Haugland R A, Cantrell M A, Beaty J S, Hanus F J, Russell S A, Evans H J
J Bacteriol. 1984 Sep;159(3):1006-12. doi: 10.1128/jb.159.3.1006-1012.1984.
Recombinant cosmids from a gene library of the DNA from Hup+ Rhizobium japonicum 122DES previously have been shown to restore hydrogenase activity when transferred by conjugation into certain Hup- mutants of R. japonicum. We generated a restriction map covering 32.2 kilobases of this cosmid DNA. At least 25.3 kilobases of the cosmid pHU1 were shown to have the same arrangement as those in the genome of strain 122DES. Analysis of Tn5 insertions into the 122DES genome indicates that hup-specific sequences occur in a region spanning about 15 kilobases of insert DNA within pHU1. Introduction of pHU1 into five out of six R. japonicum Hup- mutants resulted in a Hup+ phenotype in some transconjugants. Three of the mutations appear to be in transcriptional units completely contained within pHU1, whereas the other two must be in genes that are at least partially contained within pHU1. pBR235 derivatives containing fragments of hup DNA can be transferred into the R. japonicum Hup- mutant PJ18nal if the derivatives contain a region of homology with the R. japonicum genome. The hup mutation in strain PJ18nal appears to be dominant. The hup genes in R. japonicum strain 122DES appear to be organized in at least two, and probably three, transcriptional units.
先前已证明，来自Hup⁺日本根瘤菌122DES DNA基因文库的重组黏粒通过接合转移到某些日本根瘤菌Hup⁻突变体中时，能够恢复氢化酶活性。我们构建了一个覆盖该黏粒DNA 32.2千碱基的限制酶切图谱。结果表明，黏粒pHU1中至少25.3千碱基的排列与122DES菌株基因组中的排列相同。对122DES基因组中Tn5插入片段的分析表明，hup特异性序列出现在pHU1内插入DNA约15千碱基的区域。将pHU1导入六个日本根瘤菌Hup⁻突变体中的五个，在一些接合子中产生了Hup⁺表型。其中三个突变似乎位于完全包含在pHU1内的转录单元中，而另外两个突变必定位于至少部分包含在pHU1内的基因中。如果含有hup DNA片段的pBR235衍生物包含与日本根瘤菌基因组的同源区域，则可以将其转移到日本根瘤菌Hup⁻突变体PJ18nal中。PJ18nal菌株中的hup突变似乎是显性的。日本根瘤菌122DES菌株中的hup基因似乎至少由两个，可能由三个转录单元组成。
