MCB 140 09-21-07 1 The recombinant DNA controversy “Those who disregard the past are bound to repeat it” George Santayana.

MCB 140 09-21-07 1

The recombinant DNA controversy

“Those who disregard the past are bound to repeat it”

George Santayana

MCB 140 09-21-07 2

Herb Boyer(EcoRI)

Stanley Cohen(pSC101)

MCB 140 09-21-07 3

1953

J. Bacteriol. 64(4): 557–569 (1952)

MCB 140 09-21-07 4

http://opbs.okstate.edu/~melcher/MG/MGW3/MG331.html

Werner Arber (1965): met-depleted E. coli don’t generate “modified” phage CH3 of phage DNA!!

MCB 140 09-21-07 5

The restriction/modification system:a bacterial pathway for defence against viruses

MCB 140 09-21-07 6

Dan Nathans Ham Smith

MCB 140 09-21-07 7

“How restriction enzymes became the workhorses of molecular biology”

A significant breakthrough came in 1970 when the first of two papers from Smith's laboratory described an enzyme, endonuclease R, that was able to cleave bacteriophage T7 DNA into specific fragments (2). This was the first type II restriction enzyme, the sort that now populates our freezers, because it recognize specific sequences and also gives rise to very specific cleavage. Smith had been looking for an enzyme that might be involved in site-specific recombination in Haemophilus influenzae and thought at first that endonuclease R might be his long-sought quarry. With Tom Kelly, he went on to determine the DNA sequence recognized by endonuclease R and reported it as GTY RAC (11). This sequence seemed too short for a recombination enzyme, and during correspondence with his close friend Nathans, who ran the neighboring laboratory but was away on sabbatical, it became clear that this enzyme might have very practical uses for the analysis of DNA.

R. Roberts – PNAS 2005

MCB 140 09-21-07 8

Radioautogram of 14C-labeled SV40 DNA cleaved with endonuclease R showing 11 distinct fragments. Figure 3 from:

Danna, K. & Nathans, D. (1971) Proc. Natl. Acad. Sci. USA 68, 2913-2917

MCB 140 09-21-07 9

plasmids

9.7

MCB 140 09-21-07 109.12 1.8 million

MCB 140 09-21-07 11

MCB 140 09-21-07 12

SV40(primate polyomavirus)

E. coli(resident of human GI tract)

Michael Rogers “Biohazard” (1977)

The question, quite simply, concerned the wisdom of transplanting SV40 genes, conceivably coding for tumor production, into a bacterium that not only lacks that capacity to begin with, but which lives in virtually every human gut on the planet.

+ = ?1972:

Berg: “ ‘My God! – people said – ‘You cannot put SV40 into E. coli! I think I was upset by the criticism at first, but then I went out and started to talk about the problem with a lot of people. … I realized that I’d been wrong many, many times before in predicting the outcome of an experiment, and that if I was wrong about my assessment of the risk in this experiment, then the consequences were not something that I would want to live with.”

Berg cancelled the experiment.

MCB 140 09-21-07 13

Science, September 1973: letter to the NAS from participants at Gordon conference on nucleic acids.

“We are writing … on behalf of a number of scientists to communicate a matter of deep concern. .We presently have the technical ability to join together, covalently, DNA molecules from diverse sources... This technique could be used, for example, to combine DNA from animal viruses with bacterial DNA... In this way, new kinds of hybrid plasmids or viruses, with biological activity of unpredictable nature, may eventually be created. These experiments offer exciting and interesting potential, both for advancing knowledge of fundamental biological processes, and for alleviation of human health problems. Certain such hybrid molecules may prove hazardous to laboratory workers and to the public. Although no hazard has yet been established, prudence suggests that the potential hazard be seriously considered.” Maxine Singer (Carnegie) Dieter Söll (Yale)

MCB 140 09-21-07 14

September 1974 – a moratorium?

New plasmids: novel antibiotic resistance markers into E. coli; Xenopus and Drosophila genomic DNA into E. coli.

Norton Zinder: “If we had any guts at all, we’d tell people to not do these experiments!”

MCB 140 09-21-07 15

MCB 140 09-21-07 16

MCB 140 09-21-07 17

Sept. ’74 – Feb’ 75

“Until the potential hazards of such recombinant DNA molecules have been better evaluated or until adequate methods are developed for preventing their spread, scientists throughout the world join with the members of this committee in voluntarily deferring the following experiments” – cloning new antibiotic resistance genes in currently naïve hosts and cloning fragments of oncoviral genomes into bacterial plasmids.

MCB 140 09-21-07 18

“an epidemic of cancer”?

MCB 140 09-21-07 19

Asilomar conference, February 1975

Paul Berg, David Baltimore, Sydney Brenner, Mike Bishop, Don Brown, Ron Davis, James Watson, Phil Sharp, Herb Boyer, Joshua Lederberg + 150 more people

+ Nature, Science, NY Times, Washington Post, Boston Globe, SF Chronicle, etc

MCB 140 09-21-07 20

(Left to right) Maxine Singer, Norton Zinder, Sydney Brenner, and Paul Berg

MCB 140 09-21-07 21

The measures

1. Moratorium lifted.2. Experiments will proceed under two levels of

containment, biological (use weakened host cell), and physical, the level of which will be gauged by the level of risk presented by the organism created (minimal, low, moderate, high):

1. Stringent, commonsense cleanliness (P1)2. Similar to surgical operating theatre (P2)3. Giant isolator (P3)4. Comparable to what one uses in biological warfare

research (P4)

MCB 140 09-21-07 22

Stringent, commonsense cleanliness (P1)

Similar to surgical operating theatre (P2)

Giant isolator (P3)

Comparable to what one uses in biological warfare research (P4)

invertebrates(including flies)

vertebrates

Where to draw the cutoff line, idea #1

MCB 140 09-21-07 23

Stringent, commonsense cleanliness (P1)

Similar to surgical operating theatre (P2)

Giant isolator (P3)

Comparable to what one uses in biological warfare research (P4)

invertebratesand cold-blooded

vertebrates (including frogs)

Warm-bloodedvertebrates

Where to draw the cutoff line, idea #2

MCB 140 09-21-07 24

Enter the sceptics

Robert Sinsheimer (CalTech):

• The dangers of “shotgunning” – evolutionary jumping? – creation of chimeras with completely unforeseen new properties?

• Prokaryote-to-eukatyote gene transfer?

• Use of E. coli as a host is potentially dangerous

MCB 140 09-21-07 25

Genomic library:

MCB 140 09-21-07 26

RNA splicing removes introns

• Exons – sequences found in a gene’s DNA and mature mRNA (expressed regions)

• Introns – sequences found in DNA but not in mRNA (intervening regions)

• Some eukaryotic genes have many introns

MCB 140 09-21-07 27

Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns

Fig. 8.15

MCB 140 09-21-07 28

Spliced Segments at the 5’ Terminus of Adenovirus 2 Late mRNASusan M. Berget, Claire Moore, and Phillip A. SharpProceedings of the National Academy of Sciences USA, Volume 74, 1977, pages 3171 3175

Also: Louise Chow, Richard Gelinas, Tom Broker, and Richard Roberts An amazing sequence arrangement at the 5 ends of adenovirus 2 messenger RNA, Cell 12: 1 8, 1977

MCB 140 09-21-07 29

MCB 140 09-21-07 30

MCB 140 09-21-07 31

How RNA processing splices out introns and adjoins adjacent exons

Fig. 8.16

MCB 140 09-21-07 32

• Splicing is catalyzed by spliceosomes– Ribozymes –

RNA molecules that act as enzymes

– Ensures that all splicing reactions take place in concert

Fig. 8.17

MCB 140 09-21-07 33

MCB 140 09-21-07 34

MCB 140 09-21-07 35

Chargaff E, Simring FR. “On the dangers of genetic meddling.” Science. (1976) 192:938

“What seems to have been disregarded completely is that we are dealing here much more with an ethical problem than one in public health, and that the principal question to be answered is whether we have the right to put an additional fearful load on generations that are not yet born… You can stop splitting the atom; you can stop visiting the moon… But you cannot recall a new form of life. Once you have constructed a viable E. coli cell carrying a plasmid DNA into which a piece of eukaryotic DNA has been spliced, it will survive you and your children and your children’s children. An irreversible attack on the biosphere is something so unheard of, so unthinkable to previous generations, that I could only wish that mine had not been guilty of. The hybridization of Prometheus with Herostratus is bound to give evil results. … My generation … has been the first to engage, under the leadership of exact sciences, in a destructive colonial war against nature. The future will curse us for it.

MCB 140 09-21-07 36

Enter the public, part 1

June 8, 1976: “Biohazards at Harvard: scientists will create new life forms – but how safe will they be?”

John Lear Recombinant DNA: the Untold Story

MCB 140 09-21-07 37

“Frankenstein monsters from Harvard Yard”

Mayor Alfred Vellucci: “They may come up with a disease that can’t be cured – even a monster! Is this the answer to Dr. Frankenstein’s dream?”

an open meeting of the Cambridge City Council, June 13, 1976

“Why didn’t someone advise the mayor of Cambridge and the CCC what was going on at Harvard?”

Resolution: ban all recombinant DNA research in the city, whether publicly or privately financed, for two years.

Establish Cambridge Experimentation Review Board. City Manager Sullivan chose to name an ERB consisting entirely of nonscientists.

John Lear Recombinant DNA: the Untold Story

MCB 140 09-21-07 38

Not only Cambridge

City ordinances requiring uniform compliance with city-specified controls on working with recombinant DNA:

Princeton, Emeryville, Berkeley (Sept. 77), Amherst, etc etc.

MCB 140 09-21-07 39

Enter the public, part 2

The opening session of a three-day forum in March 1977, sponsored by the National Academy of Sciences, … was disrupted by a citizens’ group, The People’s Business Commission, led by activist Jeremy Rifkin, whose members, chanting “We Will Not Be Cloned,” draped across the platform a banner with the caption, “We Will Create the Perfect Race.”

S. Wright Molecular Politics

MCB 140 09-21-07 40

MCB 140 09-21-07 41

The severe adverse events that have occurred in the subsequent 30 years of nonstop cloning of all

imaginable sorts of DNA into E. coli vectors

MCB 140 09-21-07 42

A more severe example of the society-genetics interface

MCB 140 09-21-07 43

Approximately 1.4 billion people, 25% of the world’s population, receive insufficient food to meet daily requirements.

Even with the heavy use of various pesticides, approximately 50% of the world’s food crops are destroyed in the field or in storage.

Levetin and McMahon Plants and Society

MCB 140 09-21-07 44

After the revolution of 1917, agriculture in Russia collapsed. Formerly one of the world’s greatest agricultural nations became a shadow of a shadow of its former self.

Something needed to be done.Stalin decided that the way to go

was nationalize agriculture. In 1930, some 10 million land-owning peasants were killed, and their land was transferred to the state.

Agricultural production levels were still unacceptably low, and hunger was ubiquitous. Some 5 million people perished in the Ukraine, formerly an agricultural paradise comparable to California, in the Great Famine of 1932-34.

Dmitry Mor Help – 1922

MCB 140 09-21-07 45

Technical problem

Getting better yielding, or more pest-resistant, or more cold-resistant crop varieties by conventional selection takes TIME.

MCB 140 09-21-07 46

MCB 140 09-21-07 47

Trofim Lysenko Iosif (Josef) Stalin

Nikolai VavilovPlant genetics and evolution

Sergei ChetverikovPopulation genetics

Nikolai KoltsovGenetics

See the book “Commissar Vanishes” and also:http://www.newseum.org/berlinwall/commissar_vanishes/

MCB 140 09-21-07 48

The two central tenets of Lysenkoism

1. Lamarck was right. Acquired traits can be inherited. From an agricultural perspective, this meant that plant lines with desired characteristics could be obtained by treating a set of plants a certain way, getting them to develop a desired characteristic, and then breeding them. It was then claimed that the F1 would have the parents’ trait. How could inheritance of acquired traits work?!

2. Weissman (“continuity of germ plasm”), Mendel (particulate inheritance) and Morgan (genes lie on chromosomes in the nucleus) are wrong. The entire cell (or the organism) is a carrier of hereditary information. “Gene” and “chromosome” are bourgeois, capitalist inventions.

MCB 140 09-21-07 49

Lysenko, 1936

“The most wonderful forms of animals and plants are found in Nature. … Man will be able to create similarly wonderful forms within an immeasurably shorter time frame … he will be able to create forms that have never existed and could not have appeared in Nature in a million years.”

MCB 140 09-21-07 50

1948: Apocalypse Now

Annual Meeting of the Soviet Academy of Agricultural Sciences:

Lysenko announces that “Comrade Stalin has not only read my talk, but approves of it.”

Result: genetics banned and declared a pseudoscience (“Weissmanism-Morganism-Mendelism”).

Geneticists are officially dubbed “fly lovers = people haters” (мухолюбы – человеконенавистники).

Countless geneticists go through the same experience as, earlier, Chetverikov (sent into exile, 1929) and Vavilov (who died in a prison camp, 1943).

MCB 140 09-21-07 51

Cheap populism and demagoguery:a textbook example

“Genetics, mendelism-morganism, the fruitlesness of which can be considered to be proven, has nothing to do with our plan to harvest enough grain. … There is a science [Lysenko’s “theories”] that is making a major contribution to our [goals in agriculture].

The truth is that our people, our working class and our peasants are decisively removing from science all that is decrepit, all that is against the people, all that is borne from a slave-like allegiance to the bourgeois West.

The truth is that [Lysenko’s “theories”] have been tested, appreciated, confirmed and beloved by the masses, and not only in [research institutes].”

A. Mikhalevich, speech at the 1948 agricultural academy annual meeting,cited in Nikolai Dubinin, “The History and Tragedy of Soviet Genetics.”

MCB 140 09-21-07 52

A perspective

The central phenomenon of Lysenkoism, and the enormity of the nightmare that ensued, was a blatant invasion of ideology into science.

The scientific method was abandonded, and notions were declared “right” or “wrong” based on whether they fit a certain ideology, not whether there are data supporting or refuting them.

It did not matter to Stalin’s henchmen in science, what the data showed. The only thing that mattered was whether a certain theory fit the ideology of lysenkoism. Morgan’s chromosome “theory” of inheritance, or Avery’s ‘theory” that DNA carries genetic information were proclaimed as wrong because they were the products of capitalist ideology.

MCB 140 09-21-07 53

Further reading

“Lysenko and the tragedy of Soviet science”Valery Soyfer

“The rise and fall of T. D. Lysenko”Zhores Medvedev

“The Lysenko affair”David Joravsky

MCB 140 09-21-07 54

A European Perspective on IDIt is a strange experience for a European biologist to read about the growing support in the United States for so-called "intelligent design," the current name for good old unintelligent creationism. Strangest of all, though, are the recent activities of the Kansas Board of Education. The Kansas Board's proposal to "[change] the definition of science" is unheard of in a western democracy, although similar activities have been common in dictatorships. In Nazi Germany, relativity was considered "Jewish science" and therefore unacceptable, while in the Soviet Union, modern genetics was rejected as unmarxist in favor of the ravings of the charlatan Lysenko. Is this the way the good citizens of Kansas (and the many other states where similar initiatives are seen) want to go? Obviously, there must be a profound ignorance of science and the scientific method among the U.S. public for such a thing to happen (an ignorance that intelligent design supporters evidently hope to perpetuate), and for this, scientists must be held responsible. There is too much looking down at colleagues who engage the public through popular science, such as the late Carl Sagan (1). All scientists, not just biologists, should realize that an attack on the very roots of science concerns every one of them, and accordingly, they should do their utmost to counteract it by actively participating in the debate. Ejnar J. FJERDINGSTAD Retired Professor of Anatomy, University of Aarhus, Denmark.

Science July 29, 2005

MCB 140 09-21-07 55

Wed/Fri

What if – if! – acquired characteristics could be inherited?...