Davidson J M, Shibahara S, Schafer M P, Harrison M, Leach C, Tolstoshev P, Crystal R G
Biochem J. 1984 Jun 15;220(3):643-52. doi: 10.1042/bj2200643.
A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabelled mRNAE by hydridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.
用部分纯化的、经32P标记的弹性蛋白mRNA（mRNAE）筛选了一个以噬菌体载体Charon 4A构建的包含绵羊DNA的绵羊基因组文库，以寻找弹性蛋白基因序列。通过二次和三次筛选，从几个阳性克隆中挑选出一个含有9.9千碱基（kb）插入片段的重组体，用于进一步鉴定。通过杂交选择翻译和对SE1 DNA的限制性酶切片段进行Southern印迹分析，用放射性标记的mRNAE对这个名为SE1的弹性蛋白克隆进行了阳性鉴定。mRNAE或弹性蛋白互补DNA与SE1的限制性片段杂交表明，SE1的这些片段大多含有弹性蛋白编码序列。通过短的互补弹性蛋白DNA与Charon 4A右臂相邻的限制性片段的优先杂交确定了插入片段的方向。在Southern印迹上，经切口平移的SE1与绵羊基因组DNA的相互杂交表明，SE1的两个限制性片段含有在绵羊基因组DNA的限制性内切酶-EcoR1消化物中高频重复的序列元件。在随附的论文中[戴维森、柴原、博伊德、梅森、托尔斯托舍夫和克里斯特尔（1984年）《生物化学杂志》220，653 - 663]，表明这个弹性蛋白基因的一个亚克隆片段与绵羊组织RNA中的mRNAE序列进行了定量和特异性杂交。SE1 - mRNAE杂交体的电子显微镜观察表明至少存在七个大的R环。对这些结构的测量表明，SE1可能含有少于2 kb的编码序列和超过8 kb的间隔序列，外显子平均大小为120个碱基对。因此，弹性蛋白基因分布在绵羊基因组的一个扩展区域，包含许多间隔序列和编码序列。
