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Summer Research Fellowship Programme of India's Science Academies

Enhancers and suppressors of Huntington's disease

Ms. Amrutha Krishnakumar

Integrated MSc Life Sciences, Central University of Tamil Nadu, Neelakudy, Thiruvarur, Tamil Nadu, 610005

Dr. Tania Bose

Assistant professor, Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra,411007

Abstract

Huntington's disease, a neurodegenerative disorder is caused by the expansion of CAG triplet codon repeats and thereby formation of toxic htt protein. If glutamine (Q) repeats 4-35 times, it is normal and if it repeats more than 35 times, the protein disease symptoms start to appear. This mainly affects regions of brain and cause memory lapses, clumsiness, involuntary movements etc. Saccharomyces cerevisiae species of yeast was selected as model organism as the genes in yeast and mammals encode very similar proteins. Being a eukaryote and haploid, yeast is similar to Mammalian cells at the same time easy to handle. First of all, protein expression and quantification is studied in normal (25 Q) and mutant (103 Q) strains. Then mutant 103 Q strain is again transformed (double transformation) by incorporating different plasmids which contains genes encoding chaperones. Molecular chaperones are proteins that help in folding and unfolding and assembly and disassembly of other macromolecular structures especially proteins. These chaperones may enhance or suppress the aggregation of poly Q containing protein. Bacterial competent cells (DH5- Alpha) are made and then plasmid coding for chaperon is inserted into it. Then plasmid isolation is done and these are incorporated into mutant (103 Q) yeast strains. These transformed yeast strains are spotted on selective medium plates along with control. An empty vector is used as control. Thus by this spotting assay growth of yeast cells is studied by comparing the control and our desired chaperon. Less growth indicates the enhanced aggregation of the toxic protein and more growth shows the suppressed aggregation of protein. Presence of protein aggregates can be studied by using confocal microscopy.

 

Keywords: neurodegeneration, molecular chaperone, protein folding, spotting assay, confocal microscopy

Ms. Amrutha krishnakumar

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