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G N Bennett

Publications and source records attributed to G N Bennett.

At least 91 records · Page 5Linked to original sources
PubMed1986

Construction and characterization of pBR322-derived plasmids with deletions of the RNA I region.

A region upstream from the origin of replication in ColE1-type plasmids has been shown to be necessary for replication. Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation. We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II. The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter. These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other ColE1-type plasmids. These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure. In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form.

Base Sequence↗
PubMed1986

Use of bacterial DHFR-II fusion proteins to elicit specific antibodies.

Plasmids containing the coding region of the type II dihydrofolate reductase (DHFR) specified by R388 have been used to alter the amino acid (aa) sequence at the C-terminus of this protein. These plasmids have a unique cloning site in the C-terminal portion of the 78-aa coding region. Insertions of DNA fragments into this site produced plasmids that code for proteins with 6- to 80-aa extensions. The vectors were constructed to terminate translation in all three phases beyond the position of insertion of foreign DNA. Random DNA fragments from the major sperm protein (MSP) gene of Caenorhabditis elegans produced by DNase I cleavage were inserted into these vectors. Cell extracts from colonies containing MSP sequences were examined by gel electrophoresis and immunoblotting. One of the hybrid DHFR-MSP proteins was isolated and antibody was prepared to it. This antibody preparation reacted with MSP in immunoblots of purified MSP and whole cell extracts of the worm. A rapid purification procedure for the DHFR is presented.

Amino Acid Sequence↗
PubMed1985

In vitro characterization of hybrid promoters and altered tryptophan operon promoters.

This study examines the in vitro interaction of hybrid and altered Escherichia coli promoters and other promoters with purified E. coli RNA polymerase. Three parameters of polymerase activity were examined: the time for open complex formation; the temperature of transitions; and the time required for productive initiation. The results indicate the rate of in vitro binding as measured by the filter binding technique does not completely correlate with the in vivo activities among these diverse promoters. Transition temperatures ranged from 13 to 27 degrees C with the lowest transition temperatures associated with the relatively weak in vivo beta-lactamase and anti-tet promoters. The productive initiation studies showed a dependence on labeled nucleoside triphosphate concentration when that nucleotide was present early and frequently in the transcript. Promoters containing the -10 region of the lac promoter had slow productive initiation rates while trp -10 promoter derivatives were generally very fast. In the promoters studied here, a trend was noted between the binding rate and transition temperature studies in that the promoters with the lower transition temperatures tended to bind more rapidly.

Cytidine Triphosphate↗
PubMed1984

Enzymatic digestion of operator DNA in the presence of the lac repressor tryptic core.

The trypsin-resistant core protein of the lac repressor was utilized in protecting operator DNA from two types of enzymatic digestion. Core repressor protects and enhances operator DNA digestion by DNase I in the same fashion as intact repressor, though to a lesser degree on the lower strand. DNase I patterns found for the ternary complexes (protein-sugar-operator) were consistent with the expected affinity alterations of the protein species in response to binding these ligands. The 3' boundaries obtained by exonuclease III digestion for the intact repressor-operator complex varied slightly from those reported by Shalloway et al. (1980). Asymmetric binding to operator by the core repressor fragment was suggested by differences in the 3' boundary for the core compared to intact repressor on the promoter-distal side of the complex. A composite picture of repressor structure and function emerges from the protection studies reported here and in the accompanying paper. In light of these and other results, models for repressor binding are examined.

Autoradiography↗
PubMed1984

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. I. Insertion of synthetic oligonucleotides.

To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter. Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced. These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG. Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid.

Base Sequence↗
PubMed1984

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. II. Insertion of lac operator fragments.

To examine the effect of introducing protein-binding sites around a promoter upon expression from that promoter, a series of altered tryptophan promoter plasmids have been constructed. In these derivatives of pKO-1 (a galK-expression, promoter-assay vector), the trp promoter has been introduced into the vector, such that the galK expression is dependent on the trp promoter. Unique restriction sites have been introduced adjacent to the trp promoter for the insertion of other DNA fragments. The DNA-binding site (a lac operator (lacO) fragment of 33 bp) for the lactose repressor was inserted into the trp promoter-pKO plasmids at various defined locations from -52 to +58 relative to the start site of transcription. Strains bearing tryptophan promoter-lactose operator plasmid derivatives were assayed for galactokinase activity in Escherichia coli C600 and JM103 (a lacIQ strain) to observe the effect of lac repressor binding and removal. In those plasmids where the lac repressor was downstream from the promoter, expression was diminished by the presence of the lactose repressor and galactokinase activity could be induced by addition of IPTG. An analogous lac promoter plasmid was repressed over 90%, and the plasmid containing the lacO fragment at +2 exhibited up to 80% repression; 40-50% repression was observed when the lacO was placed at positions +27 and +58. Placement of the lacO at the upstream locations -39 and -52 produced lower repression. To examine the effects of more than one operator surrounding the promoter, plasmids were constructed that had a lacO at -39 or -52 and, in addition, had an operator downstream.+2

DNA, Bacterial↗
PubMed1983

Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.

Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core. Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence. These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate. Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence. Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection. These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.

DNA, Bacterial↗
PubMed1982

Formation of alkali labile linkages in DNA by hedamycin and use of hedamycin as a probe of protein-DNA complexes.

Hedamycin forms a stable complex with DNA and introduces alkali labile linkages in the DNA. These labile linkages are located at deoxyguanosine residues and are cleaved by the treatment used for breakage at bases alkylated by dimethyl sulfate. The reaction of hedamycin with all G residues in the chain is not uniform, and certain positions, particularily those in TG tracts, are especially reactive. The reaction of hedamycin with DNA can be inhibited by ethidium bromide, suggesting that intercalation is important in positioning the reactive group of hedamycin near to the base which is modified. The low amount of hedamycin needed to produce observable breakage, its specificity for reaction with DNA and its ability to react with DNA under mild conditions make it suitable for use as a probe of protein-DNA complexes. This was shown by the ability of lac repressor and RNA polymerase to block reaction of hedamycin with the DNA of the lac regulatory region.

Anthraquinones↗
PubMed1982

Cloning of small DNA fragments containing the Escherichia coli tryptophan operon promoter and operator.

A41-bp AluI restriction fragment from the trp promoter-operator region has been cloned into the PvuII site of pBR322, regenerating PvuII sites. Transformants were selected on media that allowed the selection of trp-operator-bearing plasmids. The cloned 41-bp fragment can be released from the vector by PvuII digestion, and it possesses a functional promoter and operator as demonstrated by in vivo tests. The 41-bp fragment contains several restriction sites: HincII, TaqI, RsaI, and a HpaI site that is located at the center of the operator sequence. Two new operator derivatives, symmetrical about the HpaI site, were prepared from the 41-bp fragment by joining two right-side, or two left-side PvuII-HpaI pieces together at the HpaI site. These derivatives showed in vivo operator activity. Plasmids containing up to five copies of the 41-bp trp-promoter-operator fragment have been constructed. These plasmids should be useful in preparing large amounts of the 41-bp fragment.

Cloning, Molecular↗
PubMed1981

Characterization of the beta-lactamase promoter of pBR322.

The beta-lactamase promoter of pBR322, derived from Tn3, has been characterized using several techniques. The transcription initiation site is located 35 base pairs from the translation initiation codon of beta-lactamase. The mRNA produced in vitro has a 5' pppGpA terminus. RNA polymerase bound at this start site protects a region from about -50 to +20 from DNase I cleavage using the footprinting technique. RNA polymerase binds rapidly to the beta-lactamase promoter. The half-time of association is less than one-half minute. The half-time of dissociation is approximately 6 hr. A study of the binding of RNA polymerase at different temperatures showed a large change between 11 degrees and 15 degrees C. Comparison of these parameters with those reported for other promoters is discussed.

Base Sequence↗
PubMed1981

Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions.

Anthramycin can form a stable complex with DNA which does not dissociate upon repeated ethanol precipitations. The complex forms in less than one hour at pH 5.5. Bound anthramycin seems to be located in the minor groove of the DNA helix in the anthramycin DNA complex, since methylation of adenosine residues at N-3 by dimethylsulfate is reduced. The anthramycin-DNA complex is resistant to digestion by an excess of a number of restriction enzymes. Anthramycin can be removed from DNA by incubation at acid pH. The released DNA can then be cleaved by restriction enzymes. Anthramycin-DNA complexes can be acted upon by T4 polynucleotide ligase to form longer DNA molecules. The ability of anthramycin to form a stable but reversible complex which is not cleaved by restriction enzymes but can engage in joining reactions may allow a wider variety of DNA fragments to be more readily constructed in vitro.

Anthramycin↗
PubMed1979

The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic.

Nucleotide sequence has been determined for the restriction fragments and cloned DNA from the pL-N-tL1 region of bacteriophage lambda. A unique reading frame for the N gene is defined by the absence of natural nonsense codons and by the presence of seven nonsense codons generated by mutations in N. This reading frame is initiated at two alternative ATG codons, the second of which is probably the in vivo translation start. Reading is stopped at a single TAG codon. The protein coded is therefore 133 or, more probably, 107 amino acids long, rich in lysine, arginine and proline.

Bacteriophage lambda↗