An RFLP map of the baboon platelet-derived growth factor receptor gene.
An RFLP map of the baboon platelet-derived growth factor receptor gene.
复制标题
狒狒血小板衍生生长因子受体基因的 RFLP 图谱。
DOI：
10.1007/bf00364806
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Henkel,RD
中科院分区：
文献类型：
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作者：
Perelygina,LM;Kammerer,CM;Henkel,RD
血小板衍生生长因子（PDGF）是一种由多种细胞类型（包括血小板、内皮细胞、血管平滑肌细胞、成纤维细胞和巨噬细胞）产生的强效有丝分裂原（Ross 1986）。PDGF通过与位于靶细胞群膜上的高亲和力受体结合，刺激多种类型间充质细胞的增殖（综述见Ross et al.1986）。这些受体的配体结合通过与分子的细胞内结构域相关的酪氨酸激酶活性激活细胞信号转导途径（Marmur et al. 1992）。PDGF受体（PDGFR）的多肽亚基有两种亚型，命名为c~和c~，它们表现出独特的配体结合特性。PDGFR-c-亚基可结合PDGF的任一同种型，而13受体亚基仅结合一种同种型（Ross等，1990 a）。在人动脉病变（Rubin et al. 1988）和灵长类动物（Ross et al. 1990 b）中报告了B型PDGF受体的表达增加，提示与动脉粥样硬化相关的平滑肌细胞增殖的潜在机制。在本报告中，我们描述了10个多态性DNA标记的狒狒PDGFR-~多肽编码基因的检测和相对位置。由于狒狒被用作研究动脉粥样硬化的模型（McGill等，1981; Henkel等，1993），这些DNA标记物将用于确定PDGFR-α基因座中的遗传变异对血管损伤形成的易感性的影响。根据既定程序（Sambrook等人，1989）从狒狒肝脏和大型（n-700）纯种狒狒群体（亚种Papio hamadryus anubis和Ph cynocephalus）成员的白色血细胞中分离高分子量DNA。用不同的限制性内切酶消化DNA样品（10 μ g），所得片段在0.8%琼脂糖凝胶中电泳分离。将DNA片段转移到尼龙膜上，并与编码人PDGF受体13-多肽片段的放射性eDNA探针杂交。从美国典型培养物保藏中心获得编码2.65 kb人PDGFR-13基因的cDNA克隆（克隆RP 41）（Gronwald等，1988）。该探针在用于Southern印迹的条件下不与PDGFR-c基因交叉杂交（Majesky等，1990）。用EcoRI从质粒上切除后，用Pst Ⅰ消化，从2.65-kb克隆制备片段。用[α 32 P]-dCTP和商业随机引物标记试剂盒标记探针。杂交反应在42 ° C下在50%甲酰胺、5x SSC、5x Denhardt溶液、0.1%SDS、10%硫酸葡聚糖和50 μ g/ml鲑鱼精子DNA中进行。将膜在2x SSC/0.1%SDS中洗涤两次20分钟，然后在60 ℃下用0.2x SSC/0.1%SDS洗涤10分钟。将膜在-80 ℃下暴露于Kodak XAR胶片。通过Pearson卡方分析（Zar 1974）确定RFLP基因型之间的关联。人PDGFR-13的完整2.65-kb cDNA克隆的用途
Platelet-derived growth factor (PDGF) is a potent mitogen produced by a number of cell types including platelets, endothelial cells, vascular smooth muscle ceils, fibroblasts, and macrophages (Ross 1986). PDGF stimulates the proliferation of many types of mesenchymal cells (reviewed in Ross et al. 1986) by binding to high-affinity receptors located in the membrane of target cell populations. Ligand binding by these receptors activates cellular signal transduction pathways through the tyrosine kinase activity (Marmur et al. 1992) associated with the intracellular domain of the molecule. There are two isoforms of the polypeptide subunits of PDGF receptors (PDGFR), designated c~ and~, that exhibit distinctive ligand-binding properties. The PDGFR-c~ subunit can bind either isoform of PDGF, whereas the 13 receptor subunit binds only one isoform (Ross et al. 1990a). Increased expression of B-type PDGF receptors has been reported in human arterial lesions (Rubin et al. 1988) and in primates (Ross et al. 1990b), suggesting a potential mechanism for the proliferation of smooth muscle cells associated with atherosclerosis. In the present report, we describe the detection and relative locations of ten polymorphic DNA markers in the gene encoding the baboon PDGFR-~ polypeptide. Because the baboon is used as a model for studying atherosclerosis (McGill et al. 1981; Henkel et al. 1993), these DNA markers will be useful for determining the effects of genetic variation in the PDGFR-~ locus on the susceptibility to vascular lesion formation. High-molecular-weight DNA was isolated according to established procedures (Sambrook et al. 1989) from baboon liver, and white blood cells from members of a large (n-700) pedigreed baboon colony (subspecies Papio hamadryus anubis and Ph cynocephalus). DNA samples (10 gg) were digested by different restriction enzymes and the resulting fragments separated by electrophoresis in a 0.8% agarose gel. DNA fragments were transferred to nylon membranes mad hybridized with radioactive eDNA probes encoding fragments of the 13-polypeptide of the human PDGF receptor. A cDNA clone encoding 2.65 kb of the human PDGFR-13-gene (Gronwald et al. 1988) was obtained from the American Type Culture Collection (clone RP41). This probe does not cross-hybridize with the PDGFR-c~ gene under conditions used for Southern blotting (Majesky et al. 1990). Fragments were prepared from the 2.65-kb clone by digestion with PstI after excision from the plasmid with EcoRI. Probes were labeled with [ot32P]-dCTP and a commercial, random primer labeling kit. Hybridization reactions were carried out at 42~ in 50% formamide, 5x SSC, 5x Denhardt's solution, 0.1% SDS, 10% dextran sulfate, and 50 gg/ml salmon sperm DNA. Membranes were washed two times in 2x SSC/0.1% SDS for 20 rain followed by 0.2 x SSC/0.1% SDS for 10 min at 60~ Membranes were exposed to Kodak XAR film at-80~ Associations between RFLP genotypes were determined by Pearson's chi square analysis (Zar 1974). Use of the intact 2.65-kb eDNA clone of the human PDGFR-13