Document Type

Thesis

Date of Award

10-2024

School/College

College of Science, Engineering, and Technology (COSET)

Degree Name

MS in Biology

Committee Chairperson

Dr. Audrey Player

Committee Member 1

Dr. Mario Hollomon

Committee Member 2

Dr. Ayodotun Sodipe

Committee Member 3

Dr. Roderick Holmes

Abstract

Nearly every gene exists as transcript variants and protein isoforms. Variations of a particular gene are generated by small nuclear ribonucleoproteins which in many cases remove introns, so that exons can be joined together in varying configurations. Transcript variants are formed, which are then translated into protein isoforms. The resulting transcript variants have substantial sequence similarities; and the isoforms have substantial sequence similarities. Studies show that isoforms generated because of these processes can have different start sites and/or sizes that can lead to generations of different proteins with different biological roles in a cell. The isoforms might have different functions in different cells, or different functions in the same cell types at various developmental or disease stages. Our laboratory studies the MYBL1 gene in triple negative breast cancer, so we considered the possibility that MYBL1 isoforms are differentially expressed in non-tumor compared to breast cancer cells. In the current study we analyzed the MYBL1 protein isoform sequences to identify differences between their sequences; then we examined the possibility of the differential expression of the isoforms in non-tumor compared to tumor triple negative breast cancer cells. We identified 10 different Reference Sequence MYBL1 isoforms deposited at the National Center for Biotechnology Information, many of which had different amino acid start sites and sizes. Four of the isoforms contain amino acids that correspond to a unique exon 15 observed in the longest transcript variants. Hence the 4 isoforms were distinctly different from the remaining 6 isoforms. The unique region in the 4 protein isoforms also over-lapped with the Negative Regulatory region susceptible to post-translational modification in the MYBL1 gene. We identified commercial antibodies that allowed for detection of (a) the 4 unique MYBL1 isoforms that contain the region corresponding to the transcript variant exon 15 region and the Negative Regulatory region, and (b) a different antibody that could detect all of the 10 MYBL1 isoforms. Our data show the antibody recognizing amino acids corresponding to the exon 15 region of the longest MYBL1 isoforms shows differential detection of triple negative breast cancer cell line preparations. And the antibody that detects all MYBL1 isoforms shows differential detection in the non-tumor triple negative cell line. These data show that individual or groups of isoforms are present and subsequently detected in tumor samples compared to non-tumor samples. The various isoforms likely have different biological roles in the different breast samples.

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