Author

Carey Stuart

Document Type

Honors Capstone Project

Date of Submission

Spring 5-1-2011

Capstone Advisor

Dr. Michael Cosgrove

Honors Reader

Dr. Ramesh Raina

Capstone Major

Biology

Capstone College

Arts and Science

Audio/Visual Component

no

Capstone Prize Winner

no

Won Capstone Funding

no

Honors Categories

Sciences and Engineering

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology

Abstract

The human version of the DPY-30 protein is homologous to the DPY-30 protein in Caenorhabditis elegans (nematode), along with other DPY-30 homologous proteins in other organisms. This protein is involved in dosage compensation of X-linked genes, balancing the levels of expression of these genes between the sexes. The mechanism by which the balancing is carried out varies from organism to organism. For example, in C. elegans hermaphrodites (XX), transcript levels of the X-linked genes are cut in half. In Drosophila, the genes on the male’s (XY) X chromosome are transcribed at twice the rate of the female’s (XX) genes. In human females (XX), one of the X chromosomes in each cell is inactivated at random. If DPY-30 is absent from the organism, this can lead to XX-lethality. In humans, DPY-30 forms a complex with four other proteins: MLL1, WDR5, RbBP5, and ASH2L. This complex is responsible for methylating histones, particularly histone H3 Lysine 4 (H3K4). Methylation of H3K4 promotes transcription of genes. Similar complexes are found in other organisms. The fact that these proteins are conserved across species indicates how important they are. Within the complex itself, DPY-30 binds to ASH2L. The amino acids responsible for this interaction, however, remain unknown. The purpose of this project is to identify which amino acids are responsible for the binding between DPY-30 and ASH2L. A further extension of this project is its potential anticancer applications. When MLL1 is activated improperly and forms the complex, it can lead to the development of Acute Lymphoblastic Leukemia (ALL). It is thought that if the complex were to be disrupted and broken apart or prevented from forming, the cancerous cell would stop proliferating and die. If the amino acids responsible for the DPY-30 and ASH2L binding were identified, a drug or peptide could be designed to bind to DPY-30 or ASH2L, preventing it from completing formation of the MLL1 Core Complex.

The first step in this experiment was to compare DPY-30 homologs from different species, to see if any amino acid residues were either completely conserved or mostly conserved. Once the conserved residues were identified, one was selected to change, Arginine (R) 54. It was changed to an Alanine (A). Three other single amino acid mutations were made: Arginine (R) 76, Leucine (L) 66, and Leucine (L) 65. All three were changed to Alanines. Once primers with the correct mutation were made, the DNA sequence was put through PCR and transformed into E. coli cells. The DNA was extracted, sequenced, and transformed into another E. coli strain. A large culture was grown, expression of protein was induced, the cells were lysed, and the protein was collected. Once the mutant protein was purified, it was subjected to multiple tests to determine its binding affinity for ASH2L. For the R54A mutant, the binding interactions were weakened, but not completely inhibited. For the L66A mutant, there was no measurable effect on the binding interactions. It was concluded that Arginine 54 was a much more important residue than Leucine 66, as far as binding affinity was concerned.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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