Renata Maas

An improved colony hybridization method with significantly increased sensitivity for detection of single genes.

By introducing a simple modification of existing methods, colony hybridization has been used to detect single copy genes coding for a heat-stable enterotoxin in wild-type strains isolated from patients with diarrhea. The modification described in this communication results in an approximately 100-fold increase in sensitivity, probably by increasing the total denatured plasmid DNA fixed to the paper.

Change of Plasmid DNA Structure, Hypermethylation, and Lon-Proteolysis as Steps in a Replicative Cascade

I have defined conditions under which RepFIC plasmid DNA can be maintained in a state of lowered helical density. In exponentially growing cultures, the DNA of lowered helical density is present in small amounts but never totally absent, suggesting that it is a normal variant of plasmid maintenance. It is fully methylated at frequent sites by dam-methyltransferase, some not previously recognized, further suggesting that the variant is a precursor of replication. The low-helical density plasmid is present in dam hosts, indicating that methylation is not essential for the change in helical density. The lowered helical density is stabilized in lon hosts, suggesting that Lon-protease may remove both the protein(s) that lower the helical density and the dam-methyltransferase after each round of replication.

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86

SummaryMany plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmids replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.

Crystallization and preliminary crystallographic analysis of RepA1, a replication control protein of the RepFIC replicon of enterotoxin plasmid EntP307

RepA1 protein is essential for replication of the RepFIC replicon of enterotoxin plasmid EntP307 and is thought to interact directly with the origin of replication. We have purified RepA1 from an over‐producing expression system and have prepared single crystals using a macroseeding technique. The crystals belong to space group P212121 or P21212, with cell dimensions a = 61 Å, b = 67 Å, and c = 243 Å. They diffract X‐rays to 3.3 Å resolution and probably contain two 40,000 molecular weight RepA1 molecules per asymmetric unit.

Prereplicative Purine Methylation and Postreplicative Demethylation in Each DNA Duplication of the Escherichia coli Replication Cycle

Escherichia coli plasmid DNA activated for initiation of duplication is in a stable low linking number supercoiled conformation. Low linking number DNA is methylated at the internal purines of a frequent 5′-Pyr-Pyr-Pur-Pur tetramer with a 5′-Pyr-Pur-3′ axis of symmetry and is cut at the axis of symmetry by pneumococcal restriction enzyme DpnI when methylated in both strands. Purine methylation is of adenine in one strand and guanine in the other. Methylation of one of the two purines is removed during the cell cycle, presumably before the reverse shift to the B-supercoiled conformation. The topological transition was reconstituted in vitro only with DNA unmethylated at purines. Methylation-restriction analyses coupled with the chemical properties of low-linking number DNA and B-DNA respectively, suggest that removal of guanine methylation is essential for the low-linking number to B-DNA transition and hence for the deactivation of replication. Demethylation of methylguanine could explain the presence in E. coli of the two-member inducible operon known as ada. Characteristics of ada suggest a cascade of chemical DNA modifications that reverse prereplicative guanine methylation. Guanine demethylation could provide a model for the pivotal role played by de novo methylation in replication and for the essential role of “repair” enzyme ExoIII in demethylation leading to the reversal of replicative DNA activation and other processes that affect DNA function.