Benjamin HW, Kleckner N. Intramolecular transposition by Tn10. Cell. 1989;59 (2) :373-83.Abstract
Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.
Huisman O, Errada PR, Signon L, Kleckner N. Mutational analysis of IS10's outside end. EMBO J. 1989;8 (7) :2101-9.Abstract
We present the genetic analysis of a large number of mutations in the outside end of insertion sequence IS10. (i) The terminal inverted repeat sequence is probably the primary site of transposase binding. Mutations in this region fall into phenotypic classes which correspond to their map locations, suggesting that this region may consist of several distinct functional segments. Similarities between the organization of IS10's inverted repeat and those of other transposable elements are discussed. (ii) Base pairs 23-42 include a consensus binding sequence for one of the IS10 transposition host factors, IHF. The phenotypes of mutations in this region suggest that IHF is the major host factor for outside-end transposition activity in vivo and that base pairs throughout this region are important for the IHF interaction. (iii) Mutations in bp 43-61 do not affect outside-end transposition activity but do affect, in expected ways, previously identified determinants involved in expression and regulation of transposase. (iv) Some mutations in bp 23-42 also affect transposase expression; the possibility that IHF negatively regulates transcription initiation is discussed.
Haniford DB, Chelouche AR, Kleckner N. A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment. Cell. 1989;59 (2) :385-94.Abstract
We report the identification and characterization of a class of IS10 transposase mutants that carry out only some of the steps required for transposition. These mutants were identified among transposition-defective mutants as a specific subclass that retains the wild-type ability to induce SOS functions in the presence of transposon ends. Mutants of this class successfully promote excision of the element from its donor site, but do not promote transfer of the transposon sequences to a target site. SOS induction presumably results from the degradation of the donor site. Uniquely among transposition-defective mutants, SOS+ Tnsp- mutants promote the formation of a new product, the excised transposon fragment (ETF), which consists of the transposon excised from the original donor molecule by double-strand breaks at the transposon ends. SOS+ Tnsp- mutants identified thus far define two patches of amino acids that might correspond to regions of different function. A single additional mutation maps within a region that is highly conserved among IS element transposases. The existence of SOS+ Tnsp- mutants and the structure of the ETF provide strong support for the previously proposed nonreplicative model of Tn10/IS10 transposition.
Alani E, Subbiah S, Kleckner N. The yeast RAD50 gene encodes a predicted 153-kD protein containing a purine nucleotide-binding domain and two large heptad-repeat regions. Genetics. 1989;122 (1) :47-57.Abstract
The RAD50 gene of Saccharomyces cerevisiae is required for chromosome synapsis and recombination during meiosis and for repair of DNA damage during vegetative growth. The precise role of the RAD50 gene product in these processes is not known. Most rad50 mutant phenotypes can be explained by the proposal that the RAD50 gene product is involved in the search for homology between interacting DNA molecules or chromosomes, but there is no direct evidence for this model. We present here the nucleotide sequence of the RAD50 locus and an analysis of the predicted 153-kD RAD50 protein. The amino terminal region of the predicted protein contains residues suggestive of a purine nucleotide binding domain, most likely for adenine. The remaining 1170 amino acids consist of two 250 amino acid segments of heptad repeat sequence separated by 320 amino acids, plus a short hydrophobic carboxy-terminal tail. Heptad repeats occur in proteins such as myosin and intermediate filaments that form alpha-helical coiled coils. One of the two heptad regions in RAD50 shows similarity to the S-2 domain of rabbit myosin beyond that expected for two random coiled coil proteins.
Kittle JD, Simons RW, Lee J, Kleckner N. Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA. J Mol Biol. 1989;210 (3) :561-72.Abstract
Transposition of insertion sequence IS10 is regulated by an anti-sense RNA which inhibits transposase expression when IS10 is present in multiple copies per cell. The anti-sense RNA (RNA-OUT) consists of a stem domain topped by a flexibly paired loop; the 5' end of the target molecule, RNA-IN, is complementary to the top of the loop, and complementarity extends for 35 base-pairs down one side of RNA-OUT. We present here genetic evidence that anti-sense pairing, both in vitro and in vivo, initiates by interaction of the 5' end of RNA-IN and the loop domain of RNA-OUT; other features of the reaction are discussed. In the context of this model, we discuss features of this anti-sense system which are important for its biological effectiveness, and suggest that IS10 provides a convenient model for design of efficient artificial anti-sense RNA molecules.
Case CC, Roels SM, Jensen PD, Lee J, Kleckner N, Simons RW. The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO J. 1989;8 (13) :4297-305.Abstract
IS10 transposition is regulated by an approximately 70 nt anti-sense RNA, RNA-OUT. RNA-OUT folds into a duplex 'stem-domain' topped by a loosely paired 'loop-domain'. The loop-domain is critical for RNA-RNA pairing per se; pairing initiates by interaction of the RNA-OUT loop with the 5' end of the target mRNA. We show here that RNA-OUT is unusually stable in vivo (half-life 60 min) and that this stability is conferred by specific features of the RNA-OUT stem-domain. One critical feature is stable base-pairing: mutations that disrupt stem pairing destabilize RNA-OUT in vivo and abolish anti-sense control; combinations of mutations that restore pairing also restore both stability and control. We propose that the stem renders RNA-OUT resistant to 3' exoribonucleases. Other features of the stem-domain prevent this essential duplex from being an effective substrate for double-strand nucleases: two single base mutations disrupt antisense control by making RNA-OUT susceptible to RNase III. Mutations in the loop region have little effect on RNA-OUT stability. Implications for IS10 biology and the design of efficient anti-sense RNAs are discussed.
Simons RW, Kleckner N. Biological regulation by antisense RNA in prokaryotes. Annu Rev Genet. 1988;22 :567-600.
Campbell JL, Kleckner N. The rate of Dam-mediated DNA adenine methylation in Escherichia coli. Gene. 1988;74 (1) :189-90.
Roberts D, Kleckner N. Tn10 transposition promotes RecA-dependent induction of a lambda prophage. Proc Natl Acad Sci U S A. 1988;85 (16) :6037-41.Abstract
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.
Sen J, Septak M, Jain C, Kleckner N. Translation start of IS10 transposase protein. Nucleic Acids Res. 1988;16 (10) :4730.
Simons RW, Houman F, Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53 (1) :85-96.Abstract
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.
Alani E, Cao L, Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987;116 (4) :541-5.Abstract
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.
Huisman O, Kleckner N. A new generalizable test for detection of mutations affecting Tn10 transposition. Genetics. 1987;116 (2) :185-9.Abstract
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.
Alani E, Kleckner N. A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast. Genetics. 1987;117 (1) :5-12.Abstract
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.
Shen MM, Raleigh EA, Kleckner N. Physical analysis of Tn10- and IS10-promoted transpositions and rearrangements. Genetics. 1987;116 (3) :359-69.Abstract
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.
Huisman O, Raymond W, Froehlich KU, Errada P, Kleckner N, Botstein D, Hoyt MA. A Tn10-lacZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes. Genetics. 1987;116 (2) :191-9.Abstract
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.
Lee SY, Butler D, Kleckner N. 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. Proc Natl Acad Sci U S A. 1987;84 (22) :7876-80.Abstract
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.
Morisato D, Kleckner N. Tn10 transposition and circle formation in vitro. Cell. 1987;51 (1) :101-11.Abstract
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.
Bender J, Kleckner N. Genetic evidence that Tn10 transposes by a nonreplicative mechanism. Cell. 1986;45 (6) :801-15.Abstract
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.
Raleigh EA, Kleckner N. Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene. Proc Natl Acad Sci U S A. 1986;83 (6) :1787-91.Abstract
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.