Summary Many biologists prefer to use the same research material for years, or even throughout their professional lives. This is a logical approach because the more information that has been collected from previous investigations on one system, the greater the number of follow-up qucstions that may be posed and the more incisive are the experiments that can be designed. Thus, most E. coli geneticists continue to use this microorganism all their lives, and Drosophila investigators continue to study the same species of fruit fly for countless numbers of years. The same philosophy also applies to many naturalists and systematists ~ they take on a project to study one group of animals or plants exhaustively for as long as possible in order to gain thorough knowledge about that group. A story I heard about President Teddy Roosevelt illus- trates this spirit of thoroughness. President Roosevelt was an enthusiastic supporter of biological research, but his idea about biology was that of a naturalist of an earlier era; there- fore, he disliked experimental biology, including the use of a microscope to study life forms. Once, a famous German zoologist visited the president at the White House. The presi- dent wanted to make sure that thc visilor was a biologist.

‘Yes, Mr. President. 1 am a biologist,’ the visitor assured him.

‘Do you use a microscope to study your animals, Profes- sor‘?’

‘Yes, Mr. President.’ ‘What kind of animals do you study’?’ ‘Whales, Mr. President.’ The story did not mention whether the president’s jaw

dropped. Three years ago Dr. Robert T. Johnson asked me to write

an article for BioEssays on “My favorite cell”. I told him that I never had such a loyalty or fidelity toward any research material and therefore, did not have one favorite cell. Rather, I had often thought that my research philosophy is analogous to the movements of an amoeba. It sends out a pseudopodiurn to explore the surroundings to find out whether that direction is profitable. If not, it withdraws that pscudopodium and tries another direction; but all the time it retains its nucleus. I sup- pose that my real favorites are the chromosomes, whose ever-enchanting beauty is addictive to some microscopists, including myself. Because of this addiction, I tried to learn as much as I could about chromosomes, their structure, behav- ior, physiology, and their role in heredity and evolution.

Therefore. the cell nucleus: coincidcntally, is the ‘nucleus’ of my research interest. When a particular question arosc, I tried to find the best available material for answers. Therefore, this personal review briefly describes some of my favorite cells. and in particular their chromosomes, not one favorite ccll.

Fate forced mc to abandon Drosophila research immedi- ately after receiving my Ph.D. degree, and required that I learn mammalian cell culture when I took a postdoctoral fel- lowship in the laboratory of the late Professor Charles M. Pomerat at The University of Texas Medical Branch in Galveston, Texas. I was extremely uneasy about my future for several reasons: (1) I loved working with Drosophila, par- ticularly D. virilis; (2) I had no idea what useful information I could uncover using cultured human tissues; and (3) Most importantly, the communist rcvolution in China and the Korean war had locked my wife and child jn China, and I had a difficult time even LO find out whether they were still alive. Therefore, my first year as a posldoc was one of the most apprehensive periods of my professional and private life.

Rut Lady Luck also gave mc her sweetest smile that same year. A technician mistakenly prepared a bottle of saline with less salt than required, and apparently I used that hypotonic solution to wash cells prior to fixation. The chromosomes of the human cells that I fixed werc more beautifully spread than any cytologist had ever seen. This miraculous accident. which I later described in considerablc detail('), changed the course of my career. I had a hunch that with the combination of tissue culture and the hypotonic solution techniques, albeit primitive at that time, mammalian and human cytogenetics might be a worthy field for some adventurous plowing. Unfortunately, only two longterm cell lines, Wilton Earle’ s mouse L line and George Gey’s HeLa line, were available in the early 1950s. Both were cancer cclls with highly abnormal chromosome constitutions. I did not like either of these but for a few years I had to use them out ofnecessity.

After I moved to Houston to join a relatively new institu- tion, the University of Texas M. D. Anderson Hospital and Tumor Institute, I made attempts to establish mass cultures from tissues of several laboratory animals (mouse, rat, golden hamster, guinea pig) and found that I could at least cultivatc fibroblasts for a number of passaged2). However, none of the laboratory animal species showed acceptable karyotypes. The diploid numbers were rather high, and many chromosome pairs were morphologically indistinguishable.

In the late 1950’s and early 1960’s, human and mam- malian cytogenetics was a relatively new ficld with only a few active investigators. Most of us were looking for a species with a low diploid number, prcferably one with rec- ognizable morphology for each chromosome pair. We heard that many Australian marsupials had low chromosome num- bers, especially thc Tasmanian rat kangaroo, Potorous tri- dactylis, which might be suitable for our purposes. At that time three groups of cytogeneticists, of which I was one, were serious enough about our aim to make arrangements to obtain live specimens of Potorous. An Australian friend helped me persuade the Tasmanian Wild Life Protection Agency to trap two pairs of these creatures and ship them to Houston, for a total cost of $1 10. The animals indeed had a low diploid number (2N=? 12, d 13), but to my disappoint-

ment, the morphology of all autosomes was the same. Margery Shaw and Robert Krooth at the University of Michi- gan were also interested in establishing cell lines from this species; so I sent my unused specimens to them. They finally also abandoned working with this species for the same rea- son I had. The available Potorous cell lines, designated as Pt- K1 and Pt-K2, were established by Kirsten Whalen, then at the Naval Biological Laboratory at Oakland, California.

Although I seldom used the Potorous cells for experi- ments, Bill Brinkley and I used them to study the fine stnic- ture of the nucleolus organizer region, because Potorous har a very prominent secondary constriction on its X chromo- some. Bill developed a flat-embedding method for electron microscopyi3’, and he was able to identify the X chromosome prior to thin sectioning. We found that the secondary con- striction was really not a constriction, but a chromosome seg- ment with less dcnscly packed chromatin fibers(4) (Fig. 1). Potorous cells never gained much popularity, because practi- cally all cytologists bccaine entranced, instead, by the chro- mosomes of Chinese hamster cell lines.

Indeed. one of the mammalian species that has had a most significant impact on biomedical research, particularly cyto- genetics, was the Chinese hamster, Cricetulus griscus. These animals were first introduced to Europe as a possible labora- tory animal, and a colony was initially maintained in Har- well, UK. However, were it not for thc initial cytogenetic work by Charles E. Ford in London and George Yerganian in Boston, the Chinese hamster probably would not have gained

Fig. 1. Electron micrograph of the long arm o f the X chromosome 01 the Tasmanian rill kangaroo hhowing h e nucleolus organizer region (secondary constriction) ds a lighlly stdined chromosomal segment with less densely pdcked chromdtin lhdn the chromosome proper 16,400 x

its present status. This species had a diploid number of 22 (XX 0 , XY &)> and most chromosome pairs are morpholog- ically distinct. Yerganian kindly supplied me with two cell lines, on which I performed my first mutagenesis experiment and I used Chinese hamster cells. both in vivo and irz vitro, for more than 20 years.

In the late 1960s, The Fifth Canadian Cancer Conference invited Charles Ford to be its featured speaker. with a scicn- tist specially assigned to introduce him. This gcnlleman overpraised Charlie, saying that Dr. Ford’s contribution was immortal. This made Ford very nervous. While presenting his lecturc, he wrapped the long microphone cord so tightly around his arm that he had a hard time untangling it later. After the session, all of us gathered in the lobby to have a drink. Klaus Rothfels wryly commented over the cocktail in Ford’s hand: ‘You just forfeited your immortality.’ Charlic was so mad that he poured his martini over Rothfels’ head.

During the late 1950s and early 1960s, there was little excitement in the area of mammalian cytogenetics, so 1 decided to incorporate some aspccls of molecular biology into cylogenetics by treating cells with chemical mutagens whose molecular mechanisms or action were known, to see what type of reactions 1 could find in the chromosomes. At that timc, chemical mutagens were not as numerous as they are now, but 5-bromodeoxyuridine (or BUdR as it was called at that time) was already well known. I had a young postdoc- toral fcllow, Carolyn Somers, working with me at that time. We treated the Chinese hamster cells with BUdR for 24 hours and discovered that BUdR caused chromosome breal- ages at specific loci. This phenomenon of specific breakage loci, either spontaneously or induced by various methods, was later referred to by human cytogencticists as fragile sites. Our findings in the Chinese hamster chromosomes were regarded as the first of its kind by Suthcrland and Hechtls). Fragile sites werc cxtensively studied during the 1970s and part of the 1980s because several human congenital anom- alies wcrc found to be associated with fragile sites. There was also hope that cancer might also be associated with chromo- some damage at specilic fragile siles, but such a correlation has not been well established.

In papers published in Proc. Not1 ilcad. Sci. US.4(6) and later in Experimental Cell Research(’) 1 made a couple of speculations: ( I ) Since BUdR is a thymidine analog and is incorporated into cellular DNA, the breakage at specific chromosome loci suggesled that these loci might bc rich in adenine-thymine base pairs, implying that along inammalian chromosomes there may be clusters of AT-rich and GC-rich regions, and (2) In view of the finding that chromosomes with characteristic break sites survived one or two additional cell generations, retaining their inorphology minus the frag- ment, I proposed that these chromosomes might have inter- stitial telomeres. These speculations. especially the former, were criticized by a number of molecular biologists who worked on the genomcs of microorganisms. As subsequent studies showed, both of my speculations regarding the Chi- nese hamster situation were incorrect, because the BUdR- induced fragile sites in the Chinese hamster were neither telomeric nor highly AT-rich; but the ideas about AT-rich and GC-rich D N A and interstitial telomeres proved to be

prophetic. Nearly a decade later, by means of in situ hybrid- ization technique, Parduc and Gall(x) proved beyond any doubt that the AT-rich murine satellite DNA sequences formed the heterochroniatin blocks of mouse chromosomes.

Proof of the existcnce of inlerstilial lelomerex came more than two decades later, when Moyzis and associated9) madc an elegant investigation of the (TTAGGG),, sequence. One member of the team, Julianne Meyne, a former student of mine. was interested in a survey on the distribution of this DNA sequence in the vertebrates. She knew that I had a large collection of mainnialian cells, so she called me to see if I would supply her with selected animal cells for her study, and said that she would invite me to be a co-author when the work was finished. I told her I would be glad to help. but it was not necessary Ihr rnc to become a co-author. She thcn told Moyzis that I didn’t sound too enthusiastic and, there- fore, to whet my appetite to be involved, she sent me some of their best pictures. When I received thosc photographs and saw an interstitial teloinere at subterminal position of rat chroinosoine No. 1, I iininediately told Julie by phone that I had changed my mind about that co-authorship. Sen Pathak and 1 had suspected that the rat chromosome No. 1 was a product of tandem translocation between two chromosoines and the locus of translocation should be exactly at that point. Therefore, a relic of a telomere may be present there. The final publication was a monstrous paper with ten co- authors‘ lo).

Back in the 1960s. ultrastructural studies of cellular com- ponents dominated the discipline of cell biology, but the great majority of investigators concentrated their attention on the cytoplasm, except for a few who studied the nucleolus. It appcared that few electron microscopists were interested in the structure of chromosonies and organelles associated with chromosomes, such as the nucleolus organizer, the kineto- chorcs, and the centrioles, but I had no money to purchase an electron microscope to investigate these structurcs. A casc of mistaken identity cost me the gift of an electron microscope, and damped my initial but I wasn’t com- pletely discouraged by this setback. I offered a post-docloral position to a young PhD., B. R. Brinklcy, applied for an NIH grant, and finally got it. Bill Brinkley worked with me for more than several years and later developed into a leading cell biologist: his adventures in Cell Biology have been recounted in a ’Roots’ article in BiuEssays~”). I shall omit repeating all the stories Bill presented in his memoir, but caii- not avoid saying that the years when Rill was with me. he and I really had a great deal of excitement. We took an experi- mental, instead of the popular descriptive, approach to elec- tron microscopy, and our approach paid off handsomely. Our priiicipal experimental material was the diploid male Chi- nese hamster cell line established in my laboratory. After Bill left my laboratory, I continued some ultrastructure studies with a graduate student, Manley McGill, primarily on the effects of mitomycin C and ethidium bromide on centrioles, chromatin slructure(I2) and on mitochondria1 DNA(]”,.

For nearly 20 years, Chinese hamster cells were the prince of my laboratory. In addition to the EM investigations, I stud- ied the DNA replication sequences of this species in consid- erable detail. Ever since my Drosoplzila days, 1 have been

interested in the structure and function of heterochromatin; however, identification or heterochromatin in maminalian chromosomes by staining reaction was never very accurate. After J. Herbert Taylor perfected the 3H-thymidine labelling method, he studied the DNA replication patterns o€ the Chi- nese hamster cells supplied by George Yerganian. I was fas- cinated to see his pictures of the late-replicating Y chromo- some and the long arm of the X It gave me hope for the identification of hetcrochromatin. Therefore, I lcarned the autoradiographic technique and used my diploid Chinese hamster cell lines as n~alcrial(’~). Not only did I con- firm Herb Taylor’s observations that the Y and the long aim of the X of the Chinese hamster were heavily labelled during the very end of the S phase, but, using a pulse labelling method to analyze thc beginning of the S phase, I found that these chromosome segments were unlabelled in early S while all the autosomes had begun replicating (Fig. 2). If late replication were indeed a characteristic of heterochromatin, then in the human and Chinese hamster karyotypes, hete-

I -

Fig. 2. Duplicate autorndiugrnphs of a male Chinese hanistet cell at the early ytage or S phase Top figure, acetoorceni stain prior to autoradiography Bottoin figure, autoradiograph of the same Note unlabelled Y chromosome and the long ai ti1 of the X, both of which are the last to bnish DNA replication

Fig. 3. A. Electronmicrograph of a Per-oinysci~s ererriicus cell fol- lowing Ihe convcntional fixation and staining procedure. Note heavily condcnsed chromatin masses around the nuclear envelope. 12,300 X. B. A cell in a hypotonic fixation medium. Note the com- plete disappearance of the condensed chromatin masses in the nuclcus and the swelling of mitochondria. 12.300 x.

rochromatin seemed to be located on many chromosomes, especially the centromeric regions. Although the discovcry that within a genome DNA replication has a definite sequence led to numerous significant findings, thymidine labelling to identify Constitutive heterochromatin was not an ideal method. The resolution of 3H is still not sharp enough, and Faculhtivc and constitutive heterochromatin regions behave in the same manner. However, this was the only tech- nique available at that time, and I believe we exhausted its usefulness.

Between the mid- 1960s and mid- I970s, I was engaged in a cytogenetic survey of mammals. One source of my interest in this project was my background as a naturalist and I therefore used cytogenetics as an approach to trace some evolutionary trends. Another reason was that in doing a survey, I might find some species that had unique cytological features for me to take advantage of. Indeed, I found several useful animals species that could answer one question or another, including lhe Scba’s fruit bat, Carollia perspicillntu, which has an interesting X/autosome translocation; the Europcan field vole, Microtus agrestis, which has monstrous sex chromo- somes made of heterochromatin; the Indian muntjac, Munti- ucus miintjuk, which has the lowest diploid number of a mammal (2n = 9 6, 6 7), and the American field mice, Per- omyscus ssp, which have an enormous variation in chromo- some morphology but the same diploid number(16). But I continued to use Chinese hamster cells for various experi- mental purposes.

Late in the 1960s, Mary Lou Pardue and Joseph G. Gall developed thc in situ DNA/KNA hybridization technique@’. In addition to proving ribosomal DNA amplification in amphibian oocytes, they localized the highly repetitive murine satellite DNA at the centromeric regions of all auto- somes and the X chromosome. These studies, indeed, repre- scnted a breakthrough in molecular cytogenetics. My interest in satellite DNA and heterochromatin prompted me to apply their method to human and various mammalian species. My colleague in molecular biology, Grady F. Saunders, isolated the repetitive fractions of human DNA with a hydroxyapatite column and synthesized cRNA for our use. We successfully identified the chromosomal locations of highly repetitive (lowest Cot) DNA sequences of many mammalian species.

In conducting in situ hybridization experiments, we got a bonus, i.e., we discovered a procedure that can reveal consti- tutivc heterochromatin (C-bat~d)([~). Presumably, highly repetitive DNA sequences renature much faster than low repetitive and single-copy DNA sequences, thus taking the stain earlier in cytogenetic preparations. We used this tech- nique to identify heterochromatin of a variety of animal species, including Dvosc@ilu(18), a material that 1 hadn’t touched for 20 years.

With the information correlating highly repetitive (satel-

lite) DNA and constitutive heterochromatin established, a number of biologists and molecular biologists speculated on the function of these satellite sequences, but none of these

hypotheses have gained any significant experimental sup- port. T also speculated about these sequences, the bodyguard hypothesis( It, too, lacked substantiation. This hypothesis was presented in 1973 at a syinposiuni in the International Genetics Congress held at Berkeley. When Bill Brinkley was with me, h e and I tried to observe the crfccts of hypotonic solution on the fine structure of the nucleus. The results were nevcr published, but one phenomenon made a deep impres- sion on me, namely that in an isotonic environment. the chro- matin layer adjacent 10 the nuclear envelope is usually more electron dense than the interior. When cells were treated with a hypotonic condition, the dense outer layer disappeared and the entire nucleus was homogeneously smooth (Fig. 3). I speculated that this dense outer layer is the heterochromatin, and that it is used by thc cell to absorb the detrimental effects of mutagens. Thus, one function of the heterochromatin may be to sacrifice itself in order to protect the vital genes in the interior, analogous to a bodyguard protecting his master in case of danger, such as assassination. To support this hypoth- esis, I made a series of experiments, using cells of the cactus mouse, Perornyscus ereminus, as test malerial because it has a largc amount of heterochromatin. The cells were pulse treated with a mutagen under either isotonic or hypotonic condition and the chromosomes were examined a few hours later. Under an isotonic cnvironmcnt, the overwhelming majority of chromosome damage was limited to the hete- rochromatin segments, whereas in a hypotonic environment, damage was obscrvcd in the cuchroniatin also. Unfortu- nately, I did not follow up this problem, and no one else has either. Therefore, the bodyguard hypothesis has never been further substantiated.

After a few years of exciting work applying the banding techniques to chromosomes and developing the silver method for staining the nucleolus organizer regions, my col- league Sen Pathak and I decided that we had picked the cream of the field of comparative mammalian karyology and evolution, and that we should therefore phase out this area of research. Because of the growing concern over global envi- ronmental deterioration and its relationship with cancer, T decided to explore the possibility of mutagen sensitivity in humans. Although. through the years, I have used, in addition to BUdR, a variety of mutagens (FUdR, actino- mycin D, mitomycin C, ethidium bromide, and gentian vio- let, to name a few) to study their cffects on chromosomes, those experiments were mainly designed to analyze chromo- some structure and chromosome behavior, not as applied research in environrnental or oncological studies. After proposing a working hypothesis(I9) that hurnan individuals may be genetically different in terms of mutagcn sensitivity (and, hence, in cancer susceptibility), I have worked dili- gently with huinan material for the past ten years, apart from some excursions to study cockroach and mosquito cytology. We assayed more than 1000 human subjects for mutagen sensitivity. normal indilliduals as wcll as cancer patients, and reached the conclusion that differential mutagen sensitivity, indeed, exisls and may be a reflection of individuals’ DNA repair capabilityiZ0.”). Thus, I finally found out that huinan chromosomes, although not very exciting, are not as dull as I had previously thought.

From this brief resume of my research adventures, it is apparent that I do not have one favorite animal species or a Favorite cell. Although the Chinese hamster cells proved suit- able for a number of reqearch problems, they do not fit all my research programs. My current studies on canccr epidemiol- ogy obviously require the use of huinan material. Therefore, my favorite rcscarch object is generically the chromosome, not the chromosomes of a particular life form.

Acknowledgement I am most grateful to Dr. B. R. Brinkley for providing prints of the electron micrographs included in this paper.

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