by David Zimmerman

Mitochondria and chloroplasts, with genes of their own, raise

questions about cellular origins.

T he energy that fuels most plants and animals reaches the earth as sunlight. It is then converted into biochemical

energy by two kinds of subcellular struc­tures, or organelles. The first are the green chloroplasts of plants (and similar red and yellow structures in some algae), the sites of photosynthesis in cells. The chloroplasts use the sun's energy directly to extract car­bon from the atmosphere 's carbon dioxide and hydrogen from its water vapor. They give free oxygen back to the atmosphere and use the carbon and hydrogen to synthe­size such plant carbohydrates as glucose.

The second set of organelles, called mito­chondria, are found in both plants and animals and use free atmospheric oxygen in the con­sumption of glucose. They incorporate a share of the sugar's energy into molecules or adenosine t r iphosphate or ATP. In this form, the energy can be applied to the work of metabolism, growth, and movement. Mito­chondria then return the products of oxida­tion—water vapor and carbon dioxide—to the atmosphere for another turn through the cycle.

Chloroplasts and mitochondria, though they differ, have in common a significant but still-much-disputed measure of autonomy and self-direction. In the past two decades, both have been found to possess their own genetic material, just as the cell nucleus does. Both also have the transcribing equipment that allows them to create some, though not all, of the structural proteins they need to function. They are, in a way, cells within cells.

A radical interpretation These findings, made in recent decades,

are revolutionary. They explode the notion that the nucleus and its DNA alone control the synthesis and functioning of cellular components. Now, interdependent links be­tween the nucleus and cytoplasmic organelles are being revealed, spurring intensive interest in the origins and evolution of cell lines that contain the gene-equipped organelles. Also

of interest is the way these organelles are made within cells today.

In recent years, an accelerating interest in chloroplast and mitochondrial genetics and b i o c h e m i s t r y h a s b e g u n t o c a s t n e w l i g h t o n

important questions in basic biology and the applied sciences. Investigators are seizing upon the newly discovered genetic centers in chloroplasts and mitochondria as probes in the study of cellular biology and the manip­ulation of plant growth and other character­istics. In short, the centers may be promising tools for genetic engineering of plants.

The first evidence that chloroplasts and mitochondria might be fundamentally dif­ferent from other organelles came a century ago. Microscopists struggling then to make sense of the indistinct images delivered by their instruments noticed a remarkable simi­larity in appearance between these organelles and some simpler, free-living, single-celled organisms. Chloroplasts confined within the leaf cells of green plants looked—and be­haved—strikingly like photosynthet ic , blue-green algae. Similarly, the mitochondria within some plant, and all animal, cells looked and acted quite like Escherichia coli and other free-living, oxygen-using bacteria. Both take atmospheric oxygen and glucose from plants in much the same manner for use in making ATP. Additionally, similarities in DNA sequences have recently been found between some aerobic bacteria and the mito­chondria in animal cells.

These resemblances between free-living bacteria and the cellular components of higher organisms have given strong and fresh credi­bility to a radical hypothesis first formulated in the 1880s: that chloroplasts did not origi­nate in the primordial cells from which mod­ern organisms descended. Rather, they started out as independent, free-living organisms. At some point they entered other cells and, once within, took up residence. There they remained as symbionts, reproducing and evolving with and within the heirs of their ancient hosts.

Mitochondrial DNA. Plant research on devel­oping disease-resistant hybrids has been focused on the mitochondrial DNA.

C. S. Levings III by permission.

This hypothesis also suggests that evolution is not a straightforward process in which simple creatures slowly become complex ones through the development and selection of ever-more-specialized adaptational structures and behavior. Rather, it says that today's complex beings can represent a coalescence of two, or perhaps many, developmental lines.

This endosymbiotic hypothesis has been championed by evolutionary biologist Lynn Margulis of Boston Universi ty. She sug­gests that the free-living precursors of today's chloroplasts and mitochondria were advan­tageous to host cells because of the energy they provided; the host, in turn , supplied a secure and hospitable envi ronment for its paying guests, a comfortable relationship in which the two parties gradually became interdependent.

As Lynn Margulis reconstructs the events, they would have begun some 2.5 billion years ago, in the Archean or early Proterozoic era, in mat-like communit ies of large bac­teria something like today's thermoplasmas.

MOSAIC July/August 1981

These organisms would have been both micro-aerophilic and heterotrophic—that is, they had a rudimentary ability to use molecular oxygen and were dependent on other, smaller, prey organisms for their energy and food.

These organisms would in turn have been preyed upon by procaryotes that were more fully aerobic and thus more efficient in energy production and resistant to the het-erotrophs' strategies. Heterotrophs that en­veloped and internalized aerobic predators, then, would have found themselves hosts to organisms that both used oxygen and resisted digestion. If the two could offer each other benefits, they might survive, first as sym-bionts and then, over evolutionary time, as parts of the same complex organism.

That the complex survived, says Margulis, suggests that the host had the benefit of two energy systems: its own rudimentary one and its symbiont's more fully oxygen-exploit­ing one. In exchange for the guest 's energy contribution, the host was able to provide both food and shelter.

S t ructura l ly , Margu l i s p roposes , the aerobes outer membrane became the inner of the mitochondrion's two membranes , while the vacuole, or cavity, within which the host cell had captured and held the intruder—perhaps thereby isolating it from the host 's own digestive processes—became the mitochondrion's second, outer mem­brane. In Margulis 's view, similar sudden incorporations can account for many of the other structural and functional differences between procaryotes and eucaryotes, and so might explain how the major evolutionary divide between them was crossed.

This bold theory—unproved and perhaps not totally provable—has won many ad­herents. This is in part because it accounts well for many important developments in the history of the cell and in part, as cell biologist Patrick Echlin says, because it has "a certain elegant and intriguing fascination."

An alternative view The alternative view-—that chloroplasts

and mitochondria appeared and evolved wholly as intramural ingrowths within an­cient cell lines—has its persuasive exponents too. Among them is Harvard University bi­ologist Lawrence Bogorad. He suggests that the surprising discovery of genes outside the cell nucleus (in chloroplasts and mito­chondria, and perhaps other organelles as well) may have a simpler developmental ex­planation: that in a time before the branching of the cellular family tree, the genetic mate­rial of the cell—a long, probably looped chain

Zimmerman writes on a range of scientific

subjects.

Lynn Wargulis. David Zimmerman, by permission.

of DNA—was loose in the cell, rather than segregated by a nuclear membrane. Today's procaryotes (cells without nuclei, such as bacteria and blue-green algae) still favor this arrangement.

Then, according to what is called the clus­ter-clone hypothesis of cellular evolution, one line of cells eventually separated its genetic material into clusters. Membranes then evolved to bind each cluster, along with a bit of the cellular protoplasm, into self-replicating clones. These clones then became the nuclei, chloroplasts, and mitochondria of today's nucleated, or eucaryotic, cells. Eucaryotes include the cells of all animals and plants and some microorganisms—plankters and protozoans, for instance. Some think the difference between nucleated and nonnucle-ated cells may represent the greatest single evolutionary discontinuity evident today.

To Harvard 's Bogorad, these hypotheses about the eucaryotes' origin are less inter­esting than other quest ions: How did the parallel genetic systems in the cell nucleus and in the mitochondrion and chloroplast evolve? How do they share in the reproduc­tion and function of contemporary (and thus analyzable) eucaryotic cells? Bogorad and many other cell biologists and biochem­ists have plunged into the study of the chloro­plast and mitochondrial genomes (haploid sets of chromosomes), the structures and functions they direct, and the question—still largely unresolved—of how two or three con­trol centers within a cell can interact and coor­dinate their efforts for the cell's well-being.

Biogenesis A principal investigation along this line

is that of biochemist Nam-Ha i Chua and his

group at Rockefeller University in New York City. They are studying the way these organ­elles are assembled and maintained within the cell. They work principally with chloro­plasts from the single-celled alga Chlamy-domonas reinhardtii and from plants such as peas and spinach.

He and his colleagues are finding, Chua reports, that the production of a small p a r t -only 5 percent—of the protein types within an algal chloroplast is actually directed by the chloroplast 's DNA or made within the chloroplast itself. This represents, however, about 30 percent of the mass of that protein. The balance—and still the bulk of the pro­tein produced—is directed by genes in the cell nucleus and manufactured in the cytoplasm but outside the chloroplast. It then moves inward through the chloroplast's outer mem­brane; Chua and others have only recently begun to work out the means of transport.

Zeroing in The Rockefeller researchers have focused

their attention on one of the principal pro­teins found in the internal space, or stroma, of their algal chloroplast, as well as in chloro­plasts of plants. This protein, called RuBPCase (for ribulose-1, 5-biphosphate carboxylase), has an enormous molecular weight: a mass equivalent to that of 1.5 million hydrogen atoms (daltons). It is the most abundant of all plant proteins, accounting for 10 percent of all the earth's green-plant protein mass and, says Bogorad, two-thirds of the protein mass within the chloroplast. The RuBPCase molecule, an enzyme that attaches atmos­pheric carbon dioxide to a sugar molecule, is thus as vital to the production of living energy as the turbine is to the generation of electricity in a hydroelectric plant.

This enzyme is composed of two separately conceived subunits . The larger one is coded by chloroplast DNA and synthesized within the chloroplast. The smaller, equally essential molecular subunit is coded by DNA in the cell nucleus and is among the proteins syn­thesized in the cytoplasm but outside the chloroplast. According to Chua, this shows the close and highly coordinated relationship between nucleus and chloroplast.

When the small subuni t is synthesized in the cellular cytoplasm, it is heavier than it will be as part of the completed RuBPCase enzyme. The Rockefeller researchers have shown that the segment at first includes a large tail whose function is to gain the seg­ment 's admittance through the chloroplast's double membrane. Once inside the chloro­plast, the segment sheds the tail and joins the larger, internally synthesized subunit . Together they become the critical carbo­hydrate-forming enzyme.

3ft Mn.QAir i . ln lw/Ai i r i i i e t1 (KM

Elucidation of this sequence of events has raised several provocative questions: How do the nuclear and chloroplast genomes com­municate to produce the right molecular seg­ments at the right time? How, Chua asks, did the synthesis of so important a protein come to be divided between two separate genomes, and what is the advantage of this division for the cell and the plant? Right now the most important problem, in Chua 's view, is to learn how the cell nucleus shares control with the chloroplast and how that process is orchestrated.

Provocative suggestions Across Manha t t an from the Rockefeller

University laboratory where Chua is un­raveling chloroplast biogenesis, Columbia University biochemist Alexander Tzagoloff and his co-workers explore comparable mysteries of mitochondrial creation. By studying mutan t forms of mitochondrial genes, they have established that these genes, like those of the chloroplast, code for a small but "absolutely essential" num­ber—seven, to be precise—of the energy-producing mitochondrial proteins.

Most of these proteins are synthesized externally, in the cytoplasm. To Tzagoloff this means that "a very specific recognition system must exist to insure that only the right proteins get into the organelles." He and his colleagues have also been able to show that genes from the cell nucleus do not enter and initiate protein synthesis within the mitochondrion. The reverse is also true: Mitochondrial genes are expressed only in­ternally; they do not initiate protein syn­thesis in the cytoplasm outside the mito­chondrial membrane.

The organism the Columbia scientists have focused on is a yeast, Saccharomyces cere-visiae. O n e of their major efforts over the last several years has been analysis of the the 70,000 base pairs of DNA in this yeast's mitochondrion—a daunting task. They hope to learn, among other things, if the mito­chondrial DNA resembles that of similar pro-caryotic bacteria more than it resembles the DNA within its own cell nucleus. The work is not finished, but so far, Tzagoloff says, mitochondrial DNAs look more like bacteria than like DNAs in the nuclei of their own cells.

The yeast-mitochondria experimental mod­el has allowed Tzagoloff to ask—and answer -other provocative questions about genetic messages from outside the cell nucleus. A

Phylogenetic tree. One hypothesis of the origins and evolution of cell lines says that today's eucaryotic (nucleated) cells may represent a coalescence of two, or per­haps many, procaryotic lines.

Lynn Margulis, by permission.

Lawrence Bogorad. David Zimmerman, by permission.

key one has been whether the genetic code, renowned for its universality, is in fact uni­versal. The code requires that specific se­quences in DNA always code for production

of the same amino acid. If this phenomenon is indeed universal, a particular three-letter sequence must encode the same amino acid, whether the sequence is present in nuclear DNA or mitochondrial DNA, or whether it is in a mouse or an elephant. In fact, however, Tzagoloff and other mitochondrial bio­chemists have found that some of the mito­chondrial codons (units that constitute a code element) differ from those found in the nucleus.

(A similar determination—that the code is not universal—was made by English Nobel-ist Frederick Sanger and a group of his col­leagues in England, Germany, Australia, and the United States. They reported it in the April 9, 1981 issue of Nature, as part of the account of their sequencing of the human mitochondrial genome.)

One of the most dramatic differences found by Tzagoloff concerns the coding triplet UGA, or uracil-guanine-adenine. When this sequence appears on a nuclear strand, it is a punctuation mark, a signal that flashes: "Stop translating; this is the end of the gene!" In

Mn>o Air* . i u / A i , n , . M -mon

the mitochondrion, however, the UGA triplet is not a stop signal. Rather, it codes for the amino acid t ryptophan. Tzagoloff suggests that the differences in the codes could have evolved as a means of separating genetic messages emanating from multiple genomes within a cell, so that only the appropriate proteins will be produced in each of the cell's compartments .

Shifting responsibilities Mitochondrial DNA has other traits that

Tzagoloff believes can be used to advantage in s tudying the evolution of nucleic acid and perhaps in resolving questions of organelle biogenesis. The nuclear genome is hundreds of times as long as the mitochondrial genome. The sequences of mitochondrial DNA thus can be fully analyzed and the mitochondrial ge­netic structures of various species compared.

T h e mitochondrial genome has further been shown to differ markedly in size from species to species. This suggests, Tzagoloff says, that evolutionary changes occur in it more rapidly than in the nuclear genome, bu t not uniformly from species to species. Such a propensity to change might help ex­plain why organelles that might have started out as symbionts in early eucaryotic cells would since have lost much of their auton­omy. It might also account for the way nuclear DNA has taken over much of the synthesis of mitochondrial proteins.

If there were an advantage in having all genetic information stored in a central re­pository, a transfer of function to the nucleus could have led first to the inactivation, and eventually to the disappearance, of the re­d u n d a n t mitochondrial gene. Support for this idea comes from the discovery of a par­ticular gene in the mitochondria of yeast. In fungi of the genus Neurospora, this gene is in the cell nucleus; only an inactive remnant remains in the fungal mitochondria. This same gene, which codes for an essential sub-uni t of the enzyme that makes the energy molecule ATP, is totally absent from h u m a n mitochondrial DNA but is thought to be pres­ent in the human cell nucleus. This sequence suggests to Tzagoloff that the transfer of responsibility for mitochondrial synthesis f rom organelle to nucleus is still going on.

An application These findings are of obvious biological

importance. In particular, the discovery that mitochondrial DNA violates the genetic code has created a stir among biologists. But these findings also have important practical im­plications for plant scientists. (See ' T h e Cloning of Russet Burbank," Mosaic, Volume 11 , Number 3.)

O n e of the first links found between non-

Elizabeth EarSe. David Zimmerman, by permission.

nuclear DNA and plant pathology involves perhaps the worst plant disease epidemic ever to strike an American crop plant. The crop is corn (Zea mays). A dozen years ago, corn fields across the nation were struck by a fungal infection called southern corn leaf blight. Leaves became spotted with dead tissue, and the plants withered and died. The 1970 United States corn crop was decimated.

Researchers soon realized that the lethal blight afflicted only one type of hybrid corn: that carrying Texas cytoplasmic male sterility, or Terns, genes. Other corn lines developed by plant breeders are wholly or largely resistant to this blight. But corn grown from seed with Terns trait (the line which gives the most reliable hybrid pro­duction and which had been adopted for seed-corn production throughout the United States) is mortally sensitive.

Plant scientists strive to produce improved hybrids by deliberate cross-pollination be­tween selected strains. This means they do not want the plants to pollinate themselves, which corn ordinarily can do: The stalks may bear both pollen-producing male tassels and female, pistil-bearing flowers, or ears, and so may naturally fertilize themselves. (Corn and other grasses are among plants for which few mechanisms to prevent self-fertilization are known. Many plants have such protection. See "Preventing incest at pistil point," in this Mosaic.) To prevent this self-pollination, seed-corn producers once hired high school students to go through the corn rows during their summer vacations pulling off the tassels—a slow, labor-inten­sive, and generally unpleasant job. The dis­covery and development of corn lines hy­bridized with Terns largely ended the need for this effort; the tassels no longer had to be removed.

Of the several male sterile corn lines that

have been developed, the most consistent and useful, biologists say, was the Terns. Male sterility in Terns corn is not passed on through nuclear genes; it is cytoplasmic s te r i l i ty -inherited via cytoplasmic genes, probably mutated mitochondrial DNA. Two other types of male-sterile corn cytoplasms that are k n o w n probably also involve mitochondrial genes. Thus the cytoplasmic male sterile corn lines seem to be the first mutants de­tected in the mitochondrial DNA gene pool of plants.

The root of the blight In 1971, a toxin—extremely lethal to Terns

corn but much less so to other lines—was isolated from the fungus of corn-leaf blight by David Koeppe and Ray Miller at the Uni­versity of Illinois. This toxin, they discovered, acts to uncouple the biochemical pathways in the mitochondria of the susceptible line so that the plant cells are no longer capable of generating energy in the usual way. Levels of the energy storage molecule ATP in Terns corn fall dramatically within minutes of ex­posure to the toxin.

At Cornell, Elizabeth D. Earle and her col­leagues are trying to transplant mitochondria from corn strains resistant to the toxin into protoplasts , or wall-free cells, of the sus­ceptible line. If the inoculated cells continue to produce energy and thrive when challenged by the disease fungus, this would suggest that genetic engineering methods might be developed to treat Terns corn—now largely abandoned by plant breeders and farmers— so that it could resist blight.

Research on ways to insert new genes into plant cell lines has been focused on the mito­chondrial DNA, and particularly on that of another cytoplasmic male-sterile hybrid line, called the S-strain. At North Carolina State University in Raleigh, plant molecular genet­icist C. S. Levings, III and several co-workers are looking at this strain's botanical and agronomical potential. They have found that S~strain mitochondria have two unique DNA molecules, s-1 and S-2, that apparently are not present in other corn strains or any other plant cells. The strands of DNA are present in the mitochondria, but they are separate from the" mitochondria 's main gene bank. They appear to be linear molecules, while the principal mitochondrial DNA is arranged in loops. More important, the S-1 and S-2 molecules seem able to undo cyto­plasmic male sterility, a function that can be readily tracked.

When Levings studied examples of S-strain that had spontaneously reverted and become fertile, he discovered that S-1 and S-2 had inserted themselves into the mitochondrial DNA. This act of insertion, or natural re-

40 MOSAIC July/August 1981

combination, Levings suggests, causes the reversion to normal fertility. More impor­tant, he adds, "Here are some molecules that know how to insert themselves into other DNA, and know how to function." Can they be to plant biology what plasmids are to recombinant DNA research? (See "Re­combinant DNA: The Power of the Tool ," Mosaic, Volume 12, Number 1).

By attaching other gene sequences coded for desirable traits to S-l or S-2 fragments, Levings thinks it might be possible to insert them so securely into the mitochondrial DNA that they would function and produce their appropriate proteins in this new milieu. Since the mitochondrial genetic material tends to be passed on unchanged from generation to generation, these introduced variants might be very stable and thus of great value in breeders ' efforts to come up with better crop plants.

Weed chloroplasts The genetics of the other critical energy-

translating organelle, the chloroplast, has been at the heart of another agricultural crisis. But the crisis has generated new in­sights into this organelle's genome.

In 1970, the same year corn plants were struck down by fungal blight, a Christmas-tree nursery in Washington State had its own problem: Broad-leafed weeds that had been controlled for many years with herbi­cides called triazines suddenly stood up to the spray. They did not die.

Triazines have been uniquely valuable to farmers because they can be sprayed directly on growing corn plants and certain other agricultural crops. These plants have enzyme systems that will metabolize and thus detoxify the chemical, which is still lethal to root-crowding weeds.

Reports soon came from widely scattered locations in the United States and Canada that populations of redroot pigweed, wild mustard, ragweed, lambsquarters, goosefoot, and common groundsel all were resisting atrazine and other triazines. Resistance in some of these weeds also turned up in Europe, and black nightshade, knotweed, and blue-grass have become immune in France.

Meanwhile, at the Michigan State Uni­versity's plant research laboratory in East Lansing, cell physiologist Charles J. Arntzen and his colleagues were learning the reason for the outbreaks. Triazines, they discovered, kill weeds by entering a plant cell's cyto­plasm, where they are caught and held by a receptor, or binding, protein in the chloro­plast membrane. This protein normally func­tions as one of a series of enzymes that transfer electrons in photosynthesis. When a triazine is attached to the protein molecule,

C. S. Lewings 111. David Zimmerman, by permission

however, it retards or blocks electron t rans­fer. Photosynthesis is interrupted; the plant, starved for energy, soon dies.

In atrazine-resistant weeds, Arntzen and his co-workers discovered, the herbicide-binding protein seems to have been modi­fied; the binding site for the herbicide has vanished. The loss of this site "explains the...resistance," Arntzen says. This recently announced discovery, made with colleagues of Arntzen's at the University of Wiirzburg in West Germany and at the Shell Develop­ment Company in Modesto, California, marks the first time a herbicide receptor has been identified at the molecular level.

Non-Mendelian inheritance Herbicide resistance may signal either

random new mutations or selection in favor of a small portion of the population that carried the resistance gene. Resistant plants would proliferate rapidly, given a competitive advantage in a triazine-sprayed field. The question then arises: How is this resistance inherited? The answer reflects the special, non-Mendelian inheritance of chloroplast and mitochondrial genes.

In the fertilized seed of a eucaryotic plant, the nuclear DNA is always a combination of a male gamete from the pollen and a female one from the seed. But most male gametes lack chloroplasts or their precursors. In the preponderance of species, the females contribute to their offspring half of the nuclear genes and all the nonnuclear DNA, including that of chloroplast and mito­chondria. Cytoplasmic genes, in short, would be inherited through the maternal line. (That the mitochondrial DNA of humans is mater­nally inherited was shown recently by a team of scientists, from the Stanford Uni­versity Medical School and the University of California at Davis, headed by Stanford 's Douglas Wallace.)

Working with a group of plant breeders at Canada's University of Guelph in Ontario, Charles Arntzen made reciprocal crosses be­tween atrazine-resistant and nonresistant biotypes of wild mustard. When the maternal plant was the resistant one, all the seeds produced plants that were resistant to atra­zine; when the maternal plant was not re­sistant, the seeds produced nonresistant mustard plants. When chloroplasts were isolated from the progeny of the reciprocal crosses, only those from the nonresis tant female parent contained the triazine-binding membrane protein.

This work confirmed the matrilineal de­scent of resistance in the atrazine-binding molecule. It also showed that this molecule, unlike Chua 's RuBPCase, is coded entirely by chloroplast DNA. What is more, Bogorad at Harvard, and his colleagues Lee Mcin tosh and Katherine Steinback, now have located the gene that specifies this molecule on the chloroplast DNA.

Engineering plans These findings suggest two routes for

genetic engineers. One is to find a way to negate resistance in weed-plant chloroplasts. The second, which currently occupies Arnt­zen's attention, is to find ways to induce resistance in other broad-leafed c rop plants, particularly soybeans. This would have two significant advantages.

O n many midwestern farms, corn and soybeans are rotated annually to maintain mineral balances in the soil. But if the soy­beans are planted in a cold, d a m p spring, there is often enough atrazine residue in the soil from the previous year to s tunt soybean growth. If a resistance gene for atrazine isolated from wild mustard could be incor­porated into the DNA molecule of a soybean chloroplast, it might be fixed in the maternal cell line and so confer atrazine resistance to all descendant soybean plants. This could make soybean culture in corn fields on alter­nate years more successful and allow farmers to use triazine herbicides on soybean fields. "If we could accomplish this engineering task," Arntzen says, "we'd have a tremen­dous market for atrazine-resistant soybeans. In addition, we'd open a new method for weed control in soybeans, which is a real problem."

Rignt now Arntzen is searching for a ve­hicle—a bit of loose chloroplast DNA, or per­haps even a virus—that could carry the re­sistant gene into the chloroplast and insert it in the DNA there. "We don ' t have a defini­tive plan of what we're going to use as the vehicle," Arntzen says. "But we have a testable system, because we have a positive selection factor that prevents susceptible plants from growing in the face of atrazine

MOSAIC July/August 1981 41

challenge. With it, we can go through, and try, a lot of different vehicles until we find one that works' /7

Missing mitochondrial DNA In trying to clarify the complex relation­

ship between the nuclear and nonnuclear genomes, researchers have turned to new study methods. For example, cellular hybrids between human and mouse cells have been used by researchers at the California Insti­tute of Technology in Pasadena, collaborat­ing with Carlo Croce of Wistar Insti tute in Philadelphia.

The hybrid lines tend to be unstable, Cal-tech biologist Giuseppe Attardi points out . As they divide and redivide into succeeding generations, some of the chromosomes tend to be segregated out, or lost, from the daughter cells. The tendency is for one or the other— either the human chromosomes or the mouse chromosomes—to vanish in this way. T h e selection of the one to be lost depends on traits of the parental cell lines.

The question Attardi and his colleagues have asked is, What happens to the mito­chondrial DNA from each parental cell line as the nuclear chromosomes disappear? Using DNA hybridization techniques and other bio­chemical marker systems, they find that as the parental chromosomes are lost from the hybrid nucleus, the mitochondrial DNA of that parent becomes undetectable. Or it de­creases until it is present only in marginal amounts, in the hybrid cell mitochondria.

The hybrid line at first contains mito­chondria from both parents. Since there is no good evidence that the mitochondrial DNAs from the two sources commingle, Attardi interprets his results to mean that the disappearance of mitochondrial DNA from one parental line, which he can measure, signifies the total disappearance of that line's mitochondria. But he cannot prove directly that this has happened.

These experiments suggest that nuclear chromosomes keep some control over the replication of the mitochondrial genes. H o w they do this is not clear. The Caltech re­searchers have not, for example, been able to find any one nuclear chromosome, or even a group of them, whose loss leads pre­dictably to the loss of that parent 's mito­chondrial DNA.

"The loss of mitochondrial DNA goes in the same direction as the loss of chromo­somes," says Attardi. "However—and this is important—the loss of mitochondrial DNA is not dependent on the loss of any par­ticular chromosome."

Carrying these experiments a step further, Attardi and his colleagues discovered that in some cell hybrids in which all chromosomes

from both parents continue to be present in the nucleus, the mitochondrial DNA of one or the other parent nevertheless tends to disappear. In that type of cross, the loss is always from the mitochondrial DNA of the less-persisting parental cell line. Carlo Croce's investigation of similar hybrids has previously shown that while genes from both parents are present, only one set of nuclear ribosomal genes is being expressed—either that of the mouse or that of the h u m a n cells. The active ribosomal genes and the surviving mitochondrial DNA tend to come from the same parental line. This indicates, Attardi says, that it is not the absence of genes in the nuclear DNA that leads to the loss of mito­chondrial DNA. Rather, it is their suppression.

"The expression of the nuclear ribosomal genes and the replication of mitochondrial DNA can be suppressed," Attardi notes, "even though all nuclear genes that control these processes are there." How and why this suppression occurs is "absolutely unknown" at this time, he adds. Still undeveloped are ways to study the nuclear mechanisms that control these interactions between the cell nucleus and the organelle genomes in mito­chondria and chloroplasts.

The DNA-containing organelles in euca-ryotic cells may or may not have once been parasites or prey of the procaryotic hosts within which, if Margulis is correct, they came to dinner—and have remained since. Whatever their origins, the relationships between their genomes and those of their hosts appear to have become vastly complex. And their fates now are entwined, as Attardi and other biologists are finding.

Philosophical as well as scientific issues hang in the balance, since the endosymbiont hypothesis, if confirmed, could force a major change in the way we look at our own evolu­tionary origins—and ourselves. The standard view, as Lynn Margulis points out, has been that simple organisms grew, evolutionally, into complex ones. Her endosymbiont hy­pothesis—already supported by discoveries of evolutionary incorporation and fixation of viruses into contemporary plant and animal lines—suggests that present-day organisms are conglomerations of discrete antecedents that came together in the past.

Wha t happened once may well have hap­pened, and be happening, many times. "People, and all animals and plants," Margulis muses, "are organized communities of microbes that have fine-tuned themselves. This is a philosophical rather than a prac­tical idea!" •

The National Science Foundation supports

the research discussed in this article through

its Cell Biology Program.

42 MOSAIC July/August 1981