T. Caspersson

Identification of human chromosomes by DNA-binding fluorescent agents.

The distribution of DNA along metaphase chromosomes that are not excessively contracted can be visualized in the fluorescence microscope with the aid of fluorescent DNA-binding agents. Additional, characteristic details in the fluorescence patterns are obtained with fluorochromes that bind preferentially to certain chromosomal regions. The highly fluorescent alkylating agent quinacrine mustard (QM) effects discrete, fluorescent labeling of both plant and mammalian metaphase chromosomes, presumably by selective binding to guanine residues in DNA, and is also capable of intercalation in the DNA double helix. Chromosome regions fluorescing particularly strongly with QM have been demonstrated in human metaphase chromosomes 3, 13–15 and Y.A convenient measuring technique has been developed for the rapid and accurate recording of fluorescence patterns in human metaphase chromosomes. These photoelectric recordings of the fluorescence patterns contain far greater detail than can be seen by the human eye.The fluorescence patterns described are based on measurements of about 1,000 human metaphase chromosomes. This new technique of determining fluorescence patterns in human chromosomes should be particularly valuable for the identification of chromosomes 4–5 and the individual types in the 6–12 group. Individual, typical patterns also occur within the groups 13–15, 17–18, and 21–22.

Differential binding of alkylating fluorochromes in human chromosomes

Abstract Human metaphase chromosomes from blood cultures, treated with quinacrine mustard (QM), show a banded pattern of fluorescence, which in the chromosomes with the most strongly fluorescent regions (3, 13–15 and Y) was found to be constant and reproducible. As in plant materials studied earlier, this pattern appears so characteristic that it can be of help for chromosome identification. Improved fluorescence techniques should permit detailed studies on smaller human chromosomes as well. A certain correlation appears to exist between strong capacity for QM-binding and the distribution of heterochromatin as defined by cold treatment (according to Darlington & La Cour) or thymidine labelling-techniques.

Chemical differentiation with fluorescent alkylating agents in Vicia faba metaphase chromosomes

With the purpose of searching for chemical bases for the transfer of differentiation information through metaphase, efforts have been made cytochemically to identify chemical and physiochemical differences between different parts of metaphase chromatin in Vicia. Equipment has been developed for very high resolution quantitative spectrophotometric determinations on chromosomes and for fluorimetric work with low irradiation load on the object. A bifunctional highly fluorescent quinacrine mustard was shown to give chromosome breaks preferentially localized to heterochromatic chromosome regions (identifiable by cold treatment) in the M-chromosome. This and other fluorescent mustards of different types accumulated preferentially in the same regions, which then appear as strongly fluorescent bands. In these bands the fluorescence per unit DNA is higher than in the rest of the chromosome. Possible mechanisms for the fluorochrome-binding are discussed.

DNA-binding fluorochromes for the study of the organization of the metaphase nucleus.

Fluorescent DNA-reagents with different modes of binding have been used for the purpose of identifying chemical differences along metaphase chromosomes. The DNA-distribution pattern along the chromosomes, determined with very high resolution procedures, has been used as reference when studying the differential uptake of different chromosome regions of the compounds. For quantitative measurements of the fluorescence of very small objects a special system has been developed. Several types of plant material and also Chinese hamster have been investigated. Great and reproducible differences in the uptake of the fluorescent reagents by different chromosome regions were observed. In Trillium a good correlation was found between regions reacting with several different fluorescent compounds and heterochromatin as defined by cold treatment. The looseness of the several presently used definitions of heterochromatin is stressed.

Fluorescence microspectrophotometry of cellular catecholamines and 5-hydroxytryptamine.

Abstract The fluorescent products formed from certain biogenic monoamines, when they are treated with formaldehyde according to the method of Falck and Hillarp, have been studied in model systems and in tissue sections. The analysis of cellular monoamine fluorescence has been made possible by a recently developed fluorescence microspectrograph. The results show that when models containing catecholamines or 5-hydroxytryptamine are treated as described for the histochemical method, consistent results are obtained; catecholamines show excitation/emission maxima at 410 470 mμ, and 5-hydroxytryptamine at 385– 415 520 mμ. The same results are found for cells normally containing these amines; adrenergic neurones, rat mast cells and rabbit enterochromaffin cells are selected as examples. Tryptamine and m-tyramine can also be identified by fluorescence microspectrographic methods due to their characteristic fluorescence following formaldehyde treatment. The reaction conditions between the amines and formaldehyde must be carefully controlled since too much humidity in the formaldehyde gas will cause a shift in emission of high concentrations of primary catecholamines from the original blue-green into yellow. In the fluorescence microscope, this might be interpreted as 5-hydroxytryptamine fluorescence. With this in mind, it can be stated that it is generally possible to distinguish between catecholamines and 5-hydroxytryptamine also in an ordinary fluorescence microscope.

SCANNING MICROSCOPY TECHNIQUES FOR HIGH RESOLUTION QUANTITATIVE CYTOCHEMISTRY

Quantitative cytochemistry is concerned with the determination of the amounts of different classes of substances in the individual cell or in its smaller parts. The dimensions of the most important cell organelles lie around 0.5 to 5p-that is, close to the wave length of light in the visible spectrum. The resulting demand for a high degree of resolution in the work has meant that the most productive working procedures so far have been those involving optical analysis, mainly spectrographic and interferometric. In the ultramicrospectrographic procedures, the determination of different substances within the cell structure is effected by means of their characteristic light absorption. This can either be the absorption of the material itself or the result of treatment with substances that react, either physically or chemically, with the material. The most important working areas of these spectrographic methods are the ultraviolet, the visible spectral region, and the long-wave Roentgen region. In the ultraviolet these methods have so far been used mainly for studies of nucleic acid and protein metabolism. In the visible they have been used, for example, for studies on certain natural pigments and on chemical color reactions such as on DNA, polysaccharides, or groups containing SH. The main field for Roentgen microspectrography is the determination of the dry weight of ce! structures with the help of absorption measurements in the region of 8 to 12 A. The distribution of certain elements in the cell or tissue-in the first line sulphur and phosphoru-can also be determined on the basis of their Roentgen absorption. Ultramicrointerferometric methods permit dry-weight determinations in objects down to a size of around 0.5 p. The techniques for measurements on biological material by these different procedures are nowadays suitable also for routine use in biological laboratories, although the outlay for instruments is rather large. FIGURE 1 indicates the most important working procedures and gives some examples of the type of information they can provide. Observe that it is not enough to have access to apparatus for measurements on cell material. It is also necessary, by measurements on model systems-as indicated in the lower part of the figure-to be able to determine the values of the optical constants required for the analysis of measurements from the cell material. The main constants to be determined are the absorption coefficients and the refraction indices for concentrated solutions and for solids. Special procedures have been evolved for this purpose. These different procedures complement each other very well, and quantitative cytochemical methods offer in their present form a good avenue of approach to many different types of problems in cytochemistry. So far, work has for natural reasons predominantly concerned questions pertaining to normal and abnormal cell growth and varied problems of cell differentiation. Early quantitative cytochemical procedures were laborious, and this re-

Die Eiweissverteilung in den Strukturen des Zellkerns

Die makrochemischen Untersuchungen von Eiweiß in Kernmaterial, welche vonKossel u. a. m. ausgeführt worden sind, haben ergeben, daß im Ruhekern wahrscheinlich oft Histone vorkommen, sowie außerdem, daß während der Spermiogenese ein Umbau von höheren Eiweißsubstanzen in Histone oder Protamine herab stattfindet, welche in Salzbindung mit Thymonukleinsäure den bei weitem überwiegenden Hauptteil des Spermienkerns bilden. Es wurde eine Methode ausgearbeitet, welche es gestattet, durch Analyse der Absorptionskurven den Gehalt an Nukleotiden, Tyrosin und Tryptophan sowie die Lage des Absorptionsmaximums des Tyrosins zu bestimmen. Bei Messung einer Anzahl von Histonpräparaten wurde eine Verschiebung des letzteren nach der langwelligen Seite beobachtet, welche durch den Gehalt an basischen Aminosäuren verursacht werden dürfte. An einzelnen Teilen von Speicheldrüsenchromosomen vonDrosophila wurden Ultraviolettabsorptionsspektra aufgenommen. Dabei ergab sich folgendes: die nukleinsäurereichen Scheiben enthalten Eiweiß vom Histonabsorptionstyp sowie wahrscheinlich auch höhere Eiweißstoffe. Die Zwischenscheiben enthalten Eiweißsubstanzen mit dem Absorptionscharakter der höheren Eiweißkörper. Die heterochromatischen Regionen enthalten ebenso wie der Nukleolus große Mengen von Eiweiß vom Histonabsorptionstyp. Die Metaphasechromosomen (Omocestus, Chorthippus) enthalten im Vergleich zu dem Eiweißgehalt sehr große Mengen von Nukleinsäure. Das Eiweiß ist vom Histontyp, sowie möglicherweise von höherem Typ oder einer Zwischenform. Der Eizellkern enthält höhere Eiweißstoffe sowie große Mengen solcher vom Histontyp, die besonders an die Nukleolarsubstanz gebunden sind. Um die Resultate der oben angeführten Messungen zu erklären, muß angenommen werden, daß die Eiweißveränderungen im Zellkern während der Mitose folgende sind: Das Metaphasechromosom besteht aus Nukleinsäure und Eiweiß vom Histontyp (+ höheres Eiweiß ?) in gleichartigen Konzentrationen. Während der Telophase produzieren die gentragenden Chromosomenelemente als erstes Erzeugnis Eiweißstoffe,in den euchromatischen Teilen solche vom höheren Typ, in den heterochromatischen besonders vom Histontyp. Durch diese Produktion scheinen die Chromosomen anzuschwellen (das Produkt des Heterochromatins sammelt sich teilweise zum Nukleolus an), und da gleichzeitig oft die Nukleinsäure in den gentragenden Strukturen verschwindet, seheint der Kern immer homogener zu werden (im Speicheldrüsenkern ist die Tendenz zur Paarung so groß, daß die lineare Anordnung von nukleinsäurehaltigen gentragenden Scheiben, die mit nukleinsäurefreien Zwischenscheiben von telophasischen Eiweißsubstanzen abwechseln, beibehalten wird). In der Prophase werden diese Eiweißstoffe, welche zwischen den gentregenden Teilen des Chromomemas interkaliert sind, abgebaut, wodurch letztere einander wieder genähert werden. Die Nukleinsäure wird daraufhin angereichert, und schließlich bleibt ein eiweißarmes Metaphasechromosom übrig, welches sich gänzlich oder hauptsächlich aus Nukleinsäure und einfachem basischem Eiweiß zusammensetzt. Der Zellkernbesteht also aus dem Euchromatin und dem Heterochromatin, mit ihren telophasischen Produkten. Die Eiweiße des Euchromatins bilden die Hauptmasse des Kernes. Das Heterochromatin ist in die Produktion des Cytoplasmaeiweißes eingeschaltet. Die Arbeit wurde durch Unterstützung der Rockefeller Foundation und Stifteisen Thérèse och Johan Anderssons minne ermöglicht.

Cytochemical observations on the nucleolus-ribosome system: Effects of actinomycin D and nitrogen mustard☆

Abstract Cytochemical techniques have been developed for the quantitative determination of the DNA, RNA and proteins (dry mass) in individual cells in large cell populations. These methods have been employed for the study of the effects of nitrogen mustard and actinomycin D on the nucleolar system of Ehrlich ascites cells in vivo and a line of normal mouse fibroblasts maintained in cell culture. Parallel studies on the nucleoli of individual cells have been made with ultraviolet microscopy and spectrophotometry. Nitrogen mustard, in certain concentrations, blocked mitosis but did not block DNA synthesis, as adjudged by the accumulation of DNA to pre-mitotic levels in non-dividing cells. Concurrently, the total cellular RNA and the total cellular mass of such cells increased far beyond normal values, with an overdevelopment of the nucleolar apparatus. The experiments with actinomycin D support the conclusion that this antibiotic inhibits the total synthesis of DNA-dependent RNA, and consequently, the synthesis of cellular proteins. Exposure to effective concentrations of actinomycin D results in marked inhibition of the synthesis of nucleolar RNA and proteins, as well as cytoplasmic RNA and proteins. Cytochemical analyses of cell populations exposed to actinomycin D and nitrogen mustard, either alone or in combination, together with studies of the nucleolar apparatus in individual cells in such populations, support the concept that the nucleolus-associated chromatin is concerned primarily with the synthesis of ribosomal RNA—either directly, or by the mediation of a “messenger” fraction of RNA. Such a function is considered to be consistent with the polygenic character of the nucleolus-associated chromatin, suggesting the presence of many repeating DNA cistrons complementary to ribosomal RNA, as described recently in E. coli .

CYTOCHEMICAL EVALUATION OF METABOLIC INHIBITORS IN CELL CULTURE.

Abstract A method for the cytochemical evaluation of the mechanism of action of potential metabolic inhibitors in cell culture, utilizing the rapid-scanning, high-resolution, automatically integrating biophysical instrumentation developed for cell population analyses is described. This method permits the in situ determination of the amount of DNA, RNA, and protein in individual cells in populations of heterogeneous cells, and the effects of inhibitory agents on these biosynthetic processes. The utility of the method has been demonstrated by the cytochemical evaluation of a series of inhibitory agents selected on the basis of what is known of their mechanisms of action with respect to inhibition of mitosis, synthesis of nucleic acids, or protein. The results of cytochemical analyses of populations of cells exposed to these agents agreed in general with what is known of their mechanisms of action from other studies. In addition to the advantages provided by a method based upon the evaluation of the effects of an inhibitory agent on single cells in heterogeneous populations, cytochemical analyses allow determination of the phase of the cell growth cycle in which inhibition occurs; information which is not readily obtained from other methods of assay. The advantages of such a method for the selection of candidate inhibitory agents for sequential or combination therapy of neoplastic disease, as well as detailed studies in cell physiology, are evident.

Cytochemical differences between mammalian cell lines of normal and neoplastic origins: Correlation with heterotransplantability in Syrian hamsters

Abstract The cytochemical characteristics of uncloned cell lines derived from normal and neoplastic sources have been determined by means of recently developed biophysical “population study techniques”. Cell lines derived from neoplastic sources exhibited a greater variability than cell lines similarly derived from non-neoplastic sources with respect to the amount of cytoplasmic proteins and cytoplasmic RNA per cell, an observation which is in accord with the results of earlier studies on populations of neoplastic cells derived from in vivo sources. It is of interest that certain cytochemical attributes of populations of neoplastic cells in vivo are retained by such populations when isolated and maintained in vitro, and that these cytochemical attributes correlate with the heterotransplantability of such cell lines to the cheek pouch of the Syrian hamster. The variability of the per cell amounts of cytoplasmic proteins and cytoplasmic RNA in these populations of neoplastic cells is of such magnitude that it must represent quantitative variations in the ribosomal RNA content of neoplastic cells. In view of the evidence for the participation of the nucleolar system in the synthesis of ribosomal RNA, the cytochemical variability of neoplastic cells may reflect disturbances in the nucleolus-nucleolus-associated chromatin biosynthetic system.