PubMed Health⌕ Search

Biomedical subjects

M Zabeau

Publications and source records attributed to M Zabeau.

29 records · Page 2Linked to original sources
PubMed1986

Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI.

We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence and structural organization of an insertion sequence element (IS231) from Bacillus thuringiensis strain berliner 1715.

This paper describes the structural organization of a repetitive DNA sequence isolated from plasmids of Bacillus thuringiensis strain berliner 1715. DNA sequence analysis of this repetitive sequence (RS) revealed all the characteristic features of an insertion sequence (IS). This 1656-bp element is delineated by two 20-bp inverted repeats which are flanked by two 11-bp direct repeats. A long open reading frame spans almost the entire sequence and is preceded by potential transcriptional and translational signals. A structural homology was observed between this RS and the Escherichia coli IS4 element in the length of their direct repeats, the sequence of their inverted repeats and the sequence of their putative transposases. These data strongly suggest that this sequence is an authentic insertion sequence. Therefore the name IS231 is proposed for this first Bacillus IS element. Further relationships with other known IS sequences were also found and are discussed.

Amino Acid Sequence↗
PubMed1985

Overproduction of the EcoR V endonuclease and methylase.

Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter. The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase. A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene downstream of the lambda PL promoter. The situation in the endonuclease overproducing clone resembles that encountered in maxi-cells. The strains described here allowed a quick purification of both enzymes in sufficient amounts for crystallisation attempts.

Base Sequence↗
PubMed1985

Purification and crystallization of the EcoRV restriction endonuclease.

The type II restriction endonuclease EcoRV purified from a genetically engineered, overproducing strain has been crystallized. Four crystal forms all obtained by precipitation with polyethylene glycol 4000 have been characterized. Two of these are suitable for high resolution structure analysis. Both are orthorhombic, have space group P2(1)2(1)2(1) and have similar unit cell dimensions of a = 58.2 A, b = 71.7 A, c = 130.6 A (form A) and a = 59.9 A, b = 74.5 A, c = 121.8 A (form B). They diffract to about 2A resolution and appear to have one dimer of 2 X 29,000 daltons in the asymmetric unit.

Crystallization↗
PubMed1985

High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda.

Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors. In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the overproduced EcoRI endonuclease.

Animals↗
PubMed1984

Effect of polyamines and basic proteins on cleavage of DNA by restriction endonucleases.

We have investigated the effect of the polyamines spermine, spermidine, and putrescine and the prokaryotic histone-like proteins NS1 and NS2 on the restriction endonuclease EcoRI catalyzed cleavage of plasmid and bacteriophage DNAs. At low concentrations of spermine and spermidine, the rate of DNA cleavage by EcoRI is increased, while high concentrations of spermine as well as of spermidine are inhibitory. These phenomena are also observed with other restriction endonucleases. They are, therefore, probably due to the interaction of the polyamines with the DNA. Putrescine does not have such an effect within the concentration range investigated. Remarkably, low concentrations of spermine and spermidine very efficiently suppress EcoRI activity. An inhibition of the EcoRI-catalyzed cleavage of DNA is also observed with NS1 and NS2, an effect that can be mimicked with other basic proteins that interact with DNA. The results are discussed in terms of the mechanism of restriction in vivo.

Bacteriophage lambda↗
PubMed1984

Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli.

A plasmid encoding the recently described Eco RV restriction and modification system has been isolated and characterized. This plasmid, pLB1 , is 6.2 kb long and carries only the Eco RV genes. A subclone of 3 kb has been inserted in pBR322. The relative positions of the endonuclease and the methylase genes were determined by the construction of a set of overlapping deletions generated by Bal31 resection. The DNA sequence of a 2.2 kb fragment containing the two genes was determined. The two genes are transcribed divergently from a 310 bp region and the assignment of the coding region has been confirmed by direct aminoacid sequence analysis. Possible mechanisms of regulation of the endonuclease gene expression at the translational level are proposed and discussed.

Amino Acid Sequence↗
PubMed1982

Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed.

Bacteriophage lambda↗
PubMed1980

The ral gene of phage lambda. I. Identification of a non-essential gene that modulates restriction and modification in E. coli.

Host controlled restriction in Escherichia coli can be relieved by pre-infecting restricting cells with modified lambda helper phages. This process, in which intact unmodified phage genomes are allowed to escape restriction attack, is mediated by a newly identified lambda function called ral. The ral gene has been located by deletion mapping between cIII and N. Efficient expression of the ral gene requires the product of the regulator gene N. Polyacrylamide gel analysis of the lambda proteins specified by the cIII-N region failed to reveal the product of the ral gene, but demonstrated that protein Ea10 is encoded by a gene located immediately to the left of ral. From these results the map order cIII-Ea10-ral-TL1-N was deduced. Ral specifically alleviates restriction in E. coli K and E. coli B, but does not affect restriction systems EcoRI, EcoRII and EcoP1. In addition, ral enhances the modification activity of the EcoK and EcoB restriction enzymes: we observed that efficient modification of progeny phages obtained by propagating unmodified lambda phages in r-m+ hosts, is dependent upon the presence of ral. We thus conclude that the ral gene product acts by modulating the restriction and modification activities of the type I restriction systems in E. coli, and the possible mechanisms will be discussed.

Bacteriophage lambda↗
PubMed1980

The ral gene of phage lambda. II. Isolation and characterization of ral deficient mutants.

The lambda ral function modulates the restriction and modification activities of the Escherichia coli K12 and B restriction enzymes (Zabeau et al., 1980). In order to further analyse this function, ral deficient mutants have been isolated, using a method which exploits the property of the strong mutagen N-methyl-N'-nitro-N-nitrosoguanidine (N.G.) to induce multiple closely linked mutations. Hence, mutagenized phages carrying mutations in one locus were frequently found to contain additional mutations in adjacent loci. This very efficient mutagenesis procedure enabled us to isolate 27 independent Ral deficient mutants. Seven mutants were found to affect the ral gene directly and were located between the genes N anc cIII. Detailed mapping of two of these mutants showed that the lambda ral gene is located at position 70.6-70.9% on the physical map. The isolation and characterization of these mutants further supports the conclusion that ral is a gene different from the N gene, and demonstrates that the ral gene product is responsible for both counteracting restriction and enhancing modification.

Bacteriophage lambda↗