Sobral B W, Honeycutt R J, Atherly A G, McClelland M
California Institute of Biological Research, La Jolla 92037.
J Bacteriol. 1991 Aug;173(16):5173-80. doi: 10.1128/jb.173.16.5173-5180.1991.
The megaplasmids and the chromosome from the bacterium Rhizobium meliloti 1021 were separated in preparative quantities by using transverse alternating-field gel electrophoresis. The genetic content of each electrophoretically separated band was determined by Southern hybridization with replicon-specific probes and by comparison with Agrobacterium tumefaciens transconjugants harboring either pSym-a or pSym-b megaplasmids. Pulsed-field gel electrophoresis analyses of PacI (5'-TTAATTAA-3') and SwaI (5'-ATTTAAAT-3') digests of the whole genome and of the separated replicons were used to calculate genome sizes in two R. meliloti strains. In these strains, PacI digestion yielded only four fragments for the entire genome. The sizes of the PacI fragments from R. meliloti 1021 in megabase pairs (Mb) were 3.32 +/- 0.30, 1.42 +/- 0.13, 1.21 +/- 0.10, and 0.55 +/- 0.08, for a total genome size of 6.50 +/- 0.61 Mb. Southern hybridization with replicon-specific probes assigned one PacI fragment to the chromosome of R. meliloti 1021, one to pRme1021a, and two to pRme1021b. PacI digestion of A. tumefaciens pTi-cured, pSym transconjugants confirmed these assignments. In agreement with PacI data, the addition of the six SwaI fragments from R. meliloti 1021 gave a genome size of 6.54 +/- 0.43 Mb. pRme1021a was calculated to be 1.42 +/- 0.13 Mb, 1.34 +/- 0.09 Mb, and 1.38 +/- 0.12 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021a, respectively. pRme1021b was calculated to be 1.76 +/- 0.18 Mb, 1.65 +/- 0.10 Mb, and 1.74 +/- 0.13 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021B, respectively. The R. meliloti 1021 chromosome was calculated to be 3.32 +/- 0.30 Mb, 3.55 +/- 0.24 Mb, and 3.26 +/- 0.46 Mb on the basis of PacI data, SwaI data, and the migration of uncut chromosome, respectively.
利用横向交变电场凝胶电泳从苜蓿根瘤菌1021中分离出大量的大质粒和染色体。通过与复制子特异性探针进行Southern杂交，并与携带pSym-a或pSym-b大质粒的根癌土壤杆菌转接合子进行比较，确定了每个电泳分离条带的遗传内容。对整个基因组和分离的复制子进行PacI（5'-TTAATTAA-3'）和SwaI（5'-ATTTAAAT-3'）酶切后的脉冲场凝胶电泳分析，用于计算两种苜蓿根瘤菌菌株的基因组大小。在这些菌株中，PacI酶切整个基因组仅产生四个片段。苜蓿根瘤菌1021的PacI片段大小以百万碱基对（Mb）计分别为3.32±0.30、1.42±0.13、1.21±0.10和0.55±0.08，基因组总大小为6.50±0.61 Mb。用复制子特异性探针进行Southern杂交，将一个PacI片段定位到苜蓿根瘤菌1021的染色体上，一个定位到pRme1021a上，两个定位到pRme1021b上。对根癌土壤杆菌pTi缺失的pSym转接合子进行PacI酶切证实了这些定位。与PacI数据一致，苜蓿根瘤菌1021的六个SwaI片段相加得到的基因组大小为6.54±0.43 Mb。根据PacI酶切、SwaI酶切以及未切割的pRme1021a的迁移情况，计算出pRme1021a的大小分别为1.42±0.13 Mb、1.34±0.09 Mb和1.38±0.12 Mb。根据PacI酶切、SwaI酶切以及未切割的pRme1021B的迁移情况，计算出pRme1021b的大小分别为1.76±0.18 Mb、1.65±0.10 Mb和1.74±0.13 Mb。根据PacI数据、SwaI数据以及未切割染色体的迁移情况，计算出苜蓿根瘤菌1021染色体的大小分别为3.32±0.30 Mb、3.55±0.24 Mb和3.26±0.46 Mb。
