Physical map location of the argFGH operon of Escherichia coli.
Physical map location of the argFGH operon of Escherichia coli.
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大肠杆菌 argFGH 操纵子的物理图位置。
DOI：
10.1128/jb.174.11.3836-3837.1992
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发表时间：
1992
影响因子：
3.2
通讯作者：
Rudd,KE
中科院分区：
文献类型：
--
作者：
Hendrickson,W;Rudd,KE
araFGH操纵子编码高亲和力阿拉伯糖转运所需的蛋白质。该操纵子先前在大肠杆菌遗传图谱上定位于44.7 min（1，2）。Clark和Hogg利用P2分泌和P1共转导的方法证明了araF基因在大肠杆菌中位于his和mgl之间。coli B菌株（2）。随后，从能够补充araF缺陷的Clark-Carbon质粒中克隆araFGH操纵子，并对基因进行测序（4，14）。通过比较从DNA序列推导出的araF氨基酸序列与从纯化的阿拉伯糖结合蛋白获得的氨基酸序列，证实了克隆操纵子的同一性（14）。此外，克隆的DNA在基因组Southern印迹中与含有araFG MulacZ基因融合体的限制性片段杂交（4）。由于限制性片段大小与Kohara等人的44分钟区域图谱预测的大小不匹配，因此这些杂交实验无法确定物理图谱位置（6）。最近，一个计算机程序MapSearch（10，12，13）将DNA序列与E.大肠杆菌基因组限制性内切酶图谱，(6)开发了将长度为4，208 bp的araFGH DNA序列用于基因组限制性图谱的计算机搜索。在从遗传数据预测的44分钟区域中未发现显著匹配，但通过MapSearch发现的flhD和tyrP基因之间的最佳比对（P= 0.003）在41.9分钟（2000 kb）处（图1）。MapSearch预测了araFGH操纵子的逆时针方向，并预测araFGH将存在于Kohara小集17 F11和18 A9上，以及部分包含在噬菌体16 B12上。根据限制性内切酶图谱的相似性，该区域先前已被鉴定为araFGH的可能位置（3，7，9）。用araFGH转录起始点+1和-200 bp序列的两个寡核苷酸探针，对携带该区域的E. coli染色体进行斑点杂交。克隆8 F11、18 A9和6 B12显示与两种探针的阳性杂交。在物理图谱上（44至46分钟），10个碱基（小集克隆351至360）没有发现与2100至2200 kb染色体片段的杂交。由于这些克隆包括his至metG基因，数据表明araFGH的转录起始位点并不位于原始遗传数据所示的位置，而是实际上位于遗传图谱上的2001 kb和41.9 min处（图1）。
The araFGH operon codes for the proteins required for high-affinity arabinose transport. The operon was previously localized to 44.7 min on the Escherichia coli genetic map (1, 2). Using P2 eduction and P1 cotransduction, Clark and Hogg demonstrated that the araF gene lies between his and mgl in an E. coli B strain (2). Subsequently, the araFGH operon was cloned from a Clark-Carbon plasmid capable of complementing an araF defect, and the genes were se-quenced (4, 14). The identity of the cloned operon was confirmed by comparison of the araF amino acid sequence as deduced fromthe DNA sequence with the amino acid sequence obtained from the purified arabinose-binding pro-tein (14). In addition, the cloned DNA hybridized in genomic Southern blots to the restriction fragments containing araFG MulacZ gene fusions (4). The physical map position was not determined from these hybridization experiments because the restriction fragment sizes did not match those predicted by the map of Kohara et al. for the 44-min region (6). Recentlya computer program, MapSearch (10, 12, 13) that aligns DNA sequencesto a digital version of the E. coli genomic restriction map of Kohara et al.(6) has been developed. The araFGH DNA sequence, 4,208 bp in length, was used in a computer search of the genomic restriction map. No significant match was found in the 44-min region predicted from the genetic data, but the best alignment (P= 0.003) found by MapSearch was at 41.9 min (2000 kb), between the flhD and tyrP genes (Fig. 1). MapSearch pre-dicted a counterclockwise orientation of the araFGH operon and also predicted that araFGH would be presenton Kohara miniset phages 7F11 and 18A9, as well as partially contained on phage 16B12. This region has previously been identified as a possible location for araFGH on the basis of restriction map similarities (3, 7, 9). By using two oligonucleotide probes derived from the sequence of araFGH at+ 1 and-200 bp from the transcription start, several phages carrying this region of the E. coli chromosome were tested by dot hybridization of plaques. Clones 8F11, 18A9, and 6B12 showed positive hybridization to both probes. No hybridization was found for 10phages (miniset clones 351 to 360) with chromosomal segments spanning 2100 to 2200 kb on the physical map (44 to 46 min.). Since these clones include the his through metG genes, the data suggest that the transcription start site of araFGH is not located as indicated by the original genetic data, but is in fact located at 2001 kb and 41.9 min on the genetic map (Fig. 1).