Krieg P, Strachan R, Wallis E, Tabe L, Colman A
J Mol Biol. 1984 Dec 15;180(3):615-43. doi: 10.1016/0022-2836(84)90030-5.
Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant "shuttle" vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known "cap" site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 "promoter". No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.
将编码鸡卵清蛋白、鸡前溶菌酶以及小牛前凝乳酶原、凝乳酶原和凝乳酶的互补DNA克隆片段，插入到经修饰的重组“穿梭”载体中各种病毒启动子的下游。将卵清蛋白、前溶菌酶和前凝乳酶原构建体显微注射到非洲爪蟾卵母细胞核中，分别导致卵清蛋白、溶菌酶和凝乳酶原的合成、在膜中的分隔以及分泌到细胞外培养基中。从分子量估计判断，溶菌酶和凝乳酶原经蛋白水解正确加工，而缺乏可裂解信号序列的卵清蛋白则发生了糖基化。注射不含信号序列的凝乳酶原DNA构建体，导致凝乳酶原蛋白的合成，该蛋白仅定位于卵母细胞胞质中。注射编码成熟凝乳酶的DNA后，未检测到免疫特异性蛋白。就卵母细胞中的蛋白表达而言，单纯疱疹病毒胸苷激酶（TK）启动子的效率比猴病毒40（SV40）早期启动子高7倍，与莫洛尼鼠肉瘤病毒长末端重复元件的效率相当。在进行测试时，如果构建体中存在编码SV40小t内含子及其侧翼序列的DNA序列，卵母细胞中的蛋白表达会大幅降低。对注射含TK启动子的DNA后产生的转录本进行S1核酸酶图谱分析表明，大多数转录本在已知“帽”位点的两个碱基处或其内部起始。然而，观察到有少量转录本在此位点上游起始，其中一个（或多个）转录本负责合成一种含51个氨基酸N端延伸的卵清蛋白多肽。这种延伸蛋白保留在卵母细胞胞质溶胶中。当将卵清蛋白cDNA以与病毒启动子相反的极性插入载体时，卵母细胞中的表达导致主要合成并分泌一种具有21个氨基酸N端延伸的变异卵清蛋白，尽管也合成并分泌了一些全长卵清蛋白。S1图谱分析显示，在这些卵母细胞中，存在预测极性的转录本，其在野生型卵清蛋白起始ATG上游118个碱基处起始，位于一个先前未报道的SV40“启动子”处。将这些反向定向构建体注射到幼仓鼠肾（BHK - 21）细胞后，未检测到蛋白合成。
