Dombroski B A, Feng Q, Mathias S L, Sassaman D M, Scott A F, Kazazian H H, Boeke J D
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Mol Cell Biol. 1994 Jul;14(7):4485-92. doi: 10.1128/mcb.14.7.4485-4492.1994.
L1 elements constitute a highly repetitive human DNA family (50,000 to 100,000 copies) lacking long terminal repeats and ending in a poly(A) tail. Some L1 elements are capable of retrotransposition in the human genome (Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, Nature (London) 332:164-166, 1988). Although most are 5' truncated, a consensus sequence of complete L1 elements is 6 kb long and contains two open reading frames (ORFs) (Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, Genomics 1:113-125, 1987). The protein encoded by ORF2 has reverse transcriptase (RT) activity in vitro (Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, Science 254:1808-1810, 1991). Because L1 elements are so numerous, efficient methods for identifying active copies are required. We have developed a simple in vivo assay for the activity of L1 RT based on the system developed by Derr et al. (Derr, L. K., J. N. Strathern, and D. J. Garfinkel, Cell 67:355-364, 1991) for yeast HIS3 pseudogene formation. L1 ORF2 displays an in vivo RT activity similar to that of yeast Ty1 RT in this system and generates pseudogenes with unusual structures. Like the HIS3 pseudogenes whose formation depends on Ty1 RT, the HIS3 pseudogenes generated by L1 RT are joined to Ty1 sequences and often are part of complex arrays of Ty1 elements, multiple HIS3 pseudogenes, and hybrid Ty1/L1 elements. These pseudogenes differ from those previously described in that there are base pairs of unknown origin inserted at several of the junctions. In two of three HIS3 pseudogenes studied, the L1 RT appears to have jumped from the 5' end of a Ty1/L1 transcript to the poly(A) tract of the HIS3 RNA.
L1元件构成了一个高度重复的人类DNA家族（50000至100000个拷贝），缺乏长末端重复序列，以聚腺苷酸尾结束。一些L1元件能够在人类基因组中进行逆转录转座（Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, 《自然》（伦敦）332:164 - 166, 1988）。尽管大多数L1元件在5'端被截短，但完整L1元件的共有序列长度为6 kb，包含两个开放阅读框（ORF）（Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, 《基因组学》1:113 - 125, 1987）。由ORF2编码的蛋白质在体外具有逆转录酶（RT）活性（Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, 《科学》254:1808 - 1810, 1991）。由于L1元件数量众多，需要高效的方法来鉴定活跃拷贝。我们基于Derr等人（Derr, L. K., J. N. Strathern, and D. J. Garfinkel, 《细胞》, 67:355 - 364, 1991）开发的用于酵母HIS3假基因形成的系统，开发了一种简单的体内检测L1 RT活性的方法。在这个系统中，L1 ORF2表现出与酵母Ty1 RT相似的体内RT活性，并产生具有异常结构的假基因。与依赖Ty1 RT形成的HIS3假基因一样，由L1 RT产生的HIS3假基因与Ty1序列相连，并且常常是Ty1元件、多个HIS3假基因和Ty1/L1杂交元件的复杂阵列的一部分。这些假基因与先前描述的假基因不同之处在于，在几个连接处插入了来源不明的碱基对。在研究的三个HIS3假基因中的两个中，L1 RT似乎从Ty1/L1转录本的5'端跳到了HIS3 RNA的聚腺苷酸区域。
