Additional observations on the preparation of R banded human chromosomes with acridine orange.

A number of technical factors which affect acridine orange R banding (RFA banding) were studied. These variables included age of slide, timing of fixation, details of incubation mounting and the use of sequential technics. Optimal RFA banding was obtained between 15 and 20 days but good or very good preparations were obtained between 7 days and 2 months. Improved results were obtained in slides that were 3-4 months old by refixing the slides in ethanol acetic acid. Intermittent movement of slides during incubation in buffer as well as the details of mounting and removal of cover slips were found to be important. The best sequential banding was obtained with the sequence of Q to R but good results were obtained with the sequence G to R using ASG banding. Satisfactory results with the sequence R to C were not obtained. With careful attention to these variables good RFA binding can be obtained over a period of several months.     

An Assessment of Routine Chromosomal Staining Procedures in Radioautography

Unlabeled human chromosome preparations were treated with commonly employed chromosome stains as follows: (I) they were stained, destained, coated with liquid emulsion, developed, fixed, and restained; (II) stained and coated directly; or (III) coated and then stained. Of the stains tested, the methylene blue-eosin type (Giemsa, MacNeal's, Wright's) was useful for application after coating, although a similar stain (eosin-Stevenel's blue) caused formation of a heavy precipitate in the emulsion when so used. None of these stains could be employed before coating, however, even though they were removed with acid alcohol prior to dipping, because they caused chemographic grain formation in the emulsion. Aceto-orcein and Feulgen could not be employed after coating because the procedures removed the emulsion from the slides. Safranin was also found to be ineffective for staining coated preparations due to chemical changes caused by the photographic processing. The only stain which did not cause chemography, and hence can be used before coating slides, is aceto-orcein. Since this stain fades during radioautographic processing and cannot be employed after coating, we recommend secondary use of one of the methylene blue-eosin type stains for revisualization of the chromosome spread.     

Interaction of glyceryl trinitrate and sodium nitroprusside with bovine pulmonary vein homogenate and 10,000 x g supernatant: biotransformation and nitric oxide formation

The current proposed mechanism of action of nitrovasodilator drugs involves biotransformation to nitric oxide, which is postulated to be the active vasodilator substance. Our objective was to determine whether nitric oxide was formed from two prototype nitrovasodilator drugs, glyceryl trinitrate (GTN) and sodium nitroprusside (SNP), after incubation with bovine pulmonary vein (BPV) preparations. GTN or SNP was incubated in an argon atmosphere with phosphate buffer, BPV homogenate, or the 10,000 x g supernatant fraction of the homogenate. Nitric oxide formation, as determined by a chemiluminescence-headspace gas method, was measurable following the incubation of SNP with BPV homogenate and 10,000 x g supernatant. There was no detectable formation of nitric oxide from the incubation of GTN with the two BPV preparations, although GTN was biotransformed to glyceryl dinitrate, as determined by gas-liquid chromatography. There was decreased recovery of nitric oxide during the incubation of authentic nitric oxide with the two BPV preparations as compared with buffer. In conclusion, formation of nitric oxide was measured for the interaction of SNP, but not GTN, with BPV preparations. However, the data do not exclude the possible formation of nitric oxide from GTN, as nitric oxide was shown to be sequestered or transformed by the BPV preparations.     

An analysis of the technical variables in the production of C bands

Numerous combinations, concentrations, pH's and durations of HCl, NaOH and SSC treatments were tested for the purpose of developing an improved C banding technique for human metaphase chromosomes. Methods of slide preparation, as they affect C banding were also evaluated. — HCl and SSC treatment used separately, for all times and concentrations tested, gave no C banding. All treatment sequences which included an NaOH exposure gave at least some C banding, but also gave considerable swelling and distortion. Surprisingly, the best results were obtained from heat-dried preparations exposed to 0.2 N HCl at 25° C for 15 minutes, no NaOH and subsequently incubated in 2xSSC, pH=7.0 at 62–65° C for 18–24 hours. This technique is now being used routinely, following a G banding technique for homologue identification, to monitor C band variation in human chromosomes. — The pH of the 2xSSC incubation solution was found to be important. Slides treated as above with HCl, but with 2xSSC, pH=6.0 gave only G banding; HCl and 2xSSC, pH=8.0 gave C banding, but considerable chromosome swelling and poor uptake of stain. — Air- or ignition-dried preparations, with the HCl and 2xSSC treatment appeared undertreated and gave a mixture of G and C banding. A brief (30 second) exposure to 0.07 N NaOH between the HCl and 2xSSC steps is recommended. These results are in support of DNA-protein interaction and/or loss rather than denaturation-renaturation as a likely mechanism for C band production.     

Fluorescent C bands of human chromosomes with 33 258 Hoechst stain

Summary Air-dried preparations of human metaphase chromosomes normally exhibit a Q band fluorescence pattern with 33 258 Hoechst stain while the C band regions of 1, 9, 16, and most acrocentric short arms appear dull. If these stained and mounted slides are stored at room temperature in the dark for several days, a spontaneous change from C-negative to C-positive bands sometimes occurs. We postulate that the pH of the buffered saline mounting medium during storage of the slides causes the C band shift since the results can be duplicated experimentally by lowering the pH of the mounting buffer from 5.5 to 4.0.     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.     

Oxidation of low-density lipoprotein by copper and iron in phosphate buffer

We examined the effect of phosphate buffer on the iron- and copper-catalyzed peroxidation of low-density lipoprotein (LDL). The incubation of LDL with CuSO4 in 0.15 M NaCl led to the peroxidation of LDL as evidenced by the detection of thiobarbituric acid-reactive substances (TBARS) and lipid hydroperoxides (LPO). The peroxidation of LDL was also observed with FeSO4 and FeCl3 in 0.15 M NaCl, although there was a lag phase with FeCl3. In 10 mM phosphate buffer, the peroxidation of LDL was observed with CuSO4 to an extent similar to that in 0.15 M NaCl. However, the peroxidation induced by incubation with FeSO4 and FeCl3 was significantly inhibited in phosphate buffer. Iron and copper each formed a complex with lipoprotein during incubation with LDL in 0.15 M NaCl. Although no effect on the formation of copper-LDL complex was observed in phosphate buffer, the formation of iron-LDL complex was reduced in the buffer. These observations suggest there are marked differences in the peroxidation of LDL and in the formation of complexes with LDL between iron and copper in phosphate buffer.     

The role of chromosomal proteins in the C-banding of Allium cepa chromosomes.

When chromosomes of Allium cepa are subjected to a C-banding procedure (incubation in saturated barium hydroxide followed by phosphate buffer at 60 degrees C for 1 h) and then treated with Giemsa stain, bands appear at the telomeres of all chromosomes. Microspectrophotometric measurements of Feulgen-DNA content, demonstrated that the C-banding procedure extracted DNA from the nuclei. Staining of banded chromosomes with several DNA-specific stains showed that this loss was differential, with the band DNA exhibiting more resistance to extraction than that of the rest of the chromosome. The C-banding procedure did not extract chromosomal proteins, however, and no difference in mass per unit length could be detected by Nomarski optics between band and interband regions. Several experiments demonstrated that chromosomal proteins play a significant role in C-banding. First, treatment of chromosomes with pronase before C-banding resulted in the elimination of differential staining with Giemsa. Furthermore, in preparations where the DNA was completely hydrolysed with hot TCA, the remaining chromosomal proteins were found to exhibit a differential affinity for Giemsa stain. Amido black staining demonstrated that total chromosomal protein was uniformly distributed after the hot TCA digestion, but the proteins localized in the telomeres had a greater affinity for the Giemsa stain than the bulk of the chromosomal proteins. When the TCA-digested chromosomes were subjected to the C-banding procedure before staining, the differential affinity of the telomeres for the Giemsa stain was lost. Thus, C-banding appears to be the result of a complex interaction between protein and DNA in which the greater resistance to extraction of the band DNA is necessary to stabilize and preserve chromatin protein which exhibits a differential affinity for Giemsa stain.     

Formation of inside-out vesicles of Bacillus licheniformis. Dependence on buffer composition and lysis procedure.

1. The extent to which the cytoplasmic membrane of the Gram-positive bacterium Bacillus licheniformis formed inside-out vesicles was studied with the freeze-fracture technique. The membrane orientation appeared to be dependent on the buffer compositon as well as on the lysis procedure used. 2. By manipulating these conditions, membrane preparations were obtained with the percentage of inside-out vesicles varying from 15 to 80%. 3. More vesicles had the opposite orientation when the cells were lysed in potassium phosphate buffer than when they were lysed in sodium phosphate buffer. Tris-HCl buffer favoured the formation of inside-out vesicles more than phosphate buffer. 4. Lysis of protoplasts in hypotonic buffers resulted in more inside-out vesicles than did direct lysis of cells in hypotonic media. 5. In an attempt to explain the observed differences, experiments were performed in which the morphology of thin-sectioned lysing cells in sodium phosphate buffer was compared with that in potassium phosphate buffer. The results from these experiments indicate that the formation of inside-out vesicles is brought about by an effect on the membrane itself rather than on the cell wall, on the cell wall membrane association, or on the cytoplasm.     

Spreading and staining of human metaphase chromosomes on aminoalkylsilane-treated glass slides

Summary The properties of aminoalkylsilane-treated glass slides for the preparation of metaphase spreads and their staining quality have been studied and compared with those of slides which had only been cleaned in ethanol/ether. The parameters investigated were: (1) the average area of metaphases from cultures of blood from both healthy donors and haematology patients; (2) the influence of the positively charged coating on the quality of quinacrine- and Giemsa-banding patterns; (3) non-specific background staining for these banding methods; (4) the number of metaphases as compared to the number of interphase cell nuclei per area of preparation; and (5) the Feulgen-staining intensities of chromosomes and chicken erythrocyte nuclei.The quality of metaphase preparations and the differential staining of chromosomes is better on aminoalkysilane-treated glass slides than that of preparations on routinely cleaned normal microscope slides. In the preparations on aminoalkylsilance-treated slides, the distribution of the cells over the glass surface is more homogeneous; and no influence could be detected on the relative frequency of metaphases as compared to the number of non-divided cell nuclei; the average area per metaphase is increased by about 10% and consequently the number of overlapping chromosomes is decreased.Preparations on aminoalkylsilane-treated glass, after Q-, G- and DAPI-banding procedures, always showed less binding of the staining compounds to the glass slide (a cleaner background) than those on routinely cleaned microscope glass slides. The Feulgen-pararosaniline staining intensities of human metaphase chromosomes and chicken erythrocyte nuclei are the same on aminoalkylsilane-treated slides and on routinely cleaned glass slides. Furthermore, the reproducibility and constancy of quinacrine banding was improved by development of an equilibrium staining method which does not require a washing procedure. The medium, containing 0.002% quinacrine, allows optimal staining results to be obtained for microphotography purposes within 30 min of staining (for visual inspection at least 90 min is required) and is used as the embedding medium.In combination with aminoalkylsilane-treated glass slides, this procedure leads to a clean background and reproducible banding patterns of excellent quality, the results being better and more constant than those of methods described before.     

The Relationship of Slide Maturity and Trypsin Exposure Time in the Differential Giemsa Banding of Chromosomes

Difficulty has been reported in attaining consistent chromosome banding using trypsin-Giemsa techniques. When trypsin concentration, incubation temperature, and the duration of staining are held constant, a logarithmic relationship has been found to exist between the length of time since a slide was made and the length of trypsin treatment necessary to produce optimal banding when it is stained. Under the conditions specified in this report, ten seconds digestion was optimal for ten-day-old slides. This time approximately doubled for each additional eight days that slides were allowed to age. From a plot of the experimental results, treatment times appropriate for slides of any age from 10 to 60 days were obtained. Use of these times produced satisfactory banding in 85% of mitoses.     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.     

Membrane glycoproteins of Chlamydomonas eugametos flagella

The flagellar glycoproteins exposed on Chlamydomonas eugametos gametes were labeled by means of lactoperoxidase, diiodosulfanilic acid and chloramine T, and characterised in SDS-electrophoresis gels. The medium from gamete cultures contains particles (isoagglutinins) that agglutinate gametes of the opposite mating type. When crude preparations of these particles were subjected to isopycnic centrifugation in a caesium chloride gradient, two bands of particles were found. The lighter, active band consisted of membrane vesicles. The denser, inactive band consisted of cell wall material. The active band had the same glycoprotein composition as membrane vesicles artificially made from isolated flagella. Preparations of glagella were also separated on a caesium chloride cushion into pure flagella and cell wall material. The flagella, but not the cell wall material, isoagglutinated opposite gametes. Again the glycoprotein composition of pure flagella was similar to that of pure isoagglutinin vesicles. No difference was detected between the protein and glycoprotein compositions of flagella and isoagglutinins from both mating types.Abbreviations LPO lactoperoxidase - PB phosphate buffer - DISA diazotized ¹²⁵I-iodo-sulfanilic acid - SDS sodium dodecyl sulphate - CBD coomassie Brilliant Blue - PAS periodic acid Schiff     

Ferrous ion-induced strand breaks in the DNA plasmid pBR322 are not mediated by hydrogen peroxide

Ferrous ion-induced generation of single and multiple strand breaks in the DNA plasmid pBR322 induces the formation of two new plasmid forms with altered electrophoretic mobility. The yield of these plasmid forms, the circular relaxed and the linear forms, depended on the applied Fe²⁺ concentration. This property was independent of the presence of hydrogen peroxide in the incubation mixture indicating the lack of Fenton chemistry to explain the DNA degradation. The removal of dioxygen or the presence of superoxide dismutase diminished partially the yield of ferrous ion-induced DNA plasmid degradation, while catalase was without any effect. Autoxidation of divalent iron as followed by the formation of a coloured iron-phenanthroline complex was enhanced in a concentration-dependent manner by phosphate and bicarbonate and very efficiently using a mixture of 0.15 M NaCl, 1.2 mM phosphate, 23.8 mM bicarbonate, pH 7.4, that concentrations correspond closely to the intracellular values of buffer components. Thus, the formation of a yet unknown reactive species from Fe²⁺, and dioxygen, that is complexed to buffer components especially phosphate and its contribution in DNA plasmid degradation is more likely than the often cited formation of hydroxyl radicals in result of the Fenton reaction from Fe²⁺ and hydrogen peroxide. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m² there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). At 70–300 J/m² the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer. — BrdU substitution and the irradiation of BrdU-substituted chromosomes with 313 nm light at 1800–15000 J/m² did not block the action of restriction nucleases. On the other hand, UV irradiation of BrdU-substituted chromosomes inhibited the action of restriction enzymes at the same fluences that blocked the nuclease action in unsubstituted chromosomes. The data indicate that DNA-protein crosslinkage is the factor inhibiting DNA extraction and chromosome banding.     

Aerotaxis and chemotaxis ofAzospirillum brasilense: A note

Azospirillum brasilense was attracted to capillaries containing either phosphate buffer, distilled water, or saline. The number of bacteria in these capillaries was 3–4×10⁴, after 1 h of incubation. In the presence of phosphate buffer + attractants, the number of cells accumulated in the capillary increased only to 5×10⁴–1.1×10⁵ cells. It was not possible, therefore, to measure chemotaxis inA. brasilense as distinct from aerotaxis by the capillary method. Chemotaxis was observed in semi-solid agar plates and was determined by a growth band oriented towards the attractant. Positive chemotactic response was obtained with peptone, tryptone, yeast extract, amino acids, organic acids, arabinose and galactose.     

Effect of amino acid starvation on UV sensitivity ofLactobacillus acidophilus cells

InLactobacillus acidophilus cultures UV irradiated in the exponential phase of growth, the dosesurvival curve was of the simple exponential type, without any shoulder. If the bacteria were subjected to amino acid starvation prior to irradiation, an shoulder corresponding to a quasi-treshold dose (D_q) of about 780 ergs/mm² appeared in the curve. The administration of protein or RNA-synthesia inhibitors prior to irradiation had the same effect. The effect of pre-irradiation amino acid starvation was abolished by simultaneous thymidine starvation. It was likewise abolished if amino acid starvation was followed by incubation in the presence of amino acids (without thymidine) and then by irradiation of the cells. Post-irradiation amino acid starvation did not lead to the formation of an shoulder but if combined with thymidine starvation it did. It can be concluded from the results that post-irradiation repair processes are facilitated or promoted if, during the post-irradiation interval DNA synthesis is delayed. This delay represents a compensation of the pre-irradiation increase of cellular DNA-content, taking place during inhibition of proteosynthesis. The postirradiation administration of caffeine did not abolish the formation of the shoulder induced by pre-irradiation amino acid starvation; on the contrary, it induced its formation even in exponentially growing, irradiated control bacteria.     

Environmental influences on skeletal banding in eastern Pacific (Panama) corals

The timing of skeletal band formation and concomitant changes in calcification rates and linear skeletal extension were investigated in Pavona corals growing under two distinct thermal regimes along the Pacific coast of Panama: fluctuating, marked by seasonal upwelling (Gulf of Panama) and stable, nonupwelling (Gulf of Chiriqui). The purpose of this study was to test the hypothesis that banding in corals is largely mediated by seasonal variations in temperature (Highsmith 1979). Our results indicate that the timing of band formation is synchronous at these two environmentally distinct locations. The low density (LD) portion of the annual band is accreted over a five month period (January–June) and represents an increase in linear skeletal extension (mm/mo.) as well as a marked increase in calcification rate (g CaCO₃ · cm^-2 · mo^-1) relative to the high density portion which forms over the remaining seven month period (July through December). In contrast to the predictions of the Highsmith model these findings indicate that variations in light levels rather than fluctuation in temperature is a better correlate to changes in skeletal density. Qualitatively, banding patterns were similar at the two sites; however, higher growth rates (particularly with respect to the LD band) for Pavona clavus in the Gulf of Panama indicate that lower water temperatures and higher productivity, or both, may be responsible for quantitative differences in banding between sites. We found that formation of the HD band corresponds to lower light levels and the production of gametes. We propose that banding in corals is a complex phenomenon governed by endogenous processes (e.g. reallocation of energy from growth to reproduction) which may be mediated by exogenous factors (e.g light and productivity).     

Formamide denaturation of chromosomal DNA for in situ hybridization and C-band preparation in the guinea pig, Cavia porcellus

Cytological preparations were incubated in 0.07 N NaOH at room temperature or 90% formamide (final salt concentration 2 × SSC) at either 65 °C or 37 °C for 2.5 h to denature guinea pig chromosomes. Chromosomes treated with NaOH or formamide at 65 °C showed a large amount of DNA loss, while chromosomes treated with formamide at 37 °C showed little or no DNA loss. Repeated sequences were isolated from guinea pig DNA and [³H]cRNA was transcribed with Escherichia coli RNA polymerase for in situ hybridization. Localization of the [³H]cRNA occurred in the centromeric regions and C-band positive short arms of almost all of the chromosomes in the NaOH preparations. Chromosomes treated with formamide at 65 °C showed the same grain distribution with a decrease in the number of grains/cluster. Slides incubated in formamide at 37 °C showed localization in only a few chromosomes and the number of grains/cluster was greatly diminished. Thermal denaturation of isolated chromatin indicated that incubation of chromosomes in formamide at 37 °C did not fully denature the DNA. C-bands could be induced by treating slides in formamide at either 65 °C or 37 °C when followed by a “reassociation” in 2 × SSC at 65 °C for 16 h. If the “reassociation” step was omitted, C-bands were found in the 65 °C formamide slides but not the 37 °C formamide slides.