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D E Berg

Publications and source records attributed to D E Berg.

At least 109 records · Page 6Linked to original sources
PubMed1992

Nonrandom orientation of transposon Tn5supF insertions in phage lambda.

Transposition of mini-transposon Tn5supF to phage lambda can be selected in two ways: (i) by plaque formation on a dnaB amber strain of Escherichia coli, which requires expression of the transposon-borne suppressor tRNA gene (supF) during lytic phage growth, or (ii) by lysogenization of a strain with amber mutations in tet and amp resistance genes, and selection of TcR ApR (Sup+) transductant colonies. Tn5supF insertions in several lambda clones were isolated and mapped using a polymerase chain reaction (PCR) amplification method. Among insertions selected during lytic growth, more than 90% were oriented such that supF could be transcribed from an upstream lambda promoter. In contrast, half of those selected by transduction were in each orientation. These results indicate that Tn5supF insertion occurs with equal frequency in each orientation. However, Tn5supF insertion phages in which transcription from the lambda and supF promoters would collide tend to be lost when supF is selected during lytic growth. The tendency to recover Tn5supF insertions in only one orientation is useful in a transposon- and crossover-PCR-based method for preparing templates for DNA sequencing.

Bacterial Proteins↗
PubMed1992

The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli.

One of the most abundant modified nucleosides in tRNA is 5-methyluridine (m5U or rT, ribothymidine). The enzyme tRNA(m5U54)methyltransferase [S-adenosyl-L-methionine:tRNA (uracil-5-)-methyltransferase, EC 2.1.1.35] (the trmA gene product) catalyzes S-adenosylmethionine-dependent methylation of the uracil in position 54 (T psi C loop) in all Escherichia coli tRNAs to form m5U. Hitherto no modified nucleoside in tRNA has been shown to be essential for growth, although their importance in fine tuning the function of tRNA is well established. In this paper, we show that the structural gene trmA is essential for viability, although the known catalytic activity of the tRNA(m5U54)methyltransferase is not.

Bacterial Proteins↗
PubMed1992

Importance of mRNA folding and start codon accessibility in the expression of genes in a ribosomal protein operon of Escherichia coli.

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kilodalton protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in that order. The synthesis of the 21K and TrmD proteins is 12 and 40-fold lower, respectively, than that of the two r-proteins, although the corresponding parts of the mRNA are equally abundant. This translational control of expression of at least the 21K protein gene (21K), is mediated by a negative control element located between codons 18 and 50 of 21K. Here, we present evidence for a model in which mRNA sequences up to around 100 nucleotides downstream from the start codon of 21K fold back and base-pair to the 21K translation initiation region, thereby decreasing the translation initiation frequency. Mutations in the internal negative control element of 21K that would prevent the formation of the proposed mRNA secondary structure over both the Shine-Dalgarno (SD) sequence and the start codon increased expression up to about 20-fold, whereas mutations that would disrupt the base-pairing with the SD-sequence had only relatively small effects on expression. In addition, the expression increased 12-fold when the stop codon of the preceding gene, rpsP, was moved next to the SD-sequence of 21K allowing the ribosomes to unfold the postulated mRNA secondary structure. The expression increased up to 150-fold when that stop codon change was combined with the internal negative control element base-substitutions that derepressed translation about 20-fold. The negative control element of 21K does not seem to be responsible for the low expression of the trmD gene located downstream. However, a similar negative control element native to trmD can explain at least partly the low expression of trmD. Possibly, the two mRNA secondary structures function to decouple translation of 21K and trmD from that of the respective upstream cistron in order to achieve their independent regulation.

Base Sequence↗
PubMed1992

The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing.

Transposon gamma delta (Tn1000), a 6-kb member of the Tn3 family, is widely used for plasmid mutagenesis. A 1.8-kb derivative of gamma delta was constructed that contains the kan gene from Tn5 and the resolution (res) site from gamma delta cloned between 40-bp inverted repeats of gamma delta's delta (delta) end. This element, named m gamma delta-1, lacks the genes encoding transposase and resolvase, and therefore depends on its host to supply transposition and resolution functions. Thus, in strains lacking gamma delta, m gamma delta-1 will not transpose. The m gamma delta-1 element is shown to be useful for mutagenesis of plasmids, DNA sequencing, and allele replacement (in Streptomyces avermitilis).

Alleles↗
PubMed1992

cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth.

The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.

Aerobiosis↗
PubMed1992

Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.

Base Sequence↗
PubMed1992

Components and defects of the coagulation system.

The purpose of this review article is to present a simplified view of coagulation in five parts (vessels, platelets, cascade, natural inhibitors, and fibrinolysis), update the NP with current concepts regarding coagulation, discuss cellular and protein functions, and provide an overall understanding of how the five components work together. Discussion of disease states, genetic deficiencies, and acquired inhibitors of the coagulation system is included in this article.

Blood Coagulation↗
PubMed1991

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.

Autoradiography↗
PubMed1991

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 bp away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam- mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.

Base Sequence↗
PubMed1991

A sequence at the inside end of IS50 down regulates transposition.

Deletion analysis has shown that the segment at the IS50 inside (I) end that is needed for efficient transposition is approximately 19 bp long. Dam methylation at two 5' GATC sequences within this segment decreases I-end transposition activity. A third 5' GATC sequence is present at bp 21-24 of the I end. The comparisons presented here show that extension of the I end from 19 to 24 bp decreases its transposition activity in dam cells 5- to 50-fold, depending on the overall transposon structure.

Bacterial Proteins↗
PubMed1991

Deletions in plasmid pBR322: replication slippage involving leading and lagging strands.

We test here whether a class of deletions likely to result from errors during DNA replication arise preferentially during synthesis of either the leading or the lagging DNA strand. Deletions were obtained by reversion of particular insertion mutant alleles of the pBR322 amp gene. The alleles contain insertions of palindromic DNAs bracketed by 9-bp direct repeats of amp sequence; in addition, bp 2 to 5 in one arm of the palindrome form a direct repeat with 4 bp of adjoining amp sequence. Prior work had shown that reversion to Ampr results from deletions with endpoints in the 8- or 4-bp repeat, and that the 4-bp repeats are used preferentially because one of them is in the palindrome. To test the role of leading and lagging strand synthesis in deletion formation, we reversed the direction of replication of the amp gene by inverting the pBR322 replication origin, and also constructed new mutant alleles with a 4-bp repeat starting counterclockwise rather than clockwise of the insertion. In both cases the 4-bp repeats were used preferentially as deletion endpoints. A model is presented in which deletions arise during elongation of the strand that copies the palindrome before the adjoining 4-bp repeat, and in which preferential use of the 4-bp repeats independent of the overall direction of replication implies that deletions arise during syntheses of both leading and lagging strands.

Alleles↗
PubMed1991

Limits to the role of palindromy in deletion formation.

We tested the effect of palindromy on deletion formation. This involved a study of reversion of insertion mutations in the pBR322 amp gene at a site where deletions end either in 9-bp direct repeats or in adjoining 4-bp direct repeats. Inserts of palindromic DNAs ranging from 10 to more than 26 bp and related nonpalindromic DNAs were compared. The frequency of deletions (selected as Ampr revertants) was stimulated by palindromy only at lengths greater than 26 bp. The 4-bp direct repeats, one component of which is located in the palindromic insert, were used preferentially as deletion endpoints with palindromes of at least 18 bp but not of 16 or 10 bp. We interpret these results with a model of slippage during DNA replication. Because deletion frequency and deletion endpoint location depend differently on palindrome length, we propose that different factors commit a molecule to undergo deletion and determine exactly where deletion endpoints will be.

Alleles↗
PubMed1991

Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage.

An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails Tn5supF transposition to lambda-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup+. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.

Bacteriophage lambda↗
PubMed1991

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.

Alleles↗
PubMed1991

Recognition and management of bleeding following cardiac surgery.

The major hemorrhagic disorders are associated with malfunction of three physiologic systems either separately or in combination: (1) abnormalities of platelets, (2) abnormalities of blood vessels, and (3) abnormalities of plasma coagulation factors. Platelets contribute to hemostasis not only by participating physically by occluding rents and tears in small vessel but also by involving the coagulation process through their biochemical activities. It is small wonder that platelet dysfunction is the most common cause of nonsurgical bleeding following CPB; however, the potential for hyperfibrinolysis cannot be overlooked. New laboratory tests and therapeutic approaches have allowed for more accurate diagnosis and more appropriate therapy in a variety of bleeding events following CPB.

Blood Coagulation↗
PubMed1990

Context effects in the formation of deletions in Escherichia coli.

We have examined the frequency with which identical deletions are formed in different chromosomal contexts. A panel of six mutant bla genes containing palindrome/direct repeat structures were moved from pBR322 to three locations: at lambda att, at chromosomal lac, and at F'lac. Deletion of the palindromes and one of the direct repeats results in reversion to Ampr. The frequency of deletion for all alleles declines beyond the reduction in copy number when they are moved from the multicopy plasmid environment to a single-copy chromosome. The magnitude of the declines varies in an allele-specific and location-specific manner. Our data support the hypothesis that context can influence the frequency of mutation independent of the immediate DNA sequence.

Alleles↗
PubMed1990

Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling.

The major hot spot of transposon Tn5 insertion in plasmid pBR322 (hot spot I) is in the promoter for the tetracycline resistance gene (tet). We made a series of pBR322 derivatives with mutations in and around this promoter and assayed their effects on insertion of Tn5 into hot spot I. Those mutations which reduced transcription from the tet promoter also reduced the frequency of insertion into hot spot I. Transcription and translation of tet are thought to cause the formation of paired domains of negative and positive supercoiling in pBR322. An amber codon in tet, 345 base pairs from hot spot I, decreases the negative supercoiling of the DNA segment containing hot spot I because it terminates translation of tet prematurely. We report here that this amber mutation also reduces insertion into hot spot I. These results suggest that the ability of Tn5 to insert into its major hot spot in pBR322 depends directly on negative supercoiling of the target DNA.

Base Sequence↗