CONSEQUENCES OF HUMAN PIT-1 AND GH PROMOTER MUTATIONS

人类 PIT-1 和 GH 启动子突变的后果

• 批准号: 2145496
• 负责人: JOHN S. PARKS
• 金额: $ 18.72万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145496
• 关键词: DNA replication; HeLa cells; autosomal dominant trait; autosomal recessive trait; child (0-11); developmental genetics; family genetics; gel mobility shift assay; gene mutation; genetic polymorphism; genetic promoter element; homeobox genes; human genetic material tag; human tissue; hypopituitarism; nucleic acid sequence; pituitary dwarfism; polymerase chain reaction; prolactin; protein structure function; site directed mutagenesis; somatotropin; thyrotropin; transcription factor; transfection

Pituitary growth hormone production is essential for normal postnatal growth. Recent studies in the applicant's laboratory have linked mutations of the Pit-1 gene to a human growth disorder involving deficiencies of the anterior pituitary hormones growth hormone, prolactin and thyrotropin. Humans with a substitution of proline for alanine at amino acid 158 differ from Snell dwarf mice with a mutation at amino acid 261 in that they have normal anterior pituitary size and only mild thyrotropin deficiency. The observation that an alteration in the POU-specific domain of Pit-1 eliminates transcriptional activation of its known target genes growth hormone and prolactin, with minimal impairment of binding to these genes and no impairment of anterior pituitary growth was not predicted by studies of mutant Pit-1 proteins produced in the laboratory. It suggests that sequence variation in the Pit-1 gene and its target gene promoters may account for a broader spectrum of human growth disorders. The proposed studies address this issue through examination of Pit-1 gene sequence in a large number of families with combined GH, Prl and TSH deficiencies, autosomal recessive isolated growth hormone deficiency, or autosomal dominant isolated growth hormone deficiency. The alternative possibility of an abnormality of regulatory elements in the growth hormone gene promoter is addressed through sequencing of the growth hormone promoter in families with autosomal recessive isolated growth hormone deficiency. Sequence variation is detected through polymerase chain reaction amplification of genomic DNA or cDNA and localized through direct DNA sequencing. The functional consequences of Pit-1 gene and growth hormone promoter sequence abnormalities are assessed by mobility shift DNA-binding assays, co-transfection of activator and reporter gene constructs in HeLa cells, and assays for activity of wild type and mutant Pit-1 in Adenovirus DNA replication assays. Studies of DNA sequence variation in children with severe disorders of growth are accompanied by studies of polymorphic variation in the same sequences among subjects at the extremes for normal growth. This project will define the importance of variations in Pit-1 and its target gene sequences in the pathogenesis of anterior pituitary hormone deficiency and in the generation of differences in patterns of growth among normal individuals.

垂体生长激素的产生对正常的产后 增长申请人实验室最近的研究表明， Pit-1基因与人类生长障碍的关系， 垂体前叶激素生长激素、催乳素和促甲状腺激素。 在氨基酸158处脯氨酸取代丙氨酸的人与在氨基酸158处脯氨酸取代丙氨酸的人不同， 来自在氨基酸261处具有突变的Snell侏儒小鼠， 垂体前叶大小正常，仅有轻度促甲状腺激素缺乏。 的 观察到Pit-1的POU特异性结构域的改变 消除了其已知靶基因的转录激活生长 激素和催乳素，与这些基因结合的损害最小 垂体前叶生长无损害， 在实验室中产生的突变Pit-1蛋白的研究。 它表明 Pit-1基因及其靶基因启动子的序列变异 可能导致更广泛的人类生长障碍。 的 建议的研究通过检测Pit-1基因来解决这个问题。 序列在大量的家庭与组合GH，Prl和TSH 缺乏症、常染色体隐性孤立性生长激素缺乏症，或 常染色体显性遗传孤立性生长激素缺乏症。 替代 生长激素调节元件异常的可能性 基因启动子是通过生长激素的测序， 常染色体隐性遗传性生长激素家系启动子研究 缺陷通过聚合酶链检测序列变异 反应扩增基因组DNA或cDNA，并通过直接 DNA测序Pit-1基因与生长的功能性后果 激素启动子序列异常通过迁移率改变来评估 DNA结合试验，激活因子和报告基因的共转染 HeLa细胞中的构建体，并测定野生型和突变体的活性 腺病毒DNA复制试验中的Pit-1。 DNA序列研究 严重生长障碍儿童的变异伴随着 研究对象之间相同序列的多态性变异， 正常生长的极端。该项目将确定的重要性， Pit-1及其靶基因序列的变异在肺癌发病中的作用 垂体前叶激素缺乏症与中世代差异 在正常个体的生长模式中。