Figure 4. Splicing products of LHX3 RNA in German shepherd dwarfs.

(A) Normal splicing of intron 5 as well as retention of the intron was observed in the pituitary of one of the dwarfs (F8). The cDNA fragments were generated with Platinum pfx polymerase and primers LHX3 ex4-7 F and LHX3 intron5 R in exons 4 and 6, respectively (Table S4). The fragments were analyzed by agarose gel electrophoresis. Lane 1: 100 bp size standard, * indicates the 500 bp fragment; lane 2: fragment derived from a control dog of mixed breed without the 7 bp deletion in intron 5 or other LHX3 mutations; lane 3: fragments derived from a dwarf homozygous for the deletion in intron 5; lanes 4 and 5: as lanes 2 and 3, respectively, without reverse transcriptase. The structure of the fragments as indicated by the drawings was confirmed by DNA sequence analysis of the excised bands. Open box: exon 5, closed box: exon 6, line: intron 5. (B) The cDNA sequence of the same dwarf, obtained with primers in exons 4 and 7 and standard taq DNA polymerase, indicates that exon 5 was skipped in a proportion of the transcripts. (C) The other dwarf (F9) for which pituitary RNA was available displayed heterozygosity for an insertion of an ACA trinucleotide sequence (underlined) in exon 5. This insertion translates into insertion of an asparagine residue (underlined) in the amino acid sequence (bold).