Inhibition of fading in fluorescence microscopy of fixed cells.

Following Hirschfeld et al., dithionite was added to mounting media to inhibit fluorescence fading. Excellent response is reported for fluorescein, acridine orange, 33258 Hoechst, acriflavine, berberine (and ethidium bromide), but not for quinacrine.     

Inhibition of fading in fluorescence microscopy of fixed cells

Summary Following Hirschfeld et al., dithionite was added to mounting media to inhibit fluorescence fading. Excellent response is reported for fluorescein, acridine orange, 33258 Hoechst, acriflavine, berberine (and ethidium bromide), but not for quinacrine.Acknowledgment. The author is grateful to Dr T. Hirschfeld of Block Engineering, Cambridge, Mass., USA, for an illuminating introduction to his work.     

Resistance to ethidium bromide inAspergillus nidulans

Summary A mutant ofAspergillus nidulans resistant to ethidium bromide was isolated and the semi-dominant gene responsible for this resistance was allocated on linkage group II at 17.42±3.05 units of recombination from thewA₃ gene. The gene also confers cross-resistance to acriflavin, malachite green and crystal violet. It was also shown that riboflavin is antagonistic to the toxic effect of ethidium bromide, at certain concentrations. The mechanisms which could be responsible for the toxic effect of this drug are discussed and compared with those of acriflavin. The use of theEtb ₁ gene in genetical analysis through the parasexual cycle is suggested.Acknowledgements. The authors are thankful to the National Council for the Development of Science and Technology (CNPq) for financial support through PIG-SIP 04/053 FAPESP.     

Effect of ethidium bromide onDrosophila melanogaster andDrosophila simulans

Summary Ethidium bromide was fed toD. melanogaster andD. simulans males in order to test its toxic capacity and potency for the induction of dominant lethals. Our results show that ethidium bromide has a high toxicity and likewise produces dominant lethals to a significant extent in both species, but more effectively inD. melanogaster.     

Plasmids controlling the resistance to several antibiotics in C. perfringens type A, strain 659]

A strain of C. perfringens type A, isolated from a patient, was found to be resistant to four antibiotics: tetracycline (Tet), chloramphénicol (Chl), erythromycin (Ero) and clindamycin (Cli). Clones resistant to only two drugs (Tet-Chl or Ero-Cli), or sensitive to all drugs were found in cultures of the wild-type strain treated by acridine dyes or ethidium bromide. DNA analysis by equilibrium centrifugation confirmed that the original strain contains two resistance plasmids, one called Rip 401 (Tet-Chl) and the other Rip 402 (Ero-Cli), which represent respectively 1.8 +/- 0.2% and 2.3 +/- 0.2% of the total amount of DNA in this strain of Cl. perfrigens.     

Early detection of cell damage by supravital acridine orange staining

Summary Cell damage can be detected in living cells by acridine orange fluorescence earlier than with phase contrast microscopy or with conventional histological methods. The change in the acridine orange fluorescence from green to red indicates that the secondary structure of the DNA is altered very early during the cell death.Acknowledgments. We thank Miss Pirjo Vuori for excellent technical assistance, Engineer Charles Vane-Tempest for providing the necessary microscope equipment and the Finnish Academy for financial support.     

Effects of ethidium bromide in diploid and duplication strains of Aspergillus nidulans.

Unstable duplication and diploid strains of Aspergillus nidulans were treated with ethidium bromide, and it was shown that this drug reduces the number of sectors produced by such strains. The mechanisms which could be responsible for the partial stabilization of the strains are discussed and it is suggested that a similar mechanism is responsible for the production of sectors in both strains. It is also suggested that ethidium bromide could be useful for the reduction of instability of industrial strains.     

Effects of ethidium bromide in diploid and duplication strains of Aspergillus nidulans

Summary Unstable duplication and diploid strains of Aspergillus nidulans were treated with ethidium bromide, and it was shown that this drug reduces the number of sectors produced by such strains. The mechanisms which could be responsible for the partial stabilization of the strains are discussed and it is suggested that a similar mechanism is responsible for the production of sectors in both strains. It is also suggested that ethidium bromide could be useful for the reduction of instability of industrial strains.Acknowledgment. The authors are thankful to the National Council for the Development of Science and Technology (CNPq) for financial assistance.     

Hotspots of homologous recombination

Homologous recombination occurs at higher than average frequency at and near hotspots. Hotspots are special nucleotide sequences recognized by proteins that promote, directly or indirectly, a rate limiting step of recombination. This review focuses on two well-studied examples, the Chi sites of the bacteriumEscherichia coli and the M26 site of the fission yeastSchizosaccharomyces pombe. Chi, 5 G-C-T-G-G-T-G-G 3, is recognized by the RecBCD enzyme, which nicks the DNA near Chi and produces a 3-ended single-stranded DNA tail; this tail is a potent substrate for homologous pairing by RecA and single-stranded DNA binding proteins. M26, 5 A-T-G-A-C-G-T 3, is recognized by a heterodimeric protein and stimulates, by an as-yet-unknown mechanism, meiotic recombination at and near theade6 gene. Additional hotspots in bacteria, fungi, and mammals enhance recombination directly or indirectly via a variety of mechanisms. Although hotspots are widespread among organisms, the biological role of their localized enhancement of recombination remains a matter of speculation.     

Comparison of the effects of ethidium bromide and of the ethidium bromide-deoxyribonucleic acid complex in Ehrlich tumor cells]

When injected into the peritoneal cavity, ethidium bromide can strongly inhibit the multiplication of mouse Ehrlich ascites tumour cells. This antitumour effect is increased when ethidium bromide is linked to DNA and also injected into the peritoneal cavity. The cellular alterations are identical after a treatment with E.B. either free or bound to DNA. However, when the cells are treated with E.B.-DNA they contain E.B. for a longer period than after a treatment with E.B.     

Analysis of the immune system with transgenic mice: T cell development

Transgenic mice carrying functionally rearranged T cell receptor genes have contributed significantly to our knowledge of T cell development and thymic positive and negative selection processes. In addition, TCR-transgenic mice have been used to investigate mutations affecting thymocyte development, likescid andlpr. Gene targeting by homologous recombination will allow to analyze more specifically the molecular mechanisms underlying thymic selection and peripheral tolerance.     

Homologous recombination in fission yeast: Absence of crossover interference and synaptonemal complex

The study of homologous recombination in the fission yeastSchizosaccharomyces pombe has recently been extended to the cytological analysis of meiotic prophase. Unlike in most eukaryotes no tripartite SC structure is detectable, but linear elements resembling axial cores of other eukaryotes are retained. They may be indispensable for meiotic recombination and proper chromosome segregation in meiosis I. In addition fission yeast shows interesting features of chromosome organization in vegetative and meiotic cells: Centromeres and telomeres cluster and associate with the spindle pole body. The special properties of fission yeast meiosis correlate with the absence of crossover interference in meiotic recombination. These findings are discussed. In addition homologous recombination in fission yeast is reviewed briefly.This article is dedicated to Urs Leupold, the founder of fission yeast genetics.     

Chromosomal localization of the locus PGM (phosphoglucomutase) inDrosophila buzzatii

Summary The locus PGM ofD. buzzatii is localized in the linkage group of chromosome 4, outside the region blocked by the inversion 4s and with a recombination percentage of about 16% from the inversion breakpoint.     

Fluorescence response of acridine orange to changes in pH gradients across liposome membranes

The influence that changes in proton distribution have on the fluorescence of acridine orange was examined using negatively charged liposomes. Our results indicate that at least two mechanisms are involved: distribution of the probe between the internal aqueous phase of the liposomes and the outside medium, and binding of the probe to the liposome membranes.     

Analysis of the relationship between age of larvae at mutagen treatment and frequency and size of spots in the wing somatic mutation and recombination test inDrosophila melanogaster

The relationship between the induction of mutant clones and the time of mutagen treatment was studied in the somatic mutation and recombination test (SMART) in wing cells ofDrosophila melanogaster. Larvae trans-heterozygous for the recessive marker mutations multiple wing hairs (mwh) and flare (flr) were produced. Batches of these larvae were then treated with mutagen at different ages spanning all three larval instars. Methyl methanesulfonate was fed acutely for 2 h by immersing the larvae in a solution of the mutagen mixed with powdered cellulose. Wings of the surviving adult flies were mounted and scored for the presence of spots. The frequency and size of single and twin spots were recorded separately. Twin spots are produced exclusively by mitotic recombination, whereas single spots can results from various types of mutational and exchange events. There exists a clear correlation between time of induction and frequency as well as size of the single spots. In young larvae only few but very large spots are induced, whereas in older larvae the frequencies are considerably increased but the sizes are smaller. The twin spots show a different relationship. Practically no twin spots are found in very young and in very old larvae. The results demonstrate that in the wing spot test the optimal age of the larvae for mutagen treatment is 72 h.     

Fluorescence response of acridine orange to changes in pH gradients across liposome membranes

Summary The influence that changes in proton distribution have on the fluorescence of acridine orange was examined using negatively charged liposomes. Our results indicate that at least two mechanisms are involved: distribution of the probe between the internal aqueous phase of the liposomes and the outside medium, and binding of the probe to the liposome membranes.     

Genotoxic evaluation of ten carcinogens in theDrosophila melanogaster wing spot test

To provide further background data on the wing spot somatic mutation and recombination assay, 10 selected carcinogens (acetamide, acrylamide, benzo(a)pyrene, cyclophosphamide, diethylstilbestrol, 4-nitroquinoline N-oxide, propyleneimine, safrole, thiourea, and o-toluidine) were tested in this assay. 72-h-old third-instar larvae, trans-heterozygous for 2 recessive wing cell markers:multiple wing hairs (mwh) andflare ³ (flr ³) were fed with 3 concentrations of each carcinogen during the rest of their development until pupation, and the genotoxic effects were measured as significant increases in the appearance of visible mutant hair clones on the adult wing blade. Our results show that 6 of the carcinogens tested produce significant increases in wing spot frequency, at least at one of the concentrations assayed. Benzo(a)pyrene, diethylstilbestrol, safrole and thiourea were the compounds that did not increase the incidence of mutant clones.     

Sex linkage of malic enzyme inXenopus laevis

Genetic analysis of mME variants (mitochondrial malic enzyme, E.C. 1.1.1.40) inXenopus laevis revealed sex linkage of the mMe locus and indicated a WZ/ZZ type of sex determination. Codominant mMe alleles occur on both W and Z chromosomes, with a recombination frequency of 6.1%±1.5% between mMe and the sex-determining locus (or region).     

Genetic variability in 3 co-occurring forms of the starfish genusOthilia (=Echinaster)

Summary High levels of genetic variability have been found in 3 (grey, brown, orange) co-occurring forms of the starfishOthilia. These high levels appear to correspond to the generalistic habit of this starfish. The biochemical data, coupled with morphological observations, indicate that the grey and orange forms are morphs of the same species and that the brown form is a species separate from the grey and orange forms.     

Differences between drosophila melanogaster and its sibling species D. simulans in sensitivity to acridine orange treatment

Larvae and adults of D. melanogaster and D. simulans were fed with acridine orange, in order to test sensitivity differences between the species. Our results show that, of the two species, D. simulans is more resistant in the larval stages, and D. melanogaster is more resistant in the adult stage. Furthermore, adult males of both species are more sensitive than adult females.