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M Hofnung

Publications and source records attributed to M Hofnung.

At least 145 records · Page 8Linked to original sources
PubMed1985

The SOS Chromotest, a colorimetric bacterial assay for genotoxins: validation study with 83 compounds.

The SOS Chromotest is a simple bacterial colorimetric assay for genotoxicity. It is based on the measure of the induction of sfiA, a gene controlled by the general repressor of the SOS system in E. coli. Expression of sfiA is monitored by means of a gene fusion with lacZ, the structural gene for beta-galactosidase. We have examined 83 compounds of various chemical classes with the SOS Chromotest using a standard procedure. Comparison of the results with those obtained in the Mutatest (the Ames test) showed that most (90%) of the mutagenic compounds were also SOS inducers. For these compounds a quantitative correlation was observed between the mutagenic potency and the SOS-inducing potency (SOSIP). The case of the 10% remaining compounds giving conflicting results in the two tests is discussed. Sensitivity, specificity and accuracy for carcinogenicity prediction have been evaluated for the SOS Chromotest and the Mutatest using 73 chemicals for which carcinogenicity data were available. In spite of some differences, similar results were obtained in the two tests. The present data indicate that the SOS Chromotest has many practical advantages and may be used as a primary screening tool or as part of a battery of short-term tests for carcinogens.

Biotransformation↗
PubMed1985

Isolation of different bacteriophages using the LamB protein for adsorption on Escherichia coli K-12.

Ten phages which use the LamB protein for adsorption have been isolated from sewage waters. Nine have a shape similar to lambda and require only the LamB protein for adsorption. One has a shape similar to T phages and can use either the LamB or the OmpC protein. Preliminary characterization by a number of criteria showed that at least nine of these phages were different and also differed from other known phages which use the LamB protein, such as lambda, 21, and K10.

Adsorption↗
PubMed1984

Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12.

The sequences of the malE gene and of its mature product, the maltose-binding protein, have been determined and are in good agreement. The malE gene encodes the pre-protein (396 amino acid residues) which yields, upon cleavage of the NH2-terminal extension (26 amino acid residues), the mature maltose-binding protein (370 amino acid residues). The malE mRNA could form stable stem and loop structures, some of which may account for translational pauses observed by Randall et al. (Randall, L., Josefsson, L. G. & Hardy, S. J. S. (1980) Eur. J. Biochem. 107, 375-379). The sequence change due to an in-frame nonpolar deletion of 765 nucleotides in malE is also presented as well as homologies between the maltose-binding protein and other sugar-binding proteins.

ATP-Binding Cassette Transporters↗
PubMed1984

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12.

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.

Amino Acid Sequence↗
PubMed1984

A family of dispersed repetitive extragenic palindromic DNA sequences in E. coli.

We report the properties of 67 members of a family of dispersed repetitive palindromic extragenic bacterial DNA sequences. These sequences, called palindromic units, appear to be present at least several hundred times outside structural genes on the Escherichia coli chromosome. They are found either in clusters - as in a previously described intercistronic element - or in single occurrences. They are not only found within an operon but also between different operons, including between convergent ones. The palindromic units could yield a stem and loop structure at the level of DNA or RNA. The base of the stem is made of eight remarkably conserved base pairs while the rest varies somewhat in length and sequence. We analyse the data available on the palindromic units and we speculate on their possible roles with emphasis on transcription and mRNA stability or processing, as well as on their possible relation to transposition elements and the modular evolution of the genome.

Base Sequence↗
PubMed1984

Linker mutagenesis in the gene of an outer membrane protein of Escherichia coli, lamB.

In order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in-phase deletions or small insertions. Except for two in-phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.

Bacterial Outer Membrane Proteins↗
PubMed1984

Induction by UV light of the SOS function sfiA in Escherichia coli strains deficient or proficient in excision repair.

The influence of the nucleotide excision repair system on the induction by UV irradiation of the SOS function sfiA has been investigated. The level of sfiA expression was monitored by means of a sfiA::lacZ operon fusion in both the wild-type strain and a uvrA mutant. We found that the initial steady rate of sfiA expression was proportional to the UV dose and was identical in uvr+ and uvrA backgrounds. This suggests that the initial steady rate of sfiA expression is determined by the initial number of lesions and before any effect of excision repair. We confirmed that after 2 h of expression the net synthesis of sfiA product is, for the same UV dose, about five times lower in uvr+ than in uvrA strains. We show that this is due to earlier repression of the SOS system in uvr+ than in uvrA strains and not to different initial rates.

DNA Repair↗
PubMed1984

High-sensitivity detection of newly induced LamB protein on the Escherichia coli cell surface.

The kinetics of the appearance at the cell surface of the outer membrane LamB protein after induction were determined by using specific antibodies and radioiodinated protein A as a probe. This was done in two different induction systems. First, LamB protein was induced in a wild-type strain by the simultaneous addition of cyclic AMP and maltose. Second, an operon fusion strain in which the lamB gene is expressed under lac promoter control was used; in this system, LamB protein can be induced by isopropyl-beta-D-thiogalactopyranoside. When uninduced cells were grown in glucose minimal medium, background expression of the lamB gene was found to be ca. 10-fold lower in lac-lamB cells than in wild-type cells. The level of LamB protein present in uninduced wild-type cells could, however, be reduced by supplementing the growth medium with Casamino Acids. After induction, the LamB protein appeared at the cell surface of both strains within a few minutes, and then the LamB level per cell increased linearly. The time lag in cell surface exposure of LamB protein differed slightly under both induction conditions: the LamB protein appeared at the surface of lac-lamB cells within 3 min of induction, whereas in wild-type cells it could not be detected earlier than after 4 to 5 min of induction.

Bacterial Outer Membrane Proteins↗
PubMed1983

Genetic study of a membrane protein: DNA sequence alterations due to 17 lamB point mutations affecting adsorption of phage lambda.

Gene lamB encodes the outer membrane receptor for phage lambda in Escherichia coli K12. We have determined the DNA sequence alterations of 17 lamB point mutations which result in resistance to phage lambda h+. The mutations correspond to four phenotypic classes according to the pattern of growth of three phages which use the lambda receptor: lambda h (a one-step host-range derivative of lambda h+), lambda hh* (a two-step host-range derivative of lambda h+) and K10 (another lambdoid phage). Fourteen mutations are of the missense type and correspond to Gly to Asp changes distributed as follows. One class I mutation is at position 382 of the mature lambda receptor. Seven class I* mutations, four of which at least are independent, are at position 401. Six independent class II mutations are at position 151. The three other (class III) mutations are of the nonsense type. They change codons TGG (Trp) into TAG (amber) at positions 120 (two mutations) and 351 (one mutation). Implications of these results for the topological organization of the lambda receptor as well as possible reasons for the limited number of altered sites detected are discussed.

Adsorption↗
PubMed1983

Negative dominance in gene lamB: random assembly of secreted subunits issued from different polysomes.

lamB is the structural gene for the lambda receptor, an oligomeric outer membrane protein from Escherichia coli K12 involved in phage lambda adsorption. We show that, under certain conditions, in a strain diploid for gene lamB, all the missense lamB mutations conferring lambda resistance that we have tested are dominant with respect to wild-type. We propose a model which allows a quantitative interpretation of the data. It is based on negative complementation at the level of oligomerisation. Wild-type and mutant subunits would assemble at random forming homo- and hetero-oligomers. Only wild-type homo-oligomers would be efficient for phage inactivation. For some classes of missense mutations the hetero-oligomers would have the capacity to bind, but not to inactivate the phage. The model confirms that active lambda receptor is a trimer and implies that for this secreted protein there is no preferential assembly of subunits originating from the same polysome.

Bacterial Outer Membrane Proteins↗
PubMed1982

Sequence of the malK gene in E.coli K12.

We present the sequence of gene malK which encodes a component of the system for maltose transport in E.coli K12. We also determined the position of deletion (S50) which fuses malK to the following gene lamB; the malK-lamB protein hybrid contains all of the malK protein. The mRNA corresponding to the last two thirds of gene malK could form stable stem and loop structures. The malK protein, as deduced from the gene sequence, would include 370 residues and correspond to a molecular weight of 40700. The sequence as well as sequence comparisons with the ndh protein of E.coli are discussed in terms of the location and function of the malK protein.

ATP-Binding Cassette Transporters↗
PubMed1982

Extensive homology between membrane-associated components of histidine and maltose transport systems of Salmonella typhimurium and Escherichia coli.

A strong homology was found between the amino acid sequences, deduced from DNA nucleotide sequences, of cytoplasmic membrane-associated components of the high affinity histidine transport system of Salmonella typhimurium (coded by the hisP gene) and the maltose-maltodextrin transport system of Escherichia coli (coded by the malK gene). When the HisP protein sequence was aligned with that of the NH2-terminal two-thirds of the MalK protein, 32% of the positions were identical, and an additional 35% were occupied by functionally similar amino acid residues. These results suggest that some, and possibly many, "periplasmic-binding protein-dependent" transport systems have evolved from a common ancestral system.

ATP-Binding Cassette Transporters↗
PubMed1982

A DNA sequence containing the control regions of the malEFG and malK-lamB operons in Escherichia coli K12.

The malB region in E. coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. We have established the DNA sequence of a 600 base pair segment which contains the sites necessary for initiation of transcription of the malB operons and the translation start sites for malE and malK. We have also determined the position of a 147 base pair deletion, malB delta 100, in this sequence. The control region contains two short segments (about 35 base pairs) showing unusual distributions of bases: the GC content (85%) is higher than average and most of the purines (85%) are on one strand. We discuss the possible relevance of this and other salient features of the DNA sequence to the mechanisms by which the expression of the malB region is positively regulated.

Base Composition↗
PubMed1982

Genetic toxicology studies with 2-nitrobenzofurans and 2-nitronaphthofurans.

The genetic toxicity of benzofurans and naphthofurans was further examined. (i) Seven new 2-nitronaphthofurans tested give a positive response on strains TA1537, TA1538, TA98, TA100, but not on strain TA1535. This response depends at least partially on the bacterial nitroreductase activities and is decreased in presence of activating mixture from rat liver. (ii) A correlation is observed between the mutagenic potency assayed in the Mutatest and the phage-inducing potency assayed in the Inductest. The relevance of these results and of complementary tests on uvr + Salmonella strains and the Spermatest are briefly discussed in perspective of the possible genetic toxicity of the compounds on mammals.

Animals↗
PubMed1982

SOS chromotest, a direct assay of induction of an SOS function in Escherichia coli K-12 to measure genotoxicity.

We present and evaluate the SOS chromotest, a bacterial test for detecting DNA-damaging agents. It is a colorimetric assay based on the induction by these agents of the SOS function sfiA, whose level of expression is monitored by means of a sfiA::lacZ operon fusion. The response is rapid (a few hours), and does not require survival of the tester strain. Dose-response curves for various chemicals include a linear region. The slope of this region is taken as a measure of the SOS inducing potency. Comparison for a number of substances of known genotoxicity of the SOS inducing potency determined in the SOS chromotest with the mutagenic potency determined in the Salmonella assay (mutatest) revealed a striking quantitative correlation over more than 7 orders of magnitude. The sensitivity of the SOS chromotest (lowest amount detected) is equal to that of the mutatest and generally 4-40 times higher than that of a phage induction assay (inductest). From a practical point of view our observations contribute to the validation of the SOS chromotest as a test for detecting genotoxins and in particular genotoxic carcinogens. From a theoretical standpoint the results suggest that mutagenic potency measured in the mutatest reflects the level of induction of an SOS function and that most genotoxins are inducers of the SOS response in bacteria.

Alkaline Phosphatase↗
PubMed1982

Mutations in gene lamB: studies on structure and topology of an E. coli outer membrane protein.

Results obtained in E. coli with a set of mutations conferring tight resistance to phage lambda lead to a first identification of three residues in the lamB protein which are important for adsorption of phage lambda. Residues 151 and 382 are important for reversible adsorption while residue 401 is important for irreversible adsorption. The identification of such residues may help to identify portions of the protein located at the cell surface. Assays of lambda receptor activity in merodiploid strains heterogenote for gene lamB show that the mutations studied can have a negative dominant effect. For one class of mutations (class II) this can be interpreted by negative complementation at the level of oligomerisation. The data confirm then that active lambda receptor is a trimer.

Amino Acid Sequence↗