Molecular and cellular studies on cardiac mutant Mexican axolotls, Ambystoma mexicanum

A recessive cardiac lethal mutation, designated by gene c&barbelow;, for “cardiac nonfunction”, in the Mexican axolotl, Ambystoma mexicanum, results in an incomplete differentiation of the myocardium. Mutant hearts do not beat and electron microscopy shows that neither do they contain sarcomeric myofibrils. However, the defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or RNA from endoderm mesoderm-conditioned medium. After constructing a cDNA library from total conditioned medium RNA in a pcDNAII expression vector and screening the cDNA library by an organ culture bioassay, a single clone (Clone #4) has been isolated. The synthetic RNA from Clone #4 corrects the heart defect by promoting myofibrillogenesis. The insert size of the active clone is 166 nt in length with a unique nucleotide sequence. The antisense RNA from Clone #4 using SP6 RNA polymerase fails to rescue mutant hearts. Current data suggest that this bioactive RNA is functioning through its secondary structure instead of through its mRNA as a peptide template. RT-PCR results indicate that the expression pattern of the bioactive RNA is the same in mutant as in the normal embryos, (i.e., both normal and mutant embryos express Clone #4 during early developmental stages). However, a point mutation (G→T) has been found within this 166 nt sequence. Bioassay, confocal and electron microscopy demonstrate that the mutant RNA derived from mutant embryos cannot rescue the mutant hearts and fails to increase the tropomyosin expression in the mutant hearts after mutant Clone #4 RNA transfection. This short gene sequence has also been extended to nearly 1 kilo-base pairs with a poly (A) tail at the 3′ end of the gene. The new extended sequence turns out to be novel and does not show homology with any other gene in the NCBI GenBank. Recent results indicate that the 166 nt Clone #4 sequence may localize in multiple genes.