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Figure 3


Fig. 3. Generation of the Nesprin-2{Delta}ABD isoform in KO cells and tissues through alternative translational initiation. (A) The coding nucleotide sequences of Nesprin-2 exons 1-10 are shown and the deduced amino acid sequence is indicated beneath. Similar to other canonical CH1-CH2-type ABDs, the ABD of Nesprin-2 Giant is also encoded by seven exons (CH1-encoded protein sequence is coloured in yellow, linker region in white and CH2 in blue). Exons 2-4 (yellow) were deleted in Nesprin-2{Delta}ABD KO mice. The names and sequences of primers employed in the RT-PCR analysis are indicated in blue lettering. The K56-386 epitope in exon 9 is marked as a red box. (B) RT-PCR analysis of KO- and WT-fibroblast cDNAs using the primer sets indicated in A shows absence of the exon-2–exon-4 region (Ex2Fw/Ex4Rv set). RT-PCR employing exon-1- and exon-8-specific primers (Ex1Fw/Ex8Rv set) on WT- and KO-fibroblast RNA samples did not reveal the anticipated 130-bp exon-1&8 cDNA; instead, a 628-bp KO band was evident, compared with the expected 860-bp WT band (exons 1 through 8). Concomitant sequencing of the KO fragment indicated a splicing event of exon 1 with exon 5. The presence of these exons in the Nesprin-2{Delta}ABD transcript was verified by RT-PCR using the Ex5Fw/Ex7Rv set of primers. Because this alternative splicing event does not result in an open reading frame on the translated transcript, we searched the following exons for alternative initiation sites. Using the Chang Bioscience software (http://www.changbioscience.com/primo/ti.html), alternative KOZAK initiation-site sequences were identified in exons 7 and 8 [indicated in boldface (red) and overline in A]. (C) Semi-quantitative RT-PCR analysis of fibroblast cDNAs using the exon-1- and exon-8-specific primer set to show accumulation of the Nesprin-2{Delta}ABD transcript only in aged KO fibroblasts. WT analysis shows the expected 860-bp band (exon 1 through 8), whereas the KO displays the 628-bp band in fibroblasts of higher passages, indicating the alternative splicing event of exon 1 with exon 5. The number of passages is shown (#1, #3, #5).





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