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.

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.

DNA sequence changes of mutations in the histidine operon control region that decrease attenuation.

The DNA sequence changes of 18 (9 different) mutations in the control region of the histidine operon of Salmonella typhimurium are presented. All of these mutations increase the level of expression of the operon, presumably by decreasing transcription termination at the attenuator. Five of the mutations were previously isolated hisO mutations, and the other four were isolated here as His+ pseudorevertants of His- stop codon mutations in the leader peptide gene. Only two mutations, O1242 and O3154, directly affect the terminator stem of the leader RNA. One mutation, O1202, creates a strong new stem that would compete with the terminator stem. Most of the other mutations damage other RNA stems. Their effect can best be explained by, and they thus provide supporting evidence for, the prevailing model of attenuator regulation involving alternative, competing RNA stems in the leader RNA. Two mutations that do not appear to significantly affect an RNA stem directly, including a deletion of three of the seven consecutive histidine codons, are best explained as effects of a translating ribosome upon the RNA stem structures, even though the histidine codons are not translated in the pseudorevertants.

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.

The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion.

KlenTaq DNA polymerase is an N-terminally truncated Thermus aquaticus (Taq) DNA polymerase I. As expressed from a gene construct in Escherichia coli, translation initiates at Met236, bypassing the 5'----3' exonuclease domain of the DNA polymerase-encoding gene. A sensitive forward mutation assay was used to measure the relative number of mutations introduced into the entire lacZ gene by the polymerase chain reaction (PCR) under various conditions which allow the amplification of such a large DNA span. Two selectable markers, one at each end of the test lacZ fragment, were employed to avoid the plating and scoring of PCR artefacts such as primer initiation in the midst of the lacZ gene, and cloning artefacts such as empty vector plasmid. The measured relative mutation rate was twofold lower for KlenTaq as compared to the full-length Taq DNA polymerase.

Integration of multiple copies of a foreign sequence into the Ti plasmid of Agrobacterium tumefaciens.

A method for constructing Ti plasmids bearing multiple copies of a sequence integrated in tandem is described. A small plasmid that confers tetracycline resistance (TcR), contains homology to a Ti plasmid, and is unable to replicate in Agrobacterium tumefaciens, was mobilized from Escherichia coli to A. tumefaciens. Ti plasmids of exconjugants selected for resistance to 12-14 micrograms Tc/ml all contained multiple tandem repeats of the integrative plasmid. Tc-sensitive variants with fewer integrated copies arose spontaneously at low frequency in the absence of Tc selection, or could be enriched for by selection on Tc in combination with the bactericidal antibiotic augmentin. Variants having an increased number of integrated copies were obtained by growth on high Tc concentrations. Tandem repeats integrated between border sequences provide, in principle, a way to reproducibly introduce many linked copies of any foreign gene into plants.

Phenotypic and reversion analysis of a Salmonella typhimurium constructed to have an arginine codon at the hisG46 missense codon.

Of the 6 single-base mutations that would be predicted to change the missense mutation hisG46 away from a proline codon in the Salmonella/microsome mutagen selection assay for histidine-independent revertants, only 5 have been observed. We have used site-specific mutagenesis to make the unobserved mutant [CCC (proline)----CGC (arginine)] codon in the Salmonella genome. Experiments with this arginine mutant demonstrate that, like bacteria containing the hisG46 mutation, bacteria with the arginine missense mutation are histidine auxotrophs which are capable of reversion to histidine independence. However, unlike the ATP phosphoribosyltransferase coded by the hisG46 his G gene (with a proline), the arginine mutant enzyme is partially active. This is indicated by a histidine-independent phenotype when the arginine hisG gene is present in multiple copies.

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium.

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.

An analysis of the mutagenicity of 1,2-dibromoethane to Escherichia coli: influence of DNA repair activities and metabolic pathways.

The mutagenicity of 1,2-dibromoethane (EDB) to Escherichia coli was reduced by the UV light-induced excision repair system but unaffected by the loss of a major apurinic/apyrimidinic site repair function. At high doses, 70-90% of the EDB-induced mutations were independent of SOS-mutagenic processing and approximately 50% were independent of glutathione conjugation. The SOS-independent mutations induced by EDB were unaffected by the enzymes that repair alkylation-induced DNA lesions. EDB-induced base substitutions were dominated by GC to AT and AT to GC transitions. These results suggest that EDB-induced premutagenic lesions have some, but not all, of the characteristics of simple alkyl lesions.

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.

PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.

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.

Variable patterns of expression of luciferase in transgenic tobacco leaves.

A carboxyl-terminally modified firefly luciferase, encoded as a gene fusion to the neomycin phosphotransferase gene (which confers kanamycin resistance), was found to be enzymatically active for both enzymes when expressed in bacteria and in transgenic plants. A military-type starlight vision system was used to conveniently analyze the pattern of gene expression in transgenic tobacco plant leaves. Transgenic tobacco plants which expressed luciferase uniformly in all areas of the leaf, and assays for luciferin, demonstrated that luciferin rapidly penetrates all regions of a tobacco leaf in at least two dimensions. Depending on the test gene structure or, presumably, on the transferred DNA (T-DNA) insertional context, other transgenic plants were obtained that expressed luciferase with a wide range of nonuniform patterns from nominally the same cauliflower mosaic virus 35S promoter. For instance, the veins can be dark, while only the interveinal regions of the leaf lamina glow, or only the small capillary veins glow, or only the major veins glow. Local and/or systemic induction in response to wounding was also demonstrated.

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.

Colony probing as an alternative to standard sequencing as a means of direct analysis of chromosomal DNA to determine the spectrum of single-base changes in regions of known sequence.

We demonstrate here the means to directly analyze bacterial chromosomal DNA for all classes of single-base mutations in a specific codon in any region of known sequence through the use of DNA probing as a powerful substitute for standard sequencing techniques. With this method, chromosomal DNA from hundreds of mutants can be examined for single-base changes without any DNA cloning. This method can conveniently provide a large data base for the assessment of all classes of single-base mutations occurring spontaneously or induced by a known or suspected mutagen. The method is demonstrated for the analysis of histidine-independent (His+) revertants of hisG46, a missense mutation of Salmonella typhimurium that can revert in six or seven ways.

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition.

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels. Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA.

Effective amplification of long targets from cloned inserts and human genomic DNA.

We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.