P. van Duijn

A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labelled RNA☆

Abstract A new method has been developed for the detection of in situ hybridization by fluorescence microscopy. It is based on the covalent binding of commercially available fluorochromes to the 3′-terminus of RNA.

Bi-color detection of two target DNAs by non-radioactive in situ hybridization

SummaryA non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.

THE REPLICATIVE ORGANIZATION OF DNA IN POLYTENE CHROMOSOMES OF DROSOPHILA HYDEI.

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with3H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10-4 pg for the largest bands inDrosophila hydei. From the results of H3-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.

In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

Abstract In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.

THEORETICAL AND EXPERIMENTAL ASPECTS OF ENZYME DETERMINATION IN A CYTOCHEMICAL MODEL SYSTEM OF POLYACRYLAMIDE FILMS CONTAINING ALKALINE PHOSPHATASE

A theoretical treatment of the influence of slow substrate diffusion on the kinetics of an enzyme reaction in a cytochemical system is given. Quantitative aspects of the effect of several parameters on the diffusion of substrate were studied experimentally on a model consisting of polyacrylamide films into which alkaline phosphatase was incorporated. Spectra and staining intensity of the films were measured in a special film colorimeter. Enzyme activity in the films was determined cytochemically (with the azo dye coupling method) and biochemically. An effectiveness factor for which diffusion is not rate-limiting proved to be attainable with both methods.

HIDACSYS: Computer programs for interactive scanning cytophotometry

SummaryA description is given of a combination of three programs developed for computer-assisted stage scanning cytophotometry and cytofluorometry of isolated, close-lying, or touching objects in different types of microscopical preparations.The advantages and limitations of the individual programs are discussed, as well as the local specimen conditions determining the optimal application range of each of the programs.The applicability of these programs was investigated by determination of the integrated absorbance values of Feulgen or gallocyanin-chrome alum stained chicken erythrocytes, human leucocytes, and skin biopsy cells in imprint preparations, as well as of guinea pig peritoneal granulocytes which had been submitted to a simultaneous coupling azo dye incubation for alkaline phosphatase activity.

The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. II. Investigations on films containing condensed or swollen chicken erythrocyte nuclei.

As models for different states of chromatin compactness, nuclei from chicken erythrocytes were isolated and either osmotically swollen or kept as condensed as possible. Both types of nuclei were then fixed and incorporated into polyacrylamide films. Hydrolysis with 5 N HCl and staining with Schiffs reagent of these model films were studied using several parameters. The phosphate content of the films was analyzed as a parameter for the depolymerization losses and the staining with Schiffs reagent as a parameter for the apurinic acid (APA) content. The loss of ultraviolet absorbance from the films and the accumulation of ultraviolet absorbing substances in the hydrolyzing acid were monitored as parameters for the progress of hydrolysis. Conversion of the generated aldehyde groups to APA-Schiff chromophore is shown to take place with the same stoichiometry for both types of nuclei as well as for DNA in model films. It is further shown that the nuclei- and DNA-films are suitable models for investigating the influence of chromatin compactness on the course of the Feulgen-Schiff reaction. For the most compact form of chromatin studied, a very high reduction in staining intensity of up to 40% could be demonstrated after certain normally applied hydrolysis times. This is due primarily to a decrease with a factor of 2.3 of the depurination rate constants of these models (from 0.030/min to 0.013/min). Therefore prolonged hydrolysis periods are required to obtain the same APA concentrations, but then depolymerization processes cause losses of nuclear material. The differences in depurination rates could be explained by a decrease in [H3O]+ in the neighborhood of the purine-sugar linkages, caused by the presence of fixed positive charges form the protein components of the chromatin. These findings may explain the cytophotometrically determined differences in chromophore yield of 10-20% found in the nuclei of cells with different states of compactness of their chromatin. The descending part of the Feulgen hydrolysis curve represents the depolymerization of APA and loss by diffusion of the reaction products. In the Appendix, cytophotometric data of cells have been analyzed to show that this part of the hydrolysis curve may be used to estimate the acid stability of chromatin complexes. The depurination and depolymerization rates found closely correspond with the data obtained from the model films.

The interaction of apurinic acid aldehyde groups with pararosaniline in the Feulgen-Schiff and related staining procedures.

SummaryThe equilibrium reactions involved in the formation of the apurinic acid (APA)-Schiff chromophores in the staining phase of the Feulgen-Schiff reaction do not allow a quantitative conversion of APA to these chromophores. By modification of the sulfite and dye concentrations and the pH of the staining reagents, or by using better solvents for pararosaniline like acetic acid or dimethylsulfoxide (DMSO) a shift of these equilibria was attempted in order to obtain a higher amount of APA-bound dye. A 40% higher absorbance, when compared with the normal Schiff-staining, was obtained in model films by staining with a satured solution of pararosaniline in a 1:1 v/v mixture of DMSO and SO2-water, followed by rinsing in SO2-water. A doubling of the absorbance resulted in the same objects when a saturated solution of pararosaniline in a 2 M acetic acid/acetate buffer of pH 4.45 was used for staining, followed by a short rinse in SO2-water.Amino groups (as found in histones) are shown to compete with the amino groups of pararosaniline for the APA aldehydes. This effect, although causing lower staining intensities, is shown not to be the explanation for the differences in stain content found between more and less compact forms of chromatin.Depending on the pH, and dye and sulfite concentrations of the staining reagents, the following components are considered as possible contributors to the mixture of chromophores (Duijndam et al., 1973b) formed between APA and Schiffs reagent or its modifications:1.An acid labile component with a wavelength of maximal absorbance (λmax) near 510 nm; its structure is probably the azomethine −CH=N−;2.A relatively acid stable component with a high value of molecular absorbance (ε), an λmax near 570 nm and possibly having an enamine structure −CH=CH−NH−;3.A component with intermediate acid stability, low ε, and λmax near 540 nm, and which is probably an alkylsulfonic acid −CH(SO3H)−NH− compound. Small differences in the staining conditions in the histochemical application of the Feulgen-Schiff reaction may cause a shift in the ratio between especially components 2 and 3, resulting in variations in stain content and in λmax.

High-resolution scanning-densitometry of photographic negatives of human metaphase chromosomes

SummaryA computer-controlled cytophotometrical system is described to determine local absorbance or fluorescence values of chromosomes.The absorbance or fluorescence values within the chromosome are obtained in digital form by interactive scanning of 35 mm photomicrographic negatives. The scanning system consists of a mechanical stage scanner (Scanning Microscope Photometer SMP, Zeiss) connected to a PDP-12 computer (Digital Equipment).A computer program (CHROSCAN) enables the scanning of single chromosome images in a direction perpendicular to the length axis with 0.16 μ increments at the specimen level, and a transmission resolution of 500 linearly spaced intensity levels. Optical density values corrected for the local background of the individual chromosome, and total integrated optical density as well as the ratio of optical density in the long arm over the total optical density are calculated. Photography of a grey wedge together with the metaphase, allows determination of the gamma of each individual photomicrograph and conversion of optical density values into actual absorbance values.The program has a papertape output of the corrected integrated optical density data per lateral scanline to be used as an input for a curve analysis program. It also allows plotting of the integrated optical density distribution over the chromosome length by a Calcomp plotter.In this paper the instrumentation and experimental set up are presented, together with data concerning the reproducibility and accuracy of the photographic and the scanning procedures.

CYTOPHOTOMETRIC DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITY OF INDIVIDUAL NEUTROPHILIC LEUKOCYTES WITH A BIOCHEMICALLY CALIBRATED MODEL SYSTEM

A model system consisting of polyacrylamide films into which cell sonicate is incorporated was applied to investigate quantitative aspects of the cytochemical azo dye coupling method for alkaline phosphatase activity in neutrophilic leukocytes. The films were assayed in media containing naphthol AS-MX phosphate and 4-aminodiphenylamine diazonium sulfate. Optimal reaction conditions under which proportionality existed between the amount of reaction product and incubation time or enzyme concentration were determined. Since the enzyme activity in the films could also be measured chemically, using disodium phenyl phosphate as a substrate, a direct relation between the cytochemical and biochemical activity could be established. The azo dye coupling method was applied for the quantification of the enzyme activity. Air-dried microscopic preparations of exudate neutrophils were stained and the amount of dye formed in the cytoplasm of individual leukocytes was measured with a cytospectrophotometer based on the two-wavelength principle. By reference to the relation between biochemical and cytochemical activities in the model films, the cytochemically determined enzyme activity could be expressed in biochemical units. Independently, the average alkaline phosphatase activity per cell was determined by direct biochemical assay of the leukocyte suspension. The results showed that about 60% of the biochemically assayed activity in sonicate was demonstrated in the cell preparations by the cytochemical staining method. The discrepancy between the results of the two methods is discussed. The applicability of the model system for elucidation of the relationship between the results of cytochemical and biochemical enzyme determination in cells is stressed.A model system consisting of polyacrylamide films into which cell sonicate is incorporated was applied to investigate quantitative aspects of the cytochemical azo dye coupling method for alkaline phosphatase activity in neutrophilic leukocytes. The films were assayed in media containing naphthol AS-MX phosphate and 4-aminodiphenylamine diazonium sulfate. Optimal reaction conditions under which proportionality existed between the amount of reaction product and incubation time or enzyme concentration were determined. Since the enzyme activity in the films could also be measured chemically, using disodium phenyl phosphate as a substrate, a direct relation between the cytochemical and biochemical activity could be established. The azo dye coupling method was applied for the quantification of the enzyme activity. Air-dried microscopic preparations of exudate neutrophils were stained and the amount of dye formed in the cytoplasm of individual leukocytes was measured with a cytospectrophotometer based on the two...