An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications.

Transposons are mobile genetic elements and have been utilized as essential tools in genetics over the years. Though highly useful, many of the current transposon-based applications suffer from various limitations, the most notable of which are: (i) transposition is performed in vivo, typically species specifically, and as a multistep process; (ii) accuracy and/or efficiency of the in vivo or in vitro transposition reaction is not optimal; (iii) a limited set of target sites is used. We describe here a genetic analysis methodology that is based on bacteriophage Mu DNA transposition and circumvents such limitations. The Mu transposon tool is composed of only a few components and utilizes a highly efficient and accurate in vitro DNA transposition reaction with a low stringency of target preference. The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies. The Mu methodology is readily applicable in a variety of current and emerging transposon-based techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins.     

Mechanism of Mu DNA transposition

The study of Mu DNA transposition in vitro has resulted in a much better understanding of the biochemical details of the transposition process. An early step in transposition is the generation of a 5th structure which is the product of the strand-tansfer reaction. The polarity of the strand transfer has been determined and substantial progress has been made on the role of the individual proteins. Moreover, the strand-transfer reaction is mediated by stable protein–DNA complexes, or transposomes, and the reaction can be divided into two sequential steps. The role of the transposomes and the requirement for a supercoiled Mu DNA substrate are also discussed.     

Mini-Mu mediates deletion-inversions in vivo by intra-transposon transposition

We have shown that a mini-Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion-inversion product, which has previously been observed in vitro, is formed by non-replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini-Mu can undergo non-replicative reactions in vivo and that these generate a 5 bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition.

The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.     

Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation. In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations.

We have examined the effects of a T----C point mutation at the terminal nucleotide of the Mu ends in a mini-Mu plasmid on the early steps in the in vitro transposition reaction. These mutations inhibit the introduction of nicks at the Mu ends in a reaction with Mu A, HU, and integration host factor proteins. The presence of the point mutation at either the left end or the right end is sufficient to block the nicking reaction at both ends, indicating that the reaction is normally concerted. Addition of Mu B and ATP, however, dramatically stimulates the reaction of mutant mini-Mu plasmids carrying the mutation at one end but not at both ends. The data suggest that the Mu B protein mediates its effect through direct interaction with Mu A and that Mu B may play a role in an earlier step in the transposition process than previously proposed. In the presence of Mu B, two products are observed with the left end or right end mutant mini-Mu plasmids, a normal protein-DNA intermediate (Type 1 complex) which contains nicks at both Mu ends and an abortive product composed of free relaxed plasmid which is nicked only at the wild-type end. Furthermore, stable protein-DNA complexes characteristic of the first step in the in vitro transposition reaction are not observed in the absence of nicking or when only one end is a nicked; the introduction of nicks at both Mu ends is a prerequisite for stable transpososome assembly.     

Genetic Study of the Loss and Restoration of Mutator Transposon Activity in Maize: Evidence against Dominant-Negative Regulator Associated with Loss of Activity

The Mutator system of transposable elements is characterized by a family of transposons called Mu transposons that share common termini and are actively transposing in Robertson's Mutator (Mu) lines of maize. Mu lines lose transposition activity during propagation by either outcrossing or inbreeding. This loss of transposition activity, which can occur at non-Mendelian frequencies, is in the form of loss of forward transposition activity resulting in a decrease in the generation of new mutations, as well as the loss of mutability of Mu transposon induced mutations, and it has been correlated with hypermethylation of the Mu elements. Previous studies have concluded that restoration of Mutator transposon activity by crossing inactive lines back to active lines is incomplete or transient, and depends upon the sex of the inactive parent. Further, it has been proposed that the inactive system is dominant to the active system, with the dominance possibly mediated through a negative regulatory factor that is preferentially transmitted through the female. In this study, we have examined the frequencies of loss and restoration of Mu transposon activity using a Mu line carrying an insertion in the bronze 1 locus. We find that transmission of Mu transposon activity to non-Mu plants can occur at high rates through males and females, but individual cases of decreased transmission through the male were observed. We also find that in crosses between inactive-Mu and active-Mu plants, reactivation was efficient as well as heritable, regardless of the sex of the inactive parent. Similar results were obtained whether the inactivation occurred in an outcross or a self. In all cases examined, loss of Mu transposon activity was correlated with hypermethylation of Mu elements, and reactivation was correlated with their demethylation. Our results indicate that an inactive Mu system does not exhibit dominance over an active Mu system. We conclude that contrary to current models, inactivation and its maintenance is not obligatorily associated with a dominant negative regulatory factor whether nuclear or cytoplasmic, and we propose a revised model to account for these and other observations.     

Abnormal cointegrate structures mediated by gene B mutants of phage Mu: their implications with regard to gene function

Summary A system where the transposition of MupApl (a derivative of phage Mu carrying a determinant coding for ampicillin resistance) is followed from the small plasmid pML2 into the conjugative plasmid R388 has been used to investigate the influence on Mu transposition of B, an early Mu gene which is involved in normal phage DNA synthesis. In the absence of active B protein a low level (about 1% of normal) of transposition was detected. Roughly a third of these transpositional events was found to lead to the formation of cointegrate DNA structures which were shown to consist of R388, two complete copies of Mu and part only of pML2. The pML2 deletions vary in size but all those investigated appear to originate at an end of Mu. An explanation of these observations is proposed which envisages the B protein as part of the normal transposition complex.     

Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes

Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K. (1987) Cell 51, 493-501). Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins. Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU. The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes. Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B. However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl. Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product. Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.     

Regulation of bacteriophage Mu transposition

Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.     

Localisation of mini-Mu in its replication intermediates.

We have located Mu delta 26 sequences straddling the forks in DNA structures which appear during Mu delta 26 replication, i.e., keys, pending keys, dumb- bells , partially fused circles, and asymmetrical forks. This brings additional evidence that these structures are mini-Mu replication intermediates. The possible relationship between these structures and those predicted by the different models formulated to explain transposition in procaryotes is discussed.     

The effect of gamma-irradiation on mu DNA transposition and gene expression

Mobile genetic elements are a ubiquitous presence in the genomes of all well-studied organisms. The effect of genomic stress on the status and transposition of these elements has not, as yet, been extensively characterized. We have been using temperate, transposable bacteriophage Mu as a model system to examine the behavior of mobile genetic elements and have previously shown that many DNA-damaging agents did not induce a Mu prophage to enter the lytic cycle of multiple rounds of DNA transposition. To extend these results and to examine the possibility that they were a reflection of damage to the DNA substrate for Mu transposition, we have constructed a mini-Mu plasmid, pMD12, which contains the early region of Mu, flanked by both extremities required for transposition in cis, and the beginning of the transposase gene A fused in frame to the lacZ gene. This A'-lacZ fusion protein maintains beta-galactosidase enzymatic activity under the control of the expression of the Mu transposase A gene and thus, the capacity for Mu transposition can be easily monitored by assaying for beta-galactosidase. By measuring the amount of beta-galactosidase after various doses of gamma-irradiation, we found that doses of up to 75 krad had no effect on the expression of the Mu transposase gene A. This was confirmed by the lack of induction of a Mu prophage in strains containing a chromosomally inserted Mu genome. Although the plaque-forming units per colony-forming unit of strain CSH67, containing a chromosomally inserted lambda prophage, increased approximately 100-fold from 0 to 75 krad, no stimulation of induction of prophage Mu lytic growth was observed. We also found that plasmid pMD12 did not transpose and chromosomally associate upon gamma-irradiation. This supports the assertion that DNA-damaging agents, including gamma-rays, do not induce the transposition of prokaryotic mobile genetic elements.     

Neighboring plasmid sequences can affect Mini-Mu DNA transposition in the absence of expression of the bacteriophage Mu semi-essential early region

Bacteriophage Mu DNA, like other transposable elements, requires DNA sequences at both extremities to transpose. It has been previously demonstrated that the transposition activity of various transposons can be influenced by sequences outside their ends. We have found that alterations in the neighboring plasmid sequences near the right extremity of a Mini-Mu, inserted in the plasmid pSC101, can exert an influence on the efficiency of Mini-Mu DNA transposition when an induced helper Mu prophage contains a polar insertion in its semi-essential early region (SEER). The SEER of Mu is known to contain several genes that can affect DNA transposition, and our results suggest that some function(s), located in the SEER of Mu, may be required for optimizing transposition (and thus, replication) of Mu genomes from restrictive locations during the lytic cycle.     

Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition.

Tn10 transposes by a nonreplicative "cut and paste" mechanism. We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site. The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction. These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition. These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes. The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Mu DNA replication in vitro: Criteria for initiation

Summary An in vitro system for investigating Mu replication and transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates.Two conditions reported to specifically decrease Mu DNA replication in vivo (Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.     

Studies on a "jumping gene machine": higher-order nucleoprotein complexes in Mu DNA transposition.

Studies in my lab have focused on DNA transposition in the bacterial virus, Mu. In vitro studies have shown that Mu DNA transposition is a three-step process involving DNA breakage, strand transfer and DNA replication. In the first step, a nick is introduced at each end of the transposon. The liberated 3'-OH groups subsequently attack a target DNA molecule resulting in strand transfer. The transposon DNA, now covalently linked to the target, is finally replicated to generate the transposition end-product, referred to as a cointegrate. The DNA cleavage and strand transfer reactions are mediated by a "jumping gene machine" or transpososomes, which we discovered in 1987. They are assembled by bringing together three different DNA regions via a process involving multiple protein-DNA and protein-protein interactions. The action of four different proteins is required in addition to protein-induced DNA bending or wrapping to overcome the intrinsic stiffness of DNA, which would ordinarily prohibit the assembly of such a structure. Transpososome assembly is a gradual process involving multiple steps with an inherent flexibility whereby alternate pathways can be used in the assembly process, biasing the reaction towards completion under different conditions.     

Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains.

Two mutations within the transposase (the A protein) gene of phage Mu with distinct effects on DNA transposition have been studied. The first mutation maps to the central domain (domain II) of A, a protein consisting of three major structural domains. The variant protein is normal in synapsis and cleavage of Mu ends but is temperature-sensitive in the strand transfer reaction, joining the Mu ends to target DNA. The second mutation is a deletion at the C terminus (within domain III); on the basis of genetic studies, the mutant protein is predicted to have lost the ability to interact with the Mu B protein. The B protein, in conjunction with A, promotes efficient intermolecular transposition, while inhibiting intramolecular transposition. We show that the purified mutant protein is proficient in intramolecular, but not intermolecular transposition in vitro. The interactions between A and B proteins have been followed by a proteolysis assay. The chymotrypsin sensitivity of the interdomainal Phe221-Ser222 peptide bond within the bidomainally organized B protein is exquisitely modulated by ATP, DNA and A protein. The sensitive or "open" state of this bond in native B protein becomes partially "open" upon binding of ATP by B, attains a "closed" or resistant configuration upon binding of DNA in presence of ATP, and is rendered "open" again upon addition of the A protein. In this test for the interaction of A protein with B protein-DNA complex, the domain II mutant behaves like wild-type A protein. However, the domain III mutant fails to restore chymotrypsin susceptibility of the Phe221-Ser222 bond.     

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage λ p_L promoter. Derepression of p_L leads to a high frequency of mini-Mu transposition (5.6 × 10⁻²) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.     

Bacteriophage Mu-1: A tool to transpose and to localize bacterial genes

In a previous publication (Faelen et al., 1975), it was predicted that the temperate phage Mu-1 would mediate transposition of bacterial genes. Here we show that this is indeed the case. By mating either induced F′ strains (which carry a thermoinducible Mu prophage in the bacterial chromosome), or sensitive F′ infected with Mu, with appropriate recipients, we were able to isolate new F′ episomes which carry various lengths of bacterial DNA. The frequency of transposition of a given marker can be as high as 10^?4. The episomes which carry the transposed DNA always carry Mu as well. When this is coupled with the fact that induction or infection with Mu is necessary for transposition to occur, it is probable that both Mu enzymes and Mu DNA are required by the transposition process. Episomes selected for the presence of a given marker were analyzed for the presence of unselected markers. It was found that: (1) only markers linked to the selected marker can be cotransposed with it; (2) when two markers are simultaneously transposed, all markers lying between them on the chromosome are also transposed; (3) the frequency at which an unselected marker is cotransposed is in some way related to the distance between that marker and the selected marker; (4) the transposition process occurs in both Rec⁺ and Rec^? strains. Mu-mediated transposition offers a new way to isolate F′ episomes and to localize and order bacterial genes as far apart as three minutes.