8 Feb 2019

Sequencing the GM2A Gene: Implications for Late-Onset Tay-Sachs

Luis Balderrama '19

Late-onset Tay-Sachs (LOTS) is a neurodegenerative disease found more frequently in French Canadians, Cajuns, and Ashkenazi Jews. LOTS symptoms include neuromuscular problems such as gait issues, swallowing difficulties, and ataxia. About half of LOTS patients also present with mental health issues including anxiety, depression, and psychosis. LOTS symptoms are caused by slow accumulation of GM2 gangliosides in the lysosomes of neurons in the central nervous system. Hexosaminidase A (HEXA) is the enzyme that cleaves GM2 gangliosides to GM3 gangliosides, preventing lipid accumulation. GM2 gangliosides must first be extracted from the lysosomal membrane by GM2 activator complex (GM2A) before HEXA can cleave them. Mutated GM2A could result in build-up of GM2 gangliosides in nerve cells. In a previous study, the HEXA genes of the offspring of a deceased Ashkenazi Jewish male (SG) suspected of having LOTS were sequenced and found to be normal. In this study, the exons of the GM2A gene were sequenced in four individuals to see if it could have accounted for the LOTS symptoms in SG. DNA from SG’s daughters (JG and ME), a woman of Ashkenazi Jewish descent and the mother of SG’s offspring (AD), and a control (KM) of non-Jewish descent were sequenced for the GM2A exons. Exons 1-3 were successfully sequenced for all subjects, while the protein coding region of exon 4 was sequenced only for JG and the control KM. JG and KM shared similar mutations in exon 2 with two methionine to valine changes. Only JG had a SNP in exon 4, changing a valine to alanine. In summary, this mutation in SG’s offspring is a conservative amino acid substitution that very likely would not affect GM2A protein function. Completion of the sequencing of exon 4, however, is warranted to determine whether that mutation came from SG or from AD.

Bacteriophage HMSP-1 and its effect on Nitrogen Fixing Bacteria in Medicago tranculata

Aldina Imamovic '19

Sinorhizobium are plant-growth promoting bacteria that form symbiotic relationships with legumes. These bacteria are important in agriculture because of their ability to convert nitrogen gas to ammonia. Ammonia is used to create nucleic acids that are essential to synthesize proteins, and chlorophylls. Sinorhizobium induces the formation of root nodules in legumes, an oxygen free space, where N 2 fixation occurs. Bacteriophages are viruses that infect Sinorhizobium and decrease their soil populations, only allowing resistant strains to persist. There have been a limited number of Sinorhizobium phages isolated and sequenced; most genome sequences lack homology to known viruses. Previous research lead to the isolation of HMSP-1, a virus that infects many strains of Sinorhizobium important for N 2 fixation. The goal of this project was to carry out a molecular characterization of phage HMSP-1. First, we performed bioinformatics analysis to examine the genome sequences Sinorhizobium strains and investigate the presence of other bacteriophages and potential relatives of phage HMSP-1. Viral DNA was isolated from HMSP-1 lysates and examined for bacterial contamination with universal 16S rRNA PCR. Restriction digests showed that the HMPS-1 genome was approximately 60 kb in size. Using the sequences from phages present in other rhizobia, we designed primers and created a PCR program to amplify DNA encoding viral proteins. The results revealed that HMSP-1 lysates contained viral DNA. Lastly, we investigated the effects of bacteriophage HMSP-1 on the growth of legumes, specifically Medicago truncatula. The plant experiment showed no significant differences between the plants exposed to the phage and the controls.