--Evgeny V. Kuzmin and Tatiana Yu. Scherban
S-plasmids are autonomous genetic elements found in mitochondria of cms-S maize. Their organization as linear DNA molecules with terminal inverted repeats and 5'-termini covalently attached to protein points to the similarity with viruses. This fact implies the existence of S-plasmid-encoded proteins that potentiate their replication and transcription in a specific viral-like mode. Indeed, analysis of published sequences of S-plasmids has allowed us to identify the putative protein products of two of the largest genes (ORF3 and ORF1) as a viral type DNA polymerase and a single-subunit RNA polymerase, respectively (Kuzmin, EV and Levchenko, NAR 15:6758; Kuzmin et al., NAR 16:4177).
We have constructed recombinant plasmids where both above-mentioned S-plasmid ORFs were placed under the control of bacterial tac-promoter and SD-sequence from vector pTTQ18/19. These procedures have resulted in the addition of 10 and 12 extra codons to the N-termini of ORF3 and ORF1, respectively. Expression of both S-plasmid-specific proteins in E. coli has proved to be poorly reproducible and extremely strain-specific, possibly because of deleterious effects of these proteins on E. coli cells. Finally we have succeeded in an optimization of ORF3 expression in E. coli resulting in reproducible synthesis of 105kDa protein visible after Coomassie staining of total E. coli protein extracts. The objective of our study was to demonstrate the preservation of native features and DNA polymerase activity of the 105kDa protein synthesized by E. coli.
E. coli cultures harbouring plasmid pQF3 (ORF3 derivative) and plasmid pQ5 (ORF1 derivative) were processed in parallel (the latter culture served as control) by the following procedure: the cells were lysed with lysozyme, sonicated, the extract was clarified by centrifugation; DNA-binding proteins were precipitated with streptomycin sulfate, solubilized and further precipitated by ammonia sulfate (50% sat.); then the preparation was brought to 0.3M NH4Cl and applied to Cibacron blue-agarose, the column was subsequently eluted with 0.6M and 1.5M NH4Cl, and DNA polymerase activity of corresponding fractions was determined.
Specific DNA polymerase activities of protein fractions eluted from
blue-agarose (dpm/mkg protein):
| E. coli CA161[pQ5] | E. coli CA161[pQF3] | |
| 0.3M NH4Cl | 3400 | 4500 |
| (flow through) | ||
| 0.6M NH4Cl | 500 | 15000 |
| 1.5M NH4Cl | 1300 | 24700 |
Electrophoretic analyses (not shown) have proved quantitative precipitation
of 105kDa protein by streptomycin, its absence in 0.3M eluate and maximal
presence in 1.5M eluate. It can be seen that E. coli cells harbouring
expressible ORF3 produce a novel DNA polymerase with considerably higher
affinity to Cibacron blue than DNA polymerase I of host cells. This is
characteristic of a-type DNA polymerases which ORF3 putative protein belongs
to according to its primary structure. Whether or not S-plasmid mRNAs are
edited in maize mitochondria remains an open question. Nevertheless absence
of this editing in the E. coli system apparently does not lead to
severe changes in the ORF3 protein sequence that abolish its DNA polymerase
activity. Further studies of enzymatic properties of ORF3 protein from
recombinant E. coli and from maize mitochondria are now in progress.
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