Automated synthesis of gene fragments.

The DNA/RNA Synthesizer provides a complete and automated procedure for the synthesis of DNA sequences. Each base unit is added in a 30-minute cycle, permitting a tetradecamer to be constructed in 6 1/2 hours. The complete procedure is described, including a practical procedure for isolation and purification of the desired DNA sequence.

Resolution of an equivocal genetic element in Drosophila melanogaster: organization of the maroon-like locus.

Despite extensive prior study, an understanding of the genetic organization of the maroon-like locus (ma-l:1-64n has been elusive. A large-scale, three-point fine structure recombination experiment is described, wholse results provide documentation for an inexcapable argument that maroon-like is a single element rather than a polycistronic system.

Cycling probe technology with RNase H attached to an oligonucleotide.

A streptavidin-RNase H gene fusion was constructed by cloning the Thermus thermophilus RNase H coding sequence in the streptavidin expression vector pTSA18F. The gene was expressed in Escherichia coli, and the resulting fusion protein was purified to apparent homogeneity. The fusion protein was shown to have a molecular weight of 128 kDa and to consist of four subunits. Furthermore, heat treatment of the fusion enzyme showed that it was stable as a tetramer at 65 degrees C. The fusion enzyme was shown to have both biotin binding and RNase H catalytic properties. Using cycling probe technology (CPT), the fusion enzyme was compared to the native RNase H with a biotinylated probe at different ratios of probe:enzyme and varying amounts of synthetic target DNA. At a ratio of 1:1, the fusion enzyme was active in CPT, but the native enzyme was not; both enzymes were active at a 1:5000 ratio of probe:enzyme. The fusion enzyme was further tested using biotinylated and non-biotinylated probes and was shown to be active at a 1:1 ratio with the biotinylated probe but not with the non-biotinylated probe. These experiments show that through binding of the streptavidin-RNase H fusion enzyme to the biotinylated probe, the efficiency of the cycling probe reaction is enhanced.

Probe amplifier system based on chimeric cycling oligonucleotides.

Amplification systems are required as part of DNA probe technology, since traditional non-amplified oligonucleotide hybridization using nonradioactive detection methods have detection limits of approximately 10(8) molecules. We present a probe amplifier technology suitable for use in large-scale automated clinical diagnostic systems. It is fast, sensitive and performs at a constant temperature. The system functions by allowing a single target molecule to act as a catalyst in converting a large number of probe molecules to a unique detectable form. We refer to this catalytic amplification process as the "cycling probe reaction." The basis of the system is an oligomer probe construction consisting of a DNA-RNA-DNA sequence.

UV-induced lethal sectoring and pure mutant clones in yeast.

The induction of lethal sectoring and pure mutant clones by ultraviolet light has been studied in a homogeneous G1 population of Saccharomyces cerevisiae grown in a normal growth medium. At the lowest UV dose of 250 ergs, which corresponds to a shoulder in the survival curve, all mutants appeared as pure clones. At higher doses the frequency of mosaic mutants progressively increased. These results indicate a relationship between the highest frequency of complete mutants and the maximum repair activity. In addition, the frequency of lethal sectoring at all doses tested was too low to account for the origin of pure mutant clones.

Spermine-mediated improvement of cycling probe reaction.

Cycling probe technology (CPT) represents a simple method for detection of DNA target sequences. Cycling probe technology utilizes a chimeric DNA-RNA-DNA probe which is cleaved by RNase H when hybridized with its complementary target. Probe cleavage in the presence or absence of target generates CPT product or background, respectively. Addition of non-homologous DNA into the CPT reaction affects the background and CPT product. Low amounts of human DNA (4-40 ng) result in high background while higher amounts (40-400 ng) inhibit the reaction. The simultaneous addition of spermine and EGTA into the CPT reaction containing human DNA resulted in a significant release of the inhibition and a reduction of background. The presence of spermine alone caused an increase of probe cleavage whereas addition of EGTA increased the specificity of the CPT. A possible mechanism by which spermine could lead to this improvement of CPT has been proposed. Using a membrane-binding assay, the authors demonstrated that human DNA competes with the probe for binding to RNase H. Furthermore, by using a DNA-agarose column, it has been shown that such RNase H-DNA binding can be disrupted by spermine. Within the CPT reaction, similar spermine-mediated displacement of RNase H from human DNA could lead to an improved CPT efficiency.

Molecular and phenotypic characterization of penicillinase-producing Neisseria gonorrhoeae from Canadian sources.

The incidence of penicillinase-producing Neisseria gonorrhoeae (PPNG) infections has increased in Canada during the past 2 years. Most of these cases were imported from abroad. The PPNG strains from these cases were characterized with respect to susceptibility to 11 antibiotics, auxotype, and plasmid content. Rosaramicin and cefuroxime proved to be the most potent of the antibiotics tested. The molecular characterization of the isolates indicated that all carried a 2.6-megadalton cryptic plasmid. Most of the PPNG isolates (87%) harbored a 4.5-megadalton penicillinase-producing plasmid, whereas only 13% harbored the 3.2-megadalton penicillinase-producing plasmid. In those cases where contact tracing was possible, the correlation linking strains of Far Eastern etiology with carriage of the 4.5-megadalton plasmid was upheld. The penicillinase-producing strains were typed auxanographically in either the proline-requiring (57%) or prototrophic groups (42%). Substrate hydrolysis profiles and analytical isoelectric focusing of crude beta-lactamase extracts of several isolates has reconfirmed that these strains elaborate a type TEM-1 enzyme. Several of the penicillinase-producing plasmids were also examined for plasmid stability.

Studies on genetic organization in higher organisms. II. Complementation and fine structure of the maroon-like locus of Drosophila melanogaster.

Mutants of the maroon-like complex, representative of the five known complementation classes, were subjected to fine structure mapping experiments utilizing a nutritional selective procedure which permits the survival of rare ma-l(+) progeny from large-scale crosses. The analysis provides an internally consistent, unique map, colinear with the complementation map. Noncomplementers exhibit a polarized mapping distribution. In addition to ma-l(+) recombinants, the selective medium permitted the survival of ma-l(+) exceptionals not associated with recombination for adjacent markers. Analysis of the exceptionals favors their origin as convertants.

Effects of thiamphenicol and chloramphenicol in inhibiting Neisseria gonorrhoeae isolates.

Thiamphenicol was compared with penicillin, tetracycline, and chloramphenicol for its ability to inhibit 530 isolates of Neisseria gonorrhoeae, including 13 penicillinase-producing isolates. Thiamphenicol proved to be as active as chloramphenicol in inhibiting all of the isolates.

Temperature-sensitive mutant of Schizosaccharomyces pombe exhibiting enhanced radiation sensitivity.

A conditional lethal and radiation-sensitive mutant of Schizosaccharomyces pombe is described in which both characteristics result from a single gene mutation. Confirmation of the pleiotropic nature of this mutant was obtained by tetrad analysis and by testing the radiation sensitivity of a large number of revertants that grew normally at the restrictive temperature. The colony-forming ability of the mutant after ultraviolet radiation, gamma radiation, and ethyl methane sulfonate treatment is considerably altered by the post-treatment incubation temperature, showing higher survival at 25 than at 30degreesC. The radiosensitivity of the mutant is also influenced by the stage of growth. The difference in radiation sensitivity between the wild type and mutant is greater when log-phase cultures are compared. The characteristics of this mutant suggest that it is defective in a step common to both deoxyribonucleic acid replication and repair.

Antibiotic resistance among predominant Salmonella serovars and phagovars in Canada.

The antibiotic susceptibility of 2609 Salmonella isolates, collected during the period 1975-1976, was tested and the relationships between antibiotic-resistance pattern, source of isolation, and serovar and phagovar were determined. Of 95 serovars examined, 40 were sensitive to all of the antibiotics tested. Salmonella typhimurium was the major contributor to multiple resistance from both human and non-human sources. Multiply resistant strains were not found from animal feed sources and, in addition, S. typhimurium, one of the most predominant serovars, was found in every source but animal feeds. 90% of phagovar 10 was sensitive to all antibiotics tested whereas over 80% of phagovars 3-aerogenic, 92, and 123 were multiply resistant.

Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction.

Cycling probe technology (CPT) is a unique and simple method for the detection of specific target sequences. CPT utilizes a chimeric DNA-RNA-DNA probe providing an RNase H-sensitive scissile linkage when bound to a complementary target sequence. For this study a diagnostic assay based on CPT was developed for the detection of the 36-bp direct repeat (DR) region in Mycobacterium tuberculosis. To determine the feasibility of using the DR for detecting M. tuberculosis by CPT, a wide variety of mycobacteria were tested by Southern blot hybridization with three DR probes to verify their specificity. The entire DR region of Mycobacterium bovis 401 was sequenced, and the data were used to design a PCR assay that would allow us to estimate the number of DRs present in a variety of strains. A CPT assay which uses a probe complementary to the DR region was developed and evaluated with synthetic targets and genomic DNA from mycobacteria. In summary, the 36-bp DR provides an attractive target for detecting M. tuberculosis because the sequence is present in high copy numbers in the genome, is specific for the M. tuberculosis complex, and is found in strains that lack IS6110.