Lynn Margulis

Margulis, Lynn

Hemeroteca Cientifica Catalana

OBITUARY Hans Ris (1914-2004).Genophore, chromosomesand the bacterial origin ofchloroplastsHans Ris (Fig. 1) professor of biology at the University ofWisconsin in Madison, once described his scientific worksaying: “My research interests include levels of organizationin chromosomes, […] evolution of mitotic mechanisms, highvoltage and stereoscopic electron microscopy […] cytoplas-mic filament systems, use of low voltage-high-resolutionscanning microscopy in the study of nuclear pores, thenuclear pore complex and of cytoplasmic structure [1].” Hisstudies of the structure of genetic material led him to be the firstmodern scientist to document the simi-larity, if not identity, between thenucleoids of cyanobacteria (then called“primitive plants”, “Cyanophyceae”,“cyanophytes” or “blue-green algae”)and those of the chloroplasts of algae(e.g. Chlamydomonas [4]). Ris’s meticulous life’s work to com-pare genetic material by microscopyanalysis included studies of a wide rangeof organisms: the Rocky Mountainspotted fever and flea-borne typhusbacterium, Rickettsia; the kappa parti-cle in the cytoplasm of Parameciumaurelia (this particle is now known to bethe prokaryote Caedibacter); the ki-netoplastid-bearing protist associatedwith certain tropical disease symp-toms, Trypanosoma; an apicomplexancorrelated with fetal abnormalities, Toxoplasma. Ris’ favoritecomparisons of genetic material were of the chromosomes,nuclear membranes and cytoskeleton of eggs and sperm cellsof animals including sea urchins, frogs, toads and salaman-ders. His long experience with comparative chromosomalcytology developed his acute awareness of the differencebetween the thin (2.5 nm diameter) all-DNA fibrils compris-ing the nucleoids of bacteria and the much thicker (10–30 nmdiameter) protein-studded DNA that makes up the chromatinof animals, plants and nearly all other eukaryotes [2]. Ris was especially interested in the peculiarities and odd-ities of insect chromosome cytology. With Sally Hughes-Schrader at Columbia University in New York City he stud-ied chromosomes in an aphid species in which the offspringof fertilized eggs develop into females. Males developparthenogenetically from unfertilized eggs. In the males(which are XO), spermatocytes con-tain one oversized X chromosomeand cell division is very unequal.The chromatids (half-chromosomes)of the large X chromosomes do notseparate from each other in the firstmeiotic cell division in the testes.Rather the distinctive spermatocyte’slarge X chromosome associates withthe membranes, fibrils and micro-tubules of cytokinesis (cytoplasmicdivision). The entire whole X chro-mosome, with both chromatids stillattached to each other at the kineto-chore, stretches along the axis of thespindle fibers, parallel to them. Oneresulting offspring sperm cell winsthe competition for the X and fertil-izes the egg. The other cell productin this first meiotic cell division is very small and inevitablydies. By study of spermatogenesis in these and other sperma-tocytes, Ris established that the normal, if unusual, cycle ofevents in chromosomes in this modified male meiosis deter-mines the life history of these aphids. He was thus reinforcedin one of his strongest educational dicta: “The lesson Ilearned was that, whenever feasible, we must include theLynn MargulisDepartment of Geosciences,University of Massachusetts,Amherst, USAINTERNATIONAL MICROBIOLOGY (2005) 8:145-148www.im.microbios.orgE-mail: celeste@geo.umass.eduFig. 1. Hans Ris (1914-2004).Int. Microbiol.146 INT. MICROBIOL. Vol. 8, 2005study of living cells” (p. 3 in H. Ris, A Life Remembered,1994).He also wrote that “Dr. Dingh in biochemistry atWisconsin was studying nitrogen fixation in blue green algae[...] We decided to study their cell organization by electronmicroscopy […] Electron microscopic analysis showed astriking resemblance between chloroplast structure and cellorganization of blue-green algae.” He studied, too, “kappa” a“cytoplasmic gene” in Paramecium. This “non-nucleargenetic factor” in the cytoplasm (to which Tracy Sonneborn,genetics and zoology Professor at the University of Indianahad drawn his attention) determined the transmission of thekiller trait in these ciliates. Ris wrote of the kappa particle: “ithad a bacteria-like ultrastructure.”Persistent and dedicated comparative ultrastructural stud-ies such as these, especially in photosynthetic organisms withtheir distinctive thylakoid membranes, inspired Ris touncharacteristically generalize, as he was never prone toover-interpretation or generalization. “In 1960, at theInternational Congress of Cell Biology, I reported theseobservations, which strongly supported the old idea ofendosymbiotic origin of mitochondria and chloroplasts.” Hiswork at the time rekindled concepts from Russian andGerman literature made known in the English-speakingworld by E.B. Wilson’s book The Cell in Development andHeredity. Wilson had collected studies that extended back tothe 19th century that photosynthetic organelles of plant cellsoriginated from bacteria.In his advanced cytology graduate class, Ris used to readaloud to us, a few dedicated students, from Wilson’s book. Inthese classes and lab courses (which he taught himself), hedisplayed his deep European-style knowledge of zoology,anatomy and of course, cells science. I had studied non-Mendelian inheritance of genetic factors including the mater-nal inheritance of the green color in Chlamydomonas,Epilobium and other algae and plants. Thus when Ris read tous: “More recently Wallin (1922) has maintained that chon-driosomes [old name for mitochondria] may be regarded assymbiotic bacteria whose associations with other cytoplas-mic components may have arisen in the earliest stages ofevolution […] To many, no doubt, such consideration mayappear too fantastic to mention in polite scientific society;nevertheless, it is in the range of possibility that they maysome day call for more serious consideration… ”, the courseof my professional life was set forever!Hans Ris was born in Bern, Switzerland, in 1914, the eld-est of three sons. His father and his father’s older brother,who became a schoolteacher, had lost both their parents at ayoung age. They grew up in an orphanage near Bern. Ris’father became a businessman who, when Hans was six yearsold, bought a coal and wood delivery business. His motherwas raised on a mountain village farm where, as a child,Hans often spent his school vacations. She was a photogra-pher who eventually bought and managed an elegant hatshop, and married in her mid-thirties. Hans remembered heras a “warm, caring person, intelligent, adventurous and veryindependent”. She died when he was only 15 years old. Ris’ love of nature, interest in science generally and biol-ogy in particular began early. From age ten he spent his freetime roaming in the woods around Bern. “Observing, listen-ing, I became fascinated by the diversity and beauty of livingthings.” By the year his mother died he had built his firstmicroscope that magnified about 400 times. He made it of“cigar boxes of my father and cardboard from old schoolbooks.” Young Hans had obtained lenses and a set of instruc-tions from a German popular science magazine. “And whatmagnificent Secret Gardens it opened for me! Here was bornmy passion to explore the world beyond human vision!” Inthe early 1930s Ris discovered the writings of the artist,scholar and activist Ernst Haeckel, the champion ofDarwinism in Germany. “This led to an agonizing revolutionin my beliefs about nature, man and universe. In SundaySchool I had been taught that God created the world, plants,animals and finally, in his image ‘man’; and he told man: goand multiply and exploit my creations for your benefits. NowI learned how scientific investigations revealed a very differ-ent concept: the unity of all life. Man originated by the sameprocesses that over millions of years produced the stunningdiversity of living forms. Science teaches us that all livingcreatures are our brothers and sisters. At age 16 I decided Iwould turn to science when creating my own world view”(p. 1 in: H. Ris, A Life Remembered, 1994). At Bern University Ris earned a “Diploma of High SchoolScience” so that he might be able to teach even though his realgoal was to become a Naturforscher (a naturalist). Fritz Baltzer,an outstanding teacher of his, did two things to excite his curios-ity for experimental biology: Baltzer provided Ris with sala-mander sperm and eggs, showing him how the eggs might beenucleated and asked him to do experiments to help under-stand the role of the nucleus in development. “This was myintroduction to the designing of experiments to answer sig-nificant questions. Secondly, in the spring of 1937 he tookme along with the staff of the [Bern University] ZoologicalInstitute to Banyuls, a French marine biological station nearthe border of Spain and France. […] During two weeks in[Laboratoire Arago of CNRS, France at] Banyuls [-sur-Mer] wecollected and studied marine organisms, but most importantlywe obtained sea urchin eggs and sperm and observed fertiliza-tion and cleavage in life. This experience later led to my interestin chromosomes and mitosis.”MARGULIS147INT. MICROBIOL. Vol. 8, 2005His academic advisor in Bern, Ernst Hadorn, obtained afellowship for Ris to study at the University of Rochester inupstate New York. In 1939 a young instructor at Rochester(Kenneth Cooper) arranged for Ris to receive a teachingassistantship at Columbia University in New York City withCooper’s former PhD professor: Franz Schrader. Thus beganRis’ important work with Sally Hughes-Schrader, wife ofFranz and fine experimental scientist from whom Ris claimsto have learned more than he did from anyone else atColumbia. After he received his PhD in 1942 at Columbia hewas hired as an instructor at Johns Hopkins University inBaltimore. When he was 33 he spent a month at the BermudaBiological Station where he was able to obtain a large varietyof tissues, eggs and sperm from marine animals. With the useof cytophotometric methods he measured the DNA content inmany species. He found that in members of any givenspecies, the amount of DNA was the same for nearly all cells.Indeed, the amount of DNA per chromatid (half-chromo-some) for any animal or plant varied a lot but it was constantin the cells of all members of a given species. Since theamount of DNA per chromosome set was constant and thenumber of chromosome sets in egg and sperm was half thatin the somatic tissues, Ris elegantly demonstrated that spermand eggs contain half the amount of DNA as do somatic(body) cells. This and much other work he summarized in animportant review [3]. This work, together with contemporarystudies by Swift and Pollister, strongly reinforced the notionthat DNA was the genetic material, 3-4 years before theHershey-Chase experiment.From 1969 until 1984 Ris was director of the HighVoltage Electron Microscope Facility supported by theNational Center for Research Resources–National Institutesof Health (NCR) at the University of Wisconsin Madison.Many regarded him as the finest microscopist in the worldand certainly one of the most innovative. He was a founderand an active member of the American Society for CellBiology throughout his life.Ris was hired to the faculty of the Zoology Departmentin Madison in 1949 and at his death he was ProfessorEmeritus in that same institution. Although always attendingto many other projects his passion was the structure of DNAin living cells. His work in recent years with two collabora-tors: Soo Siang Lim (now at the National Science Foun-dation in Washington DC) and Marek Malecki (still at theUniversity of Wisconsin-Madison) involved details ofnuclear pores, pore complexes and their associated hollowcables. These electron micrographs that we found in hisMadison office filing cabinet drawers (he called them his“treasure-chest”) give some idea of the quality of his work.The definitive manuscript on the three-dimensional structureof the nucleus of the amphibian egg, work Ris began before1979, will be published posthumously as soon as possible byhis co-author, Malecki. The photographs show a stunningpattern of the nuclear pore complexes by low voltage scan-ning electron microscopy correlated with thin-section trans-mission electron-micrographs and stereoimages of the samepores obtained in high voltage electron micrographic images(Fig. 2). His son found the magnificent manuscript of thejoint work with Malecki nearly finished both on Ris’ com-puter and in a file labeled “manuscript” on a shelf at home[H Ris, M Malecki in Xenopus oocyte nuclei, branching hol-low cables connect the nuclear interior to the intranuclearcomponents of the pore complexes (fish traps). Manuscriptin preparation].HANS RIS (1914-2004)Fig. 2. Nuclear pore complexes and cables.(A) Newt (Notopthelmus viridescens) oocyte,external view of the egg cell membrane, lowvoltage SEM. (B) Xenopus laevis hollownuclear cables of the pore-studded nuclear eggcell membrane, thin section TEM. (C)Xenopus laevis egg-cell nucleus. Isolatedoocyte membrane shows that tops of the “fishtrap” pore-studded structure are connected bythe hollow nuclear cable system. Int. Microbiol.148 INT. MICROBIOL. Vol. 8, 2005Ris regretted, he told me on several occasions, that hisinsistence to limit the word “chromosome” to eukaryotes(because no prokaryote has them) tended to be ignored. Hestrongly urged the use of the word “genophore” which means“gene-holder” instead of “chromosome” to refer to the gene-tic behavior and correlated DNA fibrils of bacteria. In allprokaryotic cells genophores are inferred from experimentswith bacterial genetic recombination by conjugation, plasmidinsertion, viral replication and other transmission genetictechniques [5]. Ris always referred to genetic material ofprokaryotes, as seen in electron microscopic thin section, asthe “nucleoid”, a nucleus-like structure. Nucleoids are nevernuclei since they never contain chromatin nor do pore-stud-ded membranes bound them. Nuclear double-membraneswith their pore complexes surround at least two bona fidehistone protein-rich chromatin-chromosomes per eukaryoticcell. Nuclei, pores, microtubules and chromosomes are lack-ing in bacteria (whether archaebacteria or eubacteria). ForRis “chromosome” always refers to eukaryotic structuresinside nuclei that are present in numbers proportional to cellploidy. In animal and plant cells chromosomes stain pinkwith the Feulgen reaction, a Schiff-base forming reaction.Chromosomes comprised of histone and other proteins com-plexed to the DNA are entirely lacking in all bacteria at alltimes. In nucleated cells chromosomes attach to micro-tubules, made of tubulin proteins, at kinetochores. Even insome marine protists, the dinoflagellates, whose peculiarchromosomes (made of bacterial-like 25 nm-small fibrils thatare histone-depleted) standard nuclear membrane and micro-tubules accompany protist mitosis. The French investigator,Marie-Odile Soyer-Gobillard at Banyuls-Sur-Mer, worked outthe peculiarities of dinoflagellate chromatin and mitosis in col-laboration with Ris. Soyer-Gobillard notes that “without Ris’visits, his intellectual and electron microscopical aid”, shecould not have accomplished her wonderful work on dinofla-gellate mitosis. Ris enjoyed an international reputation for hisadvice and help to women and very young scientists.Ris was very disappointed when his appropriate name forthe “bacterial chromosome, the “genophore ”, a term hecoined, was not generally known or, if known, not acceptedby important microbiologists. Ris, of course, was correctabout the profound differences between the proteinaceouschromatin of eukaryotes (chromosomes) and the genophoreDNA fibers in the nucleoids of prokaryotes. Kind of tricky asthe word chromosome came from their staining by dyes thatare specific for the protein component, so bacterial nu-cleoids/genophores are not typically “chromo-”. I think hewas right to use the term “genophore”, esp. since the suffix–some (body) was not apparent as it is in the mitotic or mei-otic chromosomes of the Eukarya. In his terminology andprofound understanding of genetic systems I have alwaystried to follow his lead. “Graduate students”, he wrote, “werealways an important component of my research lab. I chosestudents who were independent and developed their ownprojects. I considered them collaborators, not members of ateam. Science is not a soccer game.”At the end of 2004 the world of biology lost a superb sci-entist, an excellent and dedicated teacher, a scholar of thefirst order and a true lover of nature. His colleagues andfriends, his former students and family sorely miss him.References1. Malecki, M (2000) Hans Ris—From chromatin fibres, through nucleartracks to nuclear pores. Trends Cell Biol 10:494-4992. Ris, H (1969) The molecular organization of chromosomes. In: Lima-de-Faria (ed) The handbook of molecular cytology. North-Hollandpress, Amsterdam3. Ris, H. Kubai, D (1970) Chromosome structure. Annu Rev Genetics4:263-2944. Ris, H Plaut, Wr (1962) Ultrastructure of DNA-containing areas in thechloroplast of Chlamydomonas. J Cell Biol 13:383-3915. Sonea, S, Mathieu, L G (2000) Prokaryotology: A coherent view.Presses Université Montréal, Montréal, Québec, CanadaMARGULIS

Hans Ris (1914-2004). Genophore, chromosomes and the bacterial origin of chloroplasts

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