Cullimore J V, Gebhardt C, Saarelainen R, Miflin B J, Idler K B, Barker R F
J Mol Appl Genet. 1984;2(6):589-99.
A recombinant plasmid (pcPvNGS-01) containing sequences related to glutamine synthetase (GS) has been identified from a cDNA library constructed from poly (A)+ RNA isolated from root nodules of Phaseolus vulgaris L. The identification of this recombinant relied on the observations that: (a) the clone hybridized strongly to purified GS mRNA; (b) in hybrid-select translation experiments, the clone selected mRNA that produced a polypeptide identical in molecular weight to purified GS subunits which was immunoprecipitated with anti-GS-antiserum; and (c) the translated nucleotide sequence of the cloned cDNA was homologous to a partial amino acid sequence of higher plant GS. The cloned cDNA hybridized to poly (A)+ RNA of different mobilities from leaves, roots, and nodules of P. vulgaris. In RNA "dot" blots washed at different stringencies, differences were observed both in the relative amounts of GS mRNA in different tissues and in the strength of their hybridization to the cDNA probe. The cloned probe hybridized to several fragments of restricted P. vulgaris DNA but not to DNA from Rhizobium phaseoli. These results suggest that GS is coded for by a small multigene family showing organ-specific expression.
从菜豆根瘤中分离的聚腺苷酸(poly(A)+)RNA构建的cDNA文库中,已鉴定出一种含有谷氨酰胺合成酶(GS)相关序列的重组质粒(pcPvNGS - 01)。该重组体的鉴定基于以下观察结果:(a)该克隆与纯化的GS mRNA强烈杂交;(b)在杂交选择翻译实验中,该克隆选择的mRNA产生的多肽分子量与用抗GS抗血清免疫沉淀的纯化GS亚基相同;(c)克隆的cDNA的翻译核苷酸序列与高等植物GS的部分氨基酸序列同源。克隆的cDNA与菜豆叶片、根和根瘤中不同迁移率的聚腺苷酸(poly(A)+)RNA杂交。在不同严谨度洗涤的RNA“点”杂交中,观察到不同组织中GS mRNA的相对含量及其与cDNA探针杂交强度的差异。克隆的探针与经酶切的菜豆DNA的几个片段杂交,但不与菜豆根瘤菌的DNA杂交。这些结果表明,GS由一个显示器官特异性表达的小多基因家族编码。