quinquecirrha's hyaluronidase was previously located in mRNA sequencing, which was then amplified from genomic DNA using primers designed off this sequence. While the primers were 674 base pairs (bp) apart in the RNA, amplification by PCR of Chrysaora quinquecirrha gDNA produced an amplicon of ca. 2300 bp. From the initial sequencing, additional primers were designed, which generated amplicons of 1100 bp. In aligning the sequences with each other and, using the original mRNA sequence as a scaffold, two introns were discovered and characterized, accounting for part of the size disparity between the RNA and DNA sequences. These represent the first introns identified and sequenced for this organism. The conservation of amino acid residues in the active site as well as several secondary sites in the putative protein support the conclusion that I have isolated a genomic clone of hyaluronidase. Additionally, the fact that hyaluronidase enzymatic activity was detected in extracts of purified cnidocysts isolated from Chrysaora quinquecirrha confirms that hyaluronidase is present in the venom of this jellyfish. The hyaluronidase protein found in other highly homologous species is longer than our present assembly encodes for, so further extension of this sequence is necessary to elucidate the entire gene. Compared to the hyaluronidase-1 from Hydra vulgaris, which is 436 amino acids in length, I have generated a translation product of 280 amino acids, or ca. 64.2%.

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