Winkfein R J, Nishikawa S, Connor W, Dixon G H
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Eur J Biochem. 1993 Jul 1;215(1):63-72. doi: 10.1111/j.1432-1033.1993.tb18007.x.
A synthetic oligonucleotide primer, designed from marsupial protamine protein-sequence data [Balhorn, R., Corzett, M., Matrimas, J. A., Cummins, J. & Faden, B. (1989) Analysis of protamines isolated from two marsupials, the ring-tailed wallaby and gray short-tailed opossum, J. Cell. Biol. 107] was used to amplify, via the polymerase chain reaction, protamine sequences from a North American opossum (Didelphis marsupialis) cDNA. Using the amplified sequences as probes, several protamine cDNA clones were isolated. The protein sequence, predicted from the cDNA sequences, consisted of 57 amino acids, contained a large number of arginine residues and exhibited the sequence ARYR at its amino terminus, which is conserved in avian and most eutherian mammal protamines. Like the true protamines of trout and chicken, the opossum protamine lacked cysteine residues, distinguishing it from placental mammalian protamine 1 (P1 or stable) protamines. Examination of the protamine gene, isolated by polymerase-chain-reaction amplification of genomic DNA, revealed the presence of an intron dividing the protamine-coding region, a common characteristic of all mammalian P1 genes. In addition, extensive sequence identity in the 5' and 3' flanking regions between mouse and opossum sequences classify the marsupial protamine as being closely related to placental mammal P1. Protamine transcripts, in both birds and mammals, are present in two size classes, differing by the length of their poly(A) tails (either short or long). Examination of opossum protamine transcripts by Northern hybridization revealed four distinct mRNA species in the total RNA fraction, two of which were enriched in the poly(A)-rich fraction. Northern-blot analysis, using an intron-specific probe, revealed the presence of intron sequences in two of the four protamine transcripts. If expressed, the corresponding protein from intron-containing transcripts would differ from spliced transcripts by length (49 versus 57 amino acids) and would contain a cysteine residue.
根据有袋动物鱼精蛋白蛋白质序列数据[巴尔霍恩，R.，科尔泽特，M.，马蒂马斯，J. A.，卡明斯，J. & 法登，B.（1989年）对从两种有袋动物环尾袋貂和灰短尾负鼠中分离的鱼精蛋白的分析，《细胞生物学杂志》107]设计的合成寡核苷酸引物，用于通过聚合酶链反应从北美负鼠（北美负鼠）cDNA中扩增鱼精蛋白序列。以扩增序列为探针，分离出几个鱼精蛋白cDNA克隆。从cDNA序列预测的蛋白质序列由57个氨基酸组成，含有大量精氨酸残基，并且在其氨基末端表现出ARYR序列，这在鸟类和大多数真兽类哺乳动物鱼精蛋白中是保守的。与鳟鱼和鸡的真正鱼精蛋白一样，负鼠鱼精蛋白缺乏半胱氨酸残基，这使其与胎盘哺乳动物鱼精蛋白1（P1或稳定型）鱼精蛋白不同。通过聚合酶链反应扩增基因组DNA分离出的鱼精蛋白基因的检查显示，存在一个将鱼精蛋白编码区分开的内含子，这是所有哺乳动物P1基因的共同特征。此外，小鼠和负鼠序列在5'和3'侧翼区域的广泛序列同一性将有袋动物鱼精蛋白归类为与胎盘哺乳动物P1密切相关。鸟类和哺乳动物中的鱼精蛋白转录本存在两种大小类别，它们的差异在于其多聚（A）尾巴的长度（短或长）。通过Northern杂交检查负鼠鱼精蛋白转录本，发现在总RNA组分中有四种不同的mRNA种类，其中两种在富含多聚（A）的组分中富集。使用内含子特异性探针的Northern印迹分析显示，在四种鱼精蛋白转录本中的两种中存在内含子序列。如果表达，来自含内含子转录本的相应蛋白质在长度上（49个与57个氨基酸）将与剪接转录本不同，并且将含有一个半胱氨酸残基。
