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R Eichenlaub

Publications and source records attributed to R Eichenlaub.

33 records · Page 2Linked to original sources
PubMed1986

The repeated sequences (incB) preceding the protein E gene of plasmid mini-F are essential for replication.

At the XhoI site (45.08F) of plasmid mini-F a deletion of 649 bp was generated employing exonuclease Bal31. By this deletion nucleotide sequences functioning as origin II and the four 19 bp direct repeats constituting the incB region in front of the E protein gene were removed from the plasmid. Analysis of proteins radioactively labelled in Escherichia coli mini-cells indicated that all mini-F encoded proteins are expressed. However, the plasmid carrying the deletion was not capable of replicating from the primary origin (origin I, 42.6F). Recently a smaller deletion at the XhoI site (45.08F) of about 300 bp, removing only the region functioning as origin II and replicating from origin I, was described by Tanimoto and Iino (1984, 1985). The data presented suggest that the incB repeats are essential for the initiation of replication from origin I, and possibly also from origin II, and seem not to be engaged in the autoregulation of E protein expression.

Bacterial Proteins↗
PubMed1985

Mutations affecting replication and copy number control in plasmid mini-F both reside in the gene for the 29-kDa protein.

We have isolated and characterized cop, copts, and repam mutants of plasmid mini-F after in vitro mutagenesis with hydroxylamine. cop mutants exhibit a copy number of about 10 per cell. The copts mutants are cold-sensitive and have, at 25 degrees C, a copy number of about 30-40 copies per cell, which drops to 4 copies at 42 degrees C. The cop and repam mutations affect the 29-kDa E protein. The Copts phenotype results from the simultaneous occurrence of two mutations, a cop mutation in the E protein and a temperature-dependent mutation (termed ecp) enhancing the Cop phenotype at low temperature. The latter new type of mutation is located within the DNA region 44.1-44.85F. Complementation experiments with plasmid cointegrates show that the wild-type gene is dominant over the cop allele. The nucleotide sequences of the cop and the repam mutations have been determined.

Base Sequence↗
PubMed1984

Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state.

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.

Chromosome Mapping↗
PubMed1981

Complementation of replication-deficient deletion derivatives of plasmid mini-F.

Deleted mini-F plasmids with defects in replication were constructed and tested to see whether they could be rescued through complementation by a helper plasmid. This allowed us to identify two genetic loci determining trans-acting functions required for stable maintenance of plasmid mini-F, one encoded by the PstI fragment from 45.7 to 47.3 F-coordinates (F) and the other most probably located in the region from 43.1 to 43.8 F. The smallest mini-F plasmid that could be established through complementation consists of the PstI fragment 44.0 to 45.7 F, encoding origin II and the incB locus.

DNA Replication↗
PubMed1980

Plasmid mini-F encoded proteins.

Proteins specified by the mini-F plasmid (EcoRI restriction fragment f5) were labeled in Escherichia coli minicells and analyzed by SDS-PAGE. Four mini-F encoded proteins could be identified, having molecular weights of 44,000 (A), 36,000 (B), 34,000 (C), and 25,300 (D) daltons. The absence of certain proteins in deleted derivatives of mini-F, generated by treatment with various restriction endonucleases, allowed mapping of the proteins. The A protein maps between F-coordinates 45.7 and 47.9 kb. The gene locus for the B protein is located between 47.2 and 49.3 kb. The C protein maps on a BamHI fragment bordered by F-coordinates 41.5 and 42.8 kb, and finally the D protein maps between 42.8 and 43.8 kb. In addition our data confirm that there are two incompatibility loci on the mini-F genome, located between 45.7 and 47.2 kb (incA) and 44.0 and 45.7 kb (incB). We suggest that (i) the C and D proteins are positive control elements, interacting with origin I and origin II, respectively, (ii) that the incB locus is involved in plasmid partitioning, and (iii) that the A protein encoded by the incA locus is a negative control element.

Bacterial Proteins↗
PubMed1979

The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis.

The nucleotide sequence recognized and cleaved by the restriction endonuclease MboI is 5' GATC and is identical to the central tetranucleotide of the restriction sites of BamHI and BglII. Experiments on the restriction of DNA from Escherichia coli dam and dam+ confirm the notion that GATC sequences are adenosyl-methylated by the dam function of E. coli and thereby are made refractory to cleavage by MboI. On the basis of this observation the degree of dam methylation of various DNAs was examined by cleavage with MboI and other restriction endonucleases. In plasmid DNA essentially all of the GATC sequences are methylated by the dam function. The DNA of phage lambda is only partially methylated, extended methylation is observed in the DNA of a substitution mutant of lambda, lambda gal8bio256, and in the lambda derived plasmid, lambdadv93, which is completely methylated. In contrast, phage T7 DNA is not methylated by dam. A suppression of dam methylation of T7 DNA appears to act only in cis dam. A suppression of dam methylation of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely methylated. The results are discussed with respect to the participation of the dam methylase in different replication systems.

Base Sequence↗
PubMed1979

Bidirectional replication of the mini-ColE1 plasmid pVH51.

Replicating molecules of the min-ColE1 plasmid pVH51 have been examined by electron microscopy after cleavage with the restriction endonuclease EcoRI. Replication apparently starts at a unique site indistinguishable from the origin of replication used by the parental plasmid ColE1. In contrast to ColE1, the structure of the majority of the replication intermediates was consistent with a bidirectional mode of replication. A minor portion of the molecules appeared to replicate unidirectionally in either direction from the same origin.

DNA Replication↗
PubMed1979

Mutants of the mini-F plasmid pML31 thermosensitive in replication.

Hydroxylamine mutagenesis was used for the induction of thermosensitive replication mutants of the mini-F plasmid pML31. Replication mutants were characterized by studying the segregation kinetics and the incorporation of [3H]-thymidine into plasmid deoxyribonucleic acid at the nonpermissive temperature. Based on these experiments two types of mutants could be distinguished. Mutants of type I are fast segregating with the kinetics expected if plasmid replication was blocked immediately. Double-label experiments showed a rapid shut-off of replication in these mutants at 42 degrees C. Mutants of type II segregate slower, showing only a partial inhibition of plasmid deoxyribonucleic acid synthesis at the nonpermissive temperature. The label incorporated at 42 degrees C was predominantly found in open circular plasmid molecules.

DNA Replication↗
PubMed1977

Bidirection replication from a unique origin in a mini-F plasmid.

Replicating molecules of the mini F-kanamycin resistance plasmid, pML31, derived from F'lac, have been isolated from Escherichia coli as covalently-closed circular DNA molecules. These molecules were examined in the electron microscope after digestion with either EcoRI or BamHI restriction endonuclease. The structure of the majority of the molecules replicating was consistent with a bidirectional mode of replication starting at a unique origin on the F-fragment. This origin is located approximately 2.3 kilobases from one of the EcoRI sites. Orientation of the F-fragment relative to the physical map of F showed the position of the origin to be at 42.6 kilobases. A small proportion of molecules appeared to be replicating unidirectionally in either direction from this origin. Termination of replication of pML31 apparently occurs in the fragment containing the locus for kanamycin resistance in a unique region opposite the origin in the circular DNA molecule.

DNA Replication↗
PubMed1974

Synthesis of colicin E1 in a cell-free system.

Colicin E1 was synthesized in a cell-free system. The in vitro synthesis was found to be dependent on the Col E1 DNA concentration and was not enhanced by the addition of mitomycin C.

Calcium↗