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A method for isolation of plasmid DNA replication intermediates from unsynchronized bacterial cultures for electron microscopy analysis

100%

Srutkowska S., Konopa G., Wegrzyn G.

Acta Biochimica Polonica

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1998

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tom 45

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nr 1

Electron microscopy is a powerful technique for analysis of DNA replication intermediates. However, isolation of replicating DNA molecules from living cells is tricky and difficult, especially in the case of small DNA molecules (such as bacterial plasmids) whose initiation of replication is not easily synchronized. Here a relatively simple and rapid method for efficient isolation of replicating plasmid molecules from unsynchronized Escherichia coli cultures is described. The efficiency of this procedure is high enough for electron microscopy analysis of plasmid replication intermediates appearing in living cells in normal growth conditions. Under optimal conditions, using standard procedures of isolation of plasmid DNA, it is possible to achieve a content of only as few as 0.02 percent of replication intermediates in a plasmid DNA sample. The described method allowed us to enrich up to 100-fold the fraction of replication intermediates suitable for microscopic analysis among all plasmid molecules.

2

Biochemical and genetic analysis of lambda W, the newly isolated lambdoid phage

100%

Wrobel B., Srutkowska S., Wegrzyn G.

Acta Biochimica Polonica

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1998

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tom 45

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nr 1

Otherwise isogenic Escherichia coli CP78 (relA+) and CP79 (relA-) strains are commonly used in studies on the stringent control, the bacterial response to amino acid starvation. We found that these strains are lysogenic for a phage which is spontaneously induced with a low frequency, producing virions able to infect other E. coli strains. Genetic studies, restriction analysis of the phage DNA genome, and electron microscopy revealed that this phage is very similar to, but not identical with, bacteriophage λ. We called the newly isolated phage λW, and found that most of CP78/CP79 ancestor strains are lysogenic for this phage.

3

Escherichia coli initiator protein DnaA, cell-cycle and control of lambda plasmid replication

64%

Taylor K., Wegrzyn G., Wegrzyn A., Szalewska-Palasz A., Herman A., Obuchowski M., Srutkowska S., Konopa G.

Bulletin of the Polish Academy of Sciences. Biological Sciences

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1996

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tom 44

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nr 3-4

Ograniczanie wyników

2 Acta Biochimica Polonica

1 Bulletin of the Polish Academy of Sciences. Biological Sciences

3 Srutkowska S.

3 Wegrzyn G.

2 Konopa G.

1 Herman A.

1 Obuchowski M.

1 Szalewska-Palasz A.

1 Taylor K.

1 Wegrzyn A.

1 Wrobel B.

2 1998

1 1996

A method for isolation of plasmid DNA replication intermediates from unsynchronized bacterial cultures for electron microscopy analysis

Biochemical and genetic analysis of lambda W, the newly isolated lambdoid phage

Escherichia coli initiator protein DnaA, cell-cycle and control of lambda plasmid replication