Prasanth Supriya Gangadharan, Ali Sher
Molecular Genetics Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067 India.
DNA Cell Biol. 2003 Jul;22(7):447-56. doi: 10.1089/104454903322247325.
The pleiotropic proto-oncogene c-kit receptor, implicated in hematopoiesis and melanogenesis, is also known to play an important role in germ cell proliferation and differentiation although the mechanisms for the latter remain unknown. We studied c-kit expression by RT-PCR in various tissues of both fertile and infertile Brown Norway rats. Using different sets of primers, several regions from within the extracellular domain were amplified, cloned, and sequenced. One set of primers, in addition to the expected 352-bp amplicon, revealed a 276-bp transcript, although its biological functions remain unknown. These two transcripts showed varying levels of expression in different tissues of infertile rats against nearly uniform expression in the fertile animals. Significantly, the 352 bp testis transcript showed mutational hotspots from nucleotide 84-266 in the infertile rats. Analysis of testis and brain genomic DNA from these infertile rats showed mutations only in the testis suggesting this to be a postzygotic event. In contrast, no mutation was detected in the genomic DNA of testis and brain of the fertile rats. Protein expression studies showed complete absence of the cytoplasmic kinase domain and soluble c-kit protein in one of the infertile rats. Histological examination of testis of these infertile animals showed stem cell depletion resulting in fewer germ cells. Based on these results, we infer that 352-bp mutant mRNA transcript is implicated in the spermatogenic failure.
多效原癌基因c-kit受体与造血和黑色素生成有关，虽然其在生殖细胞增殖和分化中发挥重要作用的机制尚不清楚，但也为人所知。我们通过逆转录聚合酶链反应（RT-PCR）研究了可育和不育的棕色挪威大鼠不同组织中的c-kit表达。使用不同的引物组，对细胞外结构域内的几个区域进行了扩增、克隆和测序。一组引物除了扩增出预期的352碱基对（bp）扩增子外，还发现了一个276-bp的转录本，尽管其生物学功能尚不清楚。这两种转录本在不育大鼠的不同组织中表达水平不同，而在可育动物中表达几乎一致。值得注意的是，352 bp的睾丸转录本在不育大鼠中从核苷酸84至266处显示出突变热点。对这些不育大鼠的睾丸和脑基因组DNA分析表明，仅在睾丸中发现突变，提示这是一个合子后事件。相比之下，在可育大鼠的睾丸和脑基因组DNA中未检测到突变。蛋白质表达研究表明，其中一只不育大鼠完全没有细胞质激酶结构域和可溶性c-kit蛋白。对这些不育动物睾丸的组织学检查显示干细胞耗竭，导致生殖细胞减少。基于这些结果，我们推断352-bp突变mRNA转录本与生精失败有关。
