NOVEL PROTEIN ASSOCIATED WITH HEART DEVELOPMENT

描述：(改编自《调查者摘要》)纯合子 墨西哥Axolotls致命性心脏突变等位基因导致 发展有组织的肌原纤维和收缩的心脏。这一点已由 将突变心脏与正常前内胚层共培养，将其培养在 来自正常内胚层培养的条件培养液，或在RNA中培养 从内皮条件培养液中分离得到。构建的全长cDNA文库 从这个RNA和一个具有唯一部分核苷酸序列的克隆(PN1) 并对其推导的88个氨基酸序列进行了命名， N1.多克隆抗体是针对该蛋白的一个14肽而提出的。 成年Axolotl心脏推定N1蛋白及蛋白质印迹分析 匀浆结果表明，该蛋白存在于前内胚层(a 强大的心脏诱导组织)和阶段16内胚层(心脏诱导 阶段)位于推定的心源性中胚层附近。N1 与对照组相比，突变心脏的蛋白质染色显著减少 表明N1是一种新的蛋白质，可能在心脏中发挥作用 腹侧中胚层在心源性诱导中的重要作用 前内胚层。正在测试的假设是N1的表达式为 心脏正常发育和N1基因改变所必需的 在心脏发育过程中，表达会导致特定的异常 导致肌原纤维形成失败和相应的缺乏 收缩功能。具体目标是：(1)分离和测序 并对该序列进行计算机辅助分析 以确定推导出的序列的潜在同源性和结构域/基序结构 氨基酸序列；(2)确定的时间和空间格局 N1基因在正常胚胎和突变胚胎中的表达 从早期发育阶段开始使用突变体(c/c) 产卵获得的受精卵；(3)测定 微量注射N1表达载体研究N1对心脏发生的影响 构建到已知基因的受精卵中，实际上， (4)N1基因组的分离和测序 DNA，并分析和表征潜在的顺式元件。

DESCRIPTION: (Adapted from the Investigator's Abstract) Homozygosity for the lethal cardiac mutant allele in Mexican axolotls results in failure to develop organized myofibrils and contracting hearts. This is corrected by coculturing mutant hearts with normal anterior endoderm, culturing them in conditioned medium from normal endoderm cultures, or culturing them in RNA isolated from endodermal conditioned medium. A cDNA library constructed from this RNA and a clone (pN1) with a unique partial nucleotide sequence was identified and its deduced 88 amino acid sequence was given the name, N1. Polyclonal antibody was raised against a 14mer peptide of this presumptive N1 protein and western blot analysis of adult axolotl heart homogenates suggest that the protein was present in anterior endoderm (a potent heart inductor tissue) and in stage 16 endoderm (heart induction stage) localized adjacent to the presumptive cardiogenic mesoderm. The N1 protein staining was significantly reduced in mutant hearts when compared to normal hearts, suggesting the N1 is a novel protein that may play a significant role in cardiogenic induction of mesoderm by the ventral anterior endoderm. The hypothesis being tested is that expression of N1 is essential for normal heart development and the alteration of N1 gene expression results in specific abnormalities during heart development leading to a failure in myofibril formation and a corresponding lack of contractile function. Specific aims are to: (1) isolate and sequence the full length N1 cDNA and subject this sequence to computer assisted analysis to identify potential homologies and domain/motif structures of the deduced amino acid sequence; (2) determine the temporal and spatial patterns of expression for the N1 mRNA and protein in normal and mutant embryos beginning at the early developmental stages using the mutant (c/c) fertilized eggs obtained by spawning chimeric c/c axolotls; (3) determine the effect of N1 on cardiogenesis by microinjecting N1 expression vector constructs into fertilized axolotl eggs of known genotype, in effect, creating transgenic axolotls and; (4) isolate and sequence the N1 genomic DNA and analyze and characterize potential cis elements.