NOVEL PROTEIN ASSOCIATED WITH HEART DEVELOPMENT

与心脏发育相关的新型蛋白质

• 批准号: 6030820
• 负责人: LARRY F LEMANSKI
• 金额: $ 22.86万
• 依托单位: TEXAS ENGINEERING EXPERIMENT STATION
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-20 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6030820
• 关键词: Urodela; animal genetic material tag; biomarker; complementary DNA; computer assisted sequence analysis; embryo /fetus; embryo /fetus protein; embryogenesis; gene induction /repression; genetically modified animals; mesoderm; messenger RNA; molecular cloning; muscle proteins; myocardium; myogenesis; nonmammalian vertebrate embryology; nucleic acid sequence; protein structure function

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.

描述：（改编自研究者的摘要）纯合性 墨西哥蝾螈的致命心脏突变等位基因导致无法 发展有组织的肌原纤维和收缩的心脏。 这是通过以下方式纠正的 将突变心脏与正常前内胚层共培养 来自正常内胚层培养物的条件培养基，或在 RNA 中培养它们 从内胚层条件培养基中分离。 构建 cDNA 文库 来自该 RNA 和具有独特部分核苷酸序列的克隆 (pN1) 被鉴定出来，并且推导出来的 88 个氨基酸序列被命名为， N1。 针对此 14mer 肽产生多克隆抗体 成年蝾螈心脏的推定 N1 蛋白和蛋白质印迹分析 匀浆表明该蛋白质存在于前内胚层（a 强效心脏诱导组织）和第 16 阶段内胚层（心脏诱导 阶段）定位于邻近假定的心源性中胚层。 N1 与相比，突变心脏中的蛋白质染色显着减少 正常心脏，表明 N1 是一种新型蛋白质，可能发挥 腹侧中胚层心源性诱导中发挥重要作用 前内胚层。 正在检验的假设是 N1 的表达是 对于正常心脏发育和 N1 基因的改变至关重要 表达导致心脏发育过程中的特定异常 导致肌原纤维形成失败并相应缺乏 收缩功能。 具体目标是：（1）分离并测序 全长 N1 cDNA 并将该序列进行计算机辅助分析 识别推导的潜在同源性和域/基序结构 氨基酸序列； (2) 确定时间和空间格局 N1 mRNA 和蛋白质在正常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) 通过产卵嵌合c/c蝾螈获得的受精卵； (3)确定 显微注射N1表达载体观察N1对心脏发生的影响 构建成已知基因型的受精蝾螈卵，实际上， 创造转基因美西螈； (4) N1基因组分离并测序 DNA 并分析和表征潜在的顺式元素。