Electrophoretic separation of the three Rhizobium meliloti replicons

Abstract

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.

A BlnI restriction map of the Salmonella typhimurium LT2 genome

“…A number of restriction maps of bacterial genomes have been constructed by using endonucleases that cleave infrequently (22) and then separating the resulting fragments by pulsed-field gel electrophoresis (PFGE) (2,5,7,8,10,17,19,20,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(36)(37)(38)(39). The physical order of these fragments has usually been determined by using Southern blotting with genetically mapped probes such as cloned genes (34) or transposons (38) or by hybridization of fragments from one restriction digest to fragments from a different digest (1,4).…”

Isolation and Characterization of Nuclei from Rice Embryos.

“…The rice nucleus is 3-5 jum in diameter, relatively smaller than that of other plants, which is likely due to the smaller genome size (6-9 x 108 bp) (24). It is also known that the rice nucleus cannot be stained with acetocarmine solution, as in the case of Arabidopsis thaliana Four ml of crude homogenates from dry rice embryos were layered on 12ml of 15-50% continuous Percoll gradient and centrifuged at 6,800rpm for 30min in a Beckman SW-27.1 rotor.…”

Low-resolution sequencing of Rhodobacter sphaeroides 2.A.1T: chromosome II is a true chromosome

“…(Lin et al, 1993), Borrelia (Ferdows & Barbour, 1989), and Agrobacterium (Allardet-Servent et al, 1993). Also some bacterial species of the genera Pseudomonas (Frantz & Chakrabarty, 1986), Rhizobium (Sobral et al, 1991), and Alcaligenes (Rodley et al, 1995) harbour very large accessory genetic elements (megaplasmids) which appear to encode ' housekeeping ' functions (Finan et al, 1986;Pardo et al, 1994) ; however their 'essentiality' has not been demonstrated. These findings raise the question : what, if anything, defines a bacterial chromosome?…”