Search PubMed⌕ Search

Biomedical subjects

D E Berg

Publications and source records attributed to D E Berg.

At least 127 records · Page 7Linked to original sources
PubMed1990

Intramolecular transposition by a synthetic IS50 (Tn5) derivative.

We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences.

Base Sequence↗
PubMed1990

Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes.

The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of interest, providing a useful tool for studying virulence and other important traits of diverse bacterial species.

Adenylyl Cyclases↗
PubMed1990

Reoperation: cardiac surgery.

The subject of cardiac reoperation in general has been infrequently discussed in the medical literature and has not yet appeared in the nursing literature. Yet reoperation presents a real challenge, accentuating some problems that are also associated with primary cases and posing some considerations that are unique to the reoperative situation. The objectives of this article are to discuss the reasons prompting reoperation for coronary revascularization, identify technical problems associated with reoperative coronary artery surgery, explore potential avenues for decreasing the need for reoperation, and discuss potential patient care problems in the immediate postoperative critical care setting.

Cardiac Surgical Procedures↗
PubMed1989

Saturation mutagenesis of the inside end of insertion sequence IS50.

A 19-bp segment at the inside (I) end of IS50 (Tn5) is needed for efficient transposition. The importance of each position was assayed by making at least one base substitution at each position by either chemical-or oligodeoxyribonucleotide-directed mutagenesis. Mutant I ends were paired with a wild-type (wt) segment from the outside (O) end of IS50 and the transposase (tnp) gene was placed either between the ends or 1200 bp from the O end. The frequency of transposition of the resultant elements to bacteriophage lambda was measured. At least one substitution at each of the 19 I-end positions decreased transposition activity to less than 25% of wt, and most substitutions (25 of 28) decreased it to less than 5% of wt from one or both donor plasmids. These results show that each position in the I end is important during transposition.

Base Sequence↗
PubMed1989

Transposition effect of adenine (Dam) methylation on activity of O end mutants of IS50.

The two ends of insertion sequence IS50 (from Tn5) differ in sequence and in activity during transposition: the IS50 I end contains DNA adenine methylation (Dam) sites and is affected directly by Dam methylation, whereas the O end lacks Dam sites. The effect of Dam methylation on the transposition of IS50-derived elements with base substitution mutations in their O ends was assayed to understand better how the divergent O and I ends interact. Of 31 O end mutations tested, ten impaired transposition less, and two impaired transposition more in Dam- than in Dam+ cells. These results suggest that the interaction between the two ends in a transposition complex is affected by the sequence or the extent of methylation of one end.

Adenine↗
PubMed1989

Specificity of deletion events in pBR322.

The reversion of mutations due to inserts of identical palindromic DNAs just 1-bp apart in the amp gene of plasmid pBR322 varied up to 3000-fold (U. DasGupta, K. Weston-Hafer, and D.E. Berg (1987) Genetics 115, 41-49). The experiments reported here show that the intrinsic frequencies of deletion from these sites are truly very different. Deletions were selected by the joint loss of sacB (sucrose sensitivity) and lacZ alpa genes cloned together at these sites, without requiring restoration of the ampr allele. We found that greater than 90% of deletions at each of these sites do restore the ampr allele. This result reinforces the view that the probability of forming a particular deletion depends strongly on the DNA sequence at its prospective endpoints.

Base Sequence↗
PubMed1989

Factors affecting transposition activity of IS50 and Tn5 ends.

The partially matched I and O ends of IS50 (the insertion sequence of the transposon Tn5) are needed for transposition, probably as the sites upon which the cis-acting transposase and host proteins act. To better understand how transposition is regulated we made a series of IS50-related elements in which the positions of the ends and of the transposase gene were varied systematically. Assays of these elements showed that the I and O ends differ inherently in transposition activity. Other workers showed that methylation, at DNA N6-adenine methyltransferase (Dam) recognition sites within the I end and the transposase tnp gene promoter, inhibits transposase synthesis and also I end activity. We show that the effect of Dammediated methylation on an I end depends on the end's orientation relative to the tnp gene. Further, in dam+ cells oriented like -tnp----in relation to the first and second ends) are (O, I) greater than (O, O) greater than or equal to (I, O) greater than (I, I). In dam- cells the relative activities are (O, I) = (I, O) = (I, I) greater than (O, O). Our results are consistent with a model orginally developed for IS10, in which hemi-methylation resulting from passage of a replication fork regulates transposition.

Bacteriophage lambda↗
PubMed1989

Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda.

We constructed a derivative of transposon Tn5 called Tn5supF for insertion mutagenesis and sequencing DNAs cloned in phage lambda. This element carries a supF amber-suppressor tRNA gene. Its insertion into lambda can be selected by plaque formation by using nonsuppressing (sup0) Escherichia coli for amber mutant lambda phage and sup0 dnaB-amber E. coli for nonamber lambda phage. Tn5supF is just 264 base pairs long. It transposes efficiently and inserts quasi-randomly into DNA targets. The unique sequences near its termini can be used as primer binding sites for dideoxynucleotide DNA sequencing, thus permitting the direct sequencing of DNAs cloned in phage lambda without subcloning.

Bacteriophage lambda↗
PubMed1989

Palindromy and the location of deletion endpoints in Escherichia coli.

The contributions of direct and inverted repeats to deletion formation were studied by characterizing Ampr revertants of plasmids with a series of insertion mutations at a specific site in the pBR322 ampicillin resistance (amp) gene. The inserts at this site are palindromic, variable in length, and bracketed by 9- or 10-bp direct repeats of amp sequence. There is an additional direct repeat composed of 4 bp within the insert and 4 bp of adjoining amp sequence. DNA sequencing and colony hybridization of Ampr revertants showed that they contained either the parental amp sequence, implying deletion endpoints in the flanking 9- or 10-bp repeats, or a specific 1-bp substitution, implying endpoints in the 4-bp repeats. Although generally direct repeats seem to be used as deletion endpoints with a frequency proportional to their lengths, we found that with uninterrupted palindromes longer than 32 bp, the majority of deletions ended in the 4 bp, not the 9- or 10-bp repeats. This preferential use of the shorter direct repeats associated with palindromes is interpreted according to a DNA synthesis-error model in which hairpin structures formed by intrastrand pairing foster the slippage of nascent strands during DNA synthesis.

Alleles↗
PubMed1989

Formation of supercoiling domains in plasmid pBR322.

Twin domains of positive and negative supercoiling are thought to form in DNA molecules whenever free rotation of a transcription complex around the DNA helix is impeded. Evidence for these domains has come from findings with Escherichia coli strains that are deficient in DNA topoisomerase I (top mutants) or that have been treated with DNA gyrase inhibitors. Plasmid pBR322 is highly supercoiled in these strains, whereas some of its deletion derivatives are not. The studies of pBR322 derivatives presented here show that high negative supercoiling in top strains requires translation as well as transcription of the first 98 codons of the tet gene and does not require the divergently transcribed amp gene. The N-terminal region of the TetA protein is thought to insert into the inner membrane. Our results favor models in which supercoiling domains are created when DNA segments are anchored to a large cellular structure via coupled transcription, translation, and membrane insertion of a nascent protein.

DNA Mutational Analysis↗
PubMed1988

Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites.

The sequencing of large DNA fragments by the chain-termination method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467] has generally required extensive manipulations to bring all parts of the fragment near a specific primer-binding site, or the repeated synthesis of new oligodeoxynucleotide primers. Here we develop a more efficient approach, the use of a transposable element to insert primer binding sites at random in the DNA of interest. We constructed a Tn5 derivative called Tn5seq1 with unique DNA segments near each end so that oligodeoxynucleotides matching them could serve as primers for sequencing in each direction from any Tn5seq1 insertion site. Our experiments demonstrate the use of Tn5seq1 for sequencing in pBR322 plasmids and also in uncloned DNAs of the Escherichia coli chromosome. The unique segments near the left and right ends of Tn5seq1 are promoters from phages T7 and SP6, respectively, to permit the efficient transcription of adjacent DNAs in vivo or in vitro.

Bacteriophage lambda↗
PubMed1988

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.

Cloning, Molecular↗
PubMed1988

Transposon Tn5 target specificity: preference for insertion at G/C pairs.

The procaryotic transposon Tn5 inserts into many different sites within a single gene, but some sites (hotspots) are targeted repeatedly. Hotspots are not closely related in sequence, but most have G/C pairs at the ends of the nine base pairs duplicated by Tn5 insertion. In pBR322, the major hotspot coincides with the "-10 region" of the tet promoter. We mutated the G/C pairs at this hotspot and assayed for insertion into hotspot I, resistance to tetracycline, and plasmid supercoiling. We found that changing the G/C pairs to A/T pairs reduced the frequency of insertion into the hotspot by at least fivefold. The reduction in hotspot use caused by these G/C to A/T changes was not attributable to changes in plasmid supercoiling or tet promoter strength.

Base Composition↗
PubMed1987

Specificity of bacteriophage Mu excision.

To study the excision of bacteriophage Mu at the DNA sequence level, the Mu-derived phage lambda placMu3 was transposed to the transcribed but non-translated leader region of a plasmid-borne tetracycline (tet) resistance gene. Revertants (excision products) were then selected by Tet+ restoration of Tet+ and characterized. Of 21 independent Tet+ revertants, 17 contained simple deletions of most or all of lambda placMu3, while the other four contained more complex rearrangements in which one end of lambda placMu3 had been transposed, and most of the prophage had been deleted. The deletion endpoints were found in short direct repeats in each of the complex rearrangements and in 11 of the 17 simple deletion excisants. The results suggest models of slipped mispairing of template and nascent DNA strands facilitated by proteins of the Mu transposition machinery.

Base Sequence↗
PubMed1987

Identification of base pairs in the outside end of insertion sequence IS50 that are needed for IS50 and Tn5 transposition.

Short DNA sequences at ends of transposable elements are needed as sites for transposition. Previous deletion mapping showed that, in Tn5 and its component IS50 elements, these essential sites are about 19 base pairs long. To determine which positions are important in transposition, we made one or more sequence changes at each position in the IS50 outside (O) end and assayed the effects of these changes on transposition. Our results indicate that the specific base pairs at 18 of the 19 positions are important in transposition. A 9-base-pair segment in the O end corresponds to a binding site for the Escherichia coli DnaA protein. Comparisons of effects of mutations at different positions in this site, and also measurements of Tn5 transposition in dnaA- cells, indicate that DnaA protein participates in O-end-mediated transposition.

Bacterial Proteins↗
PubMed1987

Local DNA sequence control of deletion formation in Escherichia coli plasmid pBR322.

The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations. Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long. We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition). Revertant frequencies were found to depend on the location and the sequence of the palindromic insert. Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold. We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs.

Base Sequence↗
PubMed1986

Localization of action of the IS50-encoded transposase protein.

The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp+), and the other contained a null (tnp-) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp- IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules.

Alleles↗