A new basic metachromatic dye, I:9-dimethyl methylene blue

Synopsis The preparation and staining properties of a new basic dye 19-Dimethyl Methylene Blue, are described. It is a sensitive dye for the demonstration of metachromasia and, since it may be prepared in a pure state, the results obtained with it are consistent.     

Optical studies of the interaction of 4′-6-diamidino-2-phenylindole with DNA and metaphase chromosomes

The optical absorption and fluorescence characteristics of 4-6-diamidino-2-phenylindole (DAPI) with DNA and chromosomes were studied. There is a decrease in extinction coefficient and shift in the absorption spectra to a higher wavelength when the dye binds to DNA. The fluorescence of DAPI is enhanced by both A-T and G-C base-pairs. The enhancement by A-T rich is significantly greater than by G-C rich DNA. The dye produces a localized bright fluorescence in centromeric regions of mouse chromosomes and the constrictions of human chromosomes 1 and 16; these regions are known to contain A-T rich DNA and show dull fluorescence when treated with quinacrine. This dye may be useful for identifying A-T rich region in chromosomes. The fluorescence of DAPI bound to polynucleotides or chromosomes is partially quenched by the introduction of BrdU. This suppression of dye fluorescence allows optical detection of sister chromatid exchanges and chromosome region containing DNA with an unequal distribution of thymidine between polynucleotide chains after BrdU incorporation.     

Yeast mutants resistant to ethidium bromide

Summary Yeast mutants resistant to ethidium bromide have been isolated among sensitive grande cells (⁺) for their ability to grow on glycerol in the presence of the dye. Mutant cells are also resistant to acriflavin and do not yield petites (^-) when grown on galactose with the mutagen. Genetic analysis reveals that resistance to ethidium bromide is controlled by a cytoplasmic factor, carried by, or linked to, the determinant (mitochondrial DNA). The expression of resistance to ethidium bromide seems to be related to the presence in the cell of a product of mitochondrial protein synthesis. It is concluded that some mitochondrial DNA sequence is involved in the resistance to ethidium bromide of yeast mitochondria.     

In vivo DNA binding of bacteriophage GA-1 protein p6

Bacteriophage GA-1 infects Bacillus sp. strain G1R and has a linear double-stranded DNA genome with a terminal protein covalently linked to its 5′ ends. GA-1 protein p6 is very abundant in infected cells and binds DNA with no sequence specificity. We show here that it binds in vivo to the whole viral genome, as detected by cross-linking, chromatin immunoprecipitation, and real-time PCR analyses, and has the characteristics of a histone-like protein. Binding to DNA of GA-1 protein p6 shows little supercoiling dependency, in contrast to the ortholog protein of the evolutionary related Bacillus subtilis phage 29. This feature is a property of the protein rather than the DNA or the cellular background, since 29 protein p6 shows supercoiling-dependent binding to GA-1 DNA in Bacillus sp. strain G1R. GA-1 DNA replication is impaired in the presence of the gyrase inhibitors novobiocin and nalidixic acid, which indicates that, although noncovalently closed, the viral genome is topologically constrained in vivo. GA-1 protein p6 is also able to bind 29 DNA in B. subtilis cells; however, as expected, the binding is less supercoiling dependent than the one observed with the 29 protein p6. In addition, the nucleoprotein complex formed is not functional, since it is not able to transcomplement the DNA replication deficiency of a 29 sus6 mutant. Furthermore, we took advantage of 29 protein p6 binding to GA-1 DNA to find that the viral DNA ejection mechanism seems to take place, as in the case of 29, with a right to left polarity in a two-step, push-pull process.     

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA

Summary Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1–2 g/ml decrease the yield of transformants, without showing signs of general toxicity.PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.     

DNA primase activity from wheat embryos

DNA primase is a recently discovered enzyme capable of synthesizing short primers involved in the initiation of DNA replication.Partially purified preparations from 4 h germinated wheat embryos or commercial wheat germ are able to catalyze the ribonucleoside triphosphate dependent synthesis of DNA with poly dT and M₁₃ single stranded DNA as templates. DNA synthesis is completely dependent on the presence of template and primase. Primase activity from wheat embryos has a molecular weight of about 110000 and a sedimentation coefficient of 5S. The enzyme activity is not inhibited by -amanitin (1 mg/ml) or aphidicolin when the latter is assayed with endogeneous plant DNA polymerase activity. Alkaline hydrolysis of the product synthesized in the presence of [-³²P]dATP and poly dT generates [³²P]-labeled 3(2)AMP showing that a ribo-deoxynucleotide linkage is formed. The size of the oligoribonucleotide primer varies from 2 to 15 residues. Most of the wheat DNA polymerase activity can be eliminated by phosphocellulose chromatography, since the bulk of plant DNA primase is not retained by this resin. Nevertheless, a small but significant amoung of DNA polymerase activity is found associated with DNA primase. By using different inhibitors of DNA polymerase different templates, we have found good indications that DNA polymerase A (-like) is associated with the DNA primase. Moreover, when the previously purified DNA polymerases from wheat embryos (2) were assayed in the presence of primase activity, only DNA polymerase A was able to stimulate DNA synthesis.     

Influence of Bacteriophage PBS1 and φW-14 Deoxyribonucleic Acids on Homologous Deoxyribonucleic Acid Uptake and Transformation in Competent Bacillus subtilis

Both bacteriophage PBS1 deoxyribonucleic acid (DNA) (in which all the thymine residues are replaced by uracil) and phage W-14 DNA [in which half the thymine residues are replaced by 5-(aminobutylaminomethyl)uracil or 5-putrescinylthymine] exhibit comparable competing abilities for uptake of homologous DNA in a Bacillus subtilis competent system. But, whereas PBS1 DNA leads to a decrease in transformation frequencies compatible with its competing ability for DNA uptake, W-14 DNA decreases transformation frequencies by a factor up to eightfold higher. The effect of W-14 DNA on transformation frequencies is visible even at a concentration level that does not decrease transforming DNA uptake. No such effect was observed with heterologous DNA containing presumably ionically bound putrescine. Low concentrations of W-14 DNA decreased the number of double (nonlinked) transformants more than single transformants. The influence on transformation was abolished when W-14 DNA was added 20 min after addition of transforming DNA, i.e., when the recombination process was terminated. The putrescine-containing DNA also decreased retention of trichloroacetic acid-precipitable radioactivity of homologous DNA taken up. We conclude that W-14 DNA inhibits some intracellular process(es) at the level of recombination. In addition, there is evidence that W-14 DNA, but not heterologous DNA with ionically bound putrescine, binds also to site(s) on the cell surface other than receptors for homologous DNA.     

Effects of Ethanol on the Metachromatic Reaction of Toluidine Blue O

Ethanol abolishes the metachromatic reaction of toluidine blue O with un-combined chromotropes but not when they are in association with protein. The green colour obtained in metachromatic regions is established as not due to any green impurity of the dye by chromatographic analysis but due to the fluid dehydrants combining with the dye as dye-organic solvent mixture showed green. The loss of metachromasia is not due to a dehydration effect of ethanol alone for the following reasons: (i) Stained samples of chromotropes dried in vacuuo continued to retain the metachromatic colour, (ii) Although other dehydrating agents likewise abolished the metachromasia, alcohols which have very slight affinity to water also abolished it, (iii) Ethanol does not abolish metachromasia produced in an acid mucopolysaccharide-protein complex. This has been suggested as due to the inability of ethanol to separate the dye from such compounds and to bring about a shift to green.     

Highly efficient labeling of DNA polymerases by a binary system of photoaffinity reagents

A binary system of photoaffinity reagents was proposed earlier for highly efficient labeling of DNA polymerases by 5"-[³²P]DNA primers. In the present study we demonstrate the feasibility of this approach to increase the efficiency of DNA polymerase labeling. A photoactive 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was incorporated at the 3"-end of 5"-[³²P]DNA primers synthesized by DNA polymerase or Tte in the presence of one of the dTTP analogs—FAB-4-dUTP, FAB-9-dUTP, or FAB-4-ddUTP. The reaction mixture was irradiated by light with wavelength of 334-365 nm (direct labeling) or 365-450 nm in the presence of photosensitizer, one of dTTP analogs containing a pyrene moiety, Pyr-6-dUTP or Pyr-8-dUTP. In the case of the binary system of photoaffinity reagents, a FAB group is activated by energy transfer from sensitizer localized in the dNTP-binding site of DNA polymerase in the triple complex, comprised by reagent, DNA polymerase, and Pyr-6(8)-dUTP. Direct activation of the FAB group under these conditions is negligible. The most efficient photolabeling of DNA polymerases was observed with a primer containing a FAB-4-dUMP group at the 3"-end, and Pyr-6-dUTP as a photosensitizer. Using 10-fold molar excess of photoreagent to DNA polymerase , the labeling efficiency was shown to achieve 60%, which is 2-fold higher than the efficiency of the direct DNA polymerase labeling under harsher conditions (334-365 nm).     

Intracellular effects of phage phi W-14 DNA on transformation of Bacillus subtilis

Summary Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage W-14 DNA (in which half the thymine residues are replaced by -putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37°C after DNA uptake at 30°C in the presence of low concentrations of W-14 DNA (0.1 g/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of W-14 DNA was enhanced by an increase in its concentration (to 0.5–1 g/ml), or by increasing the temperature of uptake (to 37°C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 g/ml) or untreated with W-14 DNA, and incubated for 20 min at 37°C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with W-14 DNA. The influence of a high concentration of W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation. On exposure to low concentrations of phage DNA, such a correlation was observed only after longer periods of incubation, due to slower intracellular degradation of homologous DNA taken up. The results are consistent with the proposal that W-14 DNA-induced reduction in efficiency of transformation is due to intracellular stimulation of transforming DNA degradation, leading to a decrease in the number of donor molecules available for recombination with the recipient chromosome.     

Further studies on polyploid amphibians

The manner in which centromere regions of mitotic chromosomes are distributed with respect to the age of their DNA was studied. Cells of the Indian deer, Muntiacus muntjak, were grown in the presence of bromodeoxyuridine (BrdU) for two generations and stained with the fluorescent dye Hoechst 33258. Chromatids containing granddaughter DNA appear dim when compared with those containing grandparental DNA. The frequencies of the various anaphase patterns of bright and dim centromere regions were binomially distributed, indicating random distribution of chromatids with respect to the age of their DNA templates.     

CTAB enhancement of FRET in DNA structures

The effect of cetyl‐trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye‐conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye‐conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

     

Effects of acridines on DNA synthesis in vitro

Summary Acridines are known to induce with a very high efficiency the cytoplasmic petite mutation in yeast. Since in petite mutants the base composition of mitochondrial DNA is altered, these dyes should interfere in the replication of mitochondrial DNA. To approach this problem a detailed knowledge of the effect of acridines on DNA synthesis is necessary.The inhibitory effect of two acridines and of the related dye methylene blue on Escherichia coli DNA polymerase has been studied under different assay conditions. Several aspects of this inhibitory effect have been investigated including the extent of inhibition in the presence of several primers, the effect of acridines on the unprimed synthesis and on the addition of single nucleoside triphosphates to DNA 3-OH terminals, the effect of preincubation of DNA and of DNA polymerase with acridines and the base composition of DNA synthesized in the presence of the dyes.The extent of inhibition has not been found to be dependent on the base composition of the primer except in the case of primers very rich in A+T, while a strong effect on the unprimed synthesis is due to a substantial increase of the lag period. The A/C ratio in the product synthesized under strongly inhibitory conditions is shifted with respect to that of the primer.The results seem to indicate the preferential replication, in the presence of acridines, of A+T rich regions of DNA.     

Fluorescence metachromasia in polypeptide hormone-producing cells of the APUD series, and its significance in relation to the structure of the precursor protein

Synopsis A fluorescence metachromatic modification of the masked basophilia method is described. It is based on the acridine dye Coriphosphine O. Excitation and emission spectra of green (orthochromatic) and red (metachromatic) fluorescent tissue components are presented.When the method is applied to suitably fixed sections, metachromasia is demonstrable in cells of the polypeptide hormone-secreting APUD series, and in a few other situations.The view that side-chain carboxyl groups are demonstrated by the masked basophilia technique is considered to be accurate but inadequate. It is proposed that the technique, and its fluorescence modification, are influenced more by secondary than by primary protein structure. In particular, it is suggested that the conformation of the protein precursors of polypeptide hormones, in the storage granules of endocrine cells, is predominantly random-coil.     

Macromolecular synthesis and cell division during morphogenesis of Arthrobacter crystallopoietes

The sphere-rod-sphere morphology cycle of Arthrobacter crystallopoietes was accompanied by changes in the rate of growth and the rates of DNA, RNA and protein synthesis. The patterns of macromolecule synthesis resembled those found in other bacteria during a step-up followed by a step-down in growth rate. During the step-up in growth spherical cells grew into rods and macromolecules were synthesized in the absence of cell division. During stepdown, successive rounds of septation produced progressively smaller cells which did not separate and remained in chains. The morphology of the cells was dependent on the growth rate and could be altered by changing the dilution rate in a malate-limited chemostat. Gradual transitions in morphology and gradual increases in macromolecule content of the cells occurred as the growth rate was increased in the chemostat. Sphere to rod morphogenesis occurred when DNA synthesis was inhibited by treatment with mitomycin C or by thymine starvation. The DNA-deficient rods did not divide and eventually lysed. DNA, RNA and protein synthesis were continuously required for the reductive division of rods to spheres.Abbreviations MS mineral salts - GS mineral salts plus glucose - CA casamino acids - GSCA mineral salts plus glucose plus casamino acids - cAMP cyclic adenosine-3,5-monophosphate - RNA ribonucleic acid - DNA deoxyribonucleic acid     

Onion fly,Delia antiqua,oviposition and mating as influenced by insect age and dosage of male reproductive tract extract (Diptera: Anthomyiidae)

One hundred percent of virgin female onion flies,Delia antiqua, receiving 1/20 of a male equivalent of an aqueous extract of mature male reproductive tract remained unmated in the presence of males and began laying unfertilized eggs at a normally mated rate of about 20 eggs/female/day. The 50% behavioral response (BR₅₀) fell between 1/40 and 1/20 of a male equivalent. Sex peptide responses are not always all-or-none. Some females receiving extract at 1/40 male equivalent oviposited at an intermediate rate. Moreover, at low sex peptide dosages, some females were fully activated ovipositionally but were receiptive to mating. A low level of sex peptide was present in 1-day-old males. Sex peptide titer rose with age until plateauing by 6 days posteclosion. Males began mating at 3 days, when they first had ample mature sperm; 50% of 6-day-old males mated. The mean number of females inseminated per male exposed to an excess of virgin females over 24 h was 4.3±0.6 (±SE). Presence of mature eggs was not always a prerequisite for mating, although probability of insemination was correlated with egg maturation. One-day-old preovipositional females receiving 1/20 of a male equivalent of extract began ovipositing when they had mature eggs at 5–6 days old. Therefore, sex peptide may act early and permanently or have a long half-life and affect behaviors once females reach sexual maturity. Male flies provide females with an excess of sex peptide in many cases.D. antiqua males transferred ca. 5–10 times more sex peptide than necessary to activate females fully. We suggest this excess is related to the speed of female response. It is yet unclear whether sex peptide potency or titer in Diptera has become exaggerated by intra- or intersexual selection.     

On the mechanism of the methyl green-pyronin stain for nucleic acids

Summary The combination of pyronin, methyl green, malachite green, crystal violet, and Alcian Blue with a large number of polynucleotides and acidic polysaccharides has been investigated. The critical electrolyte concentration approach has been used to provide a measure of affinity between dye and substrate.The interaction of methyl green with DNA, RNA and heparin has been examined spectroscopically.Previously published results are re-examined, and with the new experiments permit consistent interpretations of the specificities of dye binding in terms of modern ideas of nucleic acid and dye structure.All dyestuffs except Alcian Blue bind more strongly to polynucleotides than would be expected if solely electrostatic bonds were present. Pyronin and planar monovalent cationic dyes interact best with polynucleotides in which purine and pyrimidine bases are freely accessible, as in single stranded molecules without extensive secondary structure, such as RNA, denatured DNA, etc. Non-planar triphenylmethane dyes, e.g. methyl green, malachite green etc. bind less strongly to such substrates, but because of their shape they fit well into the secondary structure of native DNA. Tumour RNA and DNA did not differ from normal RNA and DNA.By varying the electrolyte concentration, pyronin-methyl green selectivity e.g. for DNA or RNA, can be controlled, and non-nucleotide staining suppressed. The relevance of the new interpretation to the ribonuclease-pyronin technique is discussed.     

Metachromasia of basic dyes induced by mercuric chloride. I

Summary Interactions of the cationic dye methylene blue with mercuric chloride have been studied conductometrically, analytically and spectrophotometrically. Methylene blue produces red colored precipitate with mercuric chloride; in presence of large excess of mercuric chloride a strong metachromasia is induced in the dye. Metachromasia induced by mercuric chloride is more hypsochromic as well as hypochromic than that induced by chromotopes like heparin. The complexes formed between methylene blue and mercuric chloride have variable compositions, the complex responsible for the red metachromatic color of the dye has the composition 2 dye: 1 HgCl₂. A model has been proposed for the metachromatic complex consisting hexa-coordinated mercury, dye is coordinated to the mercury by donating the lone pair electrons of terminal nitrogen. The non-metachromatic dye capri blue also interacts with mercuric chloride but without any change in the visible spectrum. Potassium iodide also gives metachromatic reddish blue colored precipitate with methylene blue.University Research Scholar.     

A molecular marker associated with the H21 Hessian fly resistance gene in wheat

Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat (Triticum aestivum L.) associated with the H21 gene conferring resistance to biotype L of Hessian fly [Mayetiola destructor (Say)] larvae. Near-isogenic lines were developed by backcross introgression BC3F3:4 (Coker 797 * 4 / Hamlet) and differed by the presence or absence of H21 (on 2RL) derived from Chaupon rye (Secale cereale L.). Bulked DNA samples were prepared from near-isogenic lines and BC3F2 population individuals segregating for reaction to Hessian fly biotype L and screened for random amplified polymorphic DNA markers using 46 10mer primers. Random-amplified polymorphic DNA markers from resistant and susceptible individuals and parental lines were scored and these data were used to identify a 3 kb DNA fragment that was related to the occurrence of H21. This fragment was amplified from DNA isolated from Hamlet, a near-isogenic line carrying 2RL, and bulked DNA from resistant BC3F2 individuals, but not from the recurrent parent Coker 797 or DNA bulks from susceptible BC3F2 plants. Analysis of 111 BC3F2 segregating individuals and BC3F2:3 segregants confirmed the co-segregation of the 3 kb DNA marker with the H21 resistance gene to Hessian fly. Use of this marker could facilitate more rapid screening of plant populations for Hessian fly resistance and monitoring the introgression of H21.     

Influence of the DNA structure on the free radical induction due to proflavine and light treatment

Summary Induction of peroxide free radicals (detected by Electron Paramagnetic Resonance at 77 K) due to the photodynamic activity of proflavine was measured on bacteriophage X174 DNA either single-stranded (ss) as isolated from the virion, or double-stranded supercoiled (RFI) as isolated from the infected bacteria. Comparison was made with calf thymus DNA photosensitization.In order to use equivalent DNA-proflavine complexes, binding of the dye to the three DNA's was first determined under those conditions of high ionic strength favourable to the photodynamic reaction.Free radical induction was maximal for definite amounts of bound proflavine (which varied depending upon the DNA substrate) and at an ionic strength value of 0.5. The level of the maximal reaction increased in the following order: from Xss DNA to calf thymus DNA and finally to XRFI DNA. The conformation of the proflavine-DNA complex was thus a determinant for the efficiency of the photodynamic process. The ionic strength effect could not be explained by the evolution of the proflavine triplet state in irradiated proflavine-calf thymus DNA complexes.