Institute of Genetic Engineering, Hankyong National University, Ansung 456-749, Korea.
Reprod Biol Endocrinol. 2013 Feb 12;11:8. doi: 10.1186/1477-7827-11-8.
The members of the microtubule-associated protein 1 light chain (MAP1LC) family, especially those of the LC3 family (MAP1LC3A, B, C), are known to induce autophagy upon localization onto the autophagosomal membrane. In this regard, LC3 can be utilized as a marker for the formation of autophagosomes during the process of autophagy. The aims of this study are to clone porcine MAP1LC3A, and analyze the pattern of its expression in the ovarian tissues of normal and miniature pig ovary in an attempt to understand the distinct mode of apoptosis between two strains.
Rapid amplification of cDNA ends (RACE) were used to obtain the 5' and 3' ends of the porcine MAP1LC3A full length cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, and western blot analysis were performed to examine the expression of porcine MAP1LC3A. The localization of MAP1LC3A in the ovary was determined by In situ Hybridization and Immunohistochemical staining.
We cloned the full-length cDNA of porcine MAP1LC3A and identified an open reading frame of 980 bp encoding 121 amino acids. Based on its homology to known mammalian proteins (98%) this novel cDNA was designated as porcine MAP1LC3A and registered to the GenBank (Accession No. GU272221). We compared the expression of MAP1LC3A in the Graafian follicles of normal and miniature pigs by in situ hybridization at day 15 of the estrus cycle. While normal pigs showed a stronger expression of MAP1LC3A mRNA than miniature pigs in the theca cell area, the expression was lower in the granulosa cells. Immunofluorescence analysis of the MAP1LC3A fusion reporter protein showed the subcellular localization of porcine MAP1LC3A and ATG5 as a punctate pattern in the cytoplasm of porcine granulosa cells under stress conditions. In addition, the expressions of MAP1LC3A and ATG5 were higher in normal pigs than in miniature pigs both in the presence and absence of rapamycin.
The newly cloned porcine MAP1LC3A provides a novel autophagosomal marker in both normal and miniature pig. We demonstrated that the expression of MAP1LC3A in graafian follicle is distinct in normal and miniature pig, which may explain the unique folliculogenesis of miniature pigs.
微管相关蛋白 1 轻链（MAP1LC）家族的成员，尤其是 LC3 家族（MAP1LC3A、B、C）的成员，在定位于自噬体膜上时会诱导自噬。在这方面，LC3 可以作为自噬过程中自噬体形成的标记物。本研究的目的是克隆猪 MAP1LC3A，并分析其在正常和小型猪卵巢组织中的表达模式，试图了解两种品系之间凋亡的独特模式。
采用快速扩增 cDNA 末端（RACE）技术获得猪 MAP1LC3A 全长 cDNA 的 5'和 3'端。采用逆转录-PCR（RT-PCR）、实时 PCR 和 Western blot 分析检测猪 MAP1LC3A 的表达。通过原位杂交和免疫组织化学染色检测 MAP1LC3A 在卵巢中的定位。
我们克隆了猪 MAP1LC3A 的全长 cDNA，并鉴定出一个开放阅读框，编码 121 个氨基酸。根据其与已知哺乳动物蛋白（98%）的同源性，该新 cDNA 被命名为猪 MAP1LC3A，并注册到 GenBank（注册号 GU272221）。我们通过在发情周期第 15 天进行原位杂交比较了正常和小型猪的格拉夫氏卵泡中 MAP1LC3A 的表达。正常猪的卵泡膜细胞区 MAP1LC3A mRNA 的表达强于小型猪，而颗粒细胞区的表达较低。应激条件下猪 MAP1LC3A 和 ATG5 融合报告蛋白的免疫荧光分析显示，猪颗粒细胞中 MAP1LC3A 的亚细胞定位呈点状模式。此外，在存在和不存在雷帕霉素的情况下，正常猪的 MAP1LC3A 和 ATG5 的表达均高于小型猪。
新克隆的猪 MAP1LC3A 为正常和小型猪提供了一种新的自噬体标记物。我们证明了 MAP1LC3A 在格拉夫氏卵泡中的表达在正常和小型猪中是不同的，这可能解释了小型猪独特的卵泡发生。
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