Calarco et al 2007 - University of Toronto

More documents

Recommendations

Info

Downloaded from genesdev.cshlp.org on June 10, 2013 - Published by Cold Spring Harbor Laboratory PressCalarco et al.many specialized cell types such as the brain are associatedwith relatively complex alternative splicing patterns(Xu et al. 2002; Johnson et al. 2003; Yeo et al.2004a). Surprisingly, comparisons of human and mousetranscript sequences have revealed that 5% substitution rates were analyzed for alternativesplicing differences by RT–PCR assays using poly(A) +RNA from human and chimpanzee tissues (see main text fordetails). (Right panel) In the quantitative alternative splicingmicroarray profiling strategy, labeled cDNA from the samepoly(A) + RNA samples were hybridized to an alternative splicingmicroarray designed to monitor inclusion levels of cassettetypealternative exons. Each alternative splicing event is monitoredby a set of six oligonucleotide probes (black horizontallines) (Pan et al. 2004). Predictions for alternative splicing differencesbetween the corresponding human and chimpanzee tissueswere validated by RT–PCR assays (see Table 1; Figs. 4, 5;Supplementary Fig. 1).2964 GENES & DEVELOPMENT
Downloaded from genesdev.cshlp.org on June 10, 2013 - Published by Cold Spring Harbor Laboratory PressSurveying primate alternative splicinget al. 2003; Yeo et al. 2004b, 2007; for reviews, see Matlinet al. 2005; Blencowe 2006; Chasin 2007). Consequently,we expected that frequent nucleotide changes in exonsand the flanking intron regions would be predictive ofsplicing differences between humans and chimpanzees.Role of nucleotide substitutions in generatingdifferences between human and chimpanzeesplicing patternsHigh substitution rates were rarely found in the alignedorthologous exons and flanking intron sequences: Ofthese regions, 0.3% displayed substitution rates of >5%,and substitution rates >10% were found in 5% substitution rates are predictive of splicingdifferences, semiquantitative RT–PCR assays wereperformed using primers specific for neighboring exonsequences and poly(A) + RNA samples from human orchimpanzee frontal cortex and heart tissues. These tissueRNA samples were pooled, in each case, from severaladult individuals (Supplementary Table 2). Of 31 regionswith >5% substitution rates selected for analysis, 14contained exons that displayed evidence of alternativesplicing in humans from analysis of the correspondingEST/cDNA (complementary DNA) sequences. Five ofthe 31 regions displayed splicing level differences betweenhumans and chimpanzees in at least one of thetwo tissues (see below). Interestingly, all five of theseisoform ratio differences involved exons included amongthe 14 alternative exons identified in the test set. At thisvalidation rate, 25%in differencebetween the human and chimpanzee frontal cortex and/or heart. Of these events, 30 (81%) were validated ashaving the expected differential splicing patterns (Figs.4, 5, below; Table 1; additional data not shown). Thus,GENES & DEVELOPMENT 2965
Page 1: Downloaded from genesdev.cshlp.org
Page 5 and 6: Downloaded from genesdev.cshlp.org
Page 13: Downloaded from genesdev.cshlp.org

Calarco et al 2007 - University of Toronto

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?