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. 2005 Oct 7;102(42):15134–15139. doi: 10.1073/pnas.0503640102

Fig. 2.

Fig. 2.

Mapping of the insertion site of Minos transposon in the sj mutant genome. (A) The design of a Minos transposon used for the insertional mutagenesis. E and H indicate the restriction sites of EcoRI and HindIII, respectively. (B) Location of the Minos insertion site in the sj mutant genome. Open and filled boxes represent exons of Ci-CesA corresponding to the UTR and ORF, respectively. A gray box indicates the position of an EST (rciad083d01) in the public genome browser of C. intestinalis (http://genome.jgi-psf.org/ciona4/ciona4.home.html). H, the restriction sites of HindIII. One restriction site showed polymorphism in the Japanese population of C. intestinalis and is shown as (H). pCesA(-2080)-GFP and pCesA(-327)-GFP indicate constructs used for the promoter analyses in Fig. 3C. (C) Genomic Southern blot analyses of sj mutants. (Left) Genomic DNA from a sj heterozygous animal (sj/+) digested with EcoRI was detected with the probe shown in A (dotted line). Multiple bands indicate the formation of a tandem repeat array. (Right) Genomic DNA from wild-type animals (+/+) and sj heterozygous animals (sj/+) digested with HindIII were detected by the probe shown in B (dotted line). M indicates the size marker. sj heterozygous animals showed specific bands (arrow) that were expected from the fusion of a part of pMiTFr3dTPO-gfp and genomic DNA. (D) An example of PCR analysis showing the homozygosity of the Minos insertion in sj mutants. DNA from larvae with normal appearance without GFP signal (N), normal appearance with GFP expression (P), and sj mutants (sj) were subjected to PCR by primers shown in B (small arrows). The absence of a band indicates the homozygosity of insertion. (Lower) The results of PCR that detects gfp (positive control). (E) A dramatic reduction of Ci-CesA expression in sj mutants (sj) revealed by RT-PCR. Ci-Epi1, which is expressed in epidermis (36), was used as a positive control. The expression of ciad083d01 was not affected in sj mutants. Negative controls without reverse transcription are shown by RT – lanes.