GENE REGULATION IN MAMMALIAN SEX DETERMINATION

哺乳动物性别决定中的基因调控

• 批准号: 3331970
• 负责人: HOLLY A. INGRAHAM
• 金额: $ 13.42万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3331970
• 关键词: DNA binding protein; DNA footprinting; Mullerian duct inhibiting substance; RNase protection assay; Sertoli cells; developmental genetics; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; in situ hybridization; laboratory mouse; laboratory rat; mammalian embryology; molecular cloning; ovary; protein purification; protein sequence; sex determination; sex differentiation; testis; transfection

The process of mammalian sex determination and differentiation is of fundamental importance and remains one of the basic questions of human biology. Recently, strong genetic evidence has determined the piece of the Y-chromosome thought responsible for testes determination. with the discovery of the SRY gene, Sex-determining Region of Y, the beginning of this gene cascade may be defined, and the search for downstream target genes can begin. While the exact identification of downstream targets may prove difficult, experimental evidence suggest that the evolution of the Sertoli cell-type of the testis is intimately linked to the Y- chromosome. As the "indifferent" bipotential genital ridge begins to differentiate into testes, one can visualize the Sertoli cells aligning themselves into cord-like structures. Shortly after, a critical function of the Sertoli cell will be the expression and local release of the hormone, Mullerian inhibiting substance, MIS. During a critical time, the secretion of MIS results in the regression or "cell death" of the Mullerian duct, the anlagen of internal female genitalia. Thus, regulation of MIS gene expression is directly coupled to the differentiation of the Sertoli cell and the execution of the Y- chromosomal developmental programs. Oddly enough, MIS is also expressed at adult granulosa cell of the ovary; functions in this "sister" cell type of the Sertoli cell remain unclear. Here, it is proposed to define and isolate the developmental regulator(s) of MIS gene expression. Preliminary data suggest that a conserved element, MIS-RE-1, close to the promoter of the mouse MIS gene is bound by a Sertoli-cell specific protein. Further, point mutations that abolish binding also decrease transcription activity as measured in transfection assays using primary Sertoli cells. Binding activity of this protein is roughly correlated to the time of peak MIS transcript expression. Moreover, the same MIS- RE-1 element is also bound by an ovarian-specific protein exhibiting distinct binding specificity and a relative molecular weight different from that observed in the testes. Aim #1 will characterize the mouse MIS gene by defining the transcriptional start site and comparing its sequence with that of the human MIS gene. Standard biochemical analyses and functional studies will probe the protein-DNA interactions between the MIS-RE-1 and the testis-specific MIS-REBP (binding protein). Complete ontogeny of testes-MIS-REBP activity and ovary-specific activity will be undertaken. Aim #2 proposes to purify and clone the Sertoli- specific protein binding to the MIS-RE-1 site. The purification and cloning of the testes-MIS-REBP will follow well-established protocols for DNA-binding proteins. If the testes-MIS-REBP acts to initiate MIS-gene expression its transcript and protein expression should correlate with the onset of MIS gene activation. This hypothesis can be tested following isolation of the MIS-REBP cDNA clone. Aim #3 will attempt to characterize the developmental effects of MIS cis-elements by a simple set of transgenic mouse experiments. In choosing to study the initial activation of MIS gene expression, it is likely that these studies will shed light on basic molecular mechanisms underlying the development and maturation of reproductive endocrine organs.

哺乳动物性别决定和分化的过程是 具有根本重要性，仍然是人类的基本问题之一 生物学。 最近，强有力的遗传证据确定了这一点 Y 染色体被认为负责睾丸的决定。 与 SRY基因的发现，Y的性别决定区， 可以定义该基因级联，并搜索下游目标 基因可以开始。 同时准确识别下游目标 可能会被证明是困难的，实验证据表明 睾丸支持细胞类型与 Y 密切相关 染色体。 当“冷漠”的双电位生殖脊开始 分化为睾丸，可以看到支持细胞排列 自身变成绳状结构。 不久之后，一个关键函数 支持细胞的表达和局部释放 激素，苗勒管抑制物质，MIS。 在关键时刻， MIS 的分泌导致细胞的退化或“细胞死亡” 苗勒氏管，女性内生殖器的原基。 因此， MIS 基因表达的调控直接耦合到 支持细胞的分化和Y-的执行 染色体发育程序。 奇怪的是，MIS也表达为 在卵巢的成体颗粒细胞处；在这个“姐妹”细胞中发挥作用 支持细胞的类型仍不清楚。 这里，建议定义 并分离 MIS 基因表达的发育调节因子。 初步数据表明保守元件 MIS-RE-1 接近 小鼠 MIS 基因的启动子与支持细胞特异性结合 蛋白质。 此外，废除结合的点突变也会减少 使用初级转染测定法测量转录活性 支持细胞。 该蛋白质的结合活性大致相关 到 MIS 转录本表达峰值的时间。 此外，同样的 MIS- RE-1 元件还与卵巢特异性蛋白质结合，表现出 不同的结合特异性和相对分子量不同 从睾丸中观察到的结果。 目标 #1 将表征鼠标 MIS 基因通过定义转录起始位点并比较其 与人类 MIS 基因的序列。 标准生化分析 功能研究将探讨蛋白质-DNA 之间的相互作用 MIS-RE-1 和睾丸特异性 MIS-REBP（结合蛋白）。 睾丸的完整个体发育-MIS-REBP 活性和卵巢特异性活性 将进行。 目标#2 提出纯化和克隆 Sertoli- 与 MIS-RE-1 位点结合的特异性蛋白质。 纯化和 睾丸-MIS-REBP 的克隆将遵循既定的协议 DNA 结合蛋白。 如果睾丸-MIS-REBP 发挥作用启动 MIS-基因 其转录本和蛋白质表达的表达应与 MIS 基因激活的开始。 这个假设可以被检验 分离 MIS-REBP cDNA 克隆后。 目标 #3 将尝试 通过简单的描述 MIS 顺式元件的发育效应 一组转基因小鼠实验。 在选择学习初期 MIS 基因表达的激活，这些研究可能会 揭示了发育和发育背后的基本分子机制 生殖内分泌器官的成熟。