Search PubMed⌕ Search

Biomedical subjects

N Kleckner

Publications and source records attributed to N Kleckner.

At least 109 records · Page 6Linked to original sources
PubMed1984

Multiple IS10 rearrangements in Escherichia coli.

We have investigated the occurrence of multiple transposon-promoted chromosomal rearrangements in Escherichia coli K12 strains containing transposon Tn10. We show that a single Tn10 element, with its two closely spaced insertion sequence (IS10) elements, frequently gives rise to complex rearrangements that can be accounted for as the sum of two "classical" IS10 events. Using a strain containing differentially marked Tn10 elements at widely separated locations, we have investigated the possibility that IS10-promoted rearrangements occur in cell-wide "bursts", as expected if cells could occasionally undergo brief periods when all IS10 transposition events were activated, interspersed with longer periods of relative quiescence. We find no evidence for strong (greater than 60-fold), periodic cell-wide activation under our experimental conditions. The sensitivity of this experiment has been evaluated using an expression for the accumulation of double mutations in populations with heterogeneous, fluctuating mutation rates (see Appendix). We discuss several mechanisms by which two closely linked IS10 elements could undergo coupled double events without cell-wide activation: local activation of small chromosomal regions, periodic bursts of synthesis of cis-acting transposase protein, and/or a propensity for elements that have actually engaged in one rearrangement event to initiate a second successive event immediately thereafter. We favor the last possibility.

Base Sequence↗
PubMed1984

Transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo.

We present evidence that Tn10 transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo. Plasmids containing a shortened Tn10 element and a transposase overproducer fusion give rise, upon transposase induction, to new DNA species. The most prominent class is a circularized transposon molecule whose structure suggests that it arises from double strand breakage at the two transposon ends followed by covalent joining between the 3' and 5' ends of one of the two strands. We have used formation of the circularized transposon as a physical assay for the interaction between transposase and different mutant and wild-type termini. These experiments show that transposase protein interacts preferentially with the genetically most active termini in a way that suppresses productive interaction with weaker termini present on the same substrate molecule.

Base Sequence↗
PubMed1984

New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition.

We describe below several new variants of the tetracycline-resistance transposon Tn10 which are more useful than the wild-type transposon for many types of genetic and physical analysis of bacteria. These derivatives have one or more of the following new properties: (i) new drug resistance markers; (ii) high transposition frequencies; (iii) removal of the transposase gene to a position outside of the transposing segment; (iv) internal deletions which eliminate the ability of Tn10 to make adjacent deletion/inversions; or (v) addition of a trp-lac operon fusion segment just inside one terminus such that insertion can automatically generate a transcriptional fusion to the interrupted operon. Phage and plasmid vehicles carrying these new elements are described.

DNA Transposable Elements↗
PubMed1984

Essential sites at transposon Tn 10 termini.

We describe here point and deletion mutations that define which sequences at the termini of Tn10 are essential for transposition. We conclude that at least 13 and no more than 27 base pairs of terminal IS10 sequence are absolutely required at each end. These sequences correspond closely to the terminal inverted repeats of IS10. Sequences between base pairs 27 and 70 at each terminus and certain non-IS10 sequences can also influence transposition, but to a lesser degree. We also describe properties of many function-defective Tn10 transposition mutants and one exceptional Tn10 mutant.

Base Sequence↗
PubMed1984

Unusual alleles of recB and recC stimulate excision of inverted repeat transposons Tn10 and Tn5.

Precise and nearly precise excision of transposon Tn10 occur by host-mediated processes unrelated to transposition. Both types of excision involve interactions between short (9 or 24 base-pair) direct repeat sequences at or near the termini of the transposon and are stimulated by the large (1,329-base-pair) inverted repeats that form the ends of Tn10. We describe here three mutations of Escherichia coli K-12, designated texA, that enhance excision of Tn10 and of the structurally analogous transposon Tn5. Genetic mapping and complementation analysis show that these mutations are unusual alleles of the recB and recC genes that alter but do not abolish RecBC function. As Tn10 excision normally does not depend on RecA or RecBC functions, texA mutations appear to provide another pathway for excision that depends on altered RecBC function; for one texA allele, excision has become dependent on RecA function as well. The available evidence suggests that texA mutations alter the stimulatory interaction between the inverted repeats of Tn10.

Alleles↗
PubMed1983

Tn10 transposase acts preferentially on nearby transposon ends in vivo.

Transposition of Tn10 requires sites at the termini of the element and one essential transposon-encoded function, "transposase", which acts at those termini. Genetic complementation experiments reveal that this "transposase" function works much more efficiently on transposon ends located near the gene from which they are expressed than on transposon ends located at a distance. This property accounts for the failure of mutant Tn10 elements to be efficiently complemented in trans. The failure of transposase protein to move freely in three dimensions could be explained by one-dimensional diffusion, energy-dependent translocation and/or extreme protein lability. Additional genetic analyses demonstrate that the rate of Tn10 transposition in vivo depends upon the length of the transposon and the amount of transposase protein. Function dependence and length dependence are independent aspects of the transposition process that could correspond to the break/join and replication aspects into which transposition has been separated conceptually.

Bacteriophage lambda↗
PubMed1983

Three promoters near the termini of IS10: pIN, pOUT, and pIII.

We have identified three IS10-encoded promoters, pIN, the promoter for IS10's transposase gene, is intrinsically weak, contributing to the low frequency of IS10 transposition in vivo. Its transcripts begin near the "outside" end of IS10 and extend inward across the element. pOUT, a strong promoter just internal to and opposing pIN, directs transcription outward. Its transcripts are proposed to inhibit translation of the transposase gene in trans (accompanying paper). pOUT may also inhibit transcription from pIN in cis. pIII, a weak promoter near the "inside" end of IS10, is of unknown genetic importance. Many transposable elements activate, by adjacent insertion, silent genes lacking normal promoters. Such IS10-promoted turn-on is mediated by pOUT and results from continuation of pOUT-initiated transcripts past the IS10 terminus, into adjoining chromosomal material. Wild-type and mutant IS10 promoters have been analyzed in vitro. pIN is weaker than pOUT because of inefficient isomerization from closed to open complexes. Despite their proximity, pIN and pOUT do not interact before or during open complex formation.

Base Sequence↗
PubMed1983

Translational control of IS10 transposition.

We present genetic evidence that insertion sequence IS10, the active element in transposon Tn10, can negatively control expression of its own transposase protein at the translational level. This control process is manifested in trans in a phenomenon called "multicopy inhibition": the presence of a multicopy plasmid containing IS10 inhibits transposition of a single copy chromosomal Tn10 element by reducing its ability to express transposition functions. Fusion analysis suggests that expression is reduced at the translational and not the transcriptional level. Only the outer 180 bp of IS10-Right are required on the plasmid for full inhibition. Plasmid-encoded transposase protein is not involved. The genetic structure of the essential plasmid region and the effects of point and deletion mutations on multicopy inhibition lead us to propose that inhibition of transposase translation occurs by direct pairing between the transposase messenger RNA and a small, complementary, regulatory RNA specified by the IS10-encoded pOUT promoter.

DNA Transposable Elements↗
PubMed1982

Mutants of Escherichia coli K12 which affect excision of transposon Tn10.

We have described three illegitimate recombination events associated with, but not promoted by, transposon Tn10: precise excision, nearly precise excision, and precise excision of a nearly precise excision remnant. All three are structurally analogous: excision occurs between two short direct repeat sequences, removing all intervening material plus one copy of the direct repeat. In each case, the direct repeats border a larger inverted repeat. We report here the isolation of host mutants of Escherichia coli K12 which exhibit increased frequencies of precise excision of Tn10. Nineteen of the 39 mutants have been mapped to five distinct loci on the E. coli genetic map and have been designated texA through texE (for Tn10 excision). Mapping and genetic characterization indicate that each tex gene corresponds to a previously identified gene involved in cellular DNA metabolism: recB and/or recC, uvrD, mutH, mutS, and dam. The role of these various DNA repair and recombination genes in an illegitimate recombination process such as Tn10 excision will be discussed. In addition to an increase in precise excision frequency, all 39 tex mutants display an increased frequency for nearly precise excision. However, none of the mutants are increased for the third excision event, precise excision of a nearly precise excision remnant, supporting the idea that precise and nearly precise excision occur by closely related pathways which are distinct from those pathways which promote the third type of excision event.

Alleles↗
PubMed1982

A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity.

Transposon Tn10 inserts at many sites in the bacterial chromosome, but preferentially inserts at particular hotspots. We believe we have identified the target DNA signal responsible for this specificity. We have determined the DNA sequences of 11 Tn10 insertion sites and identified a particular 6 base pair (bp) symmetrical consensus sequence (GCTNAGC) common to those sites. The sequences at some sites differ from the consensus sequence but only in limited and well defined ways. The sequences at some sites differ from the consensus sequence than do sequences at other sites, and the consensus sequence and closely related sequences are generally absent from potential target regions where Tn10 is known not to insert. Other aspects of the target DNA can significantly influence the efficiency with which a particular target site sequence is used. The 6 bp consensus sequence is symmetrically located within the 9 bp target DNA sequence that is cleaved and duplicated during Tn10 insertion. This juxtaposition of recognition and cleavage sites plus the symmetry of the perfect consensus sequence suggest that the target DNA may be both recognized and cleaved by the symmetrically disposed subunits of a single protein, as suggested for type II restriction endonucleases. There is plausible homology between the consensus sequence and the very ends of Tn10, compatible with recognition of transposon ends and target DNA by the same protein. The sequences of actual insertion sites deviate from the perfect consensus sequence in a way which suggests that the 6 bp specificity determinant may be recognized through protein-DNA contacts along the major groove of the DNA double helix.

Base Sequence↗
PubMed1982

DNA sequence organization of IS10-right of Tn10 and comparison with IS10-left.

Tn10 is 9,300 base pairs long and has inverted repeats of an insertion sequence (IS)-like sequence (IS10) at its ends. IS10-right provides all of the Tn10-encoded functions used for normal Tn10 transposition. IS10-left can also provide these functions but at a much reduced level. We report here the complete nucleotide sequence of IS10-right and a partial sequence of IS10-left. From our analysis of this information, we draw the following conclusions. (i) IS10-right is 1,329 base pairs long. Like most IS elements, it has short (23-base pair) nearly perfect inverted repeats at its termini. We can divide these 23-base pair segments into at least two functionally distinct parts. IS10-right also shares with other elements the presence of a single long coding region that extends the entire length of the element. Genetic evidence suggests that this coding region specifies an essential IS10 transposition function. A second, overlapping, coding region may or may not be important. (ii) The "outside" end of IS10-right contains three suggestively positioned internal symmetries. Two of these (A1 and A2) are nearly identical in sequence. Symmetry A1 overlaps the terminal inverted repeat; symmetry A2 overlaps the promoter shown elsewhere to be responsible for expression of IS10 functions and lies very near a second characterized promoter that directs transcription outward across the end of IS10. Symmetries A1 and A2 may play a role in modulation of Tn10 activity and are likely to function at least in part as protein recognition sites. We propose that the third symmetry (B) acts to prevent fortuitous expression of IS10 functions from external promoters. The transcripts from such promoters can assume a stable secondary structure in which the AUG start codon of the long coding region is sequestered in a region of double-stranded mRNA formed by pairing between the two halves of symmetry B. (iii) IS10-left differs from IS10-right at many nucleotide positions in both the presumptive regulatory region and the long coding region. The available evidence suggests that Tn10 may be older than other analyzed drug-resistance transposons and thus have had more time to accumulate mutational changes.

Base Composition↗
PubMed1982

Transposon Tn10: genetic organization, regulation, and insertion specificity.

Transposon Tn10 is a composite element in which two individual insertion sequence (IS)-like sequences cooperate to mediate transposition of the intervening material. The two flanking IS10 elements are not identical; IS10-right is responsible for functions required to promote transposition, and IS10-left is defective in transposition functions. We suggest that the two IS10 elements were originally identical in sequence and have subsequently diverged. IS10-right is compactly organized with structural gene(s), promoters, and sites important for transposition and (presumably) its regulation all closely linked and, in some cases, overlapping. IS10 has a single major coding region that almost certainly encodes an essential transposition function. A pair of opposing promoters flank the start of this coding region. One of these promoters is responsible for expression in vivo of transposon-encoded transposition functions. We propose that the second promoter is involved in modulation of Tn10 transposition. Genetic analysis suggests that transposon-encoded function(s) may be preferentially cis-acting. Insertion of Tn10 into particular preferred target sites is due primarily to the occurrence of a particular six-base pair target DNA sequence. The properties of this sequence suggest that symmetrically disposed subunits of a single protein may be responsible for both recognition and cleavage of target DNA during insertion.

Base Sequence↗
PubMed1981

Genetic organization of transposon Tn10.

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a "high hopper" Tn10 element.

Base Sequence↗
PubMed1981

Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats.

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.

Base Sequence↗