2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: Mismatches function as inducing signals?

Summary The EcoK restriction of unmodified phage is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU). 2AP treatment of bacteria affects specificially the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction. 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam ^- strains. We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Characterization, sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

 The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites. Received: 25 April 1996 / Accepted: 20 June 1996     

A motif conserved among the type I restriction-modification enzymes and antirestriction proteins: a possible basis for mechanism of action of plasmid-encoded antirestriction functions.

Antirestriction proteins Ard encoded by some self-transmissible plasmids specifically inhibit restriction by members of all three families of type I restriction-modification (R-M) systems in E.coli. Recently, we have identified the amino acid region, 'antirestriction' domain, that is conserved within different plasmid and phage T7-encoded antirestriction proteins and may be involved in interaction with the type I R-M systems. In this paper we demonstrate that this amino acid sequence shares considerable similarity with a well-known conserved sequence (the Argos repeat) found in the DNA sequence specificity (S) polypeptides of type I systems. We suggest that the presence of these similar motifs in restriction and antirestriction proteins may give a structural basis for their interaction and that the antirestriction action of Ard proteins may be a result of the competition between the 'antirestriction' domains of Ard proteins and the similar conserved domains of the S subunits that are believed to play a role in the subunit assembly of type I R-M systems.     

Characterization,sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites.