Gene regulation and overlooked coding regions : the case for translation past the 3'-UGA codon in CD4
Ware, Brady Neal
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Purpose. Understanding the mechanism involved in translating the UGA codon as selenocysteine instead of “STOP” is important for understanding the human genome. Selenocysteine provides a biological basis for the growing evidence that selenium is an important micronutrient. Low serum levels of selenium is linked to development and poor prognosis of diseases ranging from AIDS to cancer. A stem loop structure (SECIS element) in the 3’ untranslated region of the mRNA is necessary for selenocysteine incorporation. There are variants of the SECIS, and proteins that interact with the SECIS. The process of selenocysteine insertion may be highly regulated and is complex. Proteins may be expressed in multiple forms and some presumed to end at a UGA codon may actually contain selenocysteine. CD4 was chosen as a model protein to study these possibilities and is assumed to end at a single UGA codon. Methods. Structural studies on the gene sequence are carried out using programs including BLAST, Scanps, ClustalW, NetPhos, and NetOGlyc. Peptides corresponding to the region past the UGA codon and the cytoplasmic tail of CD4 are prepared. Antibodies used for labeling experiments are prepared against these peptides. Microscopy studies are done using a laser confocal microscope and a TEM to locate the antibodies within H9 T cells. Results. The region past the UGA has plausible structure including a consensus sequence for protein kinase PKA. Antibodies against the cytoplasmic tail of CD4, and the putative region past the UGA codon were prepared. A suitable method for preparing the H9 T cells for antibody labeling was developed. Antibody labeling microscopy studies are underway. Conclusion. Structural studies on the sequence of the CD4 gene support the possibility that translation continues beyond the UGA codon. Satisfactory methods for antibody labeling of the CD4 receptor using both laser confocal and immunogold electron microscopy was developed.