Rothkopf G S, Telakowski-Hopkins C A, Stotish R L, Pickett C B
Biochemistry. 1986 Mar 11;25(5):993-1002. doi: 10.1021/bi00353a007.
Southern blot analysis of rat genomic DNA using glutathione S-transferase Ya and Yc cDNA probes was employed to estimate the size of the Ya/Yc multigene family. A minimum of five to seven Ya/Yc genes were detected; at least two of these are Yc genes. The presence of multiple genes was further supported by the isolation of three nonoverlapping genomic clones from a rat EcoRI library that hybridized to a Ya cDNA clone, pGTB38. However, not all EcoRI bands seen in genomic blots were represented in the clones, suggesting that not all Ya/Yc genes have been isolated. The organization of a Ya gene in one of these EcoRI genomic clones, lambda GTB38-3, and an overlapping clone, lambda GTB45-1, isolated from a HaeIII library, was investigated with 5' and 3' probes prepared from Ya and Yc cDNA clones. Restriction endonuclease mapping and hybridization studies revealed that the gene spans over 10 kilobases and contains at least three introns. Sequences upstream from the 5' untranslated region of the gene, and within an intron in the 5' coding region, were found to contain sequences homologous to a type 2 Alu repetitive element from the rat growth hormone gene [Page, G.S., Smith, S., & Goodman, H.M. (1981) Nucleic Acids Res. 9, 2087-2104]. The repetitive sequences in lambda GTB38-3 were identified by hybridization to a novel Ya cDNA clone, pGTB45. This cDNA clone was isolated from a cDNA library previously described [Telakowski-Hopkins, C.A., Rodkey, J.A., Bennett, C.D., Lu, A.Y.H., & Pickett, C.B. (1985) J. Biol. Chem. 260, 5820-5825] with nick-translated intron sequences as probes. pGTB45 is virtually identical with pGTR261 [Tu, C.-P.D., Lai, H.-C.J., Li, N.-Q., Weiss, M.J., & Reddy, C.C. (1984) J. Biol. Chem. 259, 9434-9439], except that the 3' untranslated region extends 231 base pairs beyond the polyadenylation signal of pGTR261. This elongated 3' untranslated sequence is unique in that it contains a full-length type 2 Alu repetitive element, which includes two additional, overlapping polyadenylation signals.
利用谷胱甘肽S - 转移酶Ya和Yc cDNA探针，对大鼠基因组DNA进行Southern印迹分析，以估算Ya/Yc多基因家族的大小。检测到至少五到七个Ya/Yc基因；其中至少两个是Yc基因。从大鼠EcoRI文库中分离出三个与Ya cDNA克隆pGTB38杂交的非重叠基因组克隆，进一步证明了多个基因的存在。然而，基因组印迹中看到的并非所有EcoRI条带都在这些克隆中出现，这表明并非所有的Ya/Yc基因都已被分离出来。利用从Ya和Yc cDNA克隆制备的5'和3'探针，研究了其中一个EcoRI基因组克隆lambda GTB38 - 3以及从HaeIII文库中分离出的重叠克隆lambda GTB45 - 1中一个Ya基因的结构。限制性内切酶图谱分析和杂交研究表明，该基因跨度超过10千碱基，至少包含三个内含子。在该基因5'非翻译区上游以及5'编码区内一个内含子中的序列，被发现含有与大鼠生长激素基因的2型Alu重复元件同源的序列[佩奇，G.S.，史密斯，S.，& 古德曼，H.M.（1981年）《核酸研究》9，2087 - 2104]。通过与一个新的Ya cDNA克隆pGTB45杂交，鉴定出lambda GTB38 - 3中的重复序列。该cDNA克隆是从先前描述的cDNA文库中分离得到的[特拉科夫斯基 - 霍普金斯，C.A.，罗德基，J.A.，贝内特，C.D.，卢，A.Y.H.，& 皮克特，C.B.（1985年）《生物化学杂志》260，5820 - 5825]，以切口平移的内含子序列作为探针。pGTB45与pGTR261[图，C.-P.D.，赖，H.-C.J.，李，N.-Q.，魏斯，M.J.，& 雷迪，C.C.（1984年）《生物化学杂志》259，9434 - 9439]几乎相同，只是其3'非翻译区在pGTR261的聚腺苷酸化信号之外延伸了231个碱基对。这个延长的3'非翻译序列的独特之处在于它包含一个全长的2型Alu重复元件，其中包括另外两个重叠的聚腺苷酸化信号。
