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Carolyn A Paisie Thesis.pdf (2.05 MB)

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Definition of mechanisms of mutation generation in tissues and embryonic stem cells of the constitutive Fhit knockout mouse

Paisie, Carolyn Anne
http://rave.ohiolink.edu/etdc/view?acc_num=osu1436542716

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Integrated Biomedical Science Graduate Program.
Genome instability, which can be defined as an increase in changes at both the nucleotide and chromosomal level (e.g. point mutations, chromosomal translocations), results from errors in normal biological processes that function to repair, replicate, and segregate the genome during cell division. Genomic instability is a hallmark of human neoplasia, present in varying degrees in all stages of cancer, from precancerous to advanced cancer. Genome instability is initiated due to loss of expression of the FHIT gene, located at 3p14.2; the murine Fhit gene is located at 14A2. Fhit loss occurs early in human cancer development and is frequently observed in preneoplastic lesions. The Fhit protein is a tumor suppressor and genome caretaker that modulates genome stability and level of DNA damage that accumulates beginning in precancerous lesions. To investigate the contribution of loss of Fhit expression to the generation of mutations and to define mechanisms that underlie these genome alterations, we performed an analysis of whole exome sequences from cell lines and tissues, from wildtype and Fhit-/- mice. -/- cells and tissues demonstrated increased numbers of C>T and T>C mutations and Fhit-deficient kidney cell cultures that survived dimethylbenz(a)anthracene treatment exhibited increased numbers of T>A mutations. Following determination of trinucleotide contexts of mutations, a Fhit-loss signature was proposed to consist of C>T and T>C mutations that may be due to respective increased spontaneous deamination (C>T mutations) and deoxyribonucleotide triphosphates pool imbalance (T>C mutations), a signature similar to the 'age at diagnosis' signature identified in human cancers. Increased T>C mutations in -/- exomes may be due to the imbalance in deoxyribonucleotide triphosphates, particularly thymidine triphosphate, resulting from decreased expression of Thymidine Kinase 1 in Fhit-deficient cells and tissues. Fhit-deficient kidney cell cultures that survived in vitro dimethylbenz(a)anthracene treatment additionally exhibited an increase in T>A mutations, a signature known to be generated by treatment with carcinogens such as dimethylbenz(a)anthracene, a consequence of inefficient nucleotide excision repair. We continued our examination of mutations in genomes of short-term cultured embryonic stem cells and derived differentiated cells. These studies revealed an increase in mutations in -/- embryonic stem and derived differentiated cells vs +/+ embryonic stem and derived differentiated cells. From this analysis we cannot separate mutations due to genetic drift, relative to the B6 reference, from possible accumulation of mutations in the -/- germline. In addition to point mutations, -/- kidney tissue contained more insertions and particularly more deletions compared to +/+ liver tissue; loci at regions of insertions and deletions in -/- kidney tissue contained sequence motifs (e.g. poly(C) stretches) and sequences that might participate in insertion/deletion through microhomology directed repair. In summary, the results revealed that multiple types of mutations occurred more frequently in the Fhit-/- environment, with multiple mechanisms of damage and repair involved in generating mutations. A future goal will be to follow the initiation and accumulation of mutations through tissue development and through the germline in a conditional Fhit-/- strain on pure background, after deleting Fhit in the germline and in specific organs through recombinase technology.
Kay Huebner, PhD (Advisor)
Vincenzo Coppola, MD (Committee Member)
Sissy Jhiang, PhD (Committee Member)
Yuri Pekarsky, PhD (Committee Member)
124 p.
Genetics
Genome instability; mutation signatures; constitutive knockout mouse strains; exome sequence

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Citations

  • Paisie, C. A. (2015). Definition of mechanisms of mutation generation in tissues and embryonic stem cells of the constitutive Fhit knockout mouse [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1436542716

    APA Style (7th edition)

  • Paisie, Carolyn. Definition of mechanisms of mutation generation in tissues and embryonic stem cells of the constitutive Fhit knockout mouse. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1436542716.

    MLA Style (8th edition)

  • Paisie, Carolyn. "Definition of mechanisms of mutation generation in tissues and embryonic stem cells of the constitutive Fhit knockout mouse." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1436542716

    Chicago Manual of Style (17th edition)

osu1436542716
432
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This open access ETD is published by The Ohio State University and OhioLINK.