（Department of Medical Physiology, Texas A&M University System Health Science Center,College Station, Texas 77843）

（ Department of Biology, Texas A&M University, College Station, Texas 77843;）

X.Huang

（Department of Medical Physiology, Texas A&M University System Health Science Center,College Station, Texas 77843）

R.W.Zajdel

（ Department of Biology, Texas A&M University, College Station, Texas 77843;）

S.L.Lemanski

（Department of Medical Physiology, Texas A&M University System Health Science Center,College Station, Texas 77843）

C.Zhang

（ Department of Biology, Texas A&M University, College Station, Texas 77843;）

F.Meng

（Department of Medical Physiology, Texas A&M University System Health Science Center,College Station, Texas 77843）

D.Foster

（Department of Medical Physiology, Texas A&M University System Health Science Center,College Station, Texas 77843）

Q.Li

（Department of Medical Physiology, Texas A&M University System Health Science Center,College Station, Texas 77843）

N.Erginel-Unaltuna

D.K.Dube

（Department of Anatomy and Cell Biology, State University of New York Health

Science Center, Syracuse, New York 13210）

The Mexican axolotl (Ambystoma mexicanum) provides and excellent model for studying heart development since it carries a simple recessive cardiac lethal mutation that results in a failure of mutant embryonic myocardium to contract. In cardiac mutant axolotls the hearts do not beat, apparently due to an absence of organized myofibrils. The mutant hearts can be rescued by coculturing them with the normal anterior endoderm/mesoderm tissue, by medium conditioned with normal anterior endoderm/mesoderm or by RNA isolated from the conditioned medium. We have previously isolated a single cDNA clone from a library prepared with total RNA from conditioned medium; this 166 nt long in vitro synthesized RNA, directed by the unique cDNA clone(Clone #4), has the ability to correct the heart defect and promote myofibrillogenesis in mutant hearts. The criteria for rescue include contraction of mutant hearts throughout their lengths, an increase in sarcomeric tropomyosin arrays as shown by immunofluorescent confocal microscopy, and the ultrastructural appearance of organized sarcomeric myofibrils. Quantitative PT-PCR has shown that Clone #4 RNA is specifically expressed in axolotl heart, not in other tissues or organs. Moreover, the peak expression of Clone #4 is from stage 15 to 30, corresponding to the heart induction and tube formation stages. These data indicate that this bioactive RNA plays an important role in heart development in axolotls. More recently, a point mutation (G-T) was found in Clone #4 RNA, which was derived from mutant axolotl embryos at stages 20 and 30. At present, we are searching for the full-length Clone #4 gene in an axolotl genomic library. Primer extension yields a product of ～500 bp at the 5' -end of the Clone #4 gene. The nucleotide sequence of the extended Clone #4(Ext-Clone #4) was determined and found to be unique showing no significant homology with other known sequences available from the gene databases. Interestingly, T7 sense RNA from the Ext-Clone # 4 (～500 nt RNA) shows a higher efficiency in rescuing mutant hearts than the T7 RNA from original Clone #4 (166 nt RNA). Our working hypothesis is that the bioactive RNA is a regulatory RNA that may upregulate directly or indirectly tropomyosin production in axolotl heart resulting in organized and functional myofibrils.

Supported by NIH Grants HL-58435 and HL-061246