A novel intercistronic regulatory element of prokaryotic operons.
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Base Sequence↗
Biomedical subjects
W M Barnes
Publications and source records attributed to W M Barnes.
Short direct repeats flank the T-DNA on a nopaline Ti plasmid.
Crown gall disease results from the insertion of a segment of the Agrobacterium Ti plasmid, called T-DNA, into host plant nuclear DNA. We have subjected to sequence analysis the border regions of pTi T37 (ends of T-DNA) and one left T-DNA/plant DNA border fragment isolated from BT37 tobacco teratoma by molecular cloning. These sequence studies, taken together with published sequence of a right T-DNA/plant DNA border fragment, allowed us to identify the positions of left and right borders at the DNA sequence level. Comparison of left and right border regions of the Ti plasmid revealed a "core" direct repeat of 13 of 14 bases (12 contiguous) precisely at the borders of T-DNA. An extended repeat of 21 of 25 bases overlaps this core repeat. T-DNA on the Ti plasmid exhibits no longer direct or inverted repeats in the border regions, based on Southern hybridization studies. The physical structure of T-DNA differs from that of known prokaryotic and eukaryotic transposable elements but bears a structural resemblance to the prophage of bacteriophage lambda.
Journal Article↗
Cloning and restriction map of the first part of the histidine operon of Salmonella typhimurium.
The first part of the histidine operon of Salmonella typhimurium, hisGpeaGD, has been cloned onto the vector plasmid mini-ColE1 (pVH51). The resulting plasmid, pWB91, has a single EcoRI site and is 11,500 base pairs in size. The HindII restriction map was determined by the method of two-dimensional cross-annealing between a partial digest pattern and a complete digest pattern. The restriction fragment containing the genetic control region was identified with the aid of the small (35-base pair) internal deletion 01242 and the observation that heteroduplexed restriction fragments containing this deletion have markedly reduced mobility on polyacrylamide gels. The genetic control region was then mapped in more detail with other restriction enzymes. The genetic orientation of the restriction map was determined with the aid of several deletions of integral HindII fragments generated in vitro.
Cloning, Molecular↗
Model for regulation of the histidine operon of Salmonella.
A model is proposed that accounts for regulation of the histidine operon by a mechanism involving alternative configuration of mRNA secondary structure (the alternative stem model). New evidence for the model includes sequence data on three regulatory mutations. The first (hisO1242) is a mutation that deletes sequences needed to form the attenuator mRNA stem and causes constitutive operon expression. The second mutation (hisO9654) is a His- ochre (UAA) mutation in the leader peptide gene; the existence of this mutation constitutes evidence that the leader peptide gene is translated. The third mutation (hisO9663) is remarkable. It neither generates a nonsense codon nor affects a translated sequence; yet, it is suppressible by amber suppressors. We believe this mutation causes a His- phenotype by interfering with mRNA secondary structure. The suppressibility of the mutation is probably due to disruption of the attenuator stem by ribosomes that read through the terminator codon of the leader peptide gene. This explanation is supported by the observation of derepression of a wild-type control region in the presence of an amber suppressor. Evidence is presented that hisT mutants (which lack pseudouridine in the anticodon arm of histidine tRNA) may cause derepression of the his operon by slowing protein synthesis in the leader peptide gene.
Bacterial Proteins↗
Isolation and restriction mapping of plasmids containing ribosomal DNA sequences from the rrn B cistron of E. coli.
Recombinant plasmids containing the entire 16S RNA gene from the rrn B cistron of E. coli inserted in Col E1 and pBR322 plasmid vectors have been constructed. These plasmids have been mapped using several restriction endonucleases as well as by DNA-RNA hybridization. These maps reveal previously undetected restriction sites in the rrn B cistron and in Col E1 plasmid DNA.
DNA Restriction Enzymes↗
Construction of an M13 histidine-transducing phage: a single-stranded cloning vehicle with one EcoRI site.
In order to create a ready source of single-stranded DNA for DNA sequence determination by the dideoxy chain-termination method, the promoter-proximal part of the histidine operon, the hisOGD region of Salmonella typhimurium, was cloned onto the single-stranded phage M13. Both orientations of the his DNA were cloned to supply DNA template for sequencing of each strand. Insertion was achieved at an HaeIII site in the intergenic region (IR) of M13, and a single EcoRI site was purposely regenerated at one boundary of the his DNA insert. Infected colonies, not plaques, were selected using the hisD gene as a selective marker. The single RI site and the hisD marker for auxotrophic selection represent improvements on the wild type M13 as a single-stranded vector for cloning other DNA.
Base Sequence↗
An electronic digital display system to record contrast medium injection rates on angiographic film.
The authors describe an electronic numerical digital display system which utilizes light emitting diodes to record contrast medium injection rates on serial angiographic film. The display system is conveniently mounted within the film changer. It was developed to record injection rates during an increasing rate of injection employed in the radiographic "spillover" technique or estimating blood flow.
Angiography↗
DNA sequencing by partial ribosubstitution.
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Base Sequence↗
DNA sequence from the histidine operon control region: seven histidine codons in a row.
The DNA sequence of 250 base pairs preceding the first structural gene of the histidine operon of Salmonella typhimurium was determined by the dideoxy chain-termination method. Single-stranded DNA template was provided by an M13-histidine transducing phage constructed for the purpose by in vitro recombination. The termination site for the histidine leader RNA is identified by analogy with the trp operon leader termination sequence, and is 47 nucleotides before the start codon of the first structural gene G. Beginning 150 nucleotides before the end of the presumed leader RNA is a possible short protein-coding region with seven histidine codons in a row. It is proposed that the major mechanism of histodine operon control must involve a ribosome arrested at this run of histidine codons when histidine is limiting.
Base Sequence↗
Plasmid detection and sizing in single colony lysates.
A simple and contained procedure for the rapid assay of the presence and size of plasmids similar to Col El is described. Bacteria are picked from an agar plate with a toothpick, lysed with dodecyl sulfate and heat, and placed directly on an agarose gel for electrophoresis.
Colicins↗
Genetic regulation: the Lac control region.
The nucleotide sequence of the lac promoter-operator region has been determined. The 122 base pairs comprising this region include the recognition sites for RNA polymerase, the positive regulatory protein, CAP, and the negative regulatory protein, the repressor. Identification of mutant variants of the sequence combined with the in vitro biochemical studies of others has allowed us to tentatively identify the recognition site for each of these proteins, and to suggest how CAP might act at a distance to affect the interaction of RNA polymerase with the promoter.
Base Sequence↗
Ossicles of knee menisci. Report of seven cases.
Seven cases of ossicles in the posterior horn of the medial meniscus revealed that: trauma is not necessarily an antecedent; some knees with ossicles are asymptomatic; some knees may be treated conservatively and without surgery; ossicles can be distinguished from loose bodies under fluoroscopic examination. Microscopically the ossicles are living bone. Although the etiology is not proven, perhaps merit should be given to the theory of a vestigial or sesamoid-like bone.
Adult↗
The construction of lambda transducing phages containing deletions defining regulatory elements of the lac and trp operons in E. coli.
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Base Sequence↗