Roy B, Rajput S, Raghav S, Kumar P, Verma A, Jain A, Jain T, Singh D, De S, Goswami S L, Datta T K
Animal Genomics Laboratory, National Dairy Research Institute, Karnal, India.
Zygote. 2013 May;21(2):115-24. doi: 10.1017/S0967199411000712. Epub 2012 Jan 10.
Summary In spite of emerging evidence about the vital role of GDF9 in determination of oocyte competence, there is insufficient information about its regulation of oocyte-specific expression, particularly in livestock animals. Because of the distinct prominence of buffalo as a dairy animal, the present study was undertaken to isolate and characterize GDF9 cDNA using orthologous primers based on the bovine GDF9 sequence. GDF9 transcripts were found to be expressed in oocytes irrespective of their follicular origin, and shared a single transcription start site (TSS) at -57 base pairs (bp) upstream of ATG. Assignment of the TSS is consistent with the presence of a TATA element at -23 of the TSS mapped in this study. Localization of a buffalo-specific minimal promoter within 320 bp upstream of ATG was consolidated by identification of an E-box element at -113bp. Presence of putative transcription factor binding sites and other cis regulatory elements were analyzed at ~5 kb upstream of TSS. Various germ cell-specific cis-acting regulatory elements (BNCF, BRNF, NR2F, SORY, Foxh1, OCT1, LHXF etc.) have been identified in the 5' flanking region of the buffalo GDF9 gene, including NOBOX DNA binding elements and consensuses E-boxes (CANNTG). Presence of two conserved E-boxes found on buffalo sequence at -520 and -718 positions deserves attention in view of its sequence deviation from other species. Two NOBOX binding elements (NBE) were detected at the -3471 and -203 positions. The fall of the NBE within the putative minimal promoter territory of buffalo GDF9 and its unique non-core binding sequence could have a possible role in the control of the core promoter activity.
摘要 尽管越来越多的证据表明生长分化因子9（GDF9）在决定卵母细胞能力方面起着至关重要的作用，但关于其对卵母细胞特异性表达的调控，尤其是在家畜中的相关信息却不足。由于水牛作为乳用动物具有独特的重要性，本研究基于牛GDF9序列使用同源引物分离并鉴定水牛GDF9 cDNA。研究发现，GDF9转录本在卵母细胞中表达，无论其卵泡来源如何，并且在ATG上游-57个碱基对（bp）处共享一个单一转录起始位点（TSS）。TSS的确定与本研究中定位的TSS -23处存在TATA元件一致。通过在ATG上游320 bp内鉴定一个E-box元件，巩固了水牛特异性最小启动子的定位。在TSS上游约5 kb处分析了推定的转录因子结合位点和其他顺式调控元件。在水牛GDF9基因的5'侧翼区域已鉴定出各种生殖细胞特异性顺式作用调控元件（BNCF、BRNF、NR2F、SORY、Foxh1、OCT1、LHXF等），包括NOBOX DNA结合元件和共有E-boxes（CANNTG）。鉴于水牛序列在-520和-718位置发现的两个保守E-boxes与其他物种的序列存在差异，值得关注。在-3471和-203位置检测到两个NOBOX结合元件（NBE）。NBE位于水牛GDF9推定的最小启动子区域内及其独特的非核心结合序列可能对核心启动子活性的控制具有潜在作用。
