THE BIALAPHOS BIOSYNTHETIC GENES OF STREPTOMYCES-VIRIDOCHROMOGENES - CLONING, HETEROSPECIFIC EXPRESSION, AND COMPARISON WITH THE GENES OF STREPTOMYCES-HYGROSCOPICUS
THE BIALAPHOS BIOSYNTHETIC GENES OF STREPTOMYCES-VIRIDOCHROMOGENES - CLONING, HETEROSPECIFIC EXPRESSION, AND COMPARISON WITH THE GENES OF STREPTOMYCES-HYGROSCOPICUS
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DOI：
10.1099/00221287-137-2-351
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发表时间：
1991-02-01
期刊：
影响因子：
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通讯作者：
THOMPSON, C
中科院分区：
文献类型：
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作者：
HARA, O;MURAKAMI, T;THOMPSON, C
双丙氨磷抗性基因 bar 被用作选择标记，以从绿色产色链霉菌基因组中分离双丙氨磷生产基因 (bap)。 使用 pIJ680 和 pIJ702 在双丙氨磷敏感宿主变青链球菌中将绿色产色链球菌 bar 基因克隆到重叠限制性片段上。 尽管这些片段的限制性内切核酸酶切割图与吸水链球菌的 bap 簇并不相似，但通过使用一系列先前表征的不产生双丙氨膦的吸水链球菌突变体的异源克隆实验，证明了 bar 和其他四个 bap 基因以及该簇转录激活所需的基因 (brpA) 的存在和位置。 由于尚未分离出链霉菌的重组缺陷突变体，因此由克隆的同源 DNA 提供的功能恢复是重组（标记拯救）和反式互补的结果。 与我们之前报道的同源克隆实验相反，在同源克隆实验中，我们能够通过重组来定义突变等位基因的位置，在这些异源克隆实验中，我们观察到质粒克隆基因和染色体之间几乎没有重组。 因此，这种方法使我们能够使用遗传互补测试来定义功能基因的位置和方向。 簇状的绿色产色链球菌 bap 基因的组织与相应的吸水链球菌突变体等位基因没有区别。 绿色产色链球菌转录调控基因 brpA 在吸水链球菌中发挥作用的事实表明，一些转录调控信号也可能是可互换的。 在这两种具有相当大核苷酸序列差异的链霉菌属物种中，双丙氨磷生物合成途径的复杂生化和遗传组织是保守的。
The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.