Erginel-Unaltuna N, Dube D K, Salsbury K G, Lemanski L F
Department of Anatomy and Cell Biology, SUNY Health Science Center, Syracuse 13210, USA.
Cell Mol Biol Res. 1995;41(2):117-30.
Recessive mutant gene c for "'cardiac nonfunction" in the mexican axolotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 weeks after fertilization, but eventually die from a lack of circulation. Morphological studies show that mutant hearts lack organized sarcomeric myofibrils. This abnormality can be corrected by co-culturing early mutant hearts with normal anterior endoderm/mesoderm tissues, by culturing them in a medium "conditioned" by this normal tissue, or by RNA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is constructed using this RNA. On the basis of sequence analyses on this cDNA library, it was estimated that 56% of the total RNA present in the conditioned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribosomal RNAs that showed no significant homology with other known sequences in the Genebank was examined further. An RT-PCR analysis showed that this RNA (designated "N1") is expressed in juvenile skeletal muscle, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide antibodies were produced against the most antigenic portion of the polypeptide which was deduced from this RNA. Western blot analyses of adult heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analyzed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investigated to determine its localization in normal isolated hearts at embryonic stages 35, 38, and 41 and on cross-sections through the heart regions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 using immunohistochemical techniques and confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.
墨西哥钝口螈（Ambystoma mexicanum）中导致“心脏无功能”的隐性突变基因c，会致使受影响的胚胎无法发育出可收缩的心脏。突变胚胎在受精后大约存活4周，但最终会因循环系统缺失而死亡。形态学研究表明，突变心脏缺乏有组织的肌节肌原纤维。通过将早期突变心脏与正常的前端内胚层/中胚层组织共同培养、在由这种正常组织“处理过”的培养基中培养，或者用从正常内胚层/中胚层分离的RNA培养，可以纠正这种异常。此外，从正常前端内胚层/中胚层处理过的培养基中分离的RNA能以剂量依赖的方式纠正突变心脏。利用这种RNA构建了一个cDNA文库。基于对该cDNA文库的序列分析，估计处理过的培养基中存在的总RNA中有56%是rRNA，而44%是非核糖体RNA。对其中一种与基因库中其他已知序列无显著同源性的非核糖体RNA进行了进一步研究。逆转录聚合酶链反应（RT-PCR）分析表明，这种RNA（命名为“N1”）在幼年骨骼肌、脑和心脏中大量表达，在肺中表达较少，在肝脏组织中完全不表达。针对从这种RNA推导出来的多肽中最具抗原性的部分，制备了亲和纯化的多克隆抗肽抗体。使用这些抗体对成年心脏匀浆进行蛋白质印迹分析，结果显示在67 kDa和65 kDa处有特异性的双条带染色。对这些双条带进行纯化并分析其氨基酸组成，结果表明这两条带很可能属于同一种蛋白质。利用免疫组织化学技术和共聚焦显微镜，进一步研究了N1蛋白在胚胎发育阶段35、38和41的正常分离心脏中的定位，以及在胚胎发育阶段16、33 - 34、37 - 38和41 - 42的整个正常胚胎心脏区域横切面上的定位。此外，还研究了处于37 - 38阶段的突变胚胎心脏区域横切面上N1蛋白的存在和分布情况。与正常心脏相比，突变心脏中的N1蛋白染色明显减少。
