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：（改编自研究者摘要） 墨西哥蝾螈致命的心脏突变等位基因导致 形成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培养基中培养它们， 分离自内胚层条件培养基。 cDNA文库的构建 从该RNA和具有独特的部分核苷酸序列的克隆（pN 1）， 鉴定了它，并将其推导的88个氨基酸序列命名为， N1。 针对该14聚体肽产生多克隆抗体。 蝾螈心脏的N1蛋白和蛋白质印迹分析 匀浆表明，蛋白质存在于前内胚层（a 有效的心脏诱导组织）和第16期内胚层（心脏诱导 阶段）位于推定的心源性中胚层附近。 的N1 与对照组相比， 这表明N1是一种新的蛋白质， 在腹侧皮层对中胚层的心源性诱导中的重要作用 前内胚层 正在测试的假设是，N1的表达是 正常心脏发育所必需的N1基因的改变 表达导致心脏发育过程中的特定异常 导致肌原纤维形成的失败和相应的缺乏 收缩功能 具体目标是：（1）分离和测序 全长N1 cDNA，并将该序列进行计算机辅助分析 以鉴定推断的氨基酸序列的潜在同源性和结构域/基序结构， 氨基酸序列;（2）确定时间和空间格局 正常和突变胚胎中N1 mRNA和蛋白的表达 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 微量注射N1基因表达载体对心肌发生的影响 构建到已知基因型的蝾螈受精卵中，实际上， 建立转基因蝾螈;（4）分离和测序N1基因组 DNA和分析和表征潜在的顺式元件。