Patent Docs

Coelacanth Genome Sequence Determined

By Kevin E. Noonan --

The coelacanth, an aquatic animal described as a "living fossil" when discovered in 1938, was thought to have gone extinct during late Cretaceous period, ~70 million years ago.  Only about 300 specimens of the African coelacanth, Latimeria chalumnae, are known to exist and a second species, Latimeria menadoensis, was discovered in 1997.  These animals are morphologically primitive, resembling fossils dating ~300 million years ago, which has suggested that these are "slowly evolving" species (although this is more a descriptive than an informed characterization).  There have been sporadic reports of coelacanth gene sequencing over the past decade, but last week Nature published the first report on whole genome sequencing (Ameniya et al., "The African coelacanth genome provides insighted into tetrapod evolution," Nature 496: 311-16, April 18, 2013).

The article reports that the coelacanth genome comprises 2.68 gigabases, on 48 chromosomes, with 19,033 protein-coding genes comprising 21,817 transcripts, 2,894 "short" non-coding RNAs, and 1,214 "long" non-coding RNAs.  336 of protein-encoding genes were found to have undergone gene duplication.  The authors examined transcripts from 251 genes from coelacanth and compared these genes with Protopterus annectens (the African lungfish) expressed in brain, gonad, and kidney and with 15 terrestrial vertebrate lineages (dog, human, mouse, elephant, armadillo, Tammar wallaby, opossum, platypus, chicken, turkey, zebra finch, lizard, Western clawed frog, Chinese brown frog) and five modern fish species (tilapia, pufferfish, zebra fish, spotted catshark, little skate, and elephant shark).  The results of these analysis, comprising 100,583 concatenated amino acid positions, was consistent with the lungfish being the earliest relative of modern tetrapods (four-limbed animals), answering a previously unresolved uncertainty as to the role of the coelacanth in vertebrate evolution.

Turning to the question of the status of the coelacanth as a "living fossil," the authors confirmed earlier conclusions (based on Hox genes and protocadherins) that this species evolves slowly, the authors assessed the data from the 251 genes used in their phylogenetic analyses.  This was done as follows:

Pair-wise distances between taxa were calculated from the branch lengths of the tree using the two-cluster test proposed previously to test for equality of average substitution rates.  Then, for each of the following species and species clusters (coelacanth, lungfish, chicken and mammals), we ascertained their respective mean distance to an outgroup consisting of three cartilaginous fishes (elephant shark, little skate and spotted catshark).  Finally, we tested whether there was any significant difference in the distance to the outgroup of cartilaginous fish for every pair of species and species clusters, using a Z statistic.

The coelacanth genes showed 0.89 substitutions per site, compared with 1.05 substitutions/site in the lungfish, 1.09 substitutions/site in chicken, and 1.21 substitutions/site in mammals.

The authors also ascertained the "abundance" of transposable elements in the coelacanth genome, because these elements are believed to provide "templates for exaptation," i.e., to facilitate formation of novel protein exons and regulatory elements, as well as providing targets for genomic rearrangement.  Transposable element content was "high" (~25%, which the authors consider an underestimate) and also showed a "wide variety of transposable-element superfamilies," with 14 such families being "currently active."  The authors acknowledge that these results "contrast[] with the slow protein evolution observed."

"[E]xtensive conservation of synteny" was observed in a comparison of chromosomal breakpoints in coelacanth and tetrapod genomes, and "indicate that large-scale rearrangements have occurred at a generally low rate in the coelacanth lineage."  Interchromosomal rearrangements indicated that "karyotypic evolution in the coelacanth lineage has occurred at a relatively slow rate, similar to that of non-mammalian tetrapods" (31 in coelacanth, 20 in salamander and 21 in Xenopus species).  Comparison of the two coelacanth species, L. chalumnae (Africa) and L. menadoensis (Indonesia), showed divergence rates similar to those found between humans and chimpanzees, and the authors estimated that these species diverged "slightly more than" 6-8 million years ago, based on the slower substitution rates found in coelacanth species.

The authors then looked at estimates of how vertebrates adapted to the terrestrial environment.  They identified 50 genes found in coelacanth but not terrestrial tetrapods, presumably because these genes were not needed when the animal left water for land.  These genes included "components of fibroblast growth factor (FGF) signalling, TGF-β and bone morphogenic protein (BMP) signalling, and WNT signalling pathways, as well as many transcription factor genes," and specifically that 4 genes (And1, And2, Fgf24 and Asip2) not present in tetrapod genomes were indeed present in the coelacanth genome.  These genes also included 13 genes involved in fin development, 8 genes in otolith and ear development, 7 genes for kidney development, 13 genes for eye development, and 23 genes for brain development.  In contrast, there were only slight differences in homeobox genes.  There were also changes in gene regulation, wherein 6% of "conserved non-coding elements (CNEs)" ("promoters, enhancers, repressors and insulators") had originated after divergence of the coelacanth from the ancestral lineage.  Further analysis showed that tetrapod-specific CNEs were most closely (genetically) linked to genes involved in smell perception (consistent with the recognized expansion of olfactory receptor genes in the evolution of tetrapods from teleost fishes) and "morphogenesis (radial pattern formation, hind limb morphogenesis, kidney morphogenesis) and cell differentiation (endothelial cell fate commitment, epithelial cell fate commitment)" and immunoglobulin VDJ recombination.

A particular set of genes compared in the study are genes for digits, "[a] major innovation in tetrapod evolution," and specifically Hox genes for regulating limb development in ray-finned fish, coelacanths and tetrapods (mouse).  Three of the six "cis-regulatory elements" showed sequence conservation limited to tetrapods, with one element being shared by tetrapods and coelacanth but not the ray-finned fish; this latter element could function in transgenic mouse assays to "drive reporter gene expression in a limb-specific pattern."  Another particular set of genes compared between tetrapod and coelacanth lineages were genes for the urea cycle, because "[e]xcretion of nitrogen is a major physiological challenge for terrestrial vertebrates."  Urea cycle genes involved in producing urea for nitrogenous waste disposal (such as carbamoyl phosphate synthase I) showed strong evidence of selection whereas genes (such as mitochondrial arginase) involved in arginine metabolism but not excretion did not show such selection.  The authors conclude that this is evidence of adaptive evolution in the transition from water to land.  Hox gene studies also indicated changes from the coelacanth and tetrapod lineages implicated in placental and other reproductive structures not found in animals living in an aquatic environment.  Finally, the coelacanth genome lacks genes for immunoglobuins of the M class but did possess two IgW genes previously found only in lungfish and certain cartilaginous fish.

In addition to establishing that lungfish not the coelacanth was the common ancestor to all terrestrial vertebrate, the authors also established that the coelacanth also showed a slow rate of evolutionary change, which they speculate might be due to "a static habitat and lack of predation" and promising that "[f]urther study of these changes between tetrapods and the coelacanth may provide important insights into how a complex organism like a vertebrate can markedly change its way of life."

The authors were affiliated with the following institutions:  The Broad Institute at MIT; Benaroya Research Institute and University of Washington;  University of Konstanz, Germany; Universite de Montreal; University of Oregon; Institute of Molecular and Cell Biology, Singapore; Universidade Federal do Para, Brazil; Harvard University; University of Utah; Ecole Normale Superieure de Lyon; University of Kentucky; Rhodes University, South Africa; Wellcome Trust Sanger Institute; University of Trieste; University of Liege; Victoria University, Australia; University of Tuscia; University of Hamburg; Polytechnic University of Marche, Italy; University of South Florida; University of Western Cape, South Africa; Woods Hole Oceanographic Institution; Oxford University; Universitat Leipzig; Keio University, Japan; The Graduate University for Advanced Studies, Japan; European Molecular Biology Laboratory; University of Wuerzburg, Germany; University of Illinois at Chicago; National Institute of Genetics, Japan; and Uppsala University.

Image of Latimeria chalumnae (above) by Alberto Fernandez Fernandez, from the Wikipedia Commons under the Creative Commons Attribution 3.0 Unported license.

White House Unveils BRAIN Initiative

By Donald Zuhn --

Last week, President Obama announced a new research initiative designed to advance our understanding of the human brain.  It is hoped that the new initiative, dubbed the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative, will lead to new methods for treating, curing, and preventing brain disorders such as Alzheimer's disease, Parkinson's disease, autism, epilepsy, schizophrenia, depression, and traumatic brain injury.  The initiative, one of the Administration's "Grand Challenges," aims to produce a dynamic picture of the brain that will show how individual cells and complex neural circuits interact in both time and space, thereby providing opportunities for exploring exactly how the brain enables the human body to record, process, utilize, store, and retrieve vast quantities of information.

In announcing the new initiative, the Administration noted that despite recent advances in neuroscience, the underlying causes of most neurological and psychiatric conditions remain largely unknown, due to the vast complexity of the human brain.  According to the White House release, significant breakthroughs in the treatment of neurological and psychiatric disease will require a new generation of tools that enable researchers to record signals from brain cells in much greater numbers and at even faster speeds.

The new initiative is still in the planning process, however, with a working group of the Advisory Committee to the NIH Director having been formed to articulate the scientific goals of the BRAIN initiative and develop a multi-year scientific plan for achieving those goals.  The working group will produce an interim report by the fall of this year that will contain specific recommendations on high priority investments for FY 2014, with a final report to be delivered to the NIH Director in June 2014.

Beginning in FY 2014, the National Institutes of Health (NIH), Defense Advanced Research Projects Agency (DARPA), and National Science Foundation (NSF) will provide some $100 million in funding to support the initiative.  DARPA's role in the initiative will be to develop a new set of tools to capture and process dynamic neural and synaptic activities, while the NSF will support research spanning biology, the physical sciences, engineering, computer science, and the social and behavioral sciences, and in particular, will develop molecular-scale probes that can sense and record the activity of neural networks, work on advances in "Big Data" that will be required to analyze the huge amounts of information that will be generated, and aid in the understanding of how thoughts, emotions, actions, and memories are represented in the brain.  Private foundations, including the Howard Hughes Medical Institute, the Allen Institute for Brain Science, The Kavli Foundation, and the Salk Institute for Biological Studies have also made commitments to support the new initiative.

In seeking additional support for the BRAIN initiative, the Administration noted that a previous Grand Challenge, the Human Genome Project, demonstrated that ambitious research projects can have a significant impact on the country's economy.  With respect to the Human Genome Project, for example, the Federal Government invested $3.8 billion in that initiative between 1988 and 2003, producing an economic output of $796 billion, or a return of $14 for every $1 invested (see "Report Gauges Economic Impact of Human Genome Project").

Legislation Introduced to Develop Public Access Policies for Federal Research

By Donald Zuhn --

Last month, companion bills were introduced in the House and Senate that would require Federal agencies to develop public access policies relating to research conducted by employees of the agency or using funds administered by the agency.  The Fair Access to Science and Technology Research Act of 2013 ("FASTR") was introduced in the House as H.R. 708 by Rep. Michael Doyle (D-PA), and in the Senate as S. 350 by Sen. John Cornyn (R-TX).

The legislation begins by listing the following findings of Congress:

(1) the Federal Government funds basic and applied research with the expectation that new ideas and discoveries that result from the research, if shared and effectively disseminated, will advance science and improve the lives and welfare of people of the United States and around the world;

(2) the Internet makes it possible for this information to be promptly available to every scientist, physician, educator, and citizen at home, in school, or in a library; and

(3) the United States has a substantial interest in maximizing the impact and utility of the research it funds by enabling a wide range of reuses of the peer-reviewed literature that reports the results of such research, including by enabling computational analysis by state-of-the-art technologies.

In view of these findings, the legislation would require Federal agencies with research expenditures of more than $100,000,000 "to develop a Federal research public access policy that is consistent with and advances the purposes of the Federal agency" within one year of the Act's enactment.  Public access policies developed pursuant to the Act would have to provide for the submission to the Federal agency of manuscripts accepted for publication in peer-reviewed journals that result from research that has been supported by Federal funding.  Submitted manuscripts would then be made available online to the public no later than six months after publication.

Under the Act, the public access policies would apply to researchers employed by the Federal agency whose works remain in the public domain and researchers funded by the Federal agency.  The Act, however, would exclude the following from the public access policies developed under the legislation (emphasis added):

(1) research progress reports presented at professional meetings or conferences;

(2) laboratory notes, preliminary data analyses, notes of the author, phone logs, or other information used to produce final manuscripts;

(3) classified research, research resulting in works that generate revenue or royalties for authors (such as books) or patentable discoveries, to the extent necessary to protect a copyright or patent; or

(4) authors who do not submit their work to a journal or works that are rejected by journals.

The Act also indicates that each Federal agency required to develop a public access policy would have to submit a report on the policy to the Committee on Homeland Security and Governmental Affairs of the Senate; Committee on Oversight and Government Reform of the House of Representatives; Committee on Science and Technology of the House of Representatives; Committee on Commerce, Science, and Transportation of the Senate; Committee on Health, Education, Labor, and Pensions of the Senate; and any other committee of Congress of appropriate jurisdiction.

The House bill, which was co-sponsored by Rep. Zoe Lofgren (D-CA) and Rep. Kevin Yoder (R-KS), was referred to the to the House Committee on Oversight and Government Reform, and the Senate bill, which was co-sponsored by Sen. Ron Wyden (D-OR), was referred to the Senate Committee on Homeland Security and Governmental Affairs.

In a press release issued by his office, Rep. Doyle stated that the legislation would "give the American people greater access to the important scientific research results they’ve paid for," adding that "[s]upporting greater collaboration among researchers in the sciences will accelerate scientific innovation and discovery, while giving the public a greater return on their scientific investment."  He also indicated that "taxpayers should not be required to pay twice for federally-funded research."  According to the press release, FASTR builds on the successful implementation by the National Institutes of Health (NIH) of a public access policy in 2008.

Supreme Court Denies Certiorari in Stem Cell Funding Case

By Donald Zuhn --

In an order issued earlier today, the Supreme Court denied certiorari in Sherley v. Sebelius, ending the efforts by two adult stem cell researchers to prevent the National Institutes of Health (NIH) from funding research using human embryonic stem cells (ESCs).  The dispute arose following the NIH's release in July 2009 of new guidelines governing the requirements for federal funding of research on human ESCs (see "NIH Releases New Stem Cell Rules").  The NIH's guidelines replaced rules promulgated during the Bush Administration that limited the number of funding-eligible human stem cell lines to 69.  Under the NIH's new guidelines, which were developed in response to an Executive Order issued by President Obama in March 2009 (see "President Obama to Lift Stem Cell Limits on Monday"), human ESCs that are listed on the NIH's stem cell registry (established pursuant to the guidelines) are eligible for funding.  The guidelines permitted human ESCs that were created using in vitro fertilization for reproductive purposes, but which we no longer needed for this purpose, to be listed on the registry.

In response to the NIH's promulgation of the stem cell guidelines, Dr. James Sherley and Dr. Theresa Deisher, two researchers who only use adult stem cells in their research, filed suit, along with several other plaintiffs, to enjoin the NIH from funding research using human ESCs.  In particular, the Appellees contended that by funding research projects using ESCs, the NIH violated the Dickey-Wicker Amendment, which Congress has included in the annual appropriation for the Department of Health and Human Services each year since 1996.  The Dickey-Wicker Amendment prohibits the NIH from funding:

(1) the creation of a human embryo or embryos for research purposes; or (2) research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero under 45 C.F.R. 46.204(b) and section 498(b) of the Public Health Service Act (42 U.S.C. 289g(b)).

The District Court for the District of Columbia granted the Government's motion to dismiss the suit for lack of standing, the plaintiffs appealed, and the U.S. Court of Appeals for the District of Columbia Circuit remanded, finding that Drs. Sherley and Deisher had standing because they competed with human ESC researchers for NIH funding.  On remand, the District Court granted Appellees' motion for a preliminary injunction, concluding that they were likely to succeed in showing that the NIH's guidelines violated the Dickey-Wicker Amendment.  On appeal, the U.S. Court of Appeals for the District of Columbia Circuit vacated the preliminary injunction, finding that the Appellees were unlikely to prevail because the Dickey-Wicker Amendment is ambiguous and the NIH reasonably concluded that, although the Dickey-Wicker Amendment bars funding for the destructive act of deriving a human ESC from an embryo, it does not prohibit funding a research project in which a human ESC will be used.

Spread of Laboratory-generated Antibiotic Drug Resistance in Chinese Rivers

By Kevin E. Noonan --

A generation ago, Jeremy Rifkin famously convinced the Cambridge city council to ban genetic engineering in that city, using the fear of "tinkering" with nature and producing a "superbug" that would hurt the public health and thereby (for a time) reduced progress in biological research at Harvard and elsewhere in the city environs.  Mr. Rifkin was not alone in his concern over potential problems from recombinant DNA; indeed, the scientist who first produced a recombinant plasmid, Paul Berg (Jackson et al., 1972, "Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli," Proc. Natl. Acad. Sci. U.S.A. 69(10): 2904-09), was instrumental in organizing a conference in 1975 on the issue at Asilomar, California, which resulted in guidelines (both physical and biological) from the National Institutes of Health for the safe practice of the new technology (Guidelines for research involving recombinant DNA molecules," 41 Fed. Reg. 27911-43 (1976)).  But in the intervening decades, widespread distribution of laboratory-derived recombinant DNA introduced into the environment has not been reported as a threat to humans or the environment.

Until now, that is.  In a report in the journal Environmental Science and Technology entitled "A Survey or Drug Resistance bla genes Originating from Synthetic Plasmid Vectors in Six Chinese Rivers," scientists from Sichuan University and the Chinese Institute of Health and Environmental Medicine report that drug resistance genes (specifically, the beta-lactamase (bla) gene that provides resistance to ampicillin) have been detected in the Pearl, Yangtze, Yellow, Hai He, Sunggari, and Huangpu rivers in mainland China (Chen et al., 2012, Environ. Sci. Technol. 46(24): 13448–54).  These rivers drain the majority of the Chinese mainland in all provinces and have the cities of Guangzhou, Nanjing, Ji'nan, Tianjin, Harbin, and Shanghai, respectively, along their banks.  The authors took samples from these rivers containing naturally occurring bacteria and assayed using polymerase chain reaction (PCR) and a variant, quantitative real-time PCR, to assess the frequency with which beta-lactamase DNA could be detected.  The assay was specific for the bla gene that comprises most recombinant plasmid strains, such as pBR322 (one of the oldest and most widespread plasmids used for molecular biological research) as well as pUC19.  PCR was performed using three pairs of "universal" primers for the bla gene as well as sequencing primers used for detecting exogenous DNA in cloning sites in the plasmid vectors.  In addition, the bacterial isolates obtained from the river samples were assayed for antibiotic resistance using assays recommended by the U.S. Clinical Laboratory Standards Institute.

Plasmid-derived bla-encoding DNA was detected in all six rivers assayed, detected using both the "universal" primers as well as the plasmid sequencing primers (the latter results further supporting the conclusion that the samples were of laboratory origin).  The detection rate varied from 21.9% (in the Hai He River samples) to 36.4% (in the Yangtze River samples), and the total copy number of bla copies in the samples ranged from 6.7 +/- 5.0 x 10¹ copies/mL (in the Yellow River samples) to 4.6 +/- 0.6 x 10³ copies/mL (in the Pearl River samples).  The Pearl and Hai He rivers showed the widest spectrum of cephalosporin resistance from the bla gene present in bacterial samples, extending to 3rd- and 4th-generation drugs like cefotaxime and cefoperazone, while the spectrum was narrower (e.g., cefalotin, cephazolin, cefmetazole, and cefoxitin) in samples from the other rivers tested.  Sequence analysis confirmed that sequences "neighboring" the bla sequences detected in the river samples "most frequently represented artificial or synthetic constructs, including cloning, expression, shuttle, gene-fusion, and gene trap vectors" derived from recombinant laboratory plasmid vectors, most strongly with pBR322.

Abstract, Chen et al., 2012.

An interesting ancillary result reported in the paper was that tetracycline resistance-conferring DNA was also detected in samples from all six rivers, and some river samples also showed genes that provided resistance to other commonly used laboratory antibiotic selection markers such as gentamicin.  Detection of gene sequences encoding tetracycline resistance is consistent with detection of pBR322, which was engineered to contain a tetracycline resistance gene as well as the bla gene.  The authors also report that detection of high levels of laboratory plasmid-derived antibiotic resistance genes in the Pearl River was consistent with the levels of antibiotic pollution know to exist in that river from human and animal sources.

This report raises serious questions, of course, over the consequences of widespread use of antibiotic resistance-encoding genetic elements.  It is particularly disturbing because genetic engineering is not limited to the lab anymore, being used in such technologies as biofuels, agriculture, and bioremediation.  The risk of antibiotic resistance gene contamination of the environment has been recognized previously and steps taken to minimize it; for example, Monsanto has developed vectors encoding other markers such as lacZY for use in its recombinant seed.  These efforts take on greater significance, and relevance for human and animal well-being, in view of this latest report.

BIO and ASTA Announce That GEMAA Is Now Effective

By Donald Zuhn --

The Biotechnology Industry Organization (BIO) and American Seed Trade Association (ASTA) announced today that the Generic Event Marketability and Access Agreement (GEMAA) has received four signatories, and as a result is now effective.  The GEMAA is one of two agreements -- the Data Use and Compensation Agreement (DUCA) being the other -- that form the Accord, a private-sector driven framework for dealing with the transition of regulatory and stewardship responsibilities for genetically modified seed technology (i.e., seed varieties containing "biotechnology events"), after patent expiration.  The GEMAA, which was launched late last month, has thus far been signed by five agricultural biotech companies:  BASF Plant Science, Bayer Crop Science, Dow Agro Sciences, DuPont Pioneer, and Monsanto Company.

According to BIO's statement on the GEMAA taking effect, the agreement "will provide confidence to growers, breeders and grain handlers in the use of generic biotechnology events [and] benefit the entire agriculture value chain by ensuring the maintenance of foreign regulatory authorizations and stewardship obligations following the expiration of patents for biotechnology events utilized in seed varieties."  The Accord website (www.AgAccord.org) notes that nearly 90% of cotton, corn, and soybean acreage in the U.S. is planted with seed varieties containing biotechnology events, and that grain from these crops accounts for more than $40 billion in global trading annually, making the U.S. the largest global producer and exporter of crops and grain derived from biotechnology.  With the first genetically modified seed patents set to go off patent by 2015, such patent expirations present challenges such as the maintenance of global regulatory authorizations for biotechnology events and associated stewardship obligations so that farmers can continue to cultivate seed varieties containing off-patent events without jeopardizing U.S. export markets.

Under the GEMAA, companies that have developed proprietary regulatory information to support the global authorizations for biotechnology events (known as Proprietary Regulatory Property (PRP) Holders under the agreement) will provide notice of patent expiration three years before the last patent on the biotechnology event expires, provide access to the biotechnology event at patent expiration, and opt for one of three choices with respect to regulatory responsibility.  Those choices include Independently maintaining regulatory responsibility for the event at no cost to users of the generic event for at least four years after the last sale of the product, seeking to share regulatory responsibility, or discontinuing regulatory responsibility (If no interest is expressed by other signatories).

The GEMAA will be overseen by a Committee of Signatories that will be formed, and will hold its first meeting, within three months.

Additional information regarding the Accord can be found here.

Ballot Initiative on Genetically Engineered Food Labeling Defeated in California

By Donald Zuhn --

On Tuesday, while California voters were helping President Obama secure a second term in the White House, picking the President over Republican challenger Mitt Romney by a 59.1% to 38.6% margin, they were also responsible for defeating a ballot initiative, Proposition 37, that would have required raw or processed food to be labeled if such food was made from plants or animals having genetic material changed in specified ways.  The initiative measure, entitled "The California Right to Know Genetically Engineered Food Act," would have amended the California Health and Safety Code such that "any food offered for retail sale in California [would be considered to be] misbranded if it is or may have been entirely or partially produced with genetic engineering and that fact is not disclosed."  The margin of defeat for Proposition 37 was somewhat narrower than that in the Presidential race, with voters rejecting the ballot initiative 53.7% to 46.3%.

According to Proposition 37, "genetically engineered" food was defined as:

[A]ny food that is produced from an organism or organisms in which the genetic material has been changed through the application of:
    (A) In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) techniques and the direct injection of nucleic acid into cells or organelles, or
    (B) Fusion of cells, including protoplast fusion, or hybridization techniques that overcome natural physiological, reproductive, or recombination barriers, where the donor cells/protoplasts do not fall within the same taxonomic family, in a way that does not occur by natural multiplication or natural recombination.

Under the Act, raw agricultural food that was genetically engineered would have to be labeled as "Genetically Engineered," and processed food ("any food produced from a raw agricultural commodity that has been subject to processing such as canning, smoking, pressing, cooking, freezing, dehydration, fermentation, or milling") would have to be labeled as "Partially Produced with Genetic Engineering."

At the California Secretary of State website, arguments in favor of and against the ballot initiative were presented.  In addition, voters were directed by the website to the groups California Right to Know (in favor of the initiative) and NO on 37 (against the initiative) for additional information.

Interestingly, the Ballotpedia webpage for Proposition 37 listed the California Democratic Party and the Green Party of California as supporters of the failed ballot initiative and the California Republican Party as being opposed to the initiative.  Donors to the "No on 37" campaign included Monsanto, E.I. Dupont De Nemours & Co., DOW Agrisciences, Bayer Cropscience, BASF Plant Science, Syngenta Corporation, and the Biotechnology Industry Organization (BIO).

According to a report in the San Jose Mercury News, proponents of the initiative, including California Right to Know, will now turn their efforts towards getting similar initiatives on the ballot in Washington and Oregon.

A Genetic Basis for Müllerian Mimicry in Butterflies

By Kevin E. Noonan --

Mimicry is a classic (and classical) biological phenomenon, the appreciation of which dates to the time when biology was more accurately called "natural history" and was more diversion for English country gentlemen than proper science.  Perhaps the most well-known form of mimicry is Müllerian mimicry, and the best example of that is the viceroy butterfly (Limenitis archippus), which although tasty to avian predators displays markings almost indistiguishable from the monarch butterfly (Danaus plexippus), which is not only unpalatable but actually toxic to birds (see below).  Anyone with even a rudimentary appreciation of Linnean classification will recognize another interesting feature of mimicry:  despite their visual similarities, the monarch and viceroy butterflies are not only different species but members of different genera, suggesting that genetic inheritance cannot account for the convergence in wing pattern phenotype.

Recent work employing genomic sequencing and linkage analysis (from the Heliconius Genome Project) has begun to shed light on the genetic bases for Müllerian mimicry.  This work was performed on a South American butterfly species, Heliconius melpomene (at right).  As reported in Nature (doi:10.1038/nature11041), naturally occurring species of Heliconius show significant introgression in regions related to color pattern development.  Heliconius butterflies comprise 43 species and hundreds of races and make up rapidly radiating butterfly species in South America; certain of these species exhibit classic Müllerian mimicry, wherein palatable species adopt the color pattern of other species that are unpalatable to bird predators.  The authors sequenced almost 13,000 genes (12,669 open reading frames) in the 269 megabase (Mb) butterfly genomic DNA and mapped the majority (~83%) of these genes to the 21 Heliconius chromosomes using a modern version of RFLP analysis termed restriction site DNA linkage mapping.  This work found that the chromosomal organization of Heliconius butterfly genomic DNA was "broadly conserved" since the evolutionary split from a common ancestor shared with Bombyx (silkmoth) species in the Cretaceous (~100 million years ago).  Comparison with the Bombyx mori (silkmoth) genome identified 6,010 orthologues and previously unidentified chromosomal fusions in the Heliconius chromosomal complement.  Biologically important expansion was found in families of genes related to chemosensation and body shape and form (homeobox, or Hox, genes).  The Heliconius genome showed an expected increase in ultraviolet opsins (due to the diurnal lifestyle adopted by butterflies) and an unexpected genetic complexity in olfactory genes (33 Heliconius olfaction genes, similar to what was found in monarch butterfly species having 34 olfactory genes).  The researchers also found additional Hox genes related to body shape and form, similar to what is found in other butterflies and related dipteran species.

The researchers compared genomic DNA from three co-mimicry species, Heliconius melpomene, Heliconius timareta, and Heliconius elevatus and produced a genetic tree between 84 individuals from H. melpomene and related species (see below).  In this comparison, the researchers found hybrid exchange especially at two genomic regions that control mimicry pattern based on ~4% (12Mb) of the genome.  The results obtained from Heliconius melpomene amaryllis and H. timareta ssp. were compared and the researchers report evidence of greater hybrid interbreeding between these species when compared with H. melpomene aglaope and H. timareta ssp, which do not have overlapping spatial ranges.  The researchers estimated 2-5% exchange of the genome; the introgression was not uniformly distributed over the 21 chromosomes but seems to be limited to 11 chromosomes with two chromosomes showing the strongest frequency of introgression.  These regions, comprising loci known to be associated with red pattern (B/D) and yellow (N/Yb) pattern loci, showed blocks of sequence (~100 kb in size) that are shared between species and exchanged in hybrids.  The genetic makeup of these shared blocks of sequence were more consistent with hybrid introgression (and fixation) than in other alternatives, such as convergent evolution.

The researchers concluded that these species exchange protective color pattern genes promiscuously and that their evidence of hybrid exchange and fixation indicates that this mode of gene transfer between diverse species may be a more important mechanism for explaining phenomena like mimicry than had been previously appreciated.  The authors propose that hybridization between mimicking species is a plausible explanation for "parallel evolution of multiple mimetic patterns" in these species.

Supplemental experimental details can be found here.

Image of Heliconius melpomene by Greg Hume, from the Wikipedia Commons under the Creative Commons Attribution-ShareAlike 3.0 Unported license.

Genetic Marker Map of Sunflower Species Reported

By Kevin E. Noonan --

Long before DNA sequencing technology existed (indeed, long before Watson and Crick proposed that DNA was the genetic material and proposed a structural basis for its ability to be replicated), scientists were able to study genome structure using strictly genetic approaches.  Genetic linkage maps, for example, date from the work of Thomas Hunt Morgan and Hermann Mueller on fruit flies at Columbia University and later at Cal Tech.  Traditionally, these maps have been generated using phenotypic markers, much like Mendel did with his pea plants (in relative obscurity) a generation before.

Marker density is a limitation using traditional methods, however.  But an improvement in this aspect of the methodology was achieved in the 1980's with the advent of restriction fragment length polymorphism (RFLP) methods.  These methods used physical changes in DNA sequence (specifically at restriction enzyme recognition sites) to greatly expand the scope of linkage analysis.  This is because the "phenotype" of a RFLP is a polymorphism that can be identified using electrophorectic separation of fragments having different lengths; unlike traditional phenotypes, unless the RFLP happens to be associated with an amino acid change in a protein it is unlikely to be subject to natural selection, and thus will fall within the "neutral" sequence variation that has been appreciated to exist in natural populations since the work of Motoo Kimura in the 1960's.  The RFLP method was used to great effectiveness by David Botstein, Ray White, Mark Skolnick, and Ronald Davis in identifying the chromosomal location of genes associated with, inter alia, Huntington's disease, and notably by Mary Claire King in identifying the locus of the BRCA1 gene and in forensic studies used to identify Argentines "disappeared" by the military dictatorship during the co-called "dirty war."

Since the completion of the Human Genome Project, the existence of single nucleotide polymorphisms (SNPs), which are RFLPs without being limited to restriction enzyme recognition sites, has expanded the extent and number of generic markers useful for genome mapping.  These SNPs have been used to construct a high-density linkage map for the sunflower (Helianthus annus) in a paper by John E. Bowers, Eleni Bachlava, Robert L. Brunick, Loren H. Rieseberg, Steven J. Knapp and John M. Burke from the Department of Plant Biology and Center for Applied Genetic Technologies, University of Georgia, the Department of Crop and Soil Science, Oregon State University, the Department of Botany, University of British Columbia, and the Department of Biology, Indiana University.  The study, "Development of a 10,000 Locus Genetic Map of the Sunflower Genome Based on Multiple Crosses" was reported in the online journal Genes/Genomes/Genetics (G3), 2: 721-729 (July 1, 2012).  These researchers identified 10,080 polymorphic genetic loci in the approximately 3.5 Gbp sunflower genome using a high throughput SNP genotyping array in developing their map, but also noted areas of the sunflower genome (up to 26 centiMorgans in size) having no markers that could be detected in individual crosses, the result, the authors speculate, of genetic identity between individuals in the four populations studied.  However, none of these regions were in common in the four populations as a whole, permitting a "gapless" map of the sunflower genome to be elucidated.

Interestingly, the economic importance of sunflowers (being the fourth largest source of cultivated vegetable oils worldwide; www.fas.usda.gov) is such that earlier genomic mapping technologies have been applied (including RFLP maps; Berry et al. 1995, "Molecular marker analysis of Helianthus-annuus L. 2. Construction of an RFLP linkage map for cultivated sunflower," Theor. Appl. Genet. 91: 195–99; Gentzbittel et al. 1995, "Development of a consensus linkage RFLP map of cultivated sunflower (Helianthus-annuus L)," Theor. Appl. Genet. 90: 1079–108) and others.  These efforts, however, have been directed towards "traits of agronomic importance" as well as comparisons with genomic structure of related Helianthus species according to the authors.  But these traditional efforts suffered from the same limited marker density deficiencies noted earlier (although SNPs and other markers identified using these methodologies were integrated into the high-density map).  Compared with the marker density in these earlier studies (one marker per 2 Mbps) other species (Arabidopsis, rice, sorghum) were previously mapped at a density of ~160 bp per marker (i.e., a ~12,500-fold higher marker density).  These efforts were also hampered by the fact that no plant species closely related to sunflower (family Asteraceae) have been fully sequenced (the closest sequenced relative is the potato), although other related species (tomato, lettuce) are currently subject to genomic sequencing efforts.

Markers were analyzed from multiple genetic crosses between members of the four populations.  The study reports crosses between individuals from an oilseed maintainer line with high oleic acid content and an oilseed "restorer" line, while other crosses involved individuals from the oilseed maintainer line and a wild sunflower line; an oilseed restorer line and a confectionery restorer line; and an oilseed restorer line that segregates for nuclear male sterility and a non-oilseed landrace line.  From these four populations, four individual maps of ~3500-5500 marker loci were created, falling into 17 linkage groups which was consistent with the 17 chromosomes in the sunflower species (although crosses with the landrace species showed genetic heterogeneity that required additional analysis).  Analyses of genotyping errors showed that the "raw allele calls" made from the data were "highly robust" (i.e., having an error rate ranging from ~0.14-1%).  Turning to these results, the authors report that the maps were "largely collinear with an average of 88.7% of all shared loci being syntenic in pairwise combinations."  "Not surprisingly," according to the study, "the cross that showed the most and largest regions of reduced recombination was the only cross that involved wild sunflower," a phenomenon that had been previously seen in maize (McMullen et al. 2009, "Genetic properties of the maize nested association mapping population," Science 325: 737–40).  The study also reports that 762 of the 5694 mapped loci could be ascribed to two different chromosomal locations in different crosses (and 21 markers were found at three genomic locations), a phenomenon the authors suggest could be due to the existence of multicopy genes, wherein the mapped markers were the result of detecting the different paralogs that were located at different loci in the sunflower genome.  This corresponded to <14% of all detected SNPs being associated with multicopy genes.

The combined consensus map reported in this paper comprised 10,080 loci spanning 1380 cM.  The authors state that the existence of this SNP map represented "over 5 million molecular data points" made possible by using multiple mapping populations simultaneously and that can be used to help assemble a map from the "sunflower genome project" currently underway (Kane et al. 2011, "Progress towards a reference genome for sunflower," Botany-Botanique 89: 429–37).  The relevance and utility of this marker map for understanding and improving sunflower species, subspecies and economically important variants remains a task for the future.

Dolphin Genes Show Relationships between Large Brains and Energy Metabolism Similar to Humans and Elephants

By Kevin E. Noonan --

"Genome projects" for a variety of species (comprising a complete sequence determination of a species' genomic DNA) has been underway for the past dozen or so years, in the wake of the completion of the Human Genome Project at the turn of the century.  While such a sequence determination remains the "gold standard" for a full appreciation of the gene structure and complement of a species, partial genome sequences, focusing on an animal's protein-coding sequences can also be useful in deriving phylogenetic relationships and understanding how genes are associated with (if not directly responsible for) shared phenotypic characteristics.

Such a study was reported recently for the bottlenose dolphin, Tursiops truncates in a paper by Michael McGowen, Lawrence Grossman and Derek Wildman of the Center for Molecular Medicine and Genetics, Wayne State University School of Medicine (Proceedings of the Royal Society: Biological Sciences, 279: 3643-3651).   The paper,"Dolphin genome provides evidence for adaptive evolution of nervous system genes and a molecular rate slowdown," reports the results of a comparison of 10,025 protein-coding genes from bottlenose dolphins and orthologous genes from cow (Bovis taurus, the closest living mammalian species in the Order Cetartiodactyla), horse (Equus caballus), dog (Canis familiaris) (together, these four species comprising the laurasiatherian species), mouse (Mus muscalus), elephant (Loxodonta africana), opossum (Monodelphis domestica), platypus (Ornithorhynchus anatinus), chicken (Gallus gallus) and man.  The comparison was limited to coding sequences (CDS) ranging from 26335 bp (SYNE1) to 150 bp (C20orf196 and FDPS).

Dolphin genes were found to have the slowest rates of synchronous substitution between the laurasiatherian species and comparable to rates seen in elephants and humans.  The mean ratio of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site (dN/dS) were highest in dolphins of all laurasiatherian species, and again close to human rate ratios.  Within the dolphin genome, 228 genes were identified as having  (dN/dS) >1 (shown in Table 2), which was interpreted to indicate positive selection at levels (2.26%) significantly higher than seen in other species (horse, 48 or 0.51%; cow, 32 or 0.32%; dog, 11 or 0.12%).  Moreover, while at least 46% of genes in all other lineages studied were subject to "purifying selection" (genes having a (dN/dS) <0.1), only 36.4% (or 3646 dolphin genes) were found with this ratio, compared with 44-51% in other species.

Of the 228 dolphin genes (2.26% of total genes sequenced) under positive selection (having dN/dS > 1), 27 of these genes are related to the nervous system including genes homologous to human genes known to be involved in sleep, synaptic plasticity and, when defective, cognitive disability.  These include "[s]even genes [that] are identified as being involved in intellectual disabilities and/or microcephaly (ERCC8, AP4S1, MCPH1, TTR), schizophrenia (MAL) or Alzheimer's susceptibility (AGER, RNF182)[; f]ive genes [] involved in neuroendocrine function, neuropeptide hormonal activity, or function as hormones that affect the brain (AGRP, C2orf40, EDN2, NMU, TTR)[; ] genes [that] function in the development of myelin (MAL), neuronal or brain development (CNPY1, ZNF597, PCP4L1, MCPH1), neural potassium channel function (KCNK18), neurite growth (CD47, LRFN1,CNPY2) and synaptic function (BAALC, DBI, SYPL1, AP4S1, LRFN1)."  These results were consistent with morphological characteristics of dolphin brains, including "high level of gyrification (cortical folding), expansion of the insular and cingulate cortices, specialized von Economo neurons, high glia to neuron ratio increase in synapse number, reduction of the olfactory system and the large relative size of the cerebral cortex."  This suggests to the authors that a genetic analysis might uncover evidence of "convergent evolution" among large-brained mammals.  Further, consistent with the source, the authors report that genes "putatively related to cetacean adaptations" were also identified, including "the cardiovascular system (TSPO2, EPGN, PLN, EDN2, PLA2G5, KCNJ2), sperm function and spermatogenesis (TNP1, USP26,SPAG4, NANOS3, SPATA9, CDYL, SOX30, SPATA7, AKAP4, SPATA3, GTSF1), lung development and respiratory function (SCGB3A2, PLUNC, TMPRSS11D), dermal development and function (KRTDAP, SPINK5, SPINK6, IL20, PSORS1C2, DMKN), hair (KRT84), olfaction (OR6B1, OR2AK2), vision (CRYGN), milk (CSN2), glucose and/or glycerol metabolism (OSTN, SOCS6, AQP9), vitamin B-1 and B-12 binding and metabolism (TCN1, THTPA) and a multitude of genes involved in lipid transport and metabolism (APOA2, APOC4, APOO, FABP4, SERINC4, CCDC129, PLA2G5, PNLIPRP3, RARRES2, NR1I3)."

The study also examined metabolism-associated genes (a total of 548 genes), particularly the mitochondrial cellular (genomic) component genes (i.e., genes residing in the cellular chromosome whose gene products localize in the mitochondrion).  These genes are believed to be important for energy metabolism.  8.5% of these genes (compared with 1.5-5% for other species) were found to have (dN/dS) > 0.5, including the gene for cytochrome c (CYCS).  This is consistent with the increased energy needs of mammals with large brains, it being known that "basal metabolic rates [are correlated] with relative [larger] brain size" [Isler et al., 2006, "Metabolic costs of brain size evolution," Biol. Lett. 2, 557–560] as has been observed in humans [Grossman et al., 2004, "Accelerated evolution of the electron transport chain in anthropoid primates," Trends Genet. 20, 578–585].  These results were consistent with increased brain size, which like in humans is larger than expected when body size is considered.

The authors conclude that their data is consistent with a slower rate of mutation that other species (1.4 x 10^-9 substitutions/per site per year compared with an average mammalian mutation rate of 2.2 x 10^-9 substitutions/per site per year).  These results are also consistent with "adaptive evolution" of both the dolphin nervous system and brain, and although the authors have not established a definitive link between any of the brain-related genes they identified and "morphological or behavioral" traits in dolphins.  But they point out that they identified six genes (AP4S1encoding a membrane protein associated with AMPA glutamate receptors; SYPL1encoding a synaptic molecule differentially regulated during human development; LRFN1encoding a synaptic adhesion molecule; BAALC encoding a component of postsynaptic complexes; and DBI encoding a modulator of signal transduction at type A gamma-aminobutyric acid receptors with implications in sleep regulation) that are thought to be involved in synaptic function; similar genes "have been implicated in the evolution of the brain in humans" according to the report.

Genes involved in DNA repair and associated with intellectual disabilities in humans were also identified in the study, including "ERCC8 is a gene involved in transcription-coupled DNA damage repair; mutations in this gene result in Cockayne's syndrome, whose symptoms include microcephaly, neurological defects and premature ageing [Rapin et al., 2006, "Cockayne syndrome in adults: Review with clinical and pathological study of a new case," J. Child Neurol. 21, 991–1006].  ERCC8 shows evidence of positive selection in recent human populations [Voight et al., 2006, "A map of recent positive selection in the human genome," PLoS Biol. 4, e72].  MCPH1 is another gene with evidence of function in DNA damage repair as well as cell cycle regulation.  MCPH1 dysfunction can cause microcephaly and has been documented to be under positive selection along the human lineage."

Regarding genes involved in energy metabolism, the positive selection observed by these authors is consistent with an oxygenation rate of 6.0 mL kg/min, greater than in humans or elephants.  In this regard the paper makes the following interesting observation:

During the course of primate evolution, metabolism evolved to provide a constant supply of energy to the increasing demands of a larger brain.  [Leonard et al., 2003, "Metabolic correlates of hominid brain evolution," Comp. Biochem. Physiol. A 136, 5–15].  Humans have evolved several adaptations, including an increase in visceral fat and the evolution of tissue-specific insulin resistance in the brain that protects nervous tissue from energy shortage; however, malfunctions of this system in humans can cause type 2 diabetes (Kuzawa, 2010 Beyond feast-famine: brain evolution, human life history, and the metabolic syndrome. In Human evolutionary biology (ed. Muehlenbein M.), pp. 518–527. Cambridge, UK: Cambridge University Press).  Interestingly, recent research has proposed that dolphins should be seen as a model for type 2 diabetes, as they possess all the hallmarks of the disease [Venn-Watson et al., 2011, "Dolphins as animal models for type 2 diabetes: sustained, post-prandial hyperglycemia and hyperinsulinemia," Gen. Comp. Endocrinol. 170: 193–99)].  Here, we find genomic signatures of adaptive evolution in dolphin genes related to control of food intake such as genes involved in glycerol uptake and/or glucose metabolism (AQP9, OSTN, SOCS6), and a neuropeptide AGRP that is expressed in the hypothalamus and involved in increasing appetite, decreasing metabolism and regulating leptin. In addition, we found several genes under selection related to lipid transport and metabolism [] that may be associated with the large fat reserves found in cetaceans.

This paper is just one example of how elucidating genomic DNA structure can provide insights (including, as set forth above regarding the relationship between energy metabolism and diabetes, unexpected insights) into genotype/phenotype relationships in evolutionarily related species.  These results also indicate that the "golden age" of genomics is still, really, in its infancy.

Study Finds That Foreign-Born Inventors Play Prominent Role at Top U.S. Patent-Producing Universities

By Donald Zuhn --

A study commissioned by the Partnership for a New American Economy, a bipartisan group of mayors from across the country and business leaders from all sectors of the economy seeking to raise awareness of the economic benefits of sensible immigration reform, shows that 76% of patents issued in 2011 to the top 10 patent-generating universities in the U.S. named a foreign-born inventor.  The 30-page report, entitled "Patent Pending: How Immigrants Are Reinventing the American Economy," was released last month.

Partnership Co-Chair, New York Mayor Michael Bloomberg, noted that the study was "indisputable proof of the enormous contribution of immigrants in developing the new technologies and ideas needed to renew the U.S. economy and create American jobs," adding that "American universities are doing their part in attracting and educating the world's top minds, but our broken immigration laws continue to push them to our competitors."  According to the report, the University of California led all other U.S. universities with respect to issued patents in 2011, securing 369.  The top 10 patent-producing universities in 2011 is shown below:

The study also found that 54% of the patents issued to the top 10 universities named foreign inventors that were most likely to face visa hurdles, namely students, postdoctoral fellows, and staff researchers.  With respect to pharmaceuticals, 79% of patents issued to the top universities named a foreign-born inventor.  In a press release issued by the Partnership, American Enterprise Institute Fellow Nick Schulz noted that "[e]very graduate with an advanced degree working in a STEM-related field in the United States has been shown to create on average 2.62 additional jobs for native-born workers," and therefore asserted that "[s]ending those people away doesn't protect American jobs, it jeopardizes them."

The group noted that the study highlights the clear value foreign-born graduates bring to the U.S. economy, and offered recommendations for the U.S. to fix its immigration system and compete with countries such as South Korea, Switzerland, Spain, Italy, Germany, and the United Kingdom, which admit 60-80% of immigrants on economic needs (as opposed to the U.S., which admits only 7% of immigrants on economic needs).  Among the group's recommendations are:

• Granting permanent residency (green cards) to foreign students who earn graduate degrees in STEM (science, technology, engineering and math) fields, where 99% of the patents naming a foreign-born inventor were issued.
• Creating a visa for foreign-born entrepreneurs having U.S. investors and wanting to start companies employing U.S. employees.
• Raising or removing the cap on H-1B visas, currently set at 65,000 (the group notes that the 2012 cap was hit in only 10 weeks).

More information regarding the study can be found here.

Tomato Genome Determined

By Kevin E. Noonan --

Hans Sauer, Associate General Counsel for Intellectual Property for the Biotechnology Industry Organization (BIO), frequently asks (when discussing patent-eligibility of genes):  "What about cucumber genes?  Should they be patented?"  If Hans wishes to remain au courant, however, he will need to update this question by asking "What about tomato genes?" after the disclosure, in this week's Nature, that the entire genomic DNA sequence of the tomato (Solanum lycopersicum) has been deciphered (The Tomato Genome Consortium, "The tomato genome sequence provides insights into fleshy fruit evolution," Nature 485: 635–41 (31 May 2012)).

As with other species genome project results, the reported sequence reveals interesting relationships between tomatoes and closely-related species (like the potato, Solanum tuberosum), including "wild" species (Solanum pimpinellifolium) related to the domestic tomato, as well as identifying sequence characteristics resulting from evolutionary events in the tomato pedigree.

The researchers used an inbred cultivar, "Heinz 1706," having a "predictive" genome size of 900Mb on 12 tomato chromosomes.  The tomato genomic sequence was compared with the "wild" tomato sequence as well as other domesticated species of Solanum (particularly potato).  The sequence differences between the domesticated and wild tomato amounted to 0.6% nucleotide divergence in 5.4 million single nucleotide polymorphisms (SNPs) scattered throughout the genome.

Between tomato and potato, on the other hand, 8.7% sequence divergence was found, including nine large and several smaller inversions (which can facilitate sequence and species divergence by interfering with meiotic pairing), with intergenic and heterochromatic (repeat-rich) sequences showing (not unexpectedly) > 30% sequence divergence.

Evolutionarily, the tomato sequence showed evidence of two consecutive genome triplications (a phenomenon common among plants but unknown for animal species), one recent between tomato and potato (~7.3 Myr ago), and one ancient (at about the rosid (tomato) - asterid (grape) divergence, ~71 +/- 19.4 Myr ago).  These events are believed to have provided the genetic plasticity to develop genes controlling fruit characteristics, such as color and "fleshiness."  These include transcription factors and enzymes involved in ethylene biosynthesis (used for ripening), red light photoreceptors (PHYB1/PHYB2), and lycopene synthesis (PST1/PSY2).  Interestingly, the researchers reported that "[s]everal cytochrome P450 subfamilies associated with toxic alkaloid biosynthesis show contraction or complete loss in tomato and the extant genes show negligible expression in ripe fruits," a genetic adaptation to be expected for plants that have adopted a seed-dispersal mechanism requiring that the fruit be eaten by animals and the seeds passed in their spoor.  Differential expression of genes for cell wall architecture was also observed and believed to be involved in this aspect of the tomato life cycle.

On the other hand, the observed patterns comparing tomato genes with grape orthologs (22.5% of grape genes have an orthologous region in tomato, 39.9% have two and 21.6% have three) suggested that triplication was followed by "widespread gene loss" (see Figure 2 of publication).  Synteny maps between tomato chromosomes and chromosomes from potato, eggplant (aubergine), pepper, and tobacco were prepared and showed high levels of synteny, not unexpected in view of these species' interrelatedness.

Turning to the overall chromosome structure, the tomato showed pericentric heterochromatin and distal euchromatin, with miRNA and chloroplast insertions "more evenly distributed" throughout euchromatin.  There was evidence of fewer high-copy LTR-containing retrotransposons in the tomato genome than in Sorghum or Arabidopsis genomes, with "older" average insertion age (2.8M v. 0.8M years ago); the tomato genome is "unusual among angiosperms by being largely comprise of low copy number DNA."  In view of these differences with Arabidopsis, it was paradoxical that "[c]hromosomal organization of  genes, transcripts, repeats and small RNAs (sRNAs) is very similar" between the tomato and Aribodopsis genomes.

Sequence comparisions and open reading frame analyses found from 34,727 to 35,004 protein-coding genes in the tomato genome.  Of these, 18,320 were orthologs to potato genes; comparisons of synonymous vs. non-synonymous nucleotide substitution patterns between potato and tomato with similar comparisons between sorghum and maize (11.9Myr divergence) suggested stronger "diversifying" selection in the tomato/potato pair.  Comparison of the tomato genome with the "wild" S. pimpinellifolium species genes showed 7,378 identical genes and 11,753 genes with only synonymous nucleotide sequence changes; of the rest (12,629 genes), there were not only non-synomynous changes but also gain/loss of stop codons, with implications for gross changes in functionality.  In some instances, "several" chromosomal segments (particularly from cherry tomatoes) were more similar to the S. pimpinellifolium species than to the Heinz 1076 cultivar sequence.

The tomato genome shares with the soybean and potato genomes the location of small, regulatory RNAs to "gene-rich" chromosomal regions and particularly promoters.  96 conserved miRNA species were identified in tomato compared with 120 such species in potato, grouped into 34 families (10 "highly conserved" in plants and the others "less conserved" that are more abundant in potato than tomato; some of these potato-specific miRNAs specifically target Toll interleukin receptor, nucleotide-binding site and leucine-rich repeat genes.  Protein-coding genes from tomato, potato, Arabidopsis, rice, and grape were "clustered" into 23,208 gene groups (having at least 2 members), of which "8,615 are common to all five genomes, 1,727 are confined to eudicots (tomato, potato, grape and Arabidopsis), and 727 are confined to plants with fleshy fruits (tomato, potato and grape)."

The report concludes with these lessons from the genome that reflect the natural history of the tomato:

The genome sequences of tomato and S. pimpinellifolium also provide a basis for understanding the bottlenecks that have narrowed tomato genetic diversity: the domestication of S. pimpinellifolium in the Americas, the export of a small number of genotypes to Europe in the 16th century, and the intensive breeding that followed.  Charles Rick pioneered the use of trait introgression from wild tomato relatives to increase genetic diversity of cultivated tomatoes.  Introgression lines exist for seven wild tomato species, including S. pimpinellifolium, in the background of cultivated tomato.  The genome sequences presented here and the availability of millions of SNPs will allow breeders to revisit this rich trait reservoir and identify domestication genes, providing biological knowledge and empowering biodiversity-based breeding.

The genomic data generated by the whole project is available on-line, in GenBank under accession number AEKE00000000.  The individual chromosome sequences as numbers CM001064–CM001075, and the data on expressed sequences are available in the "Sequence Read Archive" under accession number SRA049915, GSE33507, SRA050797 and SRA048144.

The Tomato Genome Consortium was responsible for the report, and reflects a vast amount of work by hundreds of researchers around the world.  Named as corresponding authors are:

Shusei Sato, Satoshi Tabata, Lukas A. Mueller, Sanwen Huang, Yongchen Du, Chuanyou Li, Zhukuan Cheng, Jianru Zuo, Bin Han, Ying Wang, Hongqing Ling, Yongbiao Xue, Doreen Ware, W. Richard McCombie, Zachary B. Lippman, Stephen M. Stack, Steven D. Tanksley, Yves Van de Peer, Klaus Mayer, Gerard J. Bishop, Sarah Butcher, Nagendra Kumar Singh, Thomas Schiex, Mondher Bouzayen, Antonio Granell, Fernando Carrari, Gianluca De Bellis, Giovanni Giuliano, Glenn Bryan, Michiel J. T. van Eijk, Hiroyuki Fukuoka, Debasis Chattopadhyay, Roeland C. H. J. van Ham, Doil Choi, Jane Rogers, Zhangjun Fei, James J. Giovannoni, Rod Wing, Heiko Schoof, Blake C. Meyers, Jitendra P. Khurana, Akhilesh K. Tyagi, Tamas Dalmay, Andrew H. Paterson, Xiyin Wang, Luigi Frusciante, Graham B. Seymour, Bruce A. Roe, Giorgio Valle, Hans H. de Jong and René M. Klein Lankhorst

Note: the complete list can be found in the Supplementary Information (Supplementary Information).

The Tomato Genome Consortium includes scientists from The Kazusa DNA Research Institute, Japan; 454 Life Sciences Co. (Roche), USA; Amplicon Express Inc., USA; Beijing Vegetable Research Center, China; National Center for Gene Research, Chinese Academy of Sciences, Shanghai, China; BGI-Shenzhen, China; BMR-Genomics SrL, Italy; Boyce Thompson Institute for Plant Research, Cornell University, USA; Centre for Biosystems Genomics, The Netherlands; Centro Nacional de Analisis Genomico, Barcelona, Spain; China Agricultural University, Beijing, China; Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences, Beijing, China; Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China; Wuhan Botanical Garden, Chinese Academy of Sciences, Beijing, China; Cold Spring Harbor Laboratory, USA; Colorado State University, USA; National Taiwan University, Taipei; Department of Plant Biology, Cornell University, USA;  Centre for Genomic Regulation, University Pompeu Fabra, Barcelona, Spain; Department of Plant Biotechnology and Bioinformatics, Ghent University, Belgium; Faculty of Agriculture, The Hebrew University of Jerusalem, Israel; Institute of Industrial Crops, Heilongjiang Academy of Agricultural Sciences, China; Institute for Bioinformatics and Systems Biology (MIPS), Helmholtz Center for Health and Environment, Germany; College of Horticulture, Henan Agricultural University, China; Department of Life Sciences, Imperial College London, UK; NRC on Plant Biotechnology, Indian Agricultural Research Institute, India; INRA, Génétique et amélioration des fruits et légumes, France; INRA, Biologie du Fruit et Pathologie, France; Unité de Biométrie et d'Intelligence Artificielle, INRA, France; INRA-CNRGV, France; Plateforme bioinformatique Genotoul, Biométrie et Intelligence Artificielle, INRA, France; Institut National Polytechnique de Toulouse - ENSAT, Université de Toulouse, France; Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Spain; Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Malaga - Consejo Superior de Investigaciones Cientificas (IHSM-UMA-CSIC), Spain; Instituto de Biotecnología, Argentina; Institute for Biomedical Technologies, National Research Council of Italy; Institute of Plant Genetics, Research Division Portici, National Research Council of Italy; ENEA, Casaccia Research Center, Italy; Scuola Superiore Sant'Anna, Italy; ENEA, Trisaia Research Center, Italy; James Hutton Institute, UK; Barcelona Supercomputing Center, Spain; Institute of Research in Biomedicine, Barcelona, Spain; ICREA, Barcelona, Spain; Keygene N.V., Wageningen, The Netherlands; Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Republic of Korea; Life Technologies, USA; Life Technologies, France; Max Planck Institute for Plant Breeding Research, Germany; School of Agriculture, Meiji University, Japan; Department of Plant Science and Plant Pathology, Montana State University, USA; NARO Institute of Vegetable and Tea Science, Japan; National Institute of Plant Genome Research, India; Plant Research International, Business Unit Bioscience, Wageningen, The Netherlands; Institute of Plant Genetic Engineering, Qingdao Agricultural University, China; Roche Applied Science, Germany; Seoul National University, Department of Plant Science and Plant Genomics and Breeding Institute, Republic of Korea; Seoul National University, Department of Agricultural Biotechnology, Republic of Korea; Seoul National University, Crop Functional Genomics Center, Republic of Korea; High-Tech Research Center, Shandong Academy of Agricultural Sciences, China; Institute of Vegetables, Shandong Academy of Agricultural Sciences, China; School of Life Sciences, Sichuan University, China; Sistemas Genomicos, Spain; College of Horticulture, South China Agricultural University, China; Syngenta Biotechnology, Inc., USA; Norwich Research Park, UK; Department of Botany, The Natural History Museum, UK; United States Department of Agriculture - Agricultural Research Service, Robert W. Holley Center, USA; Instituto de Hortofruticultura Subtropical y Mediterranea, Departamento de Biologia Molecular y Bioquimica, Spain; Centre de Regulacio Genomica, Universitat Pompeu Fabra, Spain; Arizona Genomics Institute, USA.; Crop Bioinformatics, Institute of Crop Science and Resource Conservation, University of Bonn, Germany;  Department of Plant and Soil Sciences, and Delaware Biotechnology Institute, University of Delaware, USA; Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, India.; University of East Anglia, UK; Department of Biology and the UF Genetics Institute, USA; Plant Genome Mapping Laboratory, University of Georgia, USA; Center for Genomics and Computational Biology, School of Life Sciences, and School of Sciences, Hebei United University, China; J. Craig Venter Institute, USA; University of Naples "Federico II" Department of Soil, Plant, Environmental and Animal Production Sciences, Italy; Division of Plant and Crop Sciences, University of Nottingham, UK; Department of Chemistry and Biochemistry, University of Oklahoma, USA; CRIBI, University of Padua, Italy; Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, USA.; Department of Agriculture and Environmental Sciences, University of Udine, Italy; Wageningen University, Laboratory of Genetics, Wageningen, The Netherlands; Wageningen University, Laboratory of Plant Breeding, Wageningen, The Netherlands; Wellcome Trust Sanger Institute Hinxton, UK; Ylichron SrL, Casaccia Research Center, Italy; and Plant Engineering Research Institute, Sejong University, Republic of Korea.

Obama Administration Releases National Bioeconomy Blueprint

By Kevin E. Noonan --

In April, the White House released a policy paper what was styled the "National Bioeconomy Blueprint."  Its presumptions are based on the idea that the portion of the economy "fueled by research and innovation in the biological sciences" is a "large and rapidly growing segment of the world economy that provides substantial public benefit."  This has caused innovation in the biological sciences to become a priority of the Obama Administration, promising not only economic development but to "live longer, healthier lives, reduce our dependence on oil, address key environmental challenges, transform manufacturing processes, and increase the productivity and scope of the agricultural sector while growing new jobs and industries."  Successes of the bioeconomy touted by the Blueprint include $76 billion in revenues from genetically engineered crops and $100 billion in revenues from "industrial biotechnology" (including "fuels, materials, chemicals, and industrial enzymes derived from genetically modified systems").  The Blueprint identifies genetic engineering technology, DNA sequence analysis and "automated high-throughput manipulations of biomolecules" as "foundational technologies," while identifying "synthetic biology," proteomics and bio-informatics as "emerging technologies."

The result of the "bioeconomy" becoming an Administration "priority" is a directive (Executive Memorandum (M-10-30)) to all Federal agencies to "support research to establish the foundations for a 21st century bioeconomy," an effort that the Blueprint says has made "significant early progress."  While these efforts also "raise important ethical and security issues that are also top priorities for the Administration," discussions of these concerns are expressly deemed to be outside the scope of the Blueprint itself.

The first chapter of the Blueprint sets out the broad goals, citing the National Research Council's 2009 report, A New Biology for the 21st Century, for a recognition of the importance of biological research for addressing societal needs for foods, fuel, and medicines (echoing BIO's motto of "Healing, Fueling and Feeding the World").  The Blueprint enunciates "key elements" of the recommended national effort, including:

• a full spectrum of basic and applied R&D activities performed by academic, government, and private sectors
• public-private partnerships
• a supportive commercialization system for bioinventions
• innovative regulatory policies that reflect government awareness of needs for and impediments to progress
• a skilled and creative workforce
• public support for technological advances
• flexibility to accommodate the evolving needs, discoveries, and challenges

The second chapter enumerates several examples of efforts to achieve the goals.

The Blueprint sets forth "five strategic objectives" for achieving the promised bioeconomy:

1. Support R&D investments that will provide the foundation for the future U.S. bioeconomy.

This objective includes "[c]oordinated, integrated R&D efforts will help strategically shape the national bioeconomy R&D agenda"; "[e]xpand[ing] and [d]eveloping essential bioeconomy technologies"; "[i]ntegrat[ing] approaches across fields"; and "[i]mplement[ing] Improved Funding Mechanisms."  In this effort, "the President has called for agencies to identify strategic R&D investments, as well as increase the use of flexible funding mechanisms to improve program efficiency and provide the best opportunity to enhance economic growth."  The rationale is that "[a] robust biological/biomedical R&D enterprise, backed by government, foundations, and for profit investments, is necessary to produce the new knowledge, ideas, and foundational technologies required to develop products and services that support businesses and industries and help create jobs."

Much of what is cited in the Blueprint are efforts like the NIH Center for Regenerative Medicine, to accelerate the development and testing of new clinical protocols using iPS-cell-based treatments, and the use of FDA data on drug products clinical data ("[t]he FDA currently houses one of the largest known repositories of clinical data, including safety, efficacy, and performance information, and an increasing amount of post-market safety surveillance data").  While technology-based, such efforts do raise their own questions, such as whether translational activities are the best use of NIH resources and the extent to which clinical and other data submitted in support of regulatory approval can be expropriated by the government for its own, alternative uses without raising constitutional concerns.  The Blueprint also cites several biofuels efforts by Advanced Research Projects Agency-Energy, Department of Agriculture (USDA) and Department of Energy's (DOE) Biomass Program, USDA-DOE Plant Feedstock Genomics for Bioenergy Program, National Science Foundation's (NSF) Engineering Directorate Sustainable Energy Pathways (SEP) effort, as well as government programs supporting biomanufacturing.

2. Facilitate the transition of bioinventions from research lab to market, including an increased focus on translational and regulatory sciences.

This objective includes "[a] dedicated commitment to translational efforts will accelerate movement of bioinventions out of laboratories and into markets, specifically: [a]ccelerat[ing] progress to market; enhance[ing] entrepreneurship at eniversities; and utilize[ing] federal procurement authority."  The Blueprint identifies programs intended to help start-ups negotiate the "valley of death" of investment, including the Startup America initiative, specifically naming "five areas highlighted for action: unlocking access to capital; connecting mentors with entrepreneurs; reducing regulatory barrier; tax relief; and other economic incentives for small businesses"; the National Institute of Standards and Technology (NIST) Hollings Manufacturing Extension Partnership (MEP); the National Innovation Marketplace; BioInnovation Initiative; Executive Order 13514, Federal Leadership in Environmental, Energy, and Economic Performance; reauthorization of the Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) programs; translational biomedicine efforts such as the National Center for Advancing Translational Sciences (NCATS), established in December 2011, and cooperative programs between FDA and NIH.  In addition, the Blueprint advocates enhanced entrepreneurial activities in universities and improved technology transfer, which includes exclusive licensing of NIH-developed innovation, "to companies developing drugs, vaccines, or therapeutics from NIH-patented or patent-pending technologies."  But the Bleuprint also contains statements like "[t]he America Invents Act provides entrepreneurs the tools they need to obtain patents more quickly and to defend them against litigation challenges, both at lower costs" which reduces a reader's confidence that the Administration has accurately assessed the important components of bio-innovation.

3. Develop and reform regulations to reduce barriers, increase the speed and predictability of regulatory processes, and reduce costs while protecting human and environmental health.

This objective includes "[i]mproved regulatory processes [that] will help rapidly and safely achieve the promise of the future bioeconomy, specifically: improve[ing] regulatory processes and regulations; and collaborat[ing] with stakeholders."

This initiative is actually directed to "reducing regulatory barriers":  specifically, regulations that "have become inadequate or unnecessarily restrictive because technology and its associated products and services, as well as our national interests, have evolved and regulations may not have kept pace."  The Blueprint also cites Executive Orders, Improving Regulation and Regulatory Review and Regulation and Independent Regulatory Agencies and revisions to FDA regulations that "increase transparency, consistency, and predictability of the regulatory processes and help drive medical product innovation forward."  Specific regulatory changes are directed to medical devices and regulation for "emerging technologies."

4. Update training programs and align academic institution incentives with student training for national workforce needs.

This objective includes the directive that "Federal agencies should take steps to ensure that the future bioeconomy has a sustainable and appropriately-trained workforce," specifically enumerating "employer-educator partnerships" and "reengineer[ing] training programs."  A large portion of this objective is directed towards an emphasis on minority-serving institutions (including community colleges).

5. Identify and support opportunities for the development of public-private partnerships and precompetitive collaborations -- where competitors pool resources, knowledge, and expertise to learn from successes and failures.

This objective includes the directive that "Federal agencies should provide incentives for public-private partnerships and precompetitive collaborations to benefit the bioeconomy broadly, specifically directed to "catalyz[ing] public-private partnerships."  "Models" for collaboration are listed as:

• Open-source initiatives
• Industry consortia for process innovation
• Discovery-enabling consortia
• Public–private consortia for 
knowledge creation
• Prizes
• Innovation incubators
• Industry complementors
• Virtual pharmaceutical companies

Citing "Overview of Precompetitive Collaboration for Institute of Medicine Workshop."

This portion of the Blueprint sets out public-private partnerships for "rescuing" or repurposing" abandoned pharmaceutical compounds and smarter drug design.

The next portion of the Blueprint sets forth purported "successes" of the Administration's efforts to stimulate the bioeconomy, which include:

• "Broad Spectrum Anti-Viral: Researchers at the Massachusetts Institute of Technology's Lincoln Laboratory reported in 2011 creation of a broad-spectrum antiviral technology that selectively kills any virus-infected cell but does not harm uninfected cells." ("Broad-Spectrum Antiviral Therapeutics")

• "Diesel from CO₂: Through photosynthesis, plants, algae, and some bacteria use the energy of sunlight to convert CO₂ into a variety of organic compounds needed for growth and survival A Massachusetts-based company re-engineered photosynthetic organisms to synthesize, from sunlight and CO₂, molecules that form the chemical basis of diesel fuel."

• "Designing Biological Systems for Next- Generation Biomanufacturing: Synthetic biology is enabling scientists to rapidly design organisms that can be used in the production of renewable chemicals, biofuels, renewable specialty and fine chemicals, food ingredients, and health-care products. Bioacrylic acid heralds the advent of synthetic-biology-enabled manufacturing: acrylic acid ingredients are used to make adhesives stronger, paints more durable, and diapers more absorbent, and today petroleum-based acrylic is an $8 billion global market" ("Current Uses of Synthetic Biology")

• "Allergen-Free Peanuts: Researchers at a number of institutions in the United States have made inroads towards eliminating or inactivating allergenic proteins in peanuts. If successful, these approaches could lead to significant health benefits for Americans and economic opportunities for the peanut industry."

• "Biodegradable Plastics from Biomass: A major commercial "polylactic acid" bioplastic is already made today from cellulose However, the ability to replace petroleum-based plastics with this bioproduct is constrained by the limited availability of the specific cellulosic source material. To address this limitation, USDA scientists discovered a bacterium that can ferment a broader range of cellulosic biomass materials into polylactic acid, enabling commercial production on a much larger scale. With an estimated $375 billion market for chemical, plastic, and rubber products based on petroleum, this represents a substantial bioeconomy opportunity." ("Sustainable Polymers")

• "Biosensor Pollution Monitoring: Biosensors are devices composed of a biological sensing component linked
to a signaling component, working together to reveal the presence of an element, molecule, or organism of interest. In 2011, a team at the Virginia Institute of Marine Science reported creation of a portable biosensor that could detect marine pollutants, including oil, much faster and more cheaply than current technologies. If deployed near oil facilities, such sensors could provide early warning of spills and leaks and track dispersal patterns in real time." ("Near real-time, on-site, quantitative analysis of PAHs in the aqueous environment using an antibody-based biosensor")

More naïve (or perhaps idealist) portions of the Blueprint posit "trends" in the bioeconomy such as "sharing:"

Non-traditional research collaborations that feature the
sharing of information, resources, and capabilities are
transforming the bioeconomy.  Precompetitive collabora
tions -- where "competitors" partner and pool resources -- are growing as partners seek new ways to leverage constrained resources and surmount shared problems.  Partnerships for innovation are increasingly observed as a response to changing economic and technological conditions.

In agriculture, as the public and private sectors seek increased information for the bioeconomy on potential crop characteristics, there is increased sharing, both domestically and internationally, of genetic information.  The sharing of genetic information enhances U.S. agricultural competitiveness for food, energy, chemical production in plants, and other biobased-product crop species.

In the health sector, precompetitive collaborations are having significant impacts in clinical-trial design and biomarker discovery, among other areas.  Combined industrial R&D has contributed to transformative progress despite major challenges such as increased management costs due to "outside" collaborations and the need to develop effective communication networks across companies.

Similarly idealistic (or perhaps fantastical) portions of the Bleuprint include the idea for "using the power of prizes to drive innovation," specifically with regard to enactment of the "America COMPETES Reauthorization Act[,] granting all Federal agencies broad authority to conduct prize competitions as called for by the President" in December 2011 ("Implementation of Federal Prize Authority: Progress Report").

The Blueprint contains some important information, such as the "30-year decline in new molecular entities per dollar spent on R&D":

Figure 1. 30-year decline in new molecular entities per dollar spent on R&D There has been a 30-year decline in pharmaceutical industry productivity, as measured by new molecular entities per dollar spent
on R&D, normalized to 5-year rolling average of 1970 
to 1975 While R&D costs have increased 50-fold during this time period, the output of investigational new drug candidates and new drug application products has stayed flat.  Figure Source: IOM (Institute of Medicine) 2010 Extending the spectrum of precompetitive collaboration in oncology research: Workshop summary Washington, DC: The National Academies Press.

The Blueprint interprets this data, by asserting that "[o]ne issue that government and industry must address is that increased investment in research has not resulted in a concomitant increase in approved drugs.  Figure 1 shows that the number of FDA-approved new molecular entities has not kept pace with the pharmaceutical sector's spending over time have cited internal drug pipeline productivity declines, delays in application review caused by both industry and regulators, and a decreasing number of tractable medical challenges as reasons for this decline in productivity.  Advances in translational and regulatory sciences, improvements in the transfer of technology from the public to the private sector, regulatory process improvements, Federal workforce enhancements, and innovative public-private partnerships have potential to help overcome these challenges."

On the other hand, the Blueprint identifies certain sectors of the bioeconomy that are rapidly advancing, such as genomic sequencing as illustrated in the following Figure:

Figure 2. "Cost per Genome" -- the cost of sequencing a human-sized genome Data from 2001 through October 2007 represent the costs of generating DNA sequence using first generation sequencing technology.  Beginning in January 2008, the data represent the costs of generating DNA sequence using 'second-generation' (or 'next-generation') sequencing platforms.  The change in instruments represents the rapid evolution of DNA sequencing technologies that has occurred in recent years. www genome gov/sequencingcosts.

The Blueprint asserts that "[w]hile the sequencing of the first human genome took 13 years and cost $2.7 billion, researchers can now sequence a human genome for a fraction of that cost (~$7,700) and within two weeks' time" and considers the implications of these rapid increases in speed and decreases in cost for "whole-genome" human genomic sequencing.

The Blueprint concludes:

The Administration has made great strides in harnessing biological research innovations to address national challenges in health, energy, food, environment, and manufacturing via the commercial economy.  But there is much more to be done.  The Administration plans to explore additional creative ideas for promoting U. S. leadership in the bioeconomy, such as innovative financing for translational research, increasing the impact of the SBIR program, and improving the ability of faculty and students to move from "idea to IPO," and will continue to accept public input by email at bioeconomy@ostp gov.

By strategically shaping future R&D investments, improving commercialization of bioinventions, updating workforce training programs for new bioeconomy careers, reforming regulatory processes, and building new bioeconomy public-private partnerships, the Administration will help stimulate the growth of a high-wage, high-skill sector while improving the lives of all Americans.

Politics aside, publication of the Blueprint indicates that the Obama Administration recognizes the importance of the bioeconomy to the nation's economy as a whole.  Provided that the government is receptive to the input and information from all stakeholders (and not just government policy wonks, academics or think tank, armchair entrepreneurs, the Blueprint may actually provide a roadmap for progress.

Survey Finds That Many U.S. Consumers Have Favorable Impression of Plant and Animal Biotechnology

By Donald Zuhn --

A survey on consumer perceptions regarding food technology indicates that many U.S. consumers have favorable opinions concerning the benefits offered by plant and animal biotechnology.  The survey, which was commissioned by the International Food Information Council (IFIC), a nonprofit, nonpartisan organization established in 1985 to effectively communicate science-based information about food safety and nutrition, was conducted by polling 750 U.S. adults between March 7-19, 2012 as to their perceptions on various food technology issues.

The survey found that 74% of respondents had read or heard at least "a little" about the concept of food biotechnology.  With respect to plant biotechnology in general, respondents who had somewhat or very favorable opinions concerning plant biotechnology (38%) outnumbered those who had somewhat or very unfavorable opinions (20%).  The percent of respondents having somewhat or favorable opinions was up from 32% in 2010.  Almost half (49%) of respondents had favorable opinions concerning the use of biotechnology by farmers to grow more crops in order to help meet food demand.  A majority of respondents noted that they were somewhat or very likely to purchase foods produced through biotechnology in order to provide more healthful fats (71%), avoid saturated fat (68%), or make foods taste better or fresher (69%).  Given the choice between increased pesticide use or biotechnology, 77% of respondents said they would be likely to purchase foods that had been biotechnologically engineered for their ability to reduce pesticide use.  While a majority of respondents said they would be somewhat or very likely to purchase foods produced through biotechnology to achieve a number of specific benefits, the number of respondents saying they would be likely to buy such products had dropped since 2010 (see chart below).

With respect to animal biotechnology in general, respondents who had somewhat or very favorable opinions concerning animal biotechnology (33%) outnumbered those who had somewhat or very unfavorable opinions (26%).  However, the difference in these groups was less pronounced than it was for biotechnologically engineered plants.  Half of the respondents had a somewhat or very favorable impression regarding the use of genomics to evaluate animals in order to make better breeding decisions to achieve improved meat, milk, and egg quality.  However, only 44% of respondents had a somewhat or very favorable impression regarding the use of genetic engineering to transfer beneficial traits from one animal to another in order to improve nutritional content or lessen environmental impact.  Despite this result, a majority of respondents (71%) still said they would be likely to buy meat, milk, and eggs from animals enhanced through genetic engineering, and a majority of respondents (67%) still said they would be likely to buy fish enhanced through genetic engineering, provided in both cases that the FDA determined that such products were safe (see chart below).

A majority of respondents (66%) also indicated that they supported the FDA's current food labeling policy for foods produced through biotechnology.  Only 24% of respondents believed additional information should be required on food labels, with only 3% suggesting that the additional information that was needed on labels related to biotechnology.

(Once Again) It Ain't Necessarily So

By Kevin E. Noonan --

A large part of the debate on patenting genetic diagnostic method and isolated genes has revolved around the effects of such patents on what is loosely termed "personalized medicine."  Personalized medicine can be summarized as a dream/holy grail/GATTACA future of universal genetic information -- every infant having her genomic DNA sequence determined at birth and contained on a medical identity card, to determine what diseases she may get, what drugs she should not and even who she should or should not marry (or at least mate with).  It is the most recent of the promises of the biotechnology or genetic revolution, made possible (in theory) by the fruits of the Human Genome Project; advances in genomic sequencing technology (as described in The $1,000 Genome and elsewhere) have quickened the expectations surrounding the technology.  But many have begun to wonder why this genetic fruit is taking so long to ripen (see, for example, "How Bright Promise of Genetic Testing Fell Apart"), and a recent study published in Science Translational Medicine may provide some clues.

The article, entitled "The Predictive Capacity of Personal Genomic Sequencing," was published on April 2, 2012 by Nicholas Roberts, Kenneth Kinzler, Bert Vogelstein, and Victor Velculescu from the Howard Hughes Medical Institute at Johns Hopkins University Kimmel Cancer Center; Joshua Vogelstein from the Department of Neuroscience, Johns Hopkins University; and Giovanni Parmigiani from the Department of Biostatistics and Computational Biology, Dana Farber Cancer Center and the Department of Biostatistics, Harvard School of Public Health.  The paper presents the results of a bioinformatics study on monozygotic twin pairs, using whole genome sequencing (WGS) that was interrogated for 24 different diseases.  For 23 of the 24 diseases tested, the results were negative, i.e., uninformative results for 19 of the 24, with risk of 50-80% for disease as compared with the general population.  However, 90% of the tested individuals were alerted to "a clinically significant predisposition" to at least one disease thus reflecting a silver lining if not a rainbow from the data.

The paper notes that there is an estimate of three million sequence variants per person (K. A. Frazer et al.,  2009, "Human genetic variation and its contribution to complex traits," Nat. Rev. Genet. 10: 241-51); of these, thousands of genetic variants have been associated with human disease, including Mendelian traits (e.g., sickle cell anemia), SNPs (e.g., Huntington's s disease), or by genome-wide association studies (GWAS) (examples include familial pancreatic cancer and Miller syndrome).

The paper addresses the question of what the benefit of such information would be, defining "benefit" as "receiving information indicating that the risk of disease is increased or decreased to a degree that would alter an individual's lifestyle or medical management."  The authors recognize that it is impossible to assess these benefits generally, but monozygotic twins present the possibility to make this assessment:  as they have in many other instances, the identity of genetic information make it possible that diseases and other traits with a large genetic component should be experienced in common between twins:  "If one twin of the pair has a disease, then the probability of the other twin developing that disease is dependent on the genome whenever that disease has some genetic component."  However, the paper also notes that "[t]he general public does not appear to be aware that, despite their very similar height and appearance, monozygotic twins in general do not always develop or die from the same
maladies," citing Wong et al., 2005, "Phenotypic differences in genetically identical organisms: The epigenetic perspective," Hum. Mol. Genet. 14 Spec No 1, R11-R18, and "Identical Twins Not As Identical As Believed," ScienceDaily, reflecting a limitation in the predictive power of genetic disease assessment even between individuals having almost identical genetic complements.  (Interestingly, even monozygotic twins are not necessarily identical genetic copies of one another, there being copy number variants between them; Bruder et al., 2008, "Phenotypically concordant and discordant monozygotic twins display different DNA copy-number variation profiles," Am. J. Hum. Genet. 82: 763-71.)  The twin populations were selected from "the Swedish Twin Registry, Danish Twin Registry, Finnish Twin Cohort, Norwegian National Birth Registry and the National Academy of Science – National Research World War II Veteran Twins Registry."

The authors also define "heritability" as the difference between the incidence of disease in monozygotic twins compared with dizygotic twins, "reflect[ing] the average genetic contribution to disease" in the population of twins studied.  Averages not as informative as distributions in this regard, since a given average incidence of disease could reflect either "a small fraction of twin-pairs with genometypes [(i.e., a complete genomic DNA sequence from an individual)] conferring high genetic risk or a larger fraction of twin-pairs with genometypes conferring a moderate genetic risk."  This challenge is illustrated by an example:

Suppose a woman receives a whole-genome test result indicating that she has a 90% lifetime risk (the total risk over her entire life) of developing breast cancer.  She may decide to have a prophylactic double mastectomy to prevent this outcome.  Similarly, if the test indicated an 80% or even a 50% lifetime risk of developing breast cancer, she may consider mastectomy.  On the other hand, if the test indicated only a 14% risk of developing breast cancer, then mastectomies would be considered by very few women, given that most women today do not opt for prophylactic mastectomies even though the lifetime risk of developing breast cancer in the general population is 12%.

The authors adopt the threshold of a "positive predictive value" of 10%, meaning that 10% of patients with a "positive" test result are expected to develop a disease, according to Clarke-Pearson, 2009, "Clinical practice. Screening for ovarian cancer," N. Engl. J. Med. 361: 170-77.  However, for several diseases, including chronic fatigue syndrome, gastro-esophageal reflux disorder, coronary heart disease-related death and general dystocia, this threshold is inappropriate due in part to the prevalence of these diseases in the population; for these diseases the threshold is a two-fold higher risk of disease compared with the general population.  In addition to these diseases, the study assessed the risk for coronary artery disease, stroke, cancer (bladder, breast, colorectal, lung, leukemia, ovarian, pancreatic, prostate and stomach), thyroid autoimmunity, diabetes (types 1 and 2), Alzheimer's disease, dementia, Parkinson's disease, irritable bowel syndrome, pelvic organ prolapse, and stress urinary incontinence.

The bulk of paper set forth a mathematical treatment of these biostatistics that is beyond the scope of the discussion here; the results, on the other hand, are informative.  These include:

•  "The fraction of patients that would receive a positive test varies widely from disease to disease."
•  "The majority of patients (>50%) who would ultimately develop 13 of the 27 disease categories would not test positive, even in the best-case scenario."
•  "There were four disease categories -- thyroid autoimmunity, type I diabetes, Alzheimer's disease, and coronary heart disease-related deaths in males -- for which genetic tests might identify more than 75% of the patients who ultimately develop the disease."
•  "The fraction of individuals in the population who would receive positive test results for each disease is small."
•  For 22 of the 27 disease categories studied, "a negative test would not indicate a risk that is less than half that in the general population, even in the best-case scenario" (which is probably not sufficient to warrant changes in behavior, lifestyle, or preventative medical practices).
•  An exception is Alzheimer's disease, where a negative test "could indicate as little as a ~12% relative risk of disease compared to the entire twin cohort . . . .  Knowledge of such a reduced risk might be comforting and relieve anxiety, particularly to those with a family history of Alzheimer's disease."
•  ">95% of men and >90% of women could receive at least one positive test result."
•  Many of these results represent the best-case scenarios and thus the true benefits of genetic disease testing may be overestimated.

The authors' conclusions are a dose of cold water on the hopes and expectations of many in the field (and even more laypersons outside the field, including policymakers, judges, and even Supreme Court justices):

[O]ur results suggest that genetic testing, at its best, will not be the dominant determinant of patient care and will not be a substitute for preventative medicine strategies incorporating routine checkups and risk management based on the history, physical status and life style of the patient.

The authors best state the significance of an accurate assessment of the likelihood of predictive genetic testing:  "Recognition of these merits and limits [for genetic diagnostic testing] can be useful to consumers, researchers, and industry, as they can minimize unrealistic expectations and foster fruitful investigations."  These are words policymakers should no doubt heed when considering changes to established patent and other policies based on perhaps unrealistic prospects for a brighter genetic future.

Legislators Support Labeling of Genetically Engineered Foods

By Donald Zuhn --

Last week, in a letter sent to the Commissioner of the Food and Drug Administration, Margaret Hamburg, 55 members of Congress expressed support for a recent petition to require the labeling of genetically engineered foods.  The petition, which was filed with the FDA by the Center for Food Safety (CFS) last October, is part of the "Just Label It" campaign that currently has the support of almost 400 organizations and businesses.  At the CFS' website focusing on the issue, the group has collected nearly one million comments from the public in support of its labeling efforts.

In their letter to Commissioner Hamburg, the legislators contend that the "FDA's regulatory regime for food labeling is inadequate and uses 19th century concepts to regulate 21st century food technologies."  Noting that the FDA, in a 1992 policy statement, permitted genetically engineered (GE) foods to be marketed without labeling unless "materially" different from other (non-genetically engineered) foods, and "severely limited what it considered 'material' to only changes in food that could be recognized by taste, smell, or other senses," the group argues that "[t]he use of novel food technologies like genetic engineering on a commercial scale has so far slipped underneath FDA's limited threshold for 'materiality' because such technologies make silent, genetic, and molecular changes to food that are not capable of being detected by human senses."

The legislators accuse the FDA of failing to failing to "revisit[] the scientific or legal merits" of the agency's "outdated GE food labeling policy" when the agency crafted a policy for genetically engineered animals in 2009.  The GE animals standard led to the FDA's recent consideration of a GE salmon intended for human consumption.

The group states that "[l]abeling foods doesn't imply a product is unsafe or will be confusing to consumers as some may argue," and would merely provide consumers with "the fundamental right . . . to make informed choices about the food they eat."  The letter closes by urging the Commissioner "to fairly review the facts, law, and science, and side with the American public by requiring the labeling of genetically engineered foods as is done in nearly 50 countries throughout the world."

Senators signing the letter included Barbara Boxer (D-CA), Patrick Leahy (D-VT), Bernie Sanders (I-VT), Daniel Akaka (D-HI), Dianne Feinstein (D-CA), Ron Wyden (D-OR), Mark Begich (D-AK), Jon Tester (D-MT), Richard Blumenthal (D-CT), and Jeff Merkley (D-OR).  Representatives signing the letter included Lloyd Doggett (D-TX), Sam Farr (D-CA), Rush Holt (D-NJ), Jim McDermott (D-WA), James Moran (D-VA), Jared Polis (D-CO), Suzanne Bonamici (D-OR), Howard Berman (D-CA), David Cicilline (D-RI), Steve Cohen (D-TN), Bob Filner (D-CA), Luis Gutierrez (D-IL), Michael Honda (D-CA), Zoe Lofgren (D-CA), Jan Schakowsky (D-IL), John Tierney (D-MA), Lynn Woolsey (D-CA), Grace Napolitano (D-CA), Peter Defazio (D-OR), Richard Hanna (R-NY), Dennis Kucinich (D-OH), George Miller (D-CA), Louise Slaughter (D-NY), Keith Ellison (D-MN), Raul Grijalva (D-AZ), Peter Welch (D-VT), Hansen Clarke (D-MI), Earl Blumenauer (D-OR), Anna Eshoo (D-CA), Maurice Hinchey (D-NY), Chellie Pingree (D-ME), Madeleine Bordallo (D-GU), John Olver (D-MA), Charles Rangel (D-NY), Pete Stark (D-CA), Robert Brady (D-PA), Yvette Clarke (D-NY), Diana Degette (D-CO), Barney Frank (D-MA), Janice Hahn (D-CA), Barbara Lee (D-CA), James McGovern (D-MA), Jackie Speier (D-CA), Mel Watt (D-NC), and Maxine Waters (D-CA).  For those keeping score, the vast majority of signatories (53) are Democrats, with a single independent and a single Republican also signing the letter.

"Public Interest" Groups Call for Synthetic Biology Regulation

By Kevin E. Noonan --

A broad coalition of 111 public interest groups announced today a manifesto for containing "synthetic biology," a term with a loose definition (including "extreme genetic engineering") that includes efforts such as Craig Venter's to produce novel microorganisms to more traditional biotechnology efforts in transgenic plants and animals and other genetically modified organisms (see "Not Quite Artificial Life, But We're Getting Closer: Reactions to Venter's Synthetic Cell").  The effort was the occasion for a press conference this morning, hosted by the Friends of the Earth and featuring four speakers:

• Jaydee Hanson -- Policy Director at the International Center for Technology Assessment
• Dr. Stuart Newman -- Professor of Cell Biology and Anatomy at New York Medical College and board member of the Alliance for Humane Biotechnology
• Becky McClain -- biotechnology whistleblower and Injured Workers National Network
• Silvia Ribeiro -- Latin American Director for the ETC Group

Eric Hoffman, Food & Technology Policy Campaigner at Friends of the Earth U.S., moderated the conference, introducing the principle motivation for the proposal:  that government regulators have prematurely deemed the technology safe, which poses a risk to humans, the earth, and its other inhabitants.

Dr. Hanson started the conference, pointing out the "newness" of the technology and distinguishing this iteration of the biotechnology revolution with the one 40 years ago, based on the difference between copying a gene from one organism into another and "writing" an entirely new gene (or genome).  He also cautioned against simplistic metaphors for genetic manipulation, such as bricks or computer code, in view of the undeniably more complex nature of this technology.  His message was simple:  we don't know enough about how genes or organisms work to be creating new ones.

Dr. Newman continued with this theme, noting that another difference between the first biotechnology revolution and this one is that in the 1970's, the NIH created guidelines for containing genetically modified organisms (both physically and biologically), while this revolution is aimed at "releasing" genetically modified organisms into the environment in the form of modified foodstuffs, biofuel producers, etc.  It was noted that, in addition to his scientific accomplishments and work with the Alliance for Humane Biotechnology, Dr. Newman was named as an inventor in an application filed several years ago directed to a human-pig chimera (at the behest of his friend, Jeremy Rifkin, who spearheaded attempts in Cambridge, MA and elsewhere to contain the first biotechnology revolution).  (If memory serves, the U.S. Patent and Trademark Office refused to examine Dr. Newman's patent application.)

Ms. McClain gave the most personal and most frightening testimonial, embodying all the fears of corporate misfeasance and unchecked and dangerous technology.  According to Ms McClain, she was a molecular biologist working for a drug company on recombinant microorganisms and became ill after exposure to such a recombinant.  She catalogued several unsafe practices in the workplace and how her efforts to correct them were met with resistance and finally refusal to remedy or even address the situation.  Tragically, Ms. McClain described not only the disastrous effects of her experience on her career and health, but the failure of the legal system to provide redress because the evidence of causation was lacking (according to her, having been suppressed or destroyed by her former employer).  She also reminded the audience (during the Q&A following the speakers' prepared remarks) that the nature of this technology -- novel genetically engineered organisms -- could make it impossible for any future injured worker to successfully sue, since the diseases that may result from exposure (inadvertent or otherwise) would not be something readily recognized by a doctor as a human disease.  This comment was reminiscent of the early response to HIV infection in the 1980's.

The final speaker, Ms. Ribiero, included in her remarks many of the ills of Western capitalism visited on developing and underdeveloped companies in addition to synthetic biology, correctly noting that the indigenous people in such countries are at risk for harmful consequences of corporate liasons with local governments that may enrich some at a societal cost to the majority that may not be noticed in the U.S.  These concerns are encapsulated in a quote from Ms. Ribiero in the group's e-mail announcing release of the Principles:

In addition to the risks synthetic biology poses to human health and the environment, this technology may also deepen global social and economic injustices.  Novel organisms tailored to break down biomass will enable a new bio-economy in which land, water and fertilizers used to produce food for communities in the global South will be diverted for producing biomass feed for synthetic organisms in order to produce fuels, chemicals and other high-end products for wealthy nations.

There was a lively question and answer period following the prepared remarks; the most interesting comment came in response to a question from a reporter covering the U.S. Environmental Protection Agency.  She asked how the group expected to provoke the U.S. Congress to pass needed legislation in this area in view of partisan gridlock, and the response was that it could (and, the suggestion was, would) be done through riders to appropriations bills.  Not a prospect likely to provide the type of factfinding or deliberations that the topic no doubt warrants, but certainly a politically astute strategy.

The proposal was released over the Internet today, entitled The Principles for the Oversight of Synthetic Biology, and has the following points: 

I.  Employ the Precautionary Principle
II. Require mandatory synthetic biology-specific regulations
III.  Protect public health and worker safety
IV.  Protect the environment
V.  Guarantee the right-to-know and democratic participation
VI.  Require corporate accountability and manufacturer liability
VII.  Protect economic and environmental justice

The upshot of the Principles is a call for a moratorium on "the release and commercial use of synthetic organisms and products."  The justification is that such a moratorium is necessary "to safeguard public health and the environment from the novel risks of synthetic biology and to ensure open, meaningful and full public participation in decisions regarding its uses."

The Precautionary Principle (said to be incorporated into many international conventions, including the Rio Declaration on Environment and Development, the Cartegena Protocol on Biosafety, the U.N. Framework Convention on Climate Change, the World Charter on Nature, the London Convention on the Prevention of Marine Pollution by Dumping Wastes and Other Matter, and the Agreement for the Implementation of the Provisions of the United Nations Convention on the Law of the Sea) reads as follows:

When an activity raises threats of harm to human health or the environment, precautionary measures should be taken even if some cause and effect relationships are not fully established scientifically.  In this context the proponent of an activity, rather than the public, should bear the burden of proof.  The process of applying the Precautionary Principle must be open, informed and democratic and must include potentially affected parties.  It must also involve an examination of the full range of alternatives, including no action.

In accordance with the application of this Principle espoused in the report, the coalition rejects voluntary compliance with good practices codes or other forms of self-regulation by the biotechnology industry, and proposes mandatory government regulation instead.  The coalition specifically calls for a ban on human genetic engineering and other extensions of genetic technology into the human species, saying:

The use of synthetic biology to change the human genetic makeup — including the human genome, epigenome and human microbiome — must be prohibited.

Enforcement of the "strictest levels of physical, biological and geographical" containment of synthetic biology is also proposed, and the groups’ proposal envisions a "legally enforceable right" by the public to comment (and presumably prevent) particular applications of the technology before they are implemented, with corporations and other developers of the technology being financially liable for any "harm" cause to the public or the environment.  Finally, uses of the technology that would "deepen economic and social injustice" should be prohibited (although it is difficult to see how such outcomes could be predicted reliably).

The report in its entirety can be found here, and the groups endorsing this approach are:

African Biodiversity Network
Agricultural Missions, Inc (AMI) (U.S.)
Alliance for Humane Biotechnology (U.S.)
Amberwaves (U.S.)
Amigos de la Tierra España
Asociacion ANDES (Peru)
Asociación para la Promoción y el Desarrollo de la
Comunidad CEIBA / Friends of the Earth Guatemala
Basler Appell gegen Gentechnologie" (Appeal of Basle against Genetic-Manipulation) (Switzerland)
Biofuelwatch (International)
Biotechnology Reference Group of the Canadian Council of Churches
Biowatch South Africa
Brazilian Research Network in Nanotechnology, Society, and Environment - RENANOSOMA
Bund für Umwelt und Naturschutz Deutschland / Friends of the Earth Germany
Canadian Biotechnology Action Network (CBAN)
Center for Biological Diversity (U.S.)
Center for Food Safety (U.S.)
Center for Genetics and Society (U.S.)
Center for Humans and Nature (U.S.)
Center for International Environmental Law (U.S.)
Centro Ecológico (Brazil)
Centre for Environmental Justice/Friends of the Earth Sri Lanka
CESTA - Amigos de la Tierra, El Salvador
Citizens' Environmental Coalition (U.S.)
COECOCEIBA - Friends of the Earth Costa Rica
Columban Center for Advocacy and Outreach (U.S.)
Community Alliance for Global Justice (CAGJ) (U.S.)
Development Fund (Norway)
Diverse Women for Diversity (India)
Doctors for Food Safety & Biosafety (India)
Econexus (International)
Ecoropa (Europe)
Envirocare (Tanzania)
Environmental Rights Action/Friends of the Earth Nigeria
ETC Group (International)
Ethiopian Society for Consumer Protection (ETHIOSCOP)
European Network of Scientists for Social and Environmental Responsibility (ENSSER)
Family Farm Defenders (U.S.)
Federation of German Scientists
Food Democracy Now! (U.S.)
Food & Water Watch (U.S.)
Friends of the Earth Australia
Friends of the Earth Brazil
Friends of the Earth Canada
Friends of the Earth Cyprus
Friends of the Earth Latin America and the Caribbean (ATALC)
Friends of the Earth Mauritius
Friends of the Earth U.S.
Friends of ETC Group (U.S.)
Gaia Foundation (U.K.)
Gene Ethics (Australia)
GeneWatch UK
GLOBAL 2000/FoE Austria
Global Forest Coalition (International)
GM Freeze (UK)
GMWatch (UK)
IBON International
Indian Biodiversity Forum
Indigenous Peoples Council on Biocolonialism (U.S.)
Initiative for Health & Equity in Society (India)
Injured Workers National Network (U.S.)
Institute for Agriculture and Trade Policy (U.S.)
Institute for Responsible Technology (U.S.)
International Center for Technology Assessment (U.S.)
International Peoples Health Council (South Asia)
International Union of Food, Agricultural, Hotel, Restaurant, Catering, Tobacco and Allied Workers' Associations (IUF) (International)
Jamaican Council of Churches
Karima Kaaithiegeni Ambaire (CBO) (Kenya)
Latin American Nanotechnology & Society Network (ReLANS)
Loka Institute (U.S.)
MADGE Australia Inc.
Maendeleo Endelevu Action Program (MEAP) (Kenya)
Maryknoll Office for Global Concerns (U.S.)
MELCA-Ethiopia
Midwest Environmental Justice Organization (U.S.)
Movimiento Madre Tierra (Honduras)
Mupo Foundation (South Africa)
Nanotechnology Citizen Engagement Organization (U.S.)
National Association of Professional Environmentalists (Friends of the Earth Uganda)
Navdanya (India)
NOAH Friends of the Earth Denmark
Non-GMO Project (U.S.)
No Patents on Life! (Germany)
Northeast Organic Farming Association -- Interstate Council (NOFA-IC) (U.S.)
Organic Seed Growers and Trade Association (U.S.)
Otros Mundos AC/Amigos de la Tierra México
Our Bodies Ourselves (U.S.)
Partners for the Land & Agricultural Needs of Traditional Peoples (PLANT) (U.S.)
Pesticide Action Network North America
Pro-Choice Alliance for Responsible Research (U.S.)
Pro Natura – Friends of the Earth Switzerland
Public Employees for Environmental Responsibility (PEER)
Rescope Programme (Malawi)
Research Foundation for Science, Technology, and Ecology (India)
Rural Coalition (U.S.)
Save our Seeds (Europe)
Say No to GMOs! (U.S.)
Schweizerische Arbeitsgruppe Gentechnologie SAG (Swiss Working Group on Genetic Engineering)
Science & Environmental Health Network (U.S.)
Seed Stewards Association of Turkey
Sobrevivencia – Amigos de la Tierra Paraguay
Sustainability Council of New Zealand
Sustainable Living Systems (U.S.)
Testbiotech (Germany)
Third World Network (International)
Timberwatch Coalition (South Africa)
Tree Is Life Trust (Kenya)
USC Canada
VivAgora (France)
Washington Biotechnology Action Council (U.S.)
Women in Europe for a Common Future (International)
World Rainforest Movement (International)

Genetic Heterogeneity in Tumor Samples May Explain Difficulties in Developing Personalized Medicine

By Kevin E. Noonan --

The promise of an era of "personalized medicine" has been pursued for a generation, being one of the rationales for and purported benefits of the Human Genome Project.  It has become such a sought-for goal that it has been used to drive policy:  it is something that health care reform is banking on (literally), since by making medicine more individualized, success rates and fewer failed therapies are envisioned.  It crept into the gene patenting debate, with Judge Bryson in his dissent crediting the ACLU's claim that gene patents will inhibit development of personalized medicine (they won't).  But as others have noted (for example, Nicholas Wade, "A dissenting voice as the genome is sifted to fight disease," The New York Times, September 15, 2008), a promise is all it remains:  achieving a personalized medicine future has proven (so far) to be much more daunting than its proponents believed (or told the rest of us to believe).

The results of a cancer study in the New England Journal of Medicine last week may have shed some light on what this has been so.  The report, "Intratumor heterogeneity and branched evolution revealed by multiregion sequencing," a team of physicians and scientists from the UK and Harvard revealed that the genetics of human tumors is much more complicated than previously thought (Gerlinger et al., 2012, N. Engl. J. Med. 366: 883-92).  These researchers obtained multiple biopsy samples from different regions of the same tumor (primary and metastatic) and performed multilocus genomic sequencing.  The tumors were all a particular subtype of primary renal cell carcinoma, clear cell carcinoma (CCC) from patients that had been treated with everolimus (Zortress®, an mTOR inhibitor) therapy before and after nephrectomy.  Whole exome multiregion spatial DNA sequencing (see below) was performed on extracted tissue from fresh frozen samples as well as SNP analysis and mRNA expression profiling using gene arrays.

Exome Sequencing - Part I:

Exome Sequencing - Part 2

The results showed a significant amount of genetic heterogeneity that could be related to chemotherapeutic drug resistance and differential metastatic potential.  In one CCC patient, nine regions of the primary tumor and three regions of metastatic tumors (as well as the germline DNA sequences) were assayed.  A 2 bp deletion in the von Hippel-Lindau (VHL) tumor suppressor gene was found, a genetic characteristic of CCC.  These analyses revealed 101 nonsynonymous point mutations and 32 instances of insertion or deletion (indels), with the assays showing a low false negative rate of detection.  From a total of 128 mutations detected in the various samples, 40 were "ubiquitous" mutations (found in all samples), 59 were shared by "several but not all" regions, and 29 were unique to a particular region (called "private mutations").  Of the "shared" mutations, 31 were shared by most of the primary tumor samples, and 28 shared by most of the metastatic tumor samples.  "The detection of private mutations suggested an ongoing regional clonal evolution," the authors concluded from this data.

From these results, the workers constructed a phylogenetic tree that revealed "branching rather than linear" tumor evolution.  Deeper analysis showed that, in some regions, the primary tumor shared more mutation with the metastases than with other areas of the primary tumor, suggesting the existence of two "clonal populations of progenitor cells in this region."  The study also compared these results with results from a "single" tumor biopsy study, which detected 70 somatic mutations (about 55% of the total detected using the multiregional approach).  These figures were put into context by noting that only 31-34% of all mutations detected using the multiregional sampling/sequencing were detected in all regions.  Finally, any major effect of the everolimus treatment on these results was discounted by finding that 67/71 mutations found after treatment were present in the tumor samples pretreatment, and that 64/66 chest wall metastasis mutations were found in post-treatment metastatic tumors.  These results indicated to the researchers that "the two main branches of the phylogenetic tree were present before drug treatment" and that "60% of the mutations in pretreatment samples of the primary tumor and chest-wall metastases were not shared by both biopsy samples," i.e., evidence of clonal evolution that would have required reversion of somatic mutations during treatment (not very likely).

A conventional measure of tumor heterogeneity, ploidy analysis (i.e., how many chromosomes and chromosome fragments were present in the tumor cells) was also performed.  While the primary tumor was predominantly diploid (i.e., facially "normal") there were two regions in the metastatic tumors that were subtetraploid (i.e., a few fewer than twice the [n]o limited by sample quality issues showed ubiquitous "allelic imbalance" on the short arm of the 3rd chromosome (3p) characterized by loss of heterozygosity at multiple allelic loci), including VHL and histone H3K36 methyltransferase SETD2.  Even here, "tumor regions shared identical allelic-imbalance profiles, and heterogeneity of allelic imbalance within metastases, which is probably driven by aneuploidy, indicates that chromosomal aberrations contribute to genetic intratumor heterogeneity."

The study also compared the mutational status of genes known to be mutated in CCC, including VHL, SETD2, KDM5C, and mTOR.  Only the VHL gene was ubiquitously mutated in all regions sampled, contrasted in the study by the mutational nature of SETD2:  the metastases all showed a missense mutation while one primary region had a splice site mutation and the others showed a 2 bp frameshift deletion (which was also present in the region with the frameshift mutation).  Convergent evolution was detected with regard to SETD2 histone methylation using functional assays; such convergent genetic evolution in tumor cells was also detected for the X chromosome-encoded histone methyltransferase KDM5C.

Another gene, mTOR, showed a missense mutation in the portion of the gene encoding a kinase domain; this mutation was found in all but one of the primary tumor regions tested.  The researchers also reported that a currently used test for CCC, a 110-gene signature that assesses patient prognosis, displayed anomalous results:  the metastases and one primary tumor sample showed the "good" prognostic pattern while all the other primary sites showed the "poor" prognostic pattern.  The authors caution that "prognostic gene-expression signatures may not correctly predict outcomes if they are assessed from a single region of a heterogeneous tumor."

The workers performed similar analyses on three other patients.  In one, patient 2, the researchers found 119 somatic mutations what also showed a branching pattern of clonal genetic evolution in this patient's tumor.  Here, ~31-37 of the mutations were found ubiquitously (the lower number was obtained when the metastases were included).  While no ploidy imbalance was detected in these tumor samples, allelic imbalance was found ubiquitously in all tested regions for 3p and on the long arm of chromosome 10 (10q).  In addition to some of the 3p mutations found in patent 1's tumor, mutations were found for genes residing on 10q, including PTEN.  Convergent evolution was also observed for the PTEN gene.  Similar results were obtained and briefly noted for tumor samples obtained from patients 3 and 4 (patient 4's tumors showed allelic imbalance on chromosomes 5 (5q) and6 (6q)).  However, "[t]hese early ubiquitous events were outnumbered by non-ubiquitous aberrations, indicating that the majority of chromosomal events occurred after tumors diverged, providing further evidence of branching evolution."  Patient 4 also showed tumor heterogeneity in genes like SETD2 that had been detected in other tumor samples.

The authors summarized their results by noting that they had detected genetic heterogeneity in each tumor assayed, showing "spatial separated heterogeneous somatic mutations and chromosomal imbalances."  These genetic lesions lead to phenotypic heterogeneity, with 63-69% of the mutations not detected in every tumor region sampled.  Their detection of "ubiquitous alterations on the trunk of the tumor phylogenetic tree . . . may account for the benefits of cytoreductive nephrectomy" because it reduces the "reservoir" of primary tumors cells capable of genetic instability and failure to respond to more "conventional" regions of the tumor.  Finally, the authors state that:

Genomics analyses from single tumor-biopsy specimens may underestimate the mutational burden of heterogeneous tumors.  Intratumor heterogeneity may explain the difficulties encountered in the validation of oncology biomarkers owing to sampling bias, contribute to Darwinian selection of preexisting drug-resistant clones, and predict therapeutic resistance.  Reconstructing tumor clonal architectures and the identification of common mutations located in the trunk of the phylogenetic tree may contribute to more robust biomarkers and therapeutic approaches.

These results illustrate a few things.  First, the gene patenting debate per se is anachronistic and ten if not thirty years out of date.  The complexity revealed by this study provides one reason why approaches tried thus far for implementing personalized medicine have not worked out as well as planned.  This complexity suggests that it will take far more time to produce a worthwhile personalized medicine paradigm that fulfills all its unfulfilled promises and that the "gene age" will likely be long past by that time.

This very same complexity reinforces the risk in making any broad pronouncements against the patent-eligibility of "products to nature."  With this level of complexity, the number of "false negatives" (and, presumably, false positives) may make it possible to identify diagnostic genetic markers for disease prognosis that can be protected without patents.  As noted by the authors, identification of the "trunk" mutations (shared by the largest number of tumor samples) provide the best information on the tumor for treatment, prognosis and otherwise.  The negative consequences of changing the incentives from disclosure (protected by patenting) and non-disclosure (protected, inter alia as a trade secret) has been discussed here before; this study points to ways that could be profitable for the company that develops the test at the cost of reaching the goal of personalized medical care.  Because the alternative may be no personalized medical care at all, it behooves participants in the policy debate about gene patents, genetic diagnostic testing, and innovation to consider this study to be but the first in a long series demonstrating that, indeed, we are only at the beginning of the road when it comes to developing a robust personalized medicine system.

Images of exome sequencing (above) by SarahKusala, from the Wikipedia Commons (Part I & Part 2) under the Creative Commons Attribution 3.0 Unported license.

Lowland Gorilla Genome Sequenced

By Kevin E. Noonan -- 

This week's edition of Nature contains the results of the latest (and last) of the "genome" projects for members of the Homini (humans and their closest primate ancestors):  Gorilla gorilla gorilla, the western lowland gorilla (in particular a gorilla named Kamilah, the Craig Venter of her species) (Saclly et al., "Insights into hominid evolution from the gorilla genome sequence," Nature 483: 169-75 (08 March 2012)).  Like most of the genome projects, the results reported in Nature merely scratch the surface of the wealth of sequence data produced in the experiments (comprising 54 Gbp conventional Sanger sequencing and 166.8 Gbp of Illumina sequencing).

In the big picture, genetic comparisons with humans and chimpanzees (Pan troglodytes) revealed that the human/chimpanzee branch of the primate lineage diverged about 10 (range: 8.5-12) million years ago; in contrast, comparison of genomes of western and eastern gorilla species provides an estimate of evolutionary divergence about 1.75 million years ago.

The paper details the assumptions used to arrive at these figures, based on an estimate of 0.5 – 0.6 x 10^-9 mutations per bp per year in the primate genomes.  However, these researchers acknowledge that there is a disconnect between fossil estimates of when the speciation event occurred and the genetic estimate presented here that can only be reconciled by positing changing mutation rates, that may be explained by correlations with larger body mass and lower generation times between the species.  The authors concede that alternative scenarios are possible, including the existence of a population bottleneck and a non-allopatric speciation process in the human/chimp/gorilla line.

Turning to the genetic data, "typical" stretchs of "error-free" sequences were found to be about 7.2 kbp long, with errors clustering in repetitive regions.  A total of 3,041,976,159 bp were sequenced, with 20,962 protein coding genes, 1553 pseudogenes, and 6,701 RNA genes identified.  The researchers detected selection in the area of coding regions in all three species that suppresses mutations, making these regions more similar between the three species than in the rest of the genome(s).  Loss-or-gain analysis in autosomes showed an average of ~3-7 Mbp per species, distributed "genome-wide" and also found in orangutangs and more evolutionarily-distant species.  These results were consistent in patterns of gene loss; instances of gene gain were consistent with gene duplication.  Interestingly, the paper reports that 30% of gorilla genes are closer to either human or chimp that the corresponding human and chimp genes are to each other.  In addition, there is evidence for "accelerated evolution" for around 500 genes in parallel between humans, chimps, and gorillas, particularly in genes involved in hearing:  "[s]ome [gene] fragments found only in one species overlap coding exons in annotated genes: 6 genes in human, 5 in chimpanzee and 9 in gorilla, the majority being associated with olfactory receptor proteins or other rapidly evolving functions, such as male fertility and immune response."  Rapid structural evolution was also detected in the gorilla Y chromosome, something also observed in comparisons between human and chimp Y chromosomal sequences.

Protein alignment over 11,538 ORFs indentified regions of "accelerated evolution" that focused on "enriched for functions associated with sensory perception, particularly in relation to hearing and brain development," including specifically OTOF (Otoferlin, mutations thereof being the cause of neurosensory nonsyndromic recessive deafness), LOXHD1 (expressed in the mechanosensory hair cells in the inner ear, with mutations being associated with hearing defects) and GPR98 (G-protein coupled receptor 98, expressed in the central nervous system in humans and associated with Usher syndrome), all genes associated with diseases involved in human deafness.  The gene reported to have the strongest evidence for accelerated evolution in the hominines is RNF213, a gene that in humans is associated with Moyamoya disease, where blood flow to the brain is restricted due to arterial stenosis.  The paper reports "relatively similar" numbers of genes in humans (663), chimpanzees (562), and gorillas (535) that have apparently undergone accelerated evolution.  In the gorilla, accelerated genes are enriched for developmental events in ear, hair follicle, gonad and brain development, and sensory perception of sound, with the most significantly accelerated gene in gorilla being EVPL, which encodes a component of the cornified envelope of keratinocytes.  The authors speculate that this differential evolution "may be related to increased cornification of knuckle pads in gorilla."

The paper also reports instances of pairwise parallel evolution among hominines, with human and chimpanzee showing the largest numbers of paired genes, and with gorilla showing more parallel evolution with human than with chimpanzees.  There were 84 instances of gene loss in gorilla due to the acquisition of a premature stop codon, and in "several" cases the gorilla genome encoded a single gene at a locus that in humans encodes a protein variant believed to cause inherited disease.  The authors speculate that while the reason "variants that appear to cause disease in humans might be associated with a normal phenotype in gorillas is unknown" it is possible that there "are compensatory molecular changes elsewhere, or differing environmental conditions."

While the data relating to differences in gene transcription were not extensive or definitive, the paper reports that there is about a 7% divergence in splicing patterns between species for the genes examined.

The paper closes by reporting on genetic relationships between Gorilla gorilla (the western lowland gorilla) and Gorilla beringei (the eastern lowland gorilla).  The data were consistent with the current disparity in population size between the eastern and western lowland gorilla populations; this evidence in the species DNA indicated that the disparity has existed for "many millennia" and thus is probably not the result of human predation and habitat invasion or disease occurrence such as "recent outbreaks of the Ebola virus."  Even these results can be related to human-gorilla differences:  gorillas were shown to have "greater copy number diversity" than humans, a result "consistent with previous observations in the great ape."

The authors state their conclusions best:

Since the middle Miocene -- an epoch of abundance and diversity for apes throughout Eurasia and Africa -- the prevailing pattern of ape evolution has been one of fragmentation and extinction.  The present-day distribution of non-human great apes, existing only as endangered and subdivided populations in equatorial forest refugia, is a legacy of that process.  Even humans, now spread around the world and occupying habitats previously inaccessible to any primate, bear the genetic legacy of past population crises.  All other branches of the genus Homo have passed into extinction.  It may be that in the condition of Gorilla, Pan and Pongo [orangutan] we see some echo of our own ancestors before the last 100,000 years, and perhaps a condition experienced many times over several million years of evolution.  It is notable that species within at least three of these genera continued to exchange genetic material long after separation, a disposition that may have aided their survival in the face of diminishing numbers.  As well as teaching us about human evolution, the study of the great apes connects us to a time when our existence was more tenuous, and in doing so, highlights the importance of protecting and conserving these remarkable species.

The work was performed by an international consortium of researchers working at the Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus; the Bioinformatics Research Center, Aarhus University; the Department of Genome Sciences, University of Washington School of Medicine; the European Bioinformatics Institute, Wellcome Trust Genome Campus; Department of Genetic Medicine and Development, University of Geneva Medical School; Institut de Biologia Evolutiva and Institucio Catalana de Recerca i Estudis Avançats, Barcelona; the Department of Zoology and Department of Oncology, Hutchison/MRC Research Centre, University of Cambridge; Cancer Research UK, Cambridge Research Institute; Howard Hughes Medical Institute, University of Washington; Institute of Medical Genetics, Cardiff University; Department of Anthropology, Yale University; The Genome Institute at Washington University, Washington University School of Medicine; MRC Functional Genomics Unit, University of Oxford, Department of Physiology, Anatomy and Genetics; Wellcome Trust Centre for Human Genetics, Oxford; Comparative Genomics Unit, Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health; Max Planck Institute for Evolutionary Anthropology, Primatology Department; Children's Hospital Oakland Research Institute; and the San Diego Zoo's Institute for Conservation Research.

Image of Western Lowland Gorilla at Bronx Zoo (above) by Fred Hsu, from the Wikipedia Commons under the Creative Commons license.

Impact of Advances in DNA Sequencing Technology on Genetic Diagnostic Testing

By Kevin E. Noonan --

Dr. Ellen Jorgensen of Genspace, one of the witnesses at the U.S. Patent and Trademark Office's first hearing regarding the advisability of permitting "second opinions" for patented genetic diagnostic tests without patent infringement liability, advocated "at-home" or "do-it-yourself" DNA testing as a solution (see "USPTO Holds First Hearing on 'Second Opinion' Genetic Testing").  This proposal raises a significant number of questions, particularly with regard to the potential for harm to the public due to errors that might arise from such "at-home" genetic diagnostic testing or whether precautions in interpreting results would be taken concerning the emotional consequences of finding a genetic mutation in an individual's BRCA genes.

However, the saliency of any technical objections to the idea must be considered to be significantly reduced by an announcement from Oxford Nanopore Technologies at the Advances in Genome Biology and Technology Conference at Marco Island, FL last week:  a disposable gene sequencing machine the size of a standard USB thumb drive and capable of providing a complete genomic sequence for about $900.  Terming the business model for the device "pay-as-you-go" sequencing, the chief technology officer of the company, Clive G. Brown stated that the new device eliminates the need for expensive ($50,000 - $750,000) machines currently in use for gene sequencing, and touted the use of the device for bedside genetic testing, biological field work, and food safety (e.g., for identifying pathogens in situ in real time).

The basis of the device is so-called "nanopore" sequencing (explained in greater detail for the interested in "The $1,000 Genome: The revolution in DNA sequencing and the new era of personalized medicine" by Kevin Davies).  Briefly, the technology employs alpha-hemolysin, a bacterial membrane "pore" protein, stabilized with cyclodextrin, to measure changes in electrical current as each base moves through the pore after exonuclease cleavage.  The devices take advantage of parallel processing on a chip and computer analysis of the data to create the linear sequence.  Initially each chip will contain 2,000 pores with machines using chips having 8,000 pored being developed for release in 2013.  While the sequencing capacity ("tens of thousands bases per read") is higher than with competing machines, so is its error rate (4%).  This level of error would preclude use of the device for genetic diagnostic sequencing, for example.

But machines and methodologies will get better, which raises the possibility that DNA sequencing soon may be within the reach of the consumer (much like accurate blood glucose determinations are now done with devices requiring nothing more than a pinprick of blood).  Under these circumstances, much of the current patent protection (and the IP protection model underlying it) for genetic diagnostic testing may become obsolete.  First, determination of an entire sequence does not per se infringe gene-specific DNA or method claims unless gene-specific primers are used (and even these claims are subject to some reevaluation; see "Caught in a Time Warp: the (In)validity of BRCA1 Oligonucleotide Claims").  This is one reason why the majority of the claims at issue in the Myriad case (i.e., claims to isolated genes) are not infringed by the practice of genetic diagnostic methods and why even if the plaintiffs and their ACLU masters prevail, the women will have no remedy.  Thus, the only direct infringer using these "mini-sequencer" devices would be the consumer, and unlike situations where suing consumers has been successful (like music file-sharing), the individual damage from any specific consumer defendant's infringement would be minimal.  While the damage to the patent-holder might be large cumulatively, it is unlikely that a patentee could successfully sue consumers as a class.  Inducement to infringe might also be challenging to prove since it is unlikely that Oxford Nanopore Technologies will provide instructions relating to any particular gene with specificity.  In any event, the identity of disease-related mutations is (and might continue to be) in the public record and so the consumer herself would remain the only infringer.

It may also be expected that Oxford Nanopore Technologies will provide information on genetic counselors to be consulted to assist the consumer to interpret the meaning of her deduced nucleotide sequence.  These genetic counselors will be practicing a method involving comparing the deduced consumer sequence with the canonical "normal" sequence, and should not be infringing any valid claims.  This is because the Federal Circuit unanimously held in AMP v. USPTO (the "Myriad" case) that mere "comparison" claims do not satisfy the Bilski test and are thus invalid.  In addition, under this scenario the issue of "joint infringement" would arise, because the consumer would produce the sequence and its interpretation relating to inherited propensity for disease would be performed by the genetic counselor.  Unless the Federal Circuit dramatically changes the jurisprudential landscape in deciding the McKesson and Akamai cases en banc, infringement would not lie against either Oxford Nanopore Technologies or the genetic counselors.

The development of such an eventuality provides yet one more impetus for genetic diagnostic testing to avoid patents as a protection for the technology, and to use instead trade secret protection and other means that avoid disclosure.  These prospects make it even more imperative, perhaps, that whatever actions are taken by the Office, Congress, or the courts regarding genetic diagnostic testing be done cautiously and in a limited fashion.  Otherwise, we may find that we have imposed impediments to future technologies, just as progress in the technologies we intend to regulate make the regulation obsolete.  Certainly this can't be the kind of progress the Founders had in mind.

An Oxford Nanopore Technologies video showing on the company's technology works can be viewed below: