Molecular analysis of the E<i>lmod1^rda-2J</i> mutation.

<p><b>A.</b> Southern blots of genomic DNA from mice with +/+ (lanes marked 1), +/<i>rda^2J</i> (lanes marked 2) and <i>rda^2J/rda^2J</i> (lanes marked 3) genotypes, digested with <i>Eco</i>RI (Eco), <i>Pst</i>I (Pst), <i>Pvu</i>II (Pvu), and <i>Msp</i>I (Msp) restriction enzymes. The blot was hybridized with an <i>Elmod1</i> cDNA probe corresponding to exons 7–11. Although the same quantity of DNA was loaded in each lane, the intensity of <i>Elmod1</i>-hybridizing bands is greater in in <i>Eco</i>RI- and <i>Pvu</i>II-digested DNA samples from <i>rda^2J/rda^2J</i> mice than samples from +/+ mice and exhibit additional bands (indicated by arrows) in <i>Pst</i>I and <i>Msp</i>I digested DNA. <b>B.</b> A northern blot of total RNA extracted from brains of adult C57BL/6J control (lane 1), <i>rda^2J</i>/+ heterozygotes (lanes 2 and 3), <i>rda^2J/rda^2J</i> homozygotes (lanes 4 and 5), and <i>rda/rda</i> homozygote (lane 6; negative control) was hybridized with an <i>Elmod1</i> cDNA probe. Wildtype <i>Elmod1</i> transcript (∼2600 nt) was not detected in RNA from <i>rda^2J/rda^2J</i> mice; however, a 3200 nt mutant transcript (about 600 nucleotides larger than wildtype) was abundantly expressed. <b>C.</b> PCR products from <i>rda^2J</i>–derived cDNAs indicate an intragenic duplication. cDNAs from mice with +/+ (lanes marked 1), +/<i>rda^2J</i> (lanes marked 2) and <i>rda^2J/rda^2J</i> (lanes marked 3) genotypes were used as PCR templates in combination with primers specific to <i>Elmod1</i> exons. Expected wildtype PCR products are marked by asterisks, and unexpected large PCR products unique to <i>rda^2J</i> samples are indicated by arrows. 500 bp (lane marked L1) and 100 bp (lane marked L2) ladders were used to estimate PCR product sizes, and predicted sizes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036074#pone.0036074.s004" target="_blank">Table S2B</a>. <b>D.</b> A western blot of protein extracts from adult brains of <i>rda/rda</i> (lane 1, negative control), <i>rda^2J/rda^2J</i> (lane 2), and +/+ B6 mice (lane 3), shows that the predicted 38 kDa wildtype ELMOD1 protein is absent from both <i>rda</i> and <i>rda^2J</i> mutant mice, but a larger 62 kDa mutant protein can be seen in <i>rda^2J</i> mutants (indicated by arrow in lane 2), corresponding to the predicted duplication of 202 amino acids. The polyclonal ELMOD1 antibody cross-reacted with other unknown proteins (non-specific bands, NS). <b>E.</b> The large PCR products obtained from <i>rda^2J</i>–derived cDNA (indicated by arrows in <b>C</b>) and multiple combinations of exon-specific primers were used to determine the DNA sequence and structure of the mutant <i>Elmod1</i> transcript, and this analysis indicated a duplication of exons 3–8, as shown in the diagram of the presumed mutant gene structure.