Caspersson, Torbjörn Oskar
CASPERSSON, TORBJöRN OSKAR
(b. Motala, Sweden, 15 October 1910;
d. 7 December 1997), cytochemistry, genetics, cytology.
One of the greatest challenges the biological sciences confront is the development of instruments and techniques that can provide information about the physical processes occurring in biological systems at very small dimensions. Success in developing new instruments and techniques for biological inquiry requires both skill in developing instruments and a deep understanding of biological problems so as to measure signals that reflect biological processes and avoid many potential artifacts. Caspersson pioneered in the development of techniques for identifying and measuring chemical components within biological systems from their optical characteristics—absorption of radiation, especially ultraviolet light—and made important discoveries that became the foundation for major advances, especially in cell biology and genetics. Caspersson’s career in cytology and genetics was characterized by a drive to measure biophysical materials precisely and the project of building instruments to make those measurements.
Research on Nucleic Acids for MD . Caspersson was born on 15 October 1910 in the town of Motala in southeastern Sweden. He studied medicine and biophysics at the Karolinska Institutet in Stockholm, from which he received his MD in 1936. Beginning with a lectureship, he remained at the Karolinska Institutet for his entire career, and from the mid-1940s, he directed a major laboratory in cell biology and genetics.
Caspersson carried out his initial research as a docent in the laboratory of Einar Hammarsten, who was professor of chemistry and one of the few investigators in the 1930s who focused his research on nucleic acids. Hammarsten had developed new procedures for the preparation of nucleic acids and with his collaborators applied physical and chemical techniques in the attempt to determine their constitution. Caspersson determined that the results of Hammarsten’s preparations could be trapped by various filters and ascertained “the astonishing fact that the complexes of nucleic acids must be larger than the protein molecules” (Caspersson, 1934). Hammarsten and Caspersson then collaborated with organic chemist Rudolf Signer in Bern who measured the weight by flow birefringence. In conflict with then accepted views, they concluded that DNA was a large molecule with a molecular weight between 500,000 and 1,000,000 (Signer, Caspersson, and Hammarsten, 1938).
Among Caspersson’s other early projects were developing procedures for assaying the potency of liver extracts by measuring blood formation in chicken embryos and an examination of changes in blood in florescent cells of embryonic liver. Soon, though, his primary focus was on the structure of chromosomes, which he prepared for study by digesting attached proteins with proteolytic enzymes. He then examined the remaining nucleic acids under ultraviolet light, where they appeared to be disks connected by a thread.
For his research leading to his medical degree, Caspersson turned to the question of measuring the quantities of proteins and nucleic acids in various specimens and the changes in these quantities over time as cells went through different stages of the cell cycle. Since different biological materials, especially proteins and nucleic acids, absorb maximally light of different wavelengths, he recognized that by measuring the absorption of light of various wavelengths in a given specimen he could determine the quantities of the materials present in that specimen.
The critical challenge was to be able to make precise measurement from very small objects. He argued theoretically that this should be possible on objects as small as three times the wavelength of the light whose absorption was measured (this meant that accurate measurements could be obtained to a resolution of 0.5μ with ultraviolet light). To do this he integrated a spectroscope with a microscope fitted with a quartz lens to produce a monochromatic ultraviolet microscope. He then confronted a number of problems, such as the loss of light due to scattering in the specimen, which could generate erroneous measurements. Accordingly, much of his effort went into overcoming these sources of error. He approached the problem of scatter by generating an additional apparatus to measure the quantity of scatter directly. To compensate
for the lack of homogeneity in specimens, he developed procedures for photographing specimens and performing measurements on the photographs.
Since nucleic acids absorb light very strongly at 2,600 angstroms (the mid-ultraviolet range), Caspersson was able to use the device to locate them in different parts of individual cells and measure their quantities. In these studies for his medical degree, he showed that regions on chromosomes that differentially absorbed the Feulgen stain (known as euchromatin bands) also exhibited high absorption at 2,600 angstroms (Caspersson, 1936). Caspersson determined that these regions also had a high protein content (revealed by absorption at 2,800 Å) that was dissolved when enzymes such as trypsin, known to digest proteins, were applied.
This supported the claim that chromosomes were long polypeptide chains on which molecules of nucleic acid were attached. Caspersson suggested that deoxyribonucleic acid (DNA) figured in gene replication as smaller groups polymerized into larger aggregates, providing a structure on which extended protein molecules could be reproduced (Caspersson and Schultz, 1938). Although focusing on nucleic acids, Caspersson did not at the time view DNA as the genetic material. Rather, he endorsed the widely accepted view that genes must be made of proteins “because of their inexhaustible possibility of variation” (1936, p. 138).
When Caspersson began his research in the 1930s what came to be known as DNA and ribonucleic acid (RNA) were often identified as originating in animals and plants (especially yeast) respectively, although RNA was also known to occur in some animal cells. As a result, they were often referred to as animal and plant nucleic acids respectively. One result of Caspersson’s research was the demonstration that RNA as well as DNA was a regular constituent of animal cells. Although spectroscopic measurements alone could not discriminate DNA from RNA, Caspersson used the Feulgen reaction, which was specific for DNA, to differentiate the two nucleic acids. He localized RNA to locations that did not show the Feulgen reaction but revealed spectroscopically the presence of nucleic acids.
Collaboration with Jack Schultz . Hammarsten received extensive support from the Rockefeller Foundation for his research and, as was customary for up-and-coming researchers with great promise, he had promoted Caspersson for a Rockefeller Fellowship to do further research in a laboratory in the United States. But in fall 1937, before those plans could be finalized, Jack Schultz came to the Karolinska on a Rockefeller Fellowship to spend two years working with Caspersson. Schultz had completed his PhD in Thomas Hunt Morgan’s genetics laboratory at the California Institute to Technology, where he had been working with Sturtevant and Bridges. Interested in relating genetics and development, Schultz had formulated the hypothesis that a concentration of DNA along the chromosomes would block the expression of nearby genes (assumed to consist of proteins). He sought out Caspersson’s help on the idea that spectroscopic techniques would permit the localization of DNA and hence the genes whose expression was blocked, on chromosomes in mutant fruit flies that he brought with him to Stockholm.
Regions of chromosomes stain differentially in the Feulgen reactions, with some regions (euchromatin) manifesting orderly bands of light and dark regions and others (heterochromatin) exhibiting a much more disorganized pattern. The heterochromatic bands stain most deeply in the interphase chromosome and Caspersson demonstrated that it had the greatest concentration of nucleic acid. These heterochromatin regions were also the locus of the abnormalities in gene reproduction due to chromosomal rearrangements in the different strands of Drosophila Shultz had been studying. Caspersson and Schultz now linked these regions with nucleic acid metabolism (Caspersson and Schultz, 1938).
Moreover, in female Drosophila eggs with an additional Y chromosome (XXY) Caspersson and Shultz established that the amount of nucleotides in the cytoplasm was greater than the amount found in normal (XX) females, suggesting that although the additional Y chromosome did not appear to be expressed in the phenotype, it did affect nucleic acid metabolism. Moreover, from the fact that the Y chromosome had extensive heterochromatin regions, they attributed to the heterochromatin region a role in nucleic acid synthesis, and proposed such synthesis played a role in gene replication.
In subsequent work, Caspersson and Schultz continued this line of research, ascertaining from absorption spectra that concentrations of RNA are high in the cytoplasm of rapidly growing tissues (Drosophila larva) but low in mature tissues, which instead have a high protein concentration. This pointed to a central role of RNA in cytoplasmic protein synthesis, a conclusion that Jean Brachet reached at the same time based on other techniques.
The conclusion, however, was not universally accepted. Shortly after Caspersson’s and Brachet’s research, Albert Claude, in pioneering research using the ultracentrifuge to segregate fractions from cells, isolated small particles he called microsomes, which he found to be high in RNA content. He, however, rejected suggestions that they figured in protein synthesis.
In their subsequent work, Caspersson and Schultz turned their attention to the nucleolus and the cytoplasm around the nuclear membrane, where they also found high concentrations of RNA. They carried out these measurements in diverse species—sea urchin eggs, periblem cells of spinach root tip, and salivary gland cells of Drosophila melanogaster. In all species, in the nucleolus they found local maxima in the wavelengths indicating nucleic acids and a secondary local maximum in the wavelengths indicative of proteins. In the sea urchin, the cytoplasm around the nuclear membrane showed a similar pattern, but with a higher local maximum in the protein region. In peripheral protoplasm there was only a temporary plateau in an otherwise steady drop from a high level of absorption in the shorter wavelengths, indicating low concentrations of RNA.
Caspersson and Schultz attempted to interpret their findings so as to provide a model of gene action. Although their data did not support transport of RNA from the nucleus to the cytoplasm, they viewed the existence of a gradient between the nuclear regions of the cytoplasm and the peripheral regions as supporting the claim that synthesis occurred in the cytoplasmic regions adjoining the nucleus. They also viewed the evidence as supporting the claim that “the activity of nucleoli is closely associated with an intense synthesis of the cytoplasmic ribonucleic acids” and given the additional association of RNA with protein synthesis, they concluded that “the nucleo-cytoplasmic relationships may provide some insight into the mode of action of the genes” (p. 514).
Making use of a linkage others had established between the heterochromatin region of the X and Y chromosomes and the nucleolus, Schultz, Caspersson, and Aquilonius used chromosomal rearrangements and duplicated regions to try to understand differences in absorption spectra identified in male and female Drosophila. They claimed to find a correlation between structural properties of nucleoli and nucleic acid content, indicating that both forms of nucleic acid have a role in structure formation: “the genotypic control of the structural characteristics of the nucleolus may be mediated by changes in its nucleic acid content” (p. 522). While drawing attention to the presence of nucleic acids and proposing a role for them in protein synthesis, the specific function Caspersson proposed for RNA remained auxiliary to that of the proteins, which he continued to view as the genes. He proposed that proteins migrated from the chromosomes to the membrane of the cell nucleus, where they induced the synthesis of RNA, which in turn supported the production of proteins in the cytosol (Caspersson and Thorell, 1941).
Continuing Spectrographic Studies during World War II . The beginning of World War II interrupted the further collaboration of Caspersson and Schultz (who returned to the United States) and prevented Caspersson from pursuing his delayed Rockefeller Fellowship (it had by then been arranged that he would spend such a fellowship with W.H. Lewis at Johns Hopkins University). Instead, with support from the Rockefeller Foundation, he continued his ultraviolet spectrographic studies of cells through the war.
One continuing focus of his research was measuring quantities of protein in the nucleus. From the evidence he gathered, he further articulated a complex scheme of protein synthesis. He contended that the swelling and disappearance of chromosomes during the telophase of mitosis was due to the generation of protein from the gene-carrying parts of the chromosomes. He viewed the synthesis of proteins as also providing the mass of the nucleolus. From the nucleolus, proteins were transported to the periphery of the nucleus, where they figured in the generation of ribonucleic acids which in turn supported the synthesis of cytoplasmic proteins. He concluded: “Polynucleotides are a base for the protein synthesis in the cell. A central function for the cell nucleus is to be the centre for the protein production. The heterochromatin is an [organ] regulating the production of the proteins of the cytoplasm. This regulation works via the nucleolus” (letter to Miller, program officer of the Rockefeller Foundation, 1 September 1940). He later termed heterochromatin-nucleolus-nuclear membrane as the cellular “system of protein synthesis” and protein synthesis as the prime function of the nucleus as a whole.
In addition to determining the nucleic acid and protein content of the nucleus, Caspersson set out to determine how it varied through the cell cycle. Using grasshopper testis, he traced changes in the absorption spectrum during the cell cycle, showing that nucleic acid concentrations increased during prophase and then remained constant while the ratio of nucleic acid to protein increased, indicating a loss of protein as meiosis proceeded. Although he did not construe nucleic acids as the genetic material, he viewed increases in nucleic acids as indicating when gene replication occurred.
During the war, Caspersson recruited a number of junior researchers, including Holger Hydén, Bo Norberg, Arne Engström, and Bo Thorell into his laboratory, and with them he began to apply these measurement techniques to other cell types (especially tumor cells and nerve cells) and to develop and deploy related measurement techniques such as photoelectric methods, x-ray ultramicrospectrography, and cathode-ray fluorescence. Caspersson construed tumor cells as performing protein synthesis without the operation of normal inhibitory mechanisms and thus as providing a particular type of specimen in which to investigate protein synthesis.
Creating an Institute and Developing Instruments . In 1944, Caspersson was appointed to a professorship for cell research specially created by an act of the Swedish parliament. Funding from the Nobel Institute provided funding for a new Medical Nobel Institute building to house the laboratories of Caspersson and Hugo Thorell, an enzyme chemist who was another protégé of Hammarsten. Grants from the Wallenberg Foundation and the Rockefeller Foundation provided for an extension of the building devoted to an Institute for Cell Research under Caspersson’s direction. Once he had adequate facilities, the size of Caspersson’s research group grew considerably, with upwards of fifteen scientists and twenty technicians carrying out research at a given time. In addition, Caspersson began to attract a substantial cadre of international researchers, who generally came to work for a period of nine to twelve months in his laboratory. The new laboratory also permitted introduction of new instruments, including an electron microscope, acquired for the objective of demonstrating virus particles in infected mammalian cell preparations that corroborate the results of ultraviolet measurements in such cells. Although Caspersson never performed electron microscope investigations himself, Venezuelan Humberto Fernández-Morán used the microscope extensively in his early studies of nerve structure.
Although the development of new instruments and techniques for using them had been a feature of Caspersson’s career from the beginning, it began to be a major preoccupation when he acquired the new laboratory facility and expanded his research group. Caspersson included an instrument workshop facility as a major component in the design of the building and became focused on building equipment that generated extremely precise measurements, was automated, and capable of supporting large-scale research.
In his annual reports to the Rockefeller Foundation, which continued to provide him substantial support until 1961, he routinely began with a discussion of the development of new instruments or improvements in existing ones and relegated the results of experimental studies to the latter part of the report. In his December 1953 report to the Rockefeller Foundation, he emphasized the division between the development of instrumentation and basic research:
work in the institute... has been carefully divided so that half the resources were devoted to developmental work on the side of the instruments and the other half to work on biological problems with the intracellular regulation of protein synthesis as key note. This arrangement has always very strictly been carried through, in spite of the fact that it has often been evident that the biological work on short sight would have benefited from a larger share of the efforts, what would undoubtedly also have made the work more easy to manage financially. The reason for this politics was that the biophysical techniques in question are the primary condition for the work, and furthermore they represent in my personal view one of the ways, which has to be gone sooner or later if we will ever get close to the basic problems of gene reproduction and gene function and thus a quite general approach from the beginning should prove most fruitful at the end. (p. 1)
Among the instruments on which Caspersson and his collaborators worked were a high vacuum x-ray microspectrograph, a universal microspectrophotometer, and instruments for photoelectric scanning. Although Caspersson emphasized both the accuracy and usefulness of the new instruments, they did not receive significant uptake in the research community. Nurnberger (1955) commented critically:
Caspersson’s photographic micrabsorption technique described in 1936 remains, to this day, the simplest and only readily available ultraviolet method for estimating mean or average concentrations of nucleic acid (and proteins) in large cell areas or, likewise of obtaining approximate absorption coefficient for extremely small areas of the order of 1μ2. Though Caspersson has recently developed a highly flexible photoelectric scanning apparatus to replace the photographic system, it is doubtful whether equipment of such complexity will find popularity in any except the best equipped laboratories where show facilities are adequate to the technical problems implied.
The tenor of Nurnberger’s evaluation seems to have been shared more generally, and indeed the responsible program officers at the Rockefeller Foundation expressed growing frustration over the years at his continually unfulfilled promises to focus more on basic scientific problems.
Caspersson defended his investment in development of precise and automatic quantitative measurement as providing the main avenue for understanding cellular processes such as protein synthesis. But while he was developing these cytochemical techniques, other approaches were being pursued elsewhere. The approach of cell fractionation through centrifugation, for example, made it possible to isolate ever more specific fractions from cells, with which it was possible both to perform chemical analysis as well as carry out specific chemical reactions in isolation from each other. When supplemented with radioactive tracers, these fractions provided a means to study the process of protein synthesis in a much more direct manner than Caspersson envisaged.
Collaboration with Zech on Chromosome Banding . Although for a prolonged period Caspersson’s contributions were primarily on the instrumental side, beginning in the late 1960s, he collaborated with Lore Zech in studies that treated metaphase plant chromosomes with different alkylating and intercalating substances. In the course of these studies they found distinctive light and dark banding patterns under ultraviolet light in chromosomes from Vicia faba (a variety of bean) and Trillium erectum(a perennial wildflower) stained with quinacrine mustard, a process that came to be known as Q-banding (Caspersson et al., 1968). They showed that the banding pattern could be recorded photoelectrically with fluorescence microscopes either directly or from photographs (which avoided problems with fading of the stain under ultraviolet illumination). In the wake of Caspersson and Zech’s research, a number of other staining techniques were developed that revealed similar banding patterns on chromosomes.
At the time of this research, human geneticists lacked adequate ways to distinguish human chromosomes from each other either in normal or pathological conditions. In subsequent studies Caspersson and Zech found that despite the fact that human chromosomes were much smaller than the plant chromosomes on which they had been working, they could adapt their techniques to study them (Caspersson, Lomakka, and Zech, 1971).
In very small regions of chromosomes they found very brightly fluorescing regions that also exhibited individual variation that they found to be heritable. The largest such region was found on the distal part of the long arm of the Y chromosome, which Caspersson and Zech noted would provide a means for prenatal sex determination and a tool for screening for XYY males. But more importantly, the whole length of each human chromo-some exhibited a faint fluorescence pattern that was distinctive of the particular chromosome and highly consistent across normal individuals. Caspersson and Zech presented curves resulting from passing a slit along the length of each chromosome and recording from the area within the slit, with each chromosome producing a distinctive curve. These consistent patterns provided a basis for detecting chromosomal abnormalities. For this purpose Caspersson developed a simpler, more readily used instrument for recording fluorescence patterns. This contribution had its primary uptake in medical genetics as it enabled investigators to determine which chromosomes were altered in various clinical populations, thereby giving rise to the field of human cytogenetics.
Professional and Personal Life . It is noteworthy that Caspersson adopted the name Institute for Cell Research for his laboratory beginning in 1947. This was a period in which researchers from a variety of different biological disciplines began to train their investigations on the functions of various organelles that were identified within cells. Terms such as cell research and cell biology began to be used to designate the domain of inquiry as distinct from classical cytology that had focused primarily on cell structure. In 1947, Caspersson played a central role in hosting the sixth International Congress for Experimental Cytology in Stockholm. At this meeting the congress renamed itself the International Society for Cell Biology. In 1950, he and John Runnström, head of the WennerGren Institute for Experimental Biology and Cell Research at the University of Stockholm, established Experimental Cell Research, the first of several new journals devoted to cell research and cell biology to be founded during the ensuing decade.
In 1936, Caspersson married Siv Gunnarson (1911–1999), a child psychiatrist who was for many years chief medical officer at the Child Guidance Center, Stockholm, and a pioneer in developing interdisciplinary treatment teams for disturbed children. The couple had two children: Gunnel and Lena. Caspersson received many honorary degrees, including ones from Brandeis University, the University of Giessen, the University of Ghent, the University of Helsinki, and the University of Rotterdam. He died on 7 December 1997.
Archival materials from Caspersson’s collaboration with Schultz are housed in the Library of the American Philosophical Society in Philadelphia. Archival materials relating to his support from the Rockefeller Foundation are housed in the Rockefeller Archive Center.
WORKS BY CASPERSSON
“Druckfiltrierung von Thymonucleinsäure.” Biochemische Zeitschrift270 (1934): 161–163.
“Über den chemischen Aufbau der Strukturen des Zellkernes.”Skandinavisches Archiv für Physiologie73, supplement no. 8 (1936): 1–151.
With R. Signer and E. Hammarsten. “Molecular Shape and Size of Thymonucleic Acid.” Nature141 (1938): 122.
With J. Schultz. “Nucleic Acid Metabolism of the Chromosomes in Relation to Gene Reproduction.” Nature 142 (1938): 294–295.
With J. Schultz. “Pentose Nucleotides in the Cytoplasm of Growing Tissues.” Nature 143 (1939): 602–603.
With J. Schultz. “Ribonucleic Acids in Both Nucleus and Cytoplasm, and the Function of the Nucleolus.” Proceedings of the National Academy of Sciences of the United States of America26 (1940): 507–515.
With J. Schultz and L. Aquilonius. “The Genetic Control of Nucleolar Composition.” Proceedings of the National Academy of Sciences of the United States of America26, no. 8 (1940): 515–523.
With B. Thorell. “Der endozelluläre Eiweiss- und Nukleinsäurestoffwechsel in Embryonalem Gewebe.” Chromosoma 2 (1941): 132–154.
With L. Santesson. “Studies on Protein Metabolism in the Cells of Epithelial Tumours.” Acta Radiologica 46, supplement (1942): 1–105.
“A Universal Ultramicrospectrograph for the Optical Range.”Experimental Cell Research 1 (1950): 595-598.
Cell Growth and Cell Function. New York: Norton, 1950.
With S. Farber, G. E. Foley, J. Kudynowski, E. J. Modest, E. Simonsson, et al. “Chemical Differentiation along Metaphase Chromosomes.” Experimental Cell Research 49 (1968): 219–222.
With L. Zech, C. Johansson, and E. J. Modest. “Identification of human chromosomes by DNA-binding fluorescent agents.” Chromosoma 30 (1970): 215–227.
With G. Lomakka and L. Zech. “The 24 Fluorescence Patterns of the Human Metaphase Chromosomes—Distinguishing Characters and Variability. Hereditas 67 (1971): 89–102.
Klein, G. G., and E. Klein. “Torbjörn Caspersson: Some Personal Perspectives.” Cytometry 5 (1984): 318.
Nurnberger, J. I. “Ultraviolet Microscopy and Microspectroscopy.” In Analytical Cytology: Methods for Studying Cellular Form and Function, edited by R. C. Mellors. New York: McGraw Hill, 1955.
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