Ankara : The Department of Molecular Biology and Genetics and the Institute of Engineering and Sciences of Bilkent University, 2008.Thesis (Master's) -- Bilkent University, 2008.Includes bibliographical references leaves 47-52.HANEIN-1 was first identified in a study investigating 3’ transcriptional regulatory
elements of the Na+
/I-
symporter gene. The protein is highly conserved among
eukaryotes and bears no domain similarities with any known proteins. In databases, it
is described as coiled coil containing-124 hypothetical protein and predicted to
encode a 223 amino acid-protein. In this project, our aim was to characterize this
highly conserved protein by using several bioinformatics and biochemical
methodologies. Sequence similarity search analysis showed that it had around 75%
identity in mammals, 50% identity with insects and nematodes. Expressional analysis
revealed that HANEIN-1 was expressed in all tissues ubiquitously with a remarkable
expression status in skeletal muscle. Beside providing information about expression
status of HANEIN-1, northern blotting showed that HANEIN-1 transcript size was
approximately 1000 bp. Regarding protein level expression, western blotting
revealed that HANEIN-1 encoded a 33 kDa protein and protein stability was
affected in a different way upon labeling with Flag epitope at N-ter and C-ter. Yeast
double hybrid screening performed in our laboratory showed that HANEIN-1
interacted with RASGEF1B, which was a guanine nucleotide exchange factor not
fully characterized. Expressional analysis displayed that RASGEF1B expression
profile inversely correlated with HANEIN-1. Finally, serine-scanning mutagenesis
analysis showed that site-directed mutagenesis of serine at position 194 significantly
affected the stability of the protein.Erkek, SerapM.S
