The rate of Dam-mediated DNA adenine methylation in Escherichia coli.
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Adenine↗
Biomedical subjects
N Kleckner
Publications and source records attributed to N Kleckner.
Translation start of IS10 transposase protein.
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DNA Transposable Elements↗
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.
Bacteriophage lambda↗
Biological regulation by antisense RNA in prokaryotes.
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Bacteriophage lambda↗
Tn10 transposition and circle formation in vitro.
We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.
Adenosine Triphosphatases↗
Improved single and multicopy lac-based cloning vectors for protein and operon fusions.
We describe several new vectors for the construction of operon and protein fusions to the Escherichia coli lacZ gene. In vitro constructions utilize multicopy plasmids containing suitable cloning sites located between upstream transcription terminators and downstream lac operon segments whose lacZ genes retain or lack translational start signals. Single-copy lambda prophage versions of multicopy constructs can be made genetically, without in vitro manipulation. The new vectors, both single and multicopy, are improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals. These vectors were developed for analysis of the expression of IS10's transposase gene, which is transcribed less than, once per generation, and whose transcripts are translated on average less than once each. Both single and multicopy constructs can also be used to select mutations affecting fusion expression, and mutations isolated in single-copy constructs can be crossed genetically back onto multicopy plasmids for further analysis.
Gene Expression Regulation↗
Efficient Tn10 transposition into a DNA insertion hot spot in vivo requires the 5-methyl groups of symmetrically disposed thymines within the hot-spot consensus sequence.
Transposon Tn10 inserts preferentially at particular insertion "hot spots" that share a symmetrical 6-base-pair consensus sequence: 5' GCTNAGC 3'. The protein that recognizes this sequence is not known but is likely to be the Tn10-encoded transposase protein. We present evidence that the 5-methyl groups of the two thymines in this sequence are essential for efficient transposon insertion; in their absence the sequence is still recognized, but at lower efficiency. We have reached this conclusion by examination of a specific hot spot whose sequence is 5' GCCAGGC 3'. The innermost cytosines of this sequence happen to be substrates for methylation at their 5 positions by the bacterial dcm-encoded methylase. We find that Tn10 transposes into this site 15 times more frequently in a Dcm+ host than in a Dcm- host; in the Dcm- host, insertions still occur, but at a low frequency. Thus, at this site, the absence of pyrimidine 5-methyl groups at the third positions of the consensus sequence is sufficient to convert a strong insertion hot spot into a weaker but still recognizable hot spot. This observation supports the general proposition, suggested previously by comparisons among consensus sequences, that the presence or absence of these 5-methyl groups is one major feature that can make the difference between a strong and a weak Tn10 insertion hot spot.
Base Sequence↗
A new generalizable test for detection of mutations affecting Tn10 transposition.
We describe here a new rapid screen that allows easy detection of transposon or host mutations that affect Tn10 transposition in Escherichia coli. This test involves a new Tn10 derivative called the "mini-lacZ-kanR fusion hopper" or mini-Tn10-LK for short. This element does not direct expression of beta-galactosidase when present at its original starting location on a suitably engineered plasmid or phage genome because it lacks appropriate transcription and translation start signals. However, transposition of this element into the chromosome of E. coli lacZ- bacteria leads to productive fusions in which the lacZ gene within the transposon is expressed from external chromosomal signals. Such fusions are readily detectable on MacConkey lactose indicator plates as red (Lac+) papillae inside of white (LacZ-) colonies. The length of time required to see red papillae appearing in a white colony sensitively and accurately reflects the transposition frequency of the mini-transposon within the colonies. Differences in times for color formation are sensitive enough that 10-fold differences in transposition frequency can readily be detected. This papillation assay can be used to identify mutant clones in which the frequency of Tn10 transposition is either increased or decreased. We have successfully used the assay to identify mutations in the terminal sequences of Tn10; mutations in the Tn10 transposase gene or the bacterial host can be isolated just as easily. This screen should be readily adaptable to transposable elements other than Tn10.
Bacteriophage lambda↗
A Tn10-lacZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes.
We describe here a new variant of transposon Tn10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn10-LUK system has several important advantages. Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. Under favorable circumstances, usable lacZ protein fusions are created. Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.
Base Sequence↗
Physical analysis of Tn10- and IS10-promoted transpositions and rearrangements.
We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10(-4) per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.
Chromosome Deletion↗
A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast.
We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5'-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.
Carboxy-Lyases↗
A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains.
In this paper, we describe a 3.8-kb molecular construct that we have used to disrupt yeast genes. The construct consists of a functional yeast URA3 gene flanked by 1.1-kb direct repeats of a bacterial sequence. It is straightforward to insert the 3.8-kb segment into a cloned target gene of interest and then introduce the resulting disruption into the yeast genome by integrative transformation. An appropriate DNA fragment containing the disruption plus flanking homology can be obtained by restriction enzyme digestion. After introducing such fragments into yeast by transformation, stable integrants can be isolated by selection for Ura+. The important feature of this construct that makes it especially useful is that recombination between the flanking direct repeats occurs at a high frequency (10(-4)) in vegetatively grown cultures. After excision, only one copy of the repeat sequence remains behind. Thus in the resulting strain, the Ura+ selection can be used again, either to disrupt a second gene in similar fashion or for another purpose.
Escherichia coli↗
Genetic evidence that Tn10 transposes by a nonreplicative mechanism.
We present genetic evidence that the tetracycline resistance element Tn10 transposes by a nonreplicative mechanism. Heteroduplex Tn10 elements containing three single base pair mismatches were constructed on lambda phage genomes and allowed to transpose from lambda into the bacterial chromosome. Analysis of TetR colonies resulting from such transpositions suggests that information from both strands of the transposing Tn10 element is transmitted faithfully to its transposition product. The simplest interpretation of these results is that the transposing element is excised from the donor molecule and inserted into the target molecule without being replicated. A mismatch 70 base pairs from one end of the transposon is preserved, suggesting that there is little or no replication, even at the termini of the element, during transposition in vivo.
Bacteriophage lambda↗
Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene.
We have found that IS10 transposase is synthesized in tiny amounts, about 0.15 polypeptide chain per cell per generation on average, as judged from the beta-galactosidase activity of a single chromosomal copy of a suitable transposase-lacZ gene fusion. Enzymatic activity from the fusion gene is a factor of 10 lower in a permeabilized whole cell assay than in cell extracts. Probably, most cells contain fewer than four polypeptide chains, and these chains can assemble into active tetramers only after cell disruption. This interpretation permits formulation of two equations relating enzyme activities to transcription and translation rates, solution of which reveals that the fusion gene is expressed at the average rate of only 0.25 transcript per cell per generation, with an average of only 0.58 translation product per transcript. This methodology is generally applicable to analysis of any gene from which fewer than four polypeptide chains are synthesized per cell per generation.
Bacterial Proteins↗
IS10 transposition is regulated by DNA adenine methylation.
We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.
Adenine↗
Tn10 protects itself at two levels from fortuitous activation by external promoters.
Tn10 rarely transposes, primarily because its IS10-encoded transposase protein is synthesized infrequently. Since the 5' end of the transposase gene is immediately adjacent to flanking host sequences, insertion of Tn10 into an actively transcribed operon could conceivably result in dramatically increased transposition. We show here that Tn10 is protected from such fortuitous activation; high levels of transcription from an upstream promoter actually decrease its rate of transposition. Protection operates at two levels. First, externally-initiated transcripts yield only a small amount of additional transposase protein, primarily because of inhibition at a posttranscriptional level. We suggest that the transposase gene start codon is sequestered in an mRNA secondary structure not present in transcripts initiated at the normal promoter. Second, transcription per se across an IS10 terminus inhibits its activity, thus negating any small transposase increase. These observations provide additional evidence that Tn10 has evolved specific mechanisms for keeping its transposition activity low.
DNA Transposable Elements↗
Mismatch repair mutations of Escherichia coli K12 enhance transposon excision.
Excision of the prokaryotic transposon Tn10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions. To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E. coli K12, designated tex, which increase the frequency of Tn10 precise excision. Three of these mutations (texA) have been shown to qualitatively alter RecBC function. We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam. Previously identified alleles of these genes also have a Tex phenotype.--Several other E. coli mutations affecting related functions have been analyzed for their effects on Tn10 excision. Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn10 excision. Mutations ssb-113 and mutD5, however, do increase Tn10 excision.--The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo. Thus, mutations in these genes might also enhance transposon excision by a single general mechanism. Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms.
Bacterial Proteins↗
Transposition of plasmid-borne Tn10 elements does not exhibit simple length-dependence.
The transposition frequencies of Tn10 elements from the bacterial chromosome to an F epitome decrease 40% for every kilobase increase in transposon length. The basis for this relationship is not known. We have now examined complemented transposition of defective Tn10 elements off small multicopy plasmids. We find that length dependence in this situation is either reduced or absent, depending on the specific class of transposition events involved. These observations can be interpreted as evidence against the model that chromosomal length dependence occurs because of decay of a transposition-associated replicative complex. This interpretation is consistent with unrelated experiments suggesting that Tn10 transposition is normally nonreplicative. Alternative explanations of length dependence phenomena are discussed.
Chromosome Deletion↗