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Drosophila melanogaster and its sibling species, Drosophila simulans, differ in expression of the enzyme alcohol dehydrogenase (ADH). Adult melanogaster flies that are homozygous for the Slow allozyme have approximately twice the level of ADH activity and crossreacting material as simulans adults. There is no corresponding difference in ADH mRNA, however, so this difference in ADH protein level is evidently due to a difference in the rate of translation of the two RNAs and/or to a difference in protein stability. Here we report an interspecific gene-transfer experiment, using P-element transformation, to determine whether this expression difference is due to genetic background differences between the species (trans-acting modifiers) or to cis-acting factors within the Adh gene. When the Adh genes from D. melanogaster and D. simulans are put into the same genetic background, there is no detectable difference in their level of expression. The level is relatively high in the melanogaster background and relatively low in the simulans background. Therefore, the interspecific difference in Adh expression is due entirely to trans-acting modifiers, in spite of the many sequence differences between the Adh genes of the two species, which include two amino acid substitutions.Drosophila melanogaster and its sibling species, Drosophila simulans, are essentially cosmopolitan in distribution and live in close association with man (1). Both species utilize fermenting fruits in which ethanol concentrations range up to several percent by volume (2, 3), but D. melanogaster appears to be more adapted to high alcohol environments than D. simulans. In laboratory toxicity tests, melanogaster adults consistently show a higher level of tolerance to ethanol and other alcohols than simulans adults (4)(5)(6)(7)(8). This difference in tolerance may be due to a difference in alcohol dehydrogenase (ADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1) expression between the species.The ADH enzyme of D. melanogaster clearly plays an important role in alcohol detoxification and metabolism. Flies homozygous for a null allele of Adh are extremely sensitive to the toxic effects of environmental alcohols (9). More than 90% of the ethanol that is metabolized to lipid in larvae goes through a pathway that is dependent on ADH activity (10). Furthermore, genetic variation in ADH activity levels in melanogaster, particularly the difference between allozymes, is frequently associated with variation in tolerance to ethanol and other alcohols (for review, see refs. 11 and 12).The D. melanogaster Adh gene produces two different transcripts, which are differentially expressed during development but produce identical proteins (13, 14) (see Fig. 1). We have shown that the timing of usage of the two promoters during development is very similar in melanogaster and simulans (15). We have also compared the pattern of Adh expression throughout development in the two species by measuring ADH activity, ADH-crossreacting material (CRM), and ADH mRNA for several st...

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4 degrees C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4 degrees C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.

Context.—To maximize the participation rate in population genetic studies, alternatives to invasive whole blood collection are increasing. One such alternative is buccal epithelial cell collection, which, in contrast to venipuncture and finger sticks, is painless. Buccal cells, if collected and purified efficiently, offer an acceptable source for DNA to be used in research and clinical applications. Objective.—To develop a noninvasive sampling method for collecting cells for routine DNA testing in a clinical laboratory setting. Design.—Five factors were used to evaluate several brands of mouthwash: (1) compatibility with the DNA purification chemistry, (2) DNA yield, (3) DNA quality, (4) DNA stability at room temperature, and (5) mouthwash taste. Next, an optimization study was undertaken to maximize DNA yield. Finally, a validation study was undertaken with the optimized protocol to test a panel of 14 donors for DNA yield and performance and to test for the stability of DNA held in mouthwash. Setting.—Industrial research and development laboratory. Results.—Of 5 mouthwashes tested, Scope brand mouthwash received the highest overall ranking. The addition of proteinase K and glycogen to the protocol significantly enhanced DNA yields, with a test panel (n = 14) giving a range of 12 to 60 μg of DNA per donor. In a 4-week room temperature stability study, the DNA in mouthwash samples was found to be stable for at least 2 weeks. Conclusion.—A clinically validated DNA purification chemistry was adapted to a noninvasive specimen collection method. This method used a commercially available mouthwash, Scope, to collect buccal epithelial cells for the preparation of high-quality DNA in high yield.

SummaryTwelve inbred lines derived from an M′ strain of Drosophila melanogaster were used to study the repression of P-element-mediated hybrid dysgenesis. Initial assessments indicated that the lines differed in the ability to repress gonadal dysgenesis, and that this ability was highly correlated with the ability to repress snw hypermutability. Later assessments indicated that most of the lines with low or intermediate repression potential evolved to a state of higher repression potential; however, Southern analyses failed to reveal significant changes in the array of genomic P elements that could account for this evolution. In addition, none of the lines possessed the incomplete P element known as KP, which has been proposed to explain repression in some D. melanogaster strains. One of the lines maintained intermediate repression potential throughout the period of study (52 generations), indicating that the intermediate condition was not intrinsically unstable. Genetic analyses demonstrated that in some of the lines, repression potential was influenced by factors that were inherited maternally through at least two generations; however, these factors were not as influential as those in a classic P cytotype strain. Additional tests with a dysgenesis-inducing X chromosome called T-5 indicated that repression itself was mediated by a combination of maternal effects and paternally inherited factors that were expressed after fertilization. These tests also suggested that in some circumstances, the P transposase, or its message, might be transmitted through the maternal cytoplasm.