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KMID : 0578319950050040342
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Molecules and Cells
1995 Volume.5 No. 4 p.342 ~ p.347
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Construction of Assay System for the Determination of Human Immunodeficiency Birus Type 1 Ribosomal Frameshifting Efficience in Yeast Cells
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Lee, Jae Yung
Park, Jin Mo/Lee, Byeong Jae
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Abstract
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Cellular factors are assumed to influence ribosomal frameshifting (RF) of retroviruses. To assess the effect of cellular factors on frameshifting efficiency, we developed an in vivo frameshifting assay system which can be monitored in yeast cells. Human immunodeficiency virus type 1 (HIV-1) RF window region was amplified by polymerase chain reaction and inserted into an E. coli-yeast shuttle vector, pLG669Z [lacZ fusion vector under the control of iso-1-cytochrome c (CYC1) promoter]. Two different clones were constructed; pLG669Z-3 contains HIV gag-pol frameshift region in frame to express β-galactosidase when the RF occurs, while pLG669Z-4 has two nucleotide deletion downstream of the stop codon for gag gene, and therefore, can not produce β-galactosidase, whether RF occurs or not. RF efficiencies of transformed yeast cells with these were compared by the intensity of blue color on X-gal plate. Control pLG669Z vector displayed deep blue color while negative control pLG669Z-4 showed milky color. pLG670Z which lacks upstream activating sequence (UAS) of CYC1 promoter revealed light blue color which is lighter than that of pLG669Z-3. Expression level of β-galactosidase was further quantitated by o-nitrophenylglucoside (ONPG) assay. pLG670Z revealed 2.0% of β-galactosidase activity relative to 100% by pLG669Z. Assay vector pLG669Z-3 showed 6.2% of the enzyme activity whereas pLG669Z-4 failed to direct the expression of lacZ gene (=(??)0%). This value (6.2%) coincides reasonably with the ribosomal frameshifting frequency of HIV (about 5%) so that pLG669Z-3 can be used as an assay system for searching yeast cellular factor(s) by the determination of RF efficiency.
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