Symposium presented at the Annual Meeting of the Tissue Culture Association, Miami, June 1-4, 1965, and published in: In Vitro, Vol. 1, pp. 1-105 (1965):

George Yerganian ¹ and Clyde J. Dawe ², Editors

¹ Children's Research Foundation, Boston, Massachusetts
² National Cancer Institute, Bethesda, Maryland

"Cytogenetic Mechanisms and Speciations of Mammals",
Robert Matthey

"Photometric Measurements of Individual Metaphase Chromosomes",
George T. Rudkin

"Consideration of Metaphase Chromosome Parameters Amenable to Digital Computer Analysis",
Frank H. Ruddle and R. S. Ledley

"Molecular Morphology of the Chromosome",
Hewson Swift

"Specific Effects of Viruses and Antimetabolites on Mammalian Chromosomes",
Maimon M. Cohen and Margery W. Shaw

"Chromosome Anomalies and Chromosome Structure",
T. C. Hsu and Frances E. Arrighi

"Synthesis and Turnover of Nuclear Proteins",
David M. Prescott and Gordon E. Stone

"Mechanisms of Repression and De-Repression within Interphase Chromatin",
John H. Frenster

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During the past 15 years, advances in tissue culture methodology have done much to encourage a rebirth of interest in mammalian chromosomes. It is therefore fitting for the Tissue Culture Association to take the opportunity of its annual symposium to assemble fundamental approaches to this subject, which has so much bearing on future developments in cell biology.

Since many of our working concepts and much of our present understanding of mammalian chromosomes stem from earlier studies on lower organisms, participants in the symposium have endeavored to include the pertinent relationships compiled for the more classical species. The reader must be warned that no attempt has been made to abbreviate or simplify terms regarding the structural and functional aspects of the mammalian chromosome, even though current descriptions have led many of us to the brink of making such proposals. I trust the reader will keep in mind that the perplexing dynamics embodied in the mitotic and meiotic chromosomes defy one's efforts truly to improve upon the working terminology employed at this time. However, at the present rate of progress, this area will experience drastic lexicographic revisions very shortly.

It is said that one looks into a microscope, his eyes adjust to a focal distance of infinite length. Perception is restricted to a flattened image, lacking three dimensional depth of field. Perhaps this deletion is responsible for the hypnotic spells one experiences while viewing the dense and static metaphase chromosome. Once the spell is broken, the viewer may find himself reliving the patience, curiosity, and imagination of earlier investigators in their attempts to relate functional and structural aspects of the chromosomes to visual differences in the staining capacity of distinct segments. Descriptive terms such as "positive and negative heteropycnosis" and "allocycly" continue to be confusing, because of the need to be constantly aware of the striking and contrasting changes in the appearance of a given segment or chromosome during each phase of the cell cycle.

The initial concept of sex chromosomes was based upon McClung's recognition in 1902 of differences in the allocyclic nature of the "accessory" chromosomes in meiotic cells of both sexes. By 1929 Heitz proposed the terms "euchromatin" and "heterochromatin" to describe segments of chromatin that were either genetically active or repressed, respectively. This may be regarded as the first attempt to associate structural with functional aspects of the chromosome. Because of its rigid oversimplification, usage of these terms was soon challenged by Darlington and others during the '30s. By the mid-40s, Casperrson and his associates proposed that inactivation of genes was due to associated histones, while globulins were associated with active genes. Shortly thereafter, M.J.D. White and Prokofyeva-Belgovskaya offered other interpretations. White considered heterochromatin to exist in several forms, each in association with a corresponding or specific protein structure, while Prokofyeva-Belgovskaya regarded heterochromatin and euchromatin to relect physiologic states of a given segment of chromatin. In essence, her cytogenetic analyses indicated that a chromosome segment could exist in either state, depending upon the physiological condition of the cell or tissue. Judging from present considerations, the latter concept is by far the more advanced and serves as an underlying theme in this symposium.

The first meiotic division is, perhaps, the most vital source of information for systematic cytotaxonomy. Although the atlas of mammalian chromosome numbers is presently being compiled rapidly (and accurately, thanks to advantages of tissue culture methods ), the vast compilation prior to 1955 was due mainly to the efforts of a few individuals, notable among them Professors R. Matthey of Lausanne, and S. Makino of Sapporo. We are most fortunate in having Professor Matthey present to us his recent findings on the African pigmy mice, of the genus Leggada. The polymorphic chromosomal features noted in his sampling fulfill the Robertsonian scheme for speciation among closely related members of a genus. With rod-shaped chromosomes joining to form V-shaped or metacentric chromosomes, and vice versa, Matthey takes us on a cytological safari to view the latest patterns of active speciation and evolution among the wild rodents of Africa.

Although similar chromosomal findings are witnessed in clonal lines of cultured cells, those of us in the laboratory can only interpret such changes to reflect a greater viability among selected normal derivatives, or a more rapid progression among tumor cells. The fact that Matthey fails to note phenotypic expressions accompanying varying Robertsonian arrangements illustrates the function that chromosome cytology continues to serve in reconciling taxonomic lumpers and splitters.

The need to include studies of meiotic division, whenever feasible, is clearly indicated by the work of Ohno and his associates, who noted a decided discrepancy, resulting from fusion and fission of centromeres, in the forms and numbers of chromosomes in meiotic as opposed to somatic cells of salmon.

Ever increasing data on the effects of viruses and antimetabolites on chromosomes have been conveniently compiled for us by Drs. Cohen and Shaw. In many of their notations, the secondary constrictions or nucleolar organizing regions were prominently implicated as preferred sites of breakage or distortion. In their studies with mitomycin C, Cohen and Shaw noted metaphase configurations suggesting somatic crossing-over between homologues. To what extent these represented actual crossovers, rather than artifacts resulting from the retention of nucleolar substance between homologues sharing the formation of a nucleolus, remains to be ascertained. We must now face up to the task of getting to know more about nucleoli and their immediate products. Until this information is on hand, fundamental differences between normal, neoplastic, and virus-altered cells will continue to remain obscure. An imbalanced production or utilization of a new family of nucleolar ribosomes may be all that is required for the initiation of cell transforming processes.

The select response of early DNA replicating segments of chromosomes to actinomycin D, as described by Hsu and Arrighi, is most enlightening. Their description of morphological alterations fits exceedingly well with the predictable sequence of events following the geometric alignment of actinomycin D with the despiraled chromatin. More recently, others conducting identical trials with cells having numerous and smaller chromosomes failed to note such a high degree of specificity among different chromosome types. Consequently, the value of employing cell types bearing prominent sex elements for such studies is emphasized once again.

The extent to which automation is currently being incorporated into cytophotometric and density scanning processes, as discussed by Drs. Rudkin, Ruddle, and Ledley, comes as no surprise. What is disturbing however, is the hiatus that existed in this area of study following Casperrson's and Schultz's earliest reports. I trust that the speed of automated systems and the present array of excellent sources of material will make compensatory efforts successful. Nevertheless, I cannot help but feel that the accuracy of such machinery depends upon the skill of the individual preparing the slide. If this is so, then some of us are assured continued employment as we face the social problems of automation. More seriously, we look forward to new instrumentation to facilitate, for example, the detection of localized uptake of fluorescent compounds having specific affinities for particular sequences of amino acids. Such instrumentation will integrate the varied approaches of immunology, organic chemistry, and chromosome cytology. We are guided in this direction by the depth of information our colleagues can extract from a dense metaphase chromosome, exemplified in Rudkin's estimation of nucleotidal contents of specific chromosome types.

While reviewing Dr. Swift's comprehensive esay, I found myself recalling the earlier studies of Nebel in which a multi-stranded state of the chromosome was described. Some critics discounted Nebel's concept because of the limited resolution offered by light microscopy. It appears that tritiated analogue techniques, autoradiography, and electron microscopy have yet to prove their superiority in bringing nearer an answer to this question. Much of the evidence supporting both the single-stranded and the multi-stranded concepts of chromosome structure continues to be based upon exceptional obervations with the light microscope.

It is quite evident that the condensed version of mammalian somatic chromosomes is inadequate, per se, for studies pertaining to the molecular structure of chromosomes. Although we must continue to rely upon the clarity offered by polytenic dipteran and lampbrushed amphibian chromosomes, there is one mammalian cell source that should be considered more seriously in the near future: namely, spermatocytes of species having large sex elements. Such cells in diplotene-diakinesis have large and heavily lampbrushed autosomal bivalents. In addition, current techniques now facilitate the unravelling and partial separation of the X and Y chromosomes with minimal distortion. This occurs so frequently that one can readily visualize distinct patterns of lampbrushing in the segments that make up both the X and Y elements. These patterns become accentuated by actinomycin D and are, presumbably, indicative of the specific DNA and RNA synthesis associated with events leading to spermatogenesis , as described for theY chromosome in Drosophila.

Drs. Prescott and Frenster have led us deeper into the complexities of protein and DNA synthesis. It is at this level that molecular biology proves its worth by demonstrating that structural complexities can be resolved into a number of relatively simple biochemical subsystems, which in turn owe their actions to structure at the molecular level. The elusive mechanisms of gene function are found to be inseparable from gene and chromosome structure, just as our axioms told us all along. So we have travelled the full cycle, and in so doing, the thoughts of the more progressive of the earlier investigators have been rephrased in the high-resolution terms of our newer knowledge. How much higher this resolution can be pushed in our generation can hardly be foretold, but we can be sure the motivation to increase it will continue to come from the perpetual curiosity instilled in an individual, when once he peers through a microscope and sees The Chromosome.

George Yerganian
Boston, Massachusetts