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M Hofnung

Publications and source records attributed to M Hofnung.

At least 163 records · Page 9Linked to original sources
PubMed1982

A system for genetic analysis in gene lamB: first results with lambda-resistant tight mutants.

We describe a system for genetic analysis in gene lamb. It consists of a phage which allows mapping, complementation and sequencing studies of lamB mutations and of the sequence of gene lamB. We present results obtained with this system for a set of mutations conferring tight resistant to phage lambda. This leads to a first identification of three residues in the LamB protein which are important for adsorption of phage lambda h+. Residues 151 and 382 are important for reversible adsorption while residues 401 is important for irreversible adsorption. We briefly review other tight mutations affecting gene lamB and discuss some implications of the results on phage receptor interactions, LamB-protein structure and membrane insertion.

Adsorption↗
PubMed1981

rho Mutations restore lamB expression in E. coli K12 strains with an inactive malB region.

lamB, the structural gene for lambda receptor, is the second gene of the malK-lamB operon in the malB region of the Escherichia coli K12 chromosome. lamB is essentially not expressed in the absence of an active malT gene product, the activator of the maltose regulon. A malT strain is resistant to phage lambda. We show that: (i) Introduction of rho mutations in malT mutants restores lamB expression to a level sufficient to render the strain sensitive to phage lambda; (ii) This restoration is not dependent on the main promoter of the malK lamB operon. It depends on the distal part of the malK gene. We propose that rho inactivation unmasks the activity of a promoter located near the distal end of malK. Experiments with Mu insertions in gene malK suggest that in the (-) orientation a Mu promoter is also able to allow lamB expression in a rho background.

Bacterial Outer Membrane Proteins↗
PubMed1981

Gene sequence of the lambda receptor, an outer membrane protein of E. coli K12.

The Escherichia coli K12 lambda receptor is a multifunctional outer membrane protein whose precursor, encoded in gene lamB, is cleaved during export. We present the DNA sequence of lamB and of the distal region that contains repetitive and palindromic sequences and could give rise to highly stable mRNA structures. The calculated molecular weight of the lambda receptor is 47,400. Of the 421 amino acids, 89 are charged, mostly negatively. No region devoid of charged amino acids and long enough to serve as a transmembrane portion is detected. The distribution of charges presents special features that we comment upon in relation to the structure, functions and localization of the lambda receptor. Gene lamB is followed by molA, an unidentified reading frame corresponding to a 131-amino-acid peptide with the characteristics of an exported protein.

Amino Acid Sequence↗
PubMed1981

Mutagenic activity of benzofurans and naphthofurans in the Salmonella/microsome assay: 2-nitro-7-methoxy-naphtho[2,1-b]furan (R7000), a new highly potent mutagenic agent.

A series of benzofurans and naphthofurans was examined through the Salmonella/microsome assay. (i) With one possible exception, only 2-nitro derivatives give a mutagenic response. However, it appears that the mutagenic potency depends notably on the nature and the position of the other substituents in the molecule. (ii) The mutagenic response occurs in strains TA1537, TA1538, TA98 and TA100 but not in strain TA1535. Reversion of the missense mutation of TA1535 is thus induced only in presence of the plasmid pKM101. (iii) This mutagenic response is at least partially dependent on the bacterial nitroreductase activities and is usually lower in presence of activating mixture from rat liver. (iv) One of the compounds tested, 2-nitro-7-methoxynaphtho[2,1-b]furan (R7000), may be the most potent mutagen examined so far in the Salmonella/microsome assay. It yields about 200 000 revertants/nanomole on strain TA100 in the standard plate test. The relation between structure, mutagenic potency and other biological activities of the compounds are briefly discussed.

Benzofurans↗
PubMed1981

In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12.

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.

Adsorption↗
PubMed1981

General method for fine mapping of the Escherichia coli K-12 lamB gene: localization of missense mutations affecting bacteriophage lambda adsorption.

lamB is the structural gene for the bacteriophage lambda receptor, a multifunctional protein located in the outer membrane of Escherichia coli K-12. We present a method for deletion mapping of any lamB mutations with a recognizable pheno-type. This method involves a transducing phage constructed by in vitro recombination which can also be used for complementation, deoxyribonucleic acid sequence, and in vitro protein synthesis studies with the mutated lamB gene. Using this method, we mapped 18 lamB missense mutations which confer resistance to phage lambda h+ (wild-type host range). The main results were the following. (i) None of the 18 mutations was located in the first 4 deletion intervals out of the 11 of the genetic map. (ii) These mutations were clustered according to their phenotype as follows. (a) Class I mutations, which allow growth of lambda h and lambda hh* (one-step and two-step host range mutants of lambda, respectively), were located in three regions--three in interval V, four in interval VIII-IX, and three in interval X-XI. Only the last three mutations still allowed growth of phage K10 which also uses the lambda receptor, and two of them still allowed reversible binding of lambda h+. (b) All seven class II mutations allowed only growth of lambda hh* and mapped in interval V. These results are discussed in the frame of a genetic approach to the functional topology of the lambda receptor.

Adsorption↗
PubMed1981

Functional implications of secondary structure analysis of wild type and mutant bacterial signal peptides.

The N-terminal extension found in the precursor form of many exported proteins in bacteria--the so called signal peptide--plays an essential role in the exportation process. Analysis of the secondary structure of several bacterial signal peptides by predictive methods shows that the predicted structures are essentially periodical and define a preferential axis. We assume that at least one step in export depends directly on the length of the hydrophobic core of the signal peptide measured along this axis. We call this parameter the Hydrophobic Axis Length or HAL. Examination of the available mutations located in bacterial signal peptides and blocking export permits us to define a minimum functional HAL value (the threshold HAL) under which little or no export occurs. A mutation of the lipoprotein signal peptide leaves an HaL above the threshold: this accounts readily for the fact that export of the protein is not strongly affected. The value calculated for the threshold HAL, ie, 18 A, corresponds to half the size of the lipid bilayer of the membrane.

Amino Acid Sequence↗
PubMed1980

Identification of a cytoplasmic membrane-associated component of the maltose transport system of Escherichia coli.

The maltose transport system of Escherichia coli contains at least five components, three of which, i.e. the products of lamB, malE, and malF genes, have so far been identified as constituents of the outer membrane, periplasmic space, and cytoplasmic membrane, respectively. We identified another component, a cytoplasmic membrane protein of an apparent molecular weight of 43,000, as the product of the malK gene on the basis of polyacrylamide gel electrophoretic analysis of various mutants and suppressed strains and by the incorporation of extra tyrosine residue into this proten in malK amber mutants containing the suppressor Su3+ allele. The transport of maltose thus appears to require at least two proteins associated with the cytoplasmic membrane.

Alleles↗
PubMed1980

Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli.

The maltose binding protein of Escherichia coli is secreted into the external periplasmic compartment of the cell by virtue of an amino-terminal signal sequence. Using DNA sequencing, we have determined the precise nature of mutations in the signal sequence which prevent the export of the maltose binding protein, causing it to accumulate in the cytoplasm in its precursor form. In most cases, the change of a single hydrophobic or uncharged amino acid to a charged amino acid within the signal sequence is sufficient to block the secretion process.

ATP-Binding Cassette Transporters↗
PubMed1980

Inductest and spermatest in genetic toxicology testing.

Bacterial strains have been especially devised to measure the ability of chemicals to induce phage development. This type of test named Inductest, responds in general to DNA damaging agents. Metabolic activation can be used and good sensitivities are achieved. Variations of the Inductest exist: some have short time responses (1 h or less) or allow to look at phage mutagenesis as well. Some compounds elicit a response in Inductest and not in the bacterial mutagenesis assay (Mutatest). The reverse is also true and may be more frequent. The sperm abnormality assay (Spermatest) consists in examining by visual scoring of sperm shape if an agent is able to damage in vivo mammalian germ cells. Such agents are in general able to interfere with the normal differenciation of sperm cells so that genetic damage is probably not the only end point of the assay. The spermatest yields false negative but few false positive for carcinogenicity. It can be used directly to monitor human populations. Both tests, have probably not yet reached their full development.

Animals↗
PubMed1980

Adaptation of the salmonella/mammalian microsome test to the determination of the mutagenic properties of mineral oils.

Two techniques allowing the determination of the mutagenicity of lipophilic compounds such as mineral oils with the Ames test have been developed by using benzo[a]pyrene (BP) dissolved in white oil as a synthetic reference oil. The first technique involves prior extraction of polynuclear aromatic hydrocarbons (PAH) with dimethyl sulfoxide. In the second method, which proved simpler and of more general use, the compounds to be tested are directly dispersed in aqueous medium with Tween 80. The use of these techniques made possible the study of mutagenicity of various kinds of mineral oil. Mutagenic activity was found in used crankcase oils, and also in petroleum distillates but much less in solvent-refined oils. A good correlation was observed between mutagenic activity and PAH content but not BP content of oils. Because of their peculiar response to the test, petroleum distillates were studied in more detail. When added in low amounts to pure PAH compounds such as BP, they enhanced its mutagenic activity (enhancement). When added in higher amounts, on the contrary, these oils completely inhibited BP mutagenic activity (inhibition effect). Both effects correlated well with the PAH content and the mutagenic activity of the petroleum distillates tested. These results explain the abnormal dose-response curves curves obtained with these petroleum distillates and the negative results regarding their mutagenic activity reported in earlier studies. A likely explanation is discussed for the enhancement and inhibition effects.

Dose-Response Relationship, Drug↗
PubMed1980

Synthesis and maturation of lambda receptor in Escherichia coli K-12: in vivo and in vitro expression of gene lamB under lac promoter control.

The lambda receptor is an outer membrane protein from Escherichia coli K-12 lamB, its structural gene, is part of the maltose regulon. We have cloned this gene in a phage so that it is under the control of the lac promoter. The phage was devised in such a way that it can infect lamB mutants and that chromosomal lamB mutations can be transferred to it. In vivo, the lambda receptor is expressed under lac promoter control and is exported normally to the outer membrane, independently of the expression of the other genes of the maltose regulon. In vitro, DNA of the phage allows efficient synthesis of the lamB product. The protein--or pre-lambda-receptor--made in vitro contains an NH2-terminal sequence of about 25 amino acids not found in the lambda receptor. We have detected no inactivation of phage lambda by the pre-lambda-receptor. Conversion of the pre-lambda-receptor to a form that has the apparent molecular weight of the mature lambda receptor was achieved. A lamB mutation that blocks export in vivo also blocks conversion in vitro.

Bacterial Proteins↗
PubMed1980

DNA sequence encoding the NH2-terminal peptide involved in transport of lambda receptor, an Escherichia coli secretory protein.

lamB encodes the lambda receptor of Escherichia coli, an outer membrane protein. We have identified the beginning of the lamB gene by correlating DNA nucleotide sequence with a partial sequence of the primary translation product of lamB. We show that lambda receptor is synthesized as a precursor containing an extra 25 amino acids at its NH2 terminus. These amino acids are predominately hydrophobic and probably comprise a structure required for initiation of transport of lambda receptor from the cytoplasm to the outer membrane.

Amino Acid Sequence↗