Genetic Parasites and a Whole Lot More

Photo: Ori Fragman, Hebrew University Hordeum spontaneum, the plant studied for BARE-1 retroelements. With genome sequences arriving almost as regularly as the morning paper, the public's attention is focused on genes--new genes to protect crops against pests; rogue genes that make bacteria resistant to antibiotics; faulty genes that, if fixed, could cure diseases such as muscular dystrophy. What many people don't realize is that genes account for only part of an organism's DNA, and in many cases it's a tiny fraction. If humans have 50,000 nuclear genes, as some researchers think, then 98 percent of more than three billion DNA base pairs is something else. Much of that something else is repeated sequences that litter the genome. Repetitive DNA comes in various sizes and classes. A lot of it is short, tandem repeats clustered at the centromeres--where spindle fibers attach--and the specialized chromosome ends called telo-meres. But a lot of the rest is distributed betwixt and within genes. Some even piles up on itself, piggyback style. There's so much of this filler DNA in maize--about 80 percent of the genome--that the good stuff is scattered like oases in the desert, thousands of nucleotides apart or clustered in gene islands along the chromosomes.1,2 For years some researchers thought repetitive DNA was so much useless junk, with no obvious function. Others weren't convinced because much of repetitive DNA consists of wayfaring sequences called transposable elements. By generating mutations, altering patterns of gene expression, and otherwise fueling genetic diversity, transposons may have primed evolutionary explosions of the kind that produced the primates and provided genetic material early farmers needed to breed important crops. Says University of Georgia geneticist Susan Wessler, "they shake up an otherwise conservative genome." Now, scientists are realizing that transposable elements may also have an impact on ecology by shaping plant adaptations to local environments. That excites evolutionary ecologist Johanna Schmitt of Brown University: "The time is ripe for molecular mechanisms to be considered in an ecological context, and for ecologists to understand the molecular basis of adaptations." Some Basics First described in the 1940s by pioneering maize geneticist Barbara McClintock--a discovery that earned her a 1983 Nobel Prize--transposable elements are segments of mobile DNA that move hither and yon through the genome. Found in organisms as simple as yeast and as complex as humans, they're the epitome of Richard Dawkins' selfish genes.3 Transposable elements behave like genetic parasites, competing with their host's genes for resources and in the process multiplying like tribbles on the Starship Enterprise. Transposable elements come in two basic flavors, depending on how they move. Class II elements, the type discovered by McClintock, have inverted repeats at each end. A self-encoded transposase enzyme moves the sequence within or between chromosomes. Some members of this class lack a transposase, depending instead on enzyme encoded by another element. Class I transposable elements, or retrotransposons, don't actually excise. Instead, using an RNA intermediate they breed copies of themselves that populate the genome. In the case of long terminal repeat (LTR) retrotransposons, the DNA sequence generates a messenger RNA encoding several proteins, including a reverse transcriptase and an integrase.4 The reverse transcriptase makes DNA copies of the retro mRNA, which then insert into chromosomes by way of the integrase. If that strategy sounds familiar, it's because LTR retrotransposons are structurally related to their retrovirus cousins, including HIV.4,5 Because class II elements simply excise and reintegrate, like an employee transferring between branch offices, retrotransposons are a cut above. By generating thousands of copies, they can have a much greater effect on genome architecture in a short period of time, notes Wessler, who's heading up a new National Science Foundation funded collaboration to study rice transposable elements. "They're like printing presses," remarks Wessler, which explains why "the maize genome has doubled in the last three million years."6 About 50 percent of its 2.4 billion base pairs consists of retrotransposons.4,7 Humans have nearly a million copies of just one type of retro element, called Alu, and others are present as well. By definition, retros must make mRNA to spread. But not all of them are active, and therein lies the key to recent results. When and how they amplify affects the organisms they inhabit. It also hints at an adversarial relationship between retrotransposons and their hosts--a 4-billion-year-old genetic war replete with episodic battles and a more than occasional armistice. Sowing Wild Oats, ...Errrr Barley Barley--Hordeum vulgare to botanists--and its wild relatives contain a prominent family of LTR retrotransposons called BARE-1. Functional BARE-1 elements are about 8,900 base pairs long, including a 1,809-base-pair repeat at each end. The two repeats are identical, but by virtue of its position, the upstream LTR at the beginning end of each element controls whether it's transcribed into RNA. According to Alan Schulman and co-workers at the University of Helsinki's Institute of Biotechnology, the genus Hordeum contains on average 14,000 copies of BARE-1 per chromosome set, or almost 3 percent of the haploid genome.8 But the number varies between species and even individual plants. To complicate matters, Hordeum also contains pieces of BARE-1 in the form of solo LTRs--scattered repeats minus the internal domain formed by recombination within an element. In fact, solos can exceed full-length elements by 40-50-fold. The question is, what do the differences mean, and in particular, are they physiologically, ecologically, or evolutionarily important? To answer these questions, Schulman went back to nature, joining forces with population geneticist Eviatar Nevo of the University of Haifa in Israel to investigate cultivated barley's wild parent, H. spontaneum. "Nevo is the world's leading expert on wild barley and has the best accessions anywhere for this species," explains Schulman. The international team first sampled plants at various sites across Israel, learning that BARE-1 tends to increase with temperature, dryness, and altitude.8 Unfortunately, the differences weren't statistically significant because of small sample size. Would the regional trend hold up and even solidify if more plants were sampled at a single site with diverse microclimates? The answer was yes. The researchers analyzed plants from six locations along a 300-meter transect across Evolution Canyon, at Israel's Mount Carmel.9 Straddling north- and south-facing slopes, the transect's microclimates vary from hot and dry at the top of the south-facing slope to moister conditions on lower stretches of the north-facing slope. Surprisingly, BARE-1 copy number varies threefold among individual plants across the canyon. That kind of variability is "unprecedented and remarkable," opined Wessler and Iowa State University researcher Jonathan Wendel in an enthusiastic commentary accompanying the study.7 But hidden in the variability were clear patterns. BARE-1 copy number was greater at the tops of the slopes than farther down. Plants at the top of the south-facing slope--the driest site--had more BARE-1 than anywhere else. The group also discovered that the ratio of total LTRs to full-length retrotransposons goes down with increasing elevation. That means stressed plants have fewer solos. In other words, not only does BARE-1 amplify, host attempts to eviscerate them by recombination are somehow suppressed. The wide variability in copy number indicates that BARE-1 is active, and indeed Schulman had earlier established that barley cells make BARE-1 mRNA. The new effort confirmed BARE-1 dynamics by analyzing where it inserted into wild barley DNA, using a method that assays flanking genomic sequences. Those sequences are surprisingly heterogeneous, which also shows that BARE-1 increases host genetic diversity. Says Wessler, "they have identified a genetic entity that's very active and capable of explosive amplification." BAREly Thirst Quenching? At a minimum, BARE-1 copy number seems related to the environment in which wild barley plants grow, but the results invite more specific possibilities: (1) water stress activates BARE-1, resulting in many more copies that insert in the genome; (2) the extra copies somehow enhance barley's ability to survive and reproduce under stress. The Schulman-Nevo team hinted at the latter, but Wendel and Wessler went further: "...perhaps the relationships between BARE-1 activity, water stress, and adaptation are not only correlative but causal." But how are BARE-1 and stress linked? The possibility that water stress activates BARE-1 resonates with what plant biologists already knew about retrotransposons and what Schulman's group had previously learned about BARE-1. Retro elements in tobacco and rice respond to a variety of stresses, including wounding and tissue culture, treatments that cause major metabolic and developmental changes.10,11 And promoter sequences that control transcription in certain retro elements have counterparts in genes turned on by pathogenic viruses and bacteria. It's probably no coincidence then that the BARE-1 LTR contains an ABA response element12--a sequence normally found in the promoter region of plant genes activated by the hormone abscisic acid. Plants use ABA to signal stress including drought. Since the LTR acts as a promoter controlling activation, ABA likely links water stress and BARE-1 amplification. Plants aren't alone in linking hormones to retrotransposons: Alu sequences in humans carry steroid hormone response elements. Still, Wessler is cautious: "Schulman doesn't know BARE-1 is actually activated by water stress," adding, "the effect could be indirect--for example, the result of selection of plants that just happen to have higher copy number. Or, it could be specific activation. I think that's the most likely scenario, but we have to be careful." Schulman doesn't disagree: "It would appear at this point that BAREs exploit stress response pathways via ABA to make more copies of themselves, but it's not proven experimentally." Obese is Better? Stress-induced amplification of retrotransposons is one matter; how it translates into adaptations that promote survival and reproduction in dry environments is quite another. Right now, speculation centers on two possibilities. When water stress generates extra BARE-1 copies, some probably insert near or within genes, changing the way they're expressed. Any resulting gene product that improves the plant's ability to cope will improve its chances in the reproductive lottery. In Darwinian terms, natural selection acting on the new phenotype will ensure survival of plants carrying the altered gene. What's more, an ABA response element from BARE-1, acting like a hormone response element in human Alu, could render a plant gene sensitive to water stress. Wessler's laboratory showed several years ago that plant promoters contain the remains of ancient retrotransposons that could have altered gene expression.13 It sounds reasonable, but Schulman has doubts: "We have no evidence that any particular class of genes has been affected by BARE-1." Wessler's gut feeling is that a direct effect on expression of specific genes via promoters is not what's going on. "I think it's doing something at a higher level, like chromosome organization, but that's pure speculation." Schulman and others think the something else involves bulk genome size, which increases as BARE-1 copies accumulate and elimination is suppressed. According to Wessler, it's obvious transposable elements are there because they outreproduce host genes, and in the process increase genome size. But do they actually do some good? "No one has ever presented a testable hypothesis before." Until Schulman, that is. DNA content varies 1000-fold in plants, from a relatively paltry 130 million base pairs in the diminutive mustard Arabidopsis to more than a hundred billion in the lily Fritillaria. Yet, both plants have the same basic biological complexity and do the same things--like flower--using approximately the same 25,000-43,000 genes. Biologists call it the C-value paradox, referring to the amount of DNA per genome complement. A few years ago, Purdue University molecular geneticist Jeffrey Bennetzen and evolutionary biologist Elizabeth Kellogg of the University of Missouri in St. Louis wondered whether plants "have a one-way ticket to genome obesity."14 The proposal raised hackles.15 Plant genomes tend toward the hefty side over time--the most primitive flowering plant genomes may have been quite small compared to present standards.16 The trend seems particularly obvious in the grass family--while rice, with the smallest characterized genome in the family, has 430 million base pairs, maize's genome exploded to six times that level in the last few million years, much of it due to retrotransposons.6,7 Schulman thinks the same sort of thing happened in barley with BARE-1,8 though the trend isn't irreversible--barley can also pare down its genome by eliminating retro elements.2 The problem is, plant biologists don't know why more would be better when it comes to nuclear DNA. Scientists have linked changes in genome size to such factors as light and nutritional status,17,18 but they haven't uncovered a specific function or mechanism. Skeptics wonder if the variations are even real.19 The connection between genome size and water stress in Israel's Evolution Canyon wasn't obvious either. In fact, genome size was only weakly related to slope orientation along the transect. Schulman points out that "bulk effects of genome size have been linked to cell volume--that is, expansion capacity," which could be adaptive in a Mediterranean climate when most growth occurs during the cool season. He also thinks you have to consider all the data, including those from across the entire genus, to see the trend in genome size. Says Schulman, "it is the repeated correlations on different scales that are convincing." The Next Step Evolutionary geneticist John McDonald is circumspect about the relationship between BARE-1 and water stress. "It's fairly clear that transposable elements have played a significant role in genome evolution over long periods of time," offers McDonald. "The question is whether they are relevant to local adaptations over short periods of time. There's a big step between correlation and causation. The work is suggestive." When scientists say something is suggestive, they expect more data. Schulman thinks, "we need to know more about stress in the canyon, and stress induction of BARE-1." Brown University's Schmitt says ecologists would like answers to two questions: "Is there a relationship between genome size and fitness, and is it related to stress?" Virginia Walbot, a Stanford University molecular biologist, would address those questions on site: "Take seed from materials on other aspects of the slope and grow them in a new location to determine if there is a 'quick' response." McDonald agrees that moving plants is the thing to do, but he wonders if the results would still be correlative. In other words, genome changes could still be independent of adaptations. That's why lab work might be a good idea, to monitor physiological consequences of increased BARE-1 copy number in planta. Schmitt's prescription is to plant a range of genotypes in an experimental plot, vary the watering, then assess growth, seed production, and gene frequencies. In a prescient 1984 article,20 McClintock marveled at the way plant genomes respond to a variety of stresses, including chromosome breakage and regeneration from tissue culture, resulting in altered gene activity. She predicted that "in the future, attention undoubtedly will be centered on the genome, with greater appreciation of its significance as a highly sensitive organ of the cell that monitors genomic activities, ......senses unusual and unexpected events, and responds to them, often by restructuring the genome." McClintock thought transposable elements are key to restructuring: "In any one strain of maize the number of silent but potentially transposable elements, as well as other repetitious DNA, may change, and most probably in response to challenges not yet recognized." Says Wessler, "I consider her the first genomicist--the first person who thought about the genome in holistic terms, responding to environmental cues in an organized, global fashion. She was ahead of her time. It took the world years to catch up." If Alan Schulman is right, barley and its BARE-1 family of retrotransposons could affirm Barbara McClintock's foresight.S Barry A. Palevitz (palevitz@dogwood.botany.uga.edu) is a contributing editor for The Scientist. References 1. P. SanMiguel et al., "Nested retrotransposons in the intergenic regions of the maize genome," Science, 274:765-8, 1996. 2. K. Shirasu et al., "A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion," Genome Research, 10:908-15, July 2000. 3. Dawkins, R., The Selfish Gene, Oxford, U.K.: Oxford University Press, 1976. 4. A. Kumar, J.L. Bennetzen, "Plant retrotransposons," Annual Review of Genetics, 33:479-532, 1999. 5. N.J. Bowen, J.F. McDonald, "Genomic analysis of Caenorhabditis elegans reveals ancient families of retroviral-like elements," Genome Research, 9:924-35, 1999. 6. P. SanMiguel et al., "The paleontology of intergene retrotransposons of maize," Nature Genetics, 20:43-5, 1998. 7. J. F. Wendel, S.R. Wessler, "Retrotransposon-mediated genome evolution on a local ecological scale," Proceedings of the National Academy of Sciences, 97:6250-2, June 6, 2000. 8. C.M. Vicient et al., "Retrotransposon BARE-1 and its role in genome evolution in the genus Hordeum," The Plant Cell, 11:1769-84, 1999. 9. R. Kalendar et al., "Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimate divergence," PNAS, 97:6603-7, June 6, 2000. 10. S.M. Wessler, "Plant retrotransposons: turned on by stress," Current Biology, 6:959-61, 1996. 11. M.-A. Grandbastien, "Activation of plant retrotransposons under stress conditions," Trends in Plant Science, 3:181-7, 1998. 12. A. Suoniemi et al., "The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays," Plant Molecular Biology, 31:295-306, 1996. 13. S.E. White et al., "Retrotransposons in the flanking regions of normal plant genes: a role for copia-like elements in the evolution of gene structure and expression," PNAS, 91:11792-6, 1994. 14. J.L. Bennetzen, E.A. Kellogg, "Do plants have a one-way ticket to genomic obesity?" The Plant Cell, 9:1509-14, 1997. 15. D. Petrov, "Slow but steady: reduction of genome size through biased mutation," The Plant Cell, 9:1900-1, 1997. 16. I.J. Leitch et al., "Phylogenetic analysis of DNA C-value provides evidence for a small ancestral genome size in flowering plants," Annals of Botany, 82 (Suppl. A):85-94, 1998. 17. V. Walbot, C.A. Cullis, "Rapid genomic change in higher plants," Annual Review of Plant Physiology, 36:367-96, 1984. 18. H.J. Price et al., "Environmentally correlated variation in 2C nuclear DNA content measurements in Helianthus annuus L.," Annals of Botany, 82 (Suppl. A):95-8, 1998. 19. J. Greilhuber, "Intraspecific variation in genome size: a critical reassessment," Annals of Botany, 82 (Suppl. A):27-35, 1998. 20. B. McClintock, "The significance of responses of the genome to challenge," Science, 226:792-801, 1984. Legacies of the Founders That the plant stress hormone ABA may control the activity of BARE-1 retrotransposons in barley strikes a familiar chord with Wanda Reynolds of the Sydney Kimmel Cancer Center in San Diego. Reynolds studies a family of short (around 300 base pairs) retrotransposons called Alu common in primates. Nearly a million Alu elements populate the human genome, accounting for about 5 percent of human DNA. Scattered among human genes, Alus sort into subfamilies that "appear to have arisen from a lineage of a very few founder genes," says Reynolds. The founder genes in turn probably arose from a highly conserved cellular gene called 7SL that encodes a metabolically important RNA. If an Alu inserts into an organism's essential genes, it can cause serious genetic disorders such as neurofibromatosis. Reynolds' team discovered that certain Alus contain a hormone response element in their promoter, a portion of the DNA that regulates Alu activity. The element recognizes receptors for steroid hormones such as retinoic acid and estrogen.1-3 Slight differences in the response element determines which receptor binds. Once binding occurs, though, RNA synthesis kicks into high gear. In other words, some Alus may have spread by taking advantage of host physiology. Remarks Reynolds, "A good attribute of a founder would be a high level of transcription in order to make copies that insert throughout the genome. The earliest founders had the SP1 [transcription factor] site, then a base change made the retinoic acid and thyroid hormone response elements, then a deletion made another retinoic acid response element. Basically, these sites had to benefit the transcriptional prowess of the Alu founder, therefore there was selection for mutations making better and better hormone response elements." Unfortunately, hormonally sensitive Alus also play havoc with the genome. If Alu inserts next to a native gene, it can transfer hormone sensitivity, thereby altering gene activity--and that could increase disease risk. Reynolds' group has identified steroid sensitive Alus in the promoter region of the human myeloperoxidase gene. The myeloperoxidase enzyme is implicated in leukemia, Alzheimer's disease and multiple sclerosis. Since promoters control gene transcription, a nesting Alu packs quite a wallop. Glucocorticoids bind to receptors in the same steroid family as the estrogen receptor. What's more, the receptors recognize similar response elements in gene promoters. Because glucocorticoid mediates stress physiology, an Alu sensitive to its receptor could impact the way we manage crises. --Barry A. Palevitz References 1. G. Vansant, W.F. Reynolds, "The consensus sequence of a major Alu subfamily contains a functional retinoic acid response element," Proceedings of the National Academy of Sciences, 92:8229-33, 1995. 2. F.J. Piedrafita et al., "An Alu element in the myeloperoxidase promoter contains a composite SP1-thyroid hormone-retinoic acid response element," Journal of Biological Chemistry, 271:14412-20, 1996. 3. W.F. Reynolds et al., "MPO and ApoEe4 polymorphisms interact to increase risk for Alzheimer's disease in Finnish males," Neurology, in press. Rice Research Continues to Advance As part of an international effort to sequence the rice genome, Rod Wing of Clemson University, in collaboration with the Novartis Agricultural Discovery Institute in San Diego, recently inventoried 73,000 sequence tagged connectors, or STCs, for transposable elements.1 The STCs, accounting for more than 10 percent of the 430 million base pair rice genome, are located at the ends of plant DNA segments incorporated into bacterial artificial chromosomes, which when aligned will spell out the entire rice genome. Recently, St. Louis-based Monsanto Co., now a subsidiary of Pharmacia, made a rough draft of the rice sequence available to researchers taking part in the International Rice Genome Sequencing Project.2 Monsanto also posted it on a new Web site (www.riceresearch.org). Using probes to known sequences, Wing's group found that almost 10 percent of the STCs contained transposable elements, or 4.5 percent of the sequenced nucleotides. Although the census revealed an assortment of elements, 88 percent were retrotransposons. While transposable elements seemed to be randomly distributed along rice chromosomes, one kind called Stowaway preferentially sits near or within genes. Wing's results came four months after Antoni Rafalski and coworkers at DuPont Agricultural Biotechnology-Genomics in Newark, Del., reported that repetitive DNA accounts for more than 28 percent of a 340,000 base pair section of rice chromosome 11, and most of that consists of transposable elements.3 Retrotransposons alone contribute half, or about 14 percent. That share is still far short of the approximately 50 percent of the much larger maize genome invested in retroelements. --Barry A. Palevitz References 1. B.A. Palevitz, "Rice genome gets a boost," The Scientist, 14[9]:1, May 1, 2000. 2. L. Mao et al., "Rice transposable elements: a survey of 73,000 sequence-tagged connectors," Genome Research, 10:982-90, July 2000. 3. R. Tarchini et al., "The complete sequence of 340 kb of DNA around the rice Adh1-Adh2 region reveals interrupted colinearity with maize chromosome 4," The Plant Cell, 12:381-91, March 2000. Barbara McClintock, as she received the Lasker Award in New York in 1981.

Genetic Parasites and a Whole Lot More

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