Jacqueline Talea Desjardin1, S Patricia Becerra1, Preeti Subramanian1. 1. Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-0608, USA.
Abstract
PURPOSE: Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. MATERIALS AND METHODS: Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length PNPLA2 cDNA or a PNPLA2 cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots. RESULTS: PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. CONCLUSIONS: The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.
PURPOSE: Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. MATERIALS AND METHODS: Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length PNPLA2 cDNA or a PNPLA2 cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots. RESULTS: PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. CONCLUSIONS: The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.
Entities:
Keywords:
Alternative splicing; PEDF; PEDF-R; Pnpla2; Retina
Authors: W James Kent; Charles W Sugnet; Terrence S Furey; Krishna M Roskin; Tom H Pringle; Alan M Zahler; David Haussler Journal: Genome Res Date: 2002-06 Impact factor: 9.043
Authors: Andrew C Lake; Ying Sun; Jian-Liang Li; Jae Eun Kim; Jeremy W Johnson; Dongmei Li; Tracy Revett; Heather H Shih; Wei Liu; Janet E Paulsen; Ruth E Gimeno Journal: J Lipid Res Date: 2005-09-08 Impact factor: 5.922
Authors: Robin E Duncan; Yuhui Wang; Maryam Ahmadian; Jennifer Lu; Eszter Sarkadi-Nagy; Hei Sook Sul Journal: J Lipid Res Date: 2009-08-19 Impact factor: 5.922