Both G W, Air G M
Eur J Biochem. 1979 May 15;96(2):363-72. doi: 10.1111/j.1432-1033.1979.tb13048.x.
After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.
在对含有基质蛋白编码信息的流感病毒RNA片段进行体外聚腺苷酸化后,可使用引物p(dT)8-dA和逆转录酶制备cDNA拷贝。通过对正负链法[Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441 - 481]的改进[Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93 - 117]以及对用于DNA测序的“双脱氧”法[Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463 - 5467]的调整,确定了cDNA的166个核苷酸序列。该cDNA序列与基质蛋白的mRNA具有相同的意义,并且在26 - 28位含有一个潜在的起始密码子d(ATG)。当用胰凝乳蛋白酶或胰蛋白酶消化从病毒颗粒中纯化的基质蛋白,并测定分离肽段的氨基酸组成时,发现了一个含有九个氨基酸的肽段,其组成与第一个甲硫氨酸密码子之后的cDNA序列预测的组成相对应,证实蛋白质合成在该位置起始。其他四个肽段的组成与从核苷酸序列预测的组成相符。在蛋白质掺入病毒颗粒之前,除了去除N端甲硫氨酸并在所得的N端丝氨酸残基上添加一个“封闭”基团外,蛋白质的N端没有加工过程。