Extension of bacteriophage lambda host range: selection, cloning, and characterization of a constitutive lambda receptor gene.

A set of plasmids has been constructed that carry a constitutive lamB gene (LamBc phenotype) from Escherichia coli and that confer functional phage lambda receptors to bacteria other than E. coli. This E. coli LamBc strain has been selected to escape both maltose-inducible and glucose-repressible control. Constitutivity results from an IS-3 insertion, carrying a mobile promoter, proximal to lamB. The LamBc DNA has been cloned into both broad and narrow host-range plasmids, and the resulting pTROY plasmids have been transferred to diverse bacteria. Both Salmonella typhimurium/pTROY and Klebsiella pneumoniae/pTROY strains efficiently adsorb phage lambda; Pseudomonas aeruginosa/pTROY strains do not. Introduction of a functional E. coli LamB protein into foreign bacterial will allow these bacteria carrying pTROY plasmids to be infected by phage lambda recombinant DNA libraries, phage lambda::Tn insertion mutagenesis vectors, and in vivo lambda-packaged cosmids.     

Excision of transposon Tn5 is dependent on the inverted repeats but not on the transposase function of Tn5.

The excision of Tn5 from sites of insertion in the Escherichia coli genome was studied by examining the reversion of lac::Tn5 insertion mutations to lac+. We find that: (i) the frequency of excision depends on the site of Tn5 insertion, (ii) excision occurs efficiently in recA- cells, (iii) excision does not require a Tn5-encoded transposition function, and (iv) efficient excision requires the inverted repeats of Tn5. We propose that excision of Tn5 is similar to the formation of spontaneous deletions and occurs by slippage during DNA synthesis.     

Detection and analysis of UV-induced mutations in mammalian cell DNA using a lambda phage shuttle vector.

In order to study mutagenesis in mammalian cells, stable mouse L-cell lines were established with multiple copies of a lambda phage vector that contains the supF gene of Escherichia coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using lambda in vitro packaging extracts was efficient (5 phage per microgram of cell DNA per copy) and yielded a negligible background of mutant phage (0 out of 54,605). From mouse cells exposed to 254-nm ultraviolet light (12J/m2), 78,510 phage were rescued, of which 8 were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Y-C) sequences, and that the most frequent mutation at this site is a C----T transition.     

Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations.

Conditional lethal mutations are valuable for analyzing essential genes. We describe here a derivative of the bacterial transposon Tn5 called Tn5tac1 and its use in an innovative strategy for making mutations with conditional phenotypes. The 4.6-kilobase Tn5tac1 element contains a strong, regulatable, outward-facing promoter (Ptac) near one end and is polar on the expression of distal genes when the inducer of Ptac [isopropyl beta-D-thiogalactoside (IPTG)] is absent. Our results show that two unusual conditional mutant phenotypes can result from Tn5tac1 insertion in Escherichia coli: one is corrected by IPTG while the other is induced by IPTG. The broad host range of Tn5 and the conditional nature of these mutant phenotypes makes Tn5tac1 well suited for identifying essential genes in diverse bacterial species.     

Viable Molecular Hybrids of Bacteriophage Lambda and Eukaryotic DNA

A bacteriophage lambda strain has been constructed and a method developed by which DNA from potentially any source can be covalently inserted through EcoRI cohesive ends into the middle of the lambda DNA. These hybrid DNAs can infect nonrestricting Escherichia coli cells and can then propagate as plaque-forming phage. A unique feature of this lambda strain is that extra DNA in the middle of its genome is required for plaque formation. A large number of such phages have been produced with E. coli DNA and Drosophila melanogaster DNA.     

Tn10 transposition promotes RecA-dependent induction of a lambda prophage.

We present evidence that Tn10 transposition, or a closely correlated event, induces expression of bacterial SOS functions. We have found that lambda prophage induction is increased in Escherichia coli lambda lysogens containing increased Tn10 transposase function plus single or multiple copies of an appropriate pair of transposon ends. This increase occurs by the normal pathway for prophage induction, which involves RecA-mediated cleavage of the phage lambda repressor protein. We also present evidence that Tn10 promotes induction of expression of the E. coli sfiA gene. Tn10 transposes by a nonreplicative mechanism. We propose that the signal for RecA protease activation and SOS induction is generated by degradation of the transposon donor molecule and suggest that SOS induction is biologically important in helping a cell undergoing transposition to repair and/or recover from damage to the transposon donor chromosome.     

DNA methylation pattern is determined by the intracellular level of the methylase.

Extrachromosomal plasmid DNA is transiently undermethylated in Escherichia coli during amplification in the presence of chloramphenicol. In addition, undermethylation of phage lambda DNA was observed after thermal induction of a lambda c1857 lysogen while the integrated lambda phage DNA was found to be fully methylated. These methylation pattern changes occur under conditions (extensive replication) in which the intracellular methylase level becomes limiting. In an E. coli strain that harbors a plasmid that carries the dam methylase gene and therefore overproduces dam methylase, there is no undermethylation of dam sites in either of the extrachromosomal DNAs. The sites that are methylated by the mec methylase in both plasmid and lambda phage DNAs were undermethylated in the dam overproducer as well. These results indicate that the intracellular level of the E. coli methylase determines the DNA methylation pattern.     

Efficient isolation of genes by using antibody probes.

A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.     

Mapping of transforming region of the Harvey murine sarcoma virus genome by using insertion-deletion mutants constructed in vitro.

Circular DNA intermediates of Harvey murine sarcoma virus (Ha-MuSV) have been cloned in lambda gtWES . lambda B and shown to be capable of transforming mouse NIH 3T3 cells [Hager, G. L., Chang, E. H., Chan, H. W., Garon, C. F., Israel, M. A., Martin, M. A., Scolnick, E. M. & Lowy, D. R. (1979) J. Virol. 31, 795-809]. By using the cloned Ha-MuSV DNA insert as a parental genome, we have constructed a series of insertion-deletion mutants by inserting an octomer containing the Sal I linker sequence (G-G-T-C-G-A-C-C) into various regions of the Ha-MuSV genome after partial digestion with Hae III. After ligation into lambda gtWES . lambda B-Sal I vector molecules, the mutant Ha-MuSV DNAs were cloned. Fourteen insertion-deletion mutants have been mapped by restriction enzyme digestion, and their biological activities have been correlated with the locations of mutations. The mutants whose lesion mapped within 3.0 kilobases (kb) frm the 3'-end of the Ha-MuSV genome retained full transforming ability. The mutants containing the Sal I linker insertion at 0.4 or 1.5 kb from the 5'-end also retained transforming ability, but the number of foci induced by the DNAs in transfection assays was greatly reduced. However, a mutant containing a deletion of 1.5 kb at the 5'-end and a mutant with a deletion of the sequences between 1.0 and 1.5 kb from the 5'-end completely lost their transforming potential. A model for the transforming region of Ha-MuSV is discussed. Furthermore, because Ha-MuSV sequences can be rescued from the mouse cells transformed by these mutants using Moloney murine leukemia virus as a helper virus, it implies that the in vitro modified DNAs may be converted into genuine mutant viruses.     

Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae.

We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae. A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination. Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E. coli strain containing an F factor derivative carrying the transposable element. The culture was grown to allow transposition and cointegrate formation and, upon conjugation, recipients were selected that contained yeast sequences with transposon insertions. The yeast DNA was removed from the vector by restriction endonuclease digestion, and the transposon insertion was transformed into yeast. The procedure required a minimum number of manipulations, and each transconjugant colony contained an independent insertion. We describe 12 transposon Tn3 derivatives for this procedure as well as several cloning vectors to facilitate the method.     

Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3).

A cloned segment of yeast DNA containing the structural gene for imidazoleglycerolphosphate dehydratase (D-erythro-imidazoleglycerolphosphate hydro-lase, EC 4.2.1.19) is transcribed and translated in Escherichia coli with sufficient fidelity to produce functional enzyme. This segment of yeast DNA was isolated as a viable molecular hybrid of bacteriophage lambda (lambdagt-Sc2601) which complements a nonrevertible hisB auxotroph of E. coli lacking dehydratase activity. The equivalent segments of DNA cloned from two independent his3 mutants of yeast lacking IGP dehydratase activity do not complement the hisB auxotroph. The two nonfunctional his3 alleles cloned in bacteriophage lambda can be recombined in E. coli to generate a hybrid phage which complements the hisB auxotroph. The dehydratase activity produced in E. coli by the cloned segment of yeast DNA strongly resembles the activity found in yeast.     

Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules.

We have developed a cell-free system from Escherichia coli for studying illegitimate recombination between nonhomologous DNA molecules. The recombination is stimulated by oxolinic acid, an inhibitor of DNA gyrase. The stimulation is abolished by coumermycin A1 and is not found in extracts of nalidixic acid-resistant (gyrA) mutants. We therefore inferred that DNA gyrase directly participates in illegitimate recombination, at least in the presence of oxolinic acid [Ikeda, H., Moriya, K. & Matsumoto, T. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 399--408]. The structure of recombinant DNA molecules formed in the presence of oxolinic acid from a cross between phage lambda and plasmid pBR322 DNAs was analyzed by heteroduplex mapping. Among nine isolates tested, two recombinants were formed by the insertion of the plasmid into the lambda genome. The seven other recombinants had more complicated genome structures. Insertion of pBR322 was accompanied by a deletion on one of the genomes. In all cases, the end points of deletions coincided with one end of the pBR322 insertion. Recombination sites seemed to be distributed randomly on the lambda and pBR322 genomes. Analysis of nucleotide sequences of the recombination junctions proved that the crossover took place between nonhomologous DNA sequences. A model for DNA gyrase-mediated illegitimate recombination is discussed.     

Method for identifying microbial antigens that stimulate specific lymphocyte responses: application to Salmonella.

Vaccine development and understanding of cellular immune stimulatory mechanisms have been impeded by the paucity of data on microbial antigens that stimulate protective immunity. We describe here a general method for identifying and isolating peptide antigens that specifically stimulate sensitized lymphocytes. First, Salmonella typhimurium C5 genomic DNA fragments were subcloned into Escherichia coli by use of the lambda gt11 expression vector. Next, antigens expressed by recombinant phage from this genomic library were tested for their capacity to stimulate proliferative responses in pooled lymphocytes obtained from BALB/c mice infected 14 days earlier with S. typhimurium. Of 2000 recombinant phages tested, 5 stimulated a polypeptide-antigen-specific proliferative response. Physical analyses of these 5 recombinant phages revealed cloned inserts of 0.5-2.4 kilobase pairs representing nonoverlapping regions of the C5 chromosome. Four of the five insert DNAs hybridized at high stringency to both S. typhimurium and Salmonella typhi total chromosomal DNA, suggesting that these pathogens of different host specificity share several antigenic determinants. Use of sensitized primary polyclonal lymphocytes provides a rapid and simple method for screening recombinant DNA libraries for clones that stimulate specific immune responses and avoids the use of cloned lymphocyte cell lines. This approach should be generally applicable to similar studies in different hosts of many other microbial pathogens.     

Cloned genomic DNA sequences from Mycoplasma hyorhinis encoding antigens expressed in Escherichia coli.

A library of cloned Mycoplasma hyorhinis genomic sequences was constructed by incorporation of EcoRI digestion fragments of mycoplasma DNA into the lambda Charon 4A bacteriophage vector. Immunological screening of recombinant phage plaques identified clones containing genes encoding mycoplasma antigenic structures expressed in an Escherichia coli host. Two such recombinant phage isolates, lambda Ch4A-MhrG1 and lambda Ch4A-MhrG28, were defined and found to contain distinct genomic sequences by analysis of restriction endonuclease fragments. Inoculation of mice with recombinant gene products from lambda Ch4A-MhrG1 yielded antiserum selectively recognizing a Mr 29,500 trypsin-sensitive mycoplasma constituent. This established a means for producing selected immunogenic mycoplasma component in a bacterial host. The cloned genomic sequences of M. hyorhinis encoding expressed mycoplasma antigens represent molecular probes that can be characterized both by specific DNA sequences and by the antigenic structure of corresponding gene products. These genomic fragments define initial physical markers of the M. hyorhinis genome and may be useful in assessing antigenic and molecular genetic relationships within the genus Mycoplasma and among other members of the class Mollicutes.     

Expression of the denV gene of bacteriophage T4 cloned in Escherichia coli

The denV gene of bacteriophage T4 has been cloned into Escherichia coli K-12 by inserting appropriate fragments of cytosine-containing T4 DNA into the Sal I site of the plasmid pBR322. The denV gene codes for an enzyme that initiates the excision repair of pyrimidine dimers produced in DNA by UV. In uvrA recA mutants, deficient in an early step in excision repair, the cloned DNA results in enhanced UV resistance that is more pronounced in stationary- than in exponential-phase cultures. The expression of the cloned DNA also results in the enhanced survival of UV-irradiated phage lambda or of a denV mutant of phage T4 and in removal of dimers from the DNA of UV-irradiated cells.     

Extensive sequence homology in the DNA coding for elongation factor Tu from Escherichia coli and the Chlamydomonas reinhardtii chloroplast.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.