Functional Analysis of Bloom Syndrome Helicase in Development and DNA Repair

Bloom Syndrome is a rare autosomal recessive disorder in humans caused by mutation of the BLM gene that leads to increased genome instability and cancer. The BLM gene codes for a helicase (BLM) that works together with Topoisomerase 3-alpha (Top3α) in homology-directed repair of DNA. Top3α directly binds to BLM and helps release the torsional stress on DNA as BLM unwinds recombination intermediates. These proteins preserve genome stability and have been shown in many organisms to operate together in the prevention of detrimental mitotic (non-meiotic) crossovers via two main DNA repair pathways, synthesis-dependent strand annealing and double Holliday junction dissolution. In Drosophila, BLM (known as Blm) also has roles in proper meiotic chromosome segregation and rapid cell cycle progression of the developing embryo. Each of these BLM functions are not well characterized and limit development of treatments for BLM-related disorders. To investigate the Blm-Top3α interaction in DNA repair, I performed a yeast 2-hybrid (Y2H) assay using the Drosophila genes. I found the interaction was specific to certain regions of Blm, with the strongest interaction observed at a C-terminal region conserved among several Drosophila species, amino acids (aa) 1381-1487. Based on these data, I created specific deletions of the Blm gene via CRISPR/Cas9 editing to characterize the various roles of Blm in vivo. First, I assessed the effects of Blm deletions on known Blm roles in meiotic chromosome segregation via a nondisjunction assay. Both aa 576-720 and N1 produced measurable defects compared to the wild type suggesting functional value of aa 576-720. Significance of this Blm region in preventing mitotic crossovers and DNA repair was evaluated by a crossover assay showcasing a lack of significant effect by aa 576-720 relative to the true null allele, N1, but still increased relative to wild type. These studies showcase the importance of aa 576-720 and other Blm regions in the roles of meiotic segregation and DNA repair. Blm aa 576-720 will be further assessed by examining the relevance of predicted ATR/ATM phosphorylation sites within the region required for proper Blm function. Additional Blm roles in embryonic development will also be explored via an embryo hatching assay. By characterizing the functions of Blm in Drosophila, we will better understand and improve BLM function within humans and the detrimental health effects associated with BLM mutations.Bachelor of Scienc

Functional Analysis of Bloom Syndrome Helicase in Development and DNA Repair

Bloom Syndrome is a rare autosomal recessive disorder in humans caused by mutation of the BLM gene that leads to increased genome instability and cancer. The BLM gene codes for a helicase (BLM) that works together with Topoisomerase 3-alpha (Top3α) in homology-directed repair of DNA. Top3α directly binds to BLM and helps release the torsional stress on DNA as BLM unwinds recombination intermediates. These proteins preserve genome stability and have been shown in many organisms to operate together in the prevention of detrimental mitotic (non-meiotic) crossovers via two main DNA repair pathways, synthesis-dependent strand annealing and double Holliday junction dissolution. In Drosophila, BLM (known as Blm) also has roles in proper meiotic chromosome segregation and rapid cell cycle progression of the developing embryo. Each of these BLM functions are not well characterized and limit development of treatments for BLM-related disorders. To investigate the Blm-Top3α interaction in DNA repair, I performed a yeast 2-hybrid (Y2H) assay using the Drosophila genes . I found the interaction was specific to certain regions of Blm, with the strongest interaction observed at a C-terminal region conserved among several Drosophila species, amino acids (aa) 1381-1487. Based on this data, I created specific deletions of the Blm gene via CRISPR/Cas9 editing to characterize the various roles of Blm in vivo. First, I assessed the effects of Blm deletions on known Blm roles in meiotic chromosome segregation via a nondisjunction assay. Both aa 576-720 and N1 produced measurable defects compared to the wild type suggesting functional value of aa 576-720. Significance of this Blm region in preventing mitotic crossovers and DNA repair was evaluated by a crossover assay showcasing a lack of significant effect by aa 576-720 relative to the true null allele, N1, but still increased relative to wild type. These studies showcase the importance of aa 576-720 and other Blm regions in the roles of meiotic segregation and DNA repair. Blm aa 576-720 will be further assessed by examining the relevance of predicted ATR/ATM phosphorylation sites within the region required for proper Blm function. Additional Blm roles in embryonic development will also be explored via an embryo hatching assay. By characterizing the functions of Blm in Drosophila, we will better understand and improve BLM function within humans and the detrimental health effects associated with BLM mutations

Identification of the Causative Organism of Tuberculous Lymphadenitis in Ethiopia by PCR

Tuberculous lymphadenitis (TBLN) is a common form of extrapulmonary tuberculosis with multiple differential diagnoses. Demonstration of the etiologic agent by smear microscopy or culture of fine needle aspirate (FNA) specimens is often unsuccessful. FNA specimens from 40 patients presenting at a rural health center in South Ethiopia and diagnosed as positive for TBLN on the basis of clinical and cytological criteria were analyzed for mycobacterial DNA by PCR. Thirty (75%) had cervical lymphadenitis and 11 (27.5%) were seropositive for human immunodeficiency virus (HIV). Three primer sets were initially used to identify the causative agent at the genus (antigen 85 complex), complex (IS6110 insertion sequence), and species (pncA gene and allelic variation) levels. Among the forty TBLN cases, 35 (87.5%) were positive by PCR at the genus and complex levels. Based on PCR for detection of allelic variation at position 169, 24 (68.6%) of the 35 were positive for Mycobacterium tuberculosis and 6 (17.1%) were positive for M. bovis. These six were positive in additional PCR assays using the JB21-JB22 primer set, which is highly specific for M. bovis. Five (14.1%) showed amplification for both M. tuberculosis and M. bovis with the allele-specific primer set. Cooccurrence of pyrazinamide (PZA)-sensitive and -resistant M. tuberculosis in those five cases was indicated, since all were negative in assays with the JB21-JB22 primer set. This feature was seen in 3 of 11 HIV-positive and 2 of 29 HIV-negative individuals (P < 0.001). Conclusion: among 35 PCR-positive cases of TBLN from southern Ethiopia, 29 (82.9%) were caused by M. tuberculosis and six (17.1%) were caused by M. bovis