Characterization of alternatively spliced and truncated forms of the Arf guanine nucleotide exchange factor GBF1 defines regions important for activity.
Analysis of multiple transcripts for the Arf-specific guanine nucleotide exchange factor GBF1 identified three positions displaying small in-frame deletions and insertions. Sequencing of genomic DNA for CHO GBF1 and analysis of the human gene established that those variations were consistent with alternate splicing events. RT-PCR analysis of CHO mRNA confirmed that these small in-frame deletions occurred at significant and similar frequencies in both WT and BFA resistant CHO cells. These splice variants behaved like GBF1 in biological assays based on the observation that GBF1 is cytotoxic at high levels but will confer resistance to BFA when moderately overexpressed. Comparison of variants with larger deletions defined regions of 75 (exons 5-7) and 412 (exons 31-39) amino acid residues that were required for cell killing but were dispensable for promoting BFA resistance.
Claude
Alejandro
A
Department of Cell Biology, University of Alberta, 5-35 Medical Sciences Building, Edmonton, AB, Canada T6G 2H7.
Zhao
Bao-Ping
BP
Melançon
Paul
P
D017398:Alternative Splicing; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D002470:Cell Survival; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005091:Exons; D017353:Gene Deletion; D015723:Gene Library; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007438:Introns; D008957:Models, Genetic; D009154:Mutation; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; C120445:GBF1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D012333:RNA, Messenger; D020126:Brefeldin A