Intron-mediated enhancement of gene expression

Most genes in eukaryotes (well, at least eukaryotes that are not Saccharomyces cerevisiae) possess introns, sequences that are transcribed by RNA polymerase II and subsequently spliced out from the primary transcript.  Introns have been the subject of tremendous interest since their discovery in the 1970’s, and have provided much insight (and grist for controversy) into subjects as disparate as junk DNA, the RNA World, and mechanisms of gene expression.  Among the still-unresolved matters today has to do with the timing of splicing – is it cotranscriptional* or does it occur after polII has released the transcript.

The case for co-transcriptional splicing has been built in part through numerous studies that reveal physical connections between splicing factors and the transcriptional complex; many (most) of these involve the so-called CTD (C-Terminal Domain) of RNA polymerase II.  (This recent review summarizes this emerging field.)  The general idea is that, owing to the association of splicing factors with the CTD of polII, they are able to bind the nascent transcript and initiate splicing before polII has completed the synthesis of the primary transcript.

Norman Ernest Borlaug (March 25, 1914 – September 12, 2009)

Norman Borlaug passed away yesterday.  Dr. Borlaug was the key contributor to the so-called Green Revolution, that brought great food security to countries such as Mexico, Pakistan, and India.  He was a clever and innovative plant breeder and a great champion for the use of high-yielding crop varieties in agriculture.

He was also an outspoken proponent of biotechnology.  As he stated in this short interview:

“I have devoted my life to the global challenge of providing adequate food production for a growing world population.  Forty years ago, a Green Revolution was started using improved seed and fertilizer, helping dramatically increase the harvest while sparing forest and natural areas from the plow.  It took both the scientific advances and the changes in economic policies by leaders to allow for the adoption of the Green Revolution technologies by millions of hungry farmers.

Over the past decade, we have been witnessing the success of plant biotechnology.  This technology is helping farmers throughout the world produce higher yield, while reducing pesticide use and soil erosion.  The benefits and safety of biotechnology has been proven over the past decade in countries with more than half of the world’s population.  What we need is courage by the leaders of those countries where farmers still have no choice but to use older and less effective methods.  The Green Revolution and now plant biotechnology are helping meet the growing demand for food production, while preserving our environment for future generations.”

From his foreword to “The Frankenfood Myth: How Protest and Politics Threaten the Biotech Revolution” by Henry Miller and Greg Conko (Praeger Publishers, 2004):

“As a plant pathologist and breeder, I have seen how the skeptics and critics of the new biotechnology wish to postpone the release of improved crop varieties in the hope that another year’s, or another decade’s, worth of testing will offer more data, more familiarity, more comfort. But more than a half-century in the agricultural sciences has convinced me that we should use the best that is at hand, while recognizing its imperfections and limitations. Far more often than not, this philosophy has worked, in spite of constant pessimism and scare-mongering by critics.

I am reminded of our using the technology at hand to defeat the specter of famine in India and Pakistan in the 1950s and early 1960s. Most “experts” thought that mass starvation was inevitable, and environmentalists like Stanford’s Paul Ehrlich predicted that hundreds of millions would die in Africa and Asia within just a few years “in spite of any crash programs embarked upon.” The funders of our work were cautioned against wasting resources on a problem that was insoluble.

Nevertheless, in 1963, the Rockefeller Foundation and the Mexican government formed the International Maize and Wheat Improvement Center (known by its Spanish acronym CIMMYT) and sent my team to South Asia to teach local farmers how to cultivate high-yield wheat varieties. As a result, Pakistan became self sufficient in wheat production by 1968 and India a few years later.

As we created what became known as the “Green Revolution,” we confronted bureaucratic chaos, resistance from local seed breeders, and centuries of farmers’ customs, habits, and superstitions. We surmounted these difficult obstacles because something new had to be done. Who knows how many would have starved if we had delayed commercializing the new high-yielding cereal varieties and improved crop management practices until we could perform tests to rule out every hypothetical problem, and test for vulnerability to every conceivable type of disease and pest? How much land for nature and wildlife habitat, and topsoil would have been lost if the more traditional, lowyield practices had not been supplanted?

At the time, Forrest Frank Hill, a Ford Foundation vice president, told me, “Enjoy this now, because nothing like it will ever happen to you again. Eventually the naysayers and the bureaucrats will choke you to death, and you won’t be able to get permission for more of these efforts.” Hill was right. His prediction anticipated the gene-splicing era that would arrive decades later. As Henry Miller and Gregory Conko describe in this volume, the naysayers and bureaucrats have now come into their own. If our new varieties had been subjected to the kinds of regulatory strictures and requirements that are being inflicted upon the new biotechnology, they would never have become available.”

Dr. Borlaug had been suggested at times to be the greatest living American.  Given the scope of his accomplishments, it’s hard to argue with this.

More gene targeting in plants

A follow-up to an entry I made a few weeks ago, showing that engineered zinc finger nucleases can be used to target gene insertion in maize.  Abstract and citation without commentary.  Enjoy.

Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable1, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus2. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD. 2009. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437-441.

Targeted gene insertion in plants

One of the strategies used by scientists to introduce foreign genes into living cells involves the targeted insertion of the foreign gene into a pre-determined chromosomal position.  This approach has been used to great effect in (for example) yeast and transgenic mice to disrupt genes (hence the term “knock-out mouse”), and thus study their functions.  Except in chloroplasts, similar strategies have been hard to perfect in plants.  However, new technologies may be bringing us closer to the day when targeted gene disruption is an accessible methodology in plant systems.  A recent report from Joe Petolino’s group at Dow Agrosciences provides an example.

Briefly, this group is developing tools that capitalize on the ability to design tailor-made DNA binding proteins that recognize specific sequences.  Over the past several years, much progress has been made in understanding and adapting the DNA binding properties of a specific class of proteins known as zinc-finger proteins.  It turns out that the chemical rules for specificity in some classes of these proteins can be delineated, and that different sequence specificities can be engineered in a rational way by linking a desired sequence of bases with amino acid side chains predicted to facilitate interactions with the base.  It also turns out that one can target double-stranded DNA cleavage to specific sequences by linking endonucleases with sequence-specific DNA-binding proteins. Putting these two things together – tailor-made DNA binding specificity and targeted double-stranded cleavage – brings us technology to introduce double-stranded breaks at pre-determined chromosomal positions.  (These chimeric proteins are known as zinc finger nucleases.)  Add to this the realization that transgene integration occurs at double-stranded breaks, and one gets to the point described in the recent paper from Dow Agrosciences.

The abstract from the paper is given beneath the fold.  What the authors did was design zinc-finger proteins that would recognize sequences in any of a number of exogenous reporters of endogenous host genes and use a battery of physiological and PCR-based assays to evaluate the efficacy of their strategies.  Thus, they showed that they could promote intra-genetic recombination so as to restore a split reporter gene (encoding green fluorescent protein, or GFP).  They then showed that they could promote intergenic recombination between two  DNA molecules introduced transiently into tobacco cells using Agrobacterium; in this instance, they assayed for the reconstitution of a transgene that confers resistance to a herbicide (Bialaphos).  Finally, they demonstrated targeted insertion of foreign DNA into a tobacco chitinase gene.  This demonstration involved the attempted insertion of a Bialaphos gene into the genome; when used in conjunction with the tailor-made zinc finger constructs, as many as 10% of the recovered Bialaphos-resistance cells has the desired insertion.

While 1 in 10 may not seem to be very efficient, it far exceeds random T-DNA insertion, and brings the possibility of targeted gene disruption within the realm of possibility in plants.  Beyond the utility of this technology in the lab, it will also provide crop scientists with ways to genetically modify plants such that unanticipated molecular consequences (inadvertent inactivation of endogenous genes by random insertion events, disruption of genetic circuits due to triggering of RNAi, to name two) are avoided.

What about agricultural science?

From an op-ed by Allen Levine in today’s Washington Post:

Like most Americans I listened intently as President Obama delivered his first address to the nation and Congress.

He outlined the economic challenges facing our country, noting “the answers to our problems don’t lie beyond our reach. They exist in our laboratories and universities; in our fields and our factories.” And he heralded the “largest investment in basic research funding in American history.” The president could not be more right. Investing in basic research will improve our global competitiveness but these investments need to occur in every area of the federal research budget.

In the blizzard of new research funding created by the federal stimulus bill, an important science was omitted: agriculture. While $10 billion was included for the National Institutes of Health, $3 billion for the National Science Foundation and $2 billion for the Energy Department, not a penny was dedicated for competitive research in the U.S. Department of Agriculture (USDA).

That’s unfortunate. Agricultural science will help us find the answers to some of our greatest problems: food safety, scarcity and cost; water quality and availability; the need for healthy soil and plants to grow food; and sustainable energy. While some of the new federal funding will find its way to agriculture-related issues like climate change and genomics, designating federal dollars to agriculture would have sent an important message.

One can read many signals into this development.  Competitive basic research has always been an afterthought with the USDA, moreso now that its competitive grants program has become more and more targeted in recent years (excluding general and basic research in favor of targeted, pre-determined areas of interest).  When it comes to a basic understanding of how plants work, there is a great deal of work to be done, a treasure trove of fascinating biology, biochemistry, and genetics awaiting resourceful and inquisitive researchers.  What is missing is the large research community, a critical mass, that drives new scientific discovery.  In my field, for example (that would be mRNA 3′ end formation in plants), there must be at least ten times as many people working on the basics in animals and yeast as in plants.  The same is true for every other aspect of basic biology in plants.  And again, from my own experience, it is safe to say that there are unique aspects in plants that cannot be teased out of, or extrapolated from, knowledge gained from research in the usual model systems. We need more, many more, basic plant scientists.

One has to wonder – given the President’s seeming intent to trim spending in part through cuts in ag subsidies, is it possible that the administration is lumping all ag spending under the “subsidy” umbrella?  Does the administration think that other agencies (NSF, DOE) may pick up the slack when it comes to plant science research?  And where were/are the USDA operatives in all of this?  Did they not advocate for more basic research?

Just wondering …

Where does all that DNA go?

In this recent essay, I discussed studies that showed a surprisingly high rate of movement of DNA from organelles to the nuclear genome.  Curious and questioning readers should have wondered about this, as one implication is that nuclear genomes should become huge mosaics of organelle DNA in a relatively short evolutionary time.  Of course, this is not the case – organelle DNA may be found in nuclear genomes, but it makes up a tiny fraction of these genomes.

If you had read my essay and wondered along these lines, you may pat yourself on the back.  For this paradox is something that has puzzled others.  A recent paper in PLoS Genetics helps to resolve things.  Briefly, Anna Sheppard and Jeremy Timmis followed up on the earlier studies , asking what happens to the organelle genes after they wander into the nucleus.  These authors found a high frequency with which the organelle DNA (at least the marker gene that was followed in these studies) is altered or deleted.  As the authors discuss (the abstract follows beneath the fold), this suggests that the overall picture is a dynamic one – DNA can move into the nucleus at a high rate, but it is also removed relatively rapidly.  The result is a sort of steady state, one that affords the creation of new nuclear genes without the burden of vast amounts of organelle DNA.

Transplastomics – a convergence of biotechnology and evolution

One of the going concerns in plant biotechnology is the matter of containment of transgenes in the field.  This concern arises from the inescapable fact that genetically-modified crop plants may, depending on the specific species involved, “disseminate” transgenes via hybridization with nearby plants (of the same species or closely-related ones).  A number of strategies have been devised to reduce or eliminate this possibility.  Among these is the approach of placing transgenes in the chloroplast genome of a recipient crop plant.  The rationale behind this approach lies in the fact that the chloroplast genome is inherited in a maternal fashion (much as are mitochondrial genomes in animals).  Consequently, pollen shed by a transplastomic plant (the jargon shorthand term for the plant that has one or more transgenes resident in the plastid genome, as opposed to the nuclear genome) should not carry or transmit the transgene, since transmission is through the female gamete.

Expressing foreign genes in the chloroplast comes with some other advantages.  Since the chloroplast is a prokaryotic genetic system, it is not “encumbered” by the presence of an elaborate and hard-to-control gene silencing system, one that affects nuclear-sited transgenes in a haphazard fashion.  This means that expression of chloroplast-situated transgenes is more consistent (and often attains higher levels) that that of similar nuclear transgenes.  The chloroplast is as well the location for some very highly-expressed proteins (plant physiology students learn early on that the chloroplast enzyme rubisco aka ribulose-1,5-bisphosphate carboxylase is the most abundant protein on earth), which means that it is feasible to attain higher protein levels in such systems than from nuclear transgenes.  (Of course, there are controls on mRNA and protein accumulation in the chloroplast, so that it is necessary to test and manipulate the specific transgene and its protein product to achieve the desired results.)

These considerations aside, the possibility of transmission of chloroplast-sited transgenes remains something of an open issue.  One matter should be familiar to readers who follow the field of human ancestry; work in this field has been complicated by the observation that mitochondrial genomes, typically assumed to be maternally-transmitted, may on occasion be inherited through the paternal gamete.  A similar concern applies to those plant species that are assumed to transmit chloroplast genomes maternally; for example, recent studies (5, 8 ) show that paternal inheritance of chloroplast-localized transgenes does occur.

Hope for school cafeterias, parents, …

…. and others who must deal with allergies to peanuts. A commodity that would be of considerable utility and value, a hypoallergenic peanut, is the goal of several laboratories. One group, consisting of labs at a USDA research unit in New Orleans and the campus of the University of Georgia in Tifton, recently published an interesting and promising approach to develop such a commodity. These researchers set out to screen wild relatives of the peanut (Arachis hypogaea L.) for variants of the principle allergen in peanuts, the seed storage protein Ara h 2.01, that did not possess the allergenic properties of the peanut protein. They used an approach called EcoTILLING (1) to screen several accessions of the wild relative Arachis duranensis for those with point mutations in the gene homologous to that encoding Ara h 2.01. In so doing, they identified several such variants, including one that had a significant reduction in IgE binding but otherwise seemed to cause a minimal structural change in the protein. This result is encouraging, as it may provide a way to replace, by standard breeding or by TILLING-assisted mutagenesis, the offending seed storage protein with one that is less allergenic.

Transgenesis vs breeding – one case study

One mantra that is repeated by anti-GMO advocates is that genetic engineering – the process of introducing foreign genes into a plant genome – has the potential to dramatically alter gene expression in the transgenic plant, and thus may lead to unanticipated and harmful changes in the plant.  The response from the scientist is that traditional breeding is logically expected to lead to much larger genome-wide changes, and that we actually will have less control over these.  With the advent of genome-wide transcription assays, it is now possible to test this directly (as opposed to trying to reason through the matter.  An example of such a study is that recently published by Baudo et al.  The bottom line of this study – gene expression is much less affected by the insertion of a transgene than by the process of traditional crop breeding.  The abstract of the study is after the fold.  As always, enjoy.

Some numbers

From a recent issue of Biotechniques, we read the following (by Douglas McCormick):

“Washington, DC, Aug 30—If elected president of the U.S., Sen. Barack Obama (D-IL) would double government funding for the National Institutes of Health and other research agencies over the next decade. Obama repeated that pledge (first made during his primary campaign) in his answers to ScienceDebate2008’s “top 14 science questions facing America.””

At first glance, this sounds like another politician throwing money at some special interests*.  But a cursory glance at some numbers suggests something else:

• 1. NIH budget for 2008 – ca. $29 billion (from the NIH web site)
• 2. Receipts of the top ten pharmaceutical companies based in the US in 2006 – $230 billion (give or take, according to Wikipedia)
• 3. Total employees of the top ten pharmaceutical companies based in the US in 2006 – 700,000 (again, according to Wikipedia)
• 4. Receipts of the top ten biotechnology firms based in the US in 2006 – about $40 billion (according to Wikipedia)
• 5. Total employees of the top ten biotech firms based in the US in 2006 – about 60,000 (again, according to Wikipedia)

Granted that these are rough figures, that this is not a rigorous scientific or statistical analysis, and that there are other factors that alter the simple equation I make in the following.  But I think that these numbers are important to listen to in this upcoming election year.

A liturgy that rolls off the tongues of candidates of both parties is that government spending is wasteful, hence the rush on both sides to pledge this and that sort of spending cut (the exact nature of which depends on your political persuasion). What we also hear about is the treasured programs that the respective sides will continue to pour money into; again, the specifics depend on your redness or blueness. But what the voting public rarely hears is an honest accounting of the returns that each party’s favorite spending actually gives. I would argue that basic biomedical research (and indeed, all basic scientific research) yields excellent returns on federal investment, and offer the above numbers in support of this idea. The two industries I mention here – pharma and biotech – are intimately interwoven with the basic biomedical research enterprise, and a significant amount of the innovation that drives these industries originates (or originated) in the NIH-funded biomedical research laboratory. In this respect, the NIH budget is an investment, and a wildly-successful one. Even if we don’t take the face-value numbers I have pulled from Wiki here (that show an annual return of some 1000%, and more than 750,000 high-paying jobs the tax receipts from which would probably pay much of the NIH tab by themselves), and instead factor in that some of these receipts and jobs are not American, it is still easy to see that basic biomedical research returns considerably more than the investment made by the government. (And this doesn’t begin to weigh the intangibles, the ways that the research enterprise gives back to society as a whole.)

Is there room for growth in the many areas that are impacted by basic life sciences research? I would argue, most definitely. And I would argue that the rate of return of investment by the government, hinted at by the above, should be the same. Think about it – multiply by a factor of four the support for biomedical research, and we may add $1 trillion to our economy. (OK, OK, I know I’m using the most optimistic interpretation of the above, but even missing by a factor of two still translates into a huge shot in the arm.) If our government is going to be spending on something, an enterprise such as this would seem to be an excellent option.

So, as the campaign rhetoric becomes more heated and disconnected from reality, remember these thoughts, and ask yourselves if your candidate has the wherewithal to both admit that some government spending can actually have a positive rate of return, and to commit to some programs that will actually help the government’s and the nation’s balance sheets.

*Important disclaimer – much of my research is, and has been, supported by federal research grants.