Experiments

Sandy Feinstein

Penn State Berks

Bryan Shawn Wang

Penn State Berks

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The student whose honors thesis I am supervising is constructing an original language in which question marks can be placed anywhere in a sentence, not just at the end as in English. Our consideration of questions below is, in some respects, a result of where we have put our interrogatory symbol. Positioning and repositioning our questions reflect different ways of thinking, of learning, of understanding, and of discovering a shared purpose, too.

While Bryan majored in biology and created DNA that could be projected onto a computer screen, I didn’t study molecular biology in high school or college; neither computers nor molecular biology was an option. Before 1975, the year I set off for graduate school, the study of DNA was largely limited to aspiring scientists. Therefore, as Bryan and I exchanged our class plans for the interdisciplinary course we developed and teach together, I would ask, “but what do you mean? Why are you doing that? How do you know?” In short, I had a series of questions. In the draft stages of our co-authored articles, we would pose other questions in the margins: I have written, for example, “But what do you see?” And Bryan would try to explain what he saw, but that wasn’t what I meant. I meant, but how can you see that (with the subtext, but I can’t see that)? 

????

When Sandy asks, “What do you see?” about the molecules of life—the proteins and nucleic acids whose interactions drive the workings of every cell—about objects that are too small to literally see, I think of the first time I saw DNA. Not DNA precipitated from a solution of high salt and cold alcohol, silky white strands spooled onto a glass rod in a high school lab demonstration, but the blurred form of a double helix in a computer monitor. As a Ph.D. student, I’d spent months learning to grow crystals of proteins and DNA, after which I had directed an X-ray beam at the crystals; measured the intensities and positions of the diffracted radiation waves; and coaxed a Silicon Graphics workstation to synthesize these measurements, calculate the structure of the molecular complex arrayed in the crystals, and display it on the screen in front of me. It was breathtaking, those atoms winding up and down in a twinned spiral shape that previously had been accessible to me only through textbooks and papers. How did I know DNA was a double helix? Now, I could say I’d seen it for myself.

Sandy’s question, however—What do you see?—is not just an occasion to reminisce, but also an opportunity to reexamine. For what the scientist (or the scientist’s computer) renders in the monitor is not the thing itself but a model, an image determined by indirect means, and therefore open to scrutiny, doubt. But what is seeing, after all? We see, in the physical sense, when light waves reflect from objects and are focused by the lens in our eyes onto photoreceptor cells, where the light stimulates a series of changes in protein conformations and interactions that ultimately results in the transmission of an electrical signal (itself produced by a series of changes in protein conformations and the movement of charged atoms across a cellular membrane) via the optic nerve to the visual cortex, where an image (by means mysterious to me) develops. In other words, vision itself is indirect. And if even this most fundamental process for data acquisition is filtered, if there is no direct method of observing natural phenomena, how may we apprehend what is real and true in the world? The point that persuades—even if it’s not the proof (even if it’s not the truth)—is in the application of the observations, the deductions.

And, yes, the questions. When Sandy hands me medieval works of literature, I receive them with awe. Neither history, languages, nor philosophy is my thing. While I can admire the beauty of the bestiaries, understand the idea of God as the ultimate authority, enjoy stories of dragons and knights, I’m not only wandering a strange land, I’m feeling completely displaced. My questions go unasked, for the most part. But perhaps not unformulated, for somehow I’ve learned something—about medievalism, and about molecular biology, too. For instance, that metaphors not only help us represent our world, but also are at the core of how we learn about it. That quests and adventures demand patience and bravery; the path to insight may be circuitous, even circular, and only dimly lit, if at all. That the question is every bit as significant as the answer.

?___?

The experiment that follows grew out of our questions to one another: the medievalist to the molecular biologist; and, sometimes, the molecular biologist to the medievalist— as well as the ones we have discovered together. How and why does one ask questions? Where and why does one look for answers? Is it different in the humanities than in the sciences? What emerges when we isolate these contrasting assumptions and approaches—and connect them?  

Sandy: Asking Questions, Seeking Truth

In graduate school, where I was a teaching assistant, a former student of mine exclaimed one day, “Men seek truth.” I didn’t call him out. I laughed. Were he a science major, I might now understand, but he was, like me, an English major. 

I would end up writing a thesis in early literature that examined how romance identity is constructed. I argued that the hero becomes his identity through cumulative actions, including serving a Lady and often marrying her.1 The hero’s actions define his masculine identity and have little to do with truth, unless he is a Grail knight like Parzival. Though Parzival wins tournaments and is knighted, he will not be completed by the love of a woman. More important to the fulfillment of his quest and, therefore, identity, is knowing he must ask a question. His asking of a question will heal Anfortas, the Grail or Fisher King, who has been wounded by a spear in his thigh, a likely euphemism suggesting the nature of the King’s transgression.2 Parzival fails the first time he comes in the presence of Anfortas. And the question he should have asked was simple, “What ails thee?”

When Parzival first fails this test, he has been invited to the Grail castle by a figure he assumes from appearances to be a fisherman; there, he witnesses an elaborate ritual and feast. The narrator is careful to say that while Parzival did not “fail to notice the richness and the great wonder, for courtesy’s sake he refrained from questions,”3 applying the lesson he was taught not long before: “Gurnemanz counseled me in all sincerity not to ask many questions,” he thought. Reflecting on this lesson, he concludes that “without any questions I shall hear how this knightly company fairs.” Immediately following this thought, a squire, bearing a rich sword and sheath, approaches and offers them to him while explaining their virtues: this presentation is the signal to ask a question that he would have had to deduce, intuit, or feel in his spirit, but he does not. As the narrator says, “Alas that he did not ask the question then! I still sorrow for him on that account. For when the sword was put into his hand, it was a sign to him that he should ask. And I pity too his sweet host whom God’s displeasure does not spare and who could have been freed from it by a question” (131). 

The question, “What ails thee,” is not intended to elicit a perfunctory response as “How are you?” does today: “I’m fine thank you. And you?” The asking of the question, and what is asked, relate, ultimately, to a spiritual truth as well as physical reality. Anfortas’s wound is symbolic; it results in the wasteland that T.S. Eliot recast of a spiritually empty post-war London that’s lost sight of the questions to ask as well. The healing of the Fisher King signifies the needed restoration of the land and of the soul. But as is characteristic in allegory, the wound is literal, too: the King is in constant pain, unable to ride, and kept alive only by the miraculous virtues of the life-giving Grail.

In Christian allegory, all must seek the Truth, for the journey is to God. The Truth is not amorphous, some vague individual goal. Nor is the Truth knowledge, except insofar as knowledge is God. Questions lead to truth, God’s Truth.

In Plato’s “Allegory of the Cave,” Socrates asks questions that challenge the substantiality, even reality, of the physical world, which is but a shadow that obscures truth. Thomas Aquinas, born after Wolfram von Eschenbach, also structures his Summa Theologia around questions to prove the existence of God and that God is Truth. 

The questions—and conclusions to be drawn from them—are themselves now contentious. Who owns truth? Do questions lead to it? 

For physicians, those in our time expected to heal our wounds, conflicts concerning the ascertaining of truth are a matter of life and death. The diagnoses and treatments are not typically spiritual or symbolic. It was salutary, then, to read how the medical doctor Michael Susmoy Sanatani addressed his hospital’s recommendation to omit physician-patient dialogue to save time and money. In “The Anfortas Question,” he begins with two questions:

Why should we now bother taking a history beyond the presenting complaint at all, if, in the age of the electronic patient record, all we have to do is click ‘copy’ for the rest? Is the skill of taking a complete, diagnostically helpful history outdated, and should we stop teaching it to the future generations of medical students?4

He answers these questions by “contemplating” what he calls “the earliest descriptions of a medical history”—or, to be more precise, the devastating outcome of not taking a history. The consequence of withholding a vital question is to extend the patient-king’s suffering and highlight the failure of the would be healer-hero. 

Sanatani recognizes that it is the question that matters, not the answer. He concludes that the purpose of the question 

I think must have been asked not just to gather information or to help him [Parzival] understand the situation, but also because he felt compassion toward the suffering patient. A fundamental way to express compassion is to enquire about the inner state of the other. And it was that component of Parzival’s brief, but effective history that had a therapeutic effect.

Poor Parzival. On the one hand, a subject is expected to answer, not question, someone of higher rank; on the other, the circumstance he, and only he faces, requires he recognize the need for an exception and speak, more specifically, that he ask the king a question. His failure to do so the first time he meets Anfortas is ascribed in the tale to his youth, to his ignorance, to being brought up in the country unaware of his noble lineage, even as one who lacks the Christian virtue of charity. Here he has eschewed presumption for humility and adhered to conventional protocols only to transgress and fail. He won’t make that mistake twice.

Is it a question of compassion or magic that will break the spell and cure the wound? What is the role or the need of an audience for verbalized questions? The “inner state” in this story is that of the soul: a matter of purity, atonement, expiation, divine forgiveness. Parzival is put in the role more of priest than physician with the expectation that he should ask a question that in Sanatani and elsewhere is translated as, “What is it that troubles you?” What sin have you to confess?

How we read Parzival’s question, and how we ask questions, reveal our subject positions: Sanatani as a physician, I as a medievalist. Sanatani is responding to having his responsibility for his patient’s well-being limited and, by implication, questioned. His reading of Parzival’s initial failure and later success is informed by his experience as a medical student and resident taking histories, asking questions and learning how to meet his patients’ needs. For me, a physician using a medieval source as a model and argument for a clinical practice is an unexpected and welcome use of medieval romance—a genre more likely to be overlooked, or worse, than to be used in support of patient treatment. 

The purpose and expectation of a question for most is an answer, the gathering of information. There is no answer in Parzival. It’s not the point. 

Parzival’s question is rhetorical in the sense that no answer is expected. But the question also lacks apparent rhetorical purpose: that is, no one is to trying to persuade anyone of anything—unless Parzival’s question is intended to show God he is worthy, or to reveal to others that he is. The term, “Rhetorical Question,” itself postdates Parzival and is first used in the eighteenth century by a lord condescending to the commons for a political purpose (OED): “To this Rhetorical Question the Commons pray they may Answer by another Question.” Parzival’s question will resolve the lineal descent, so in that sense it may be seen as political—the bumpkin knight will become king. But the urgency of the question has less to do with matters of inheritance: asking the question not only cures Anfortas but is necessary for the healing of the human spirit itself. God works in mysterious ways—even through language. The fulfilled quest is a question asked. 

Bryan: Restriction and Ligation

How does a scientist establish something about the nature of things—and then construct a persuasive argument that what has been established is true, or at least is closer to the true nature of things than what others had previously established? Questions are involved, certainly, but questions alone cannot comprise the quest.

In a sophomore-level molecular biology laboratory course, I teach students to cut DNA. Instead of scissors or knives, they employ restriction enzymes—large protein molecules that ordinarily serve as a line of defense for the bacteria that produce them. In nature, the enzymes chop up the DNA of viruses that would invade and subvert the bacteria’s cellular machinery; the enzymes thereby “restrict” the viruses’ growth. Since the 1970s, scientists have co-opted these enzymes—and others that can reconnect, or ligate, DNA fragments—to produce recombinant DNA, hybrid genetic material from different sources. It is a technology that’s integral to modern biomedical research and that held such fascination for me when I was a student that it spurred me into my career as a molecular biologist.

The students can’t actually see the enzymes or grasp them; the molecules are much too small. They can’t even see the DNA they’re cutting. 

Cut: it’s a metaphor. The phrase “cutting DNA” is shorthand for the physical separation of one collection of atoms—one region of the original DNA molecule—from another. Shorthand for how the enzyme provides a nanoenvironment for appropriate atomic assemblages to congregate, and for electrons within the reactive partners to rearrange. Shorthand for a chemical reaction that my students may or may not remember having studied a semester or two earlier. In the educational videos and textbooks, the atoms and electrons are represented by letters and dots, the reactions by lines and arrows, the molecules by ribbons and blobs. Amid these symbols, what’s real and what’s metaphorical? If we can’t see molecules, atoms, electrons, how do we know they’re there and that they behave as we’re told they do?

Sometimes science starts with an observation, something that the scientist notices, that stimulates their curiosity, a question. The question, however, isn’t sufficient. The work also requires answers. Not the answer—the answers may supplant what’s already been established (tentatively), or they themselves may prove easily discardable. These answers are conjectures put forward to explain the observations: they are hypotheses. Experiments are then conducted, yielding data to help sort through and refine those hypotheses.

The students, if they’ve done the assigned reading, have seen the observations, hypotheses, experiments—as well as the questions—about how restriction enzymes function. Conceivably, the students could replicate the reported studies, given the time, money, equipment, and know-how.

The manual explains that different bacteria produce different restriction enzymes. Each enzyme scans the double helix with an eye for a unique combination of four possible atomic clusters called nucleotides: adenine, cytosine, guanine, and thymine. Each enzyme reads the DNA for a specific sequence of the nucleotide letters A, C, G, and T. For instance, the enzyme EcoRI looks for the six-letter word GAATTC and cuts the DNA between the G and the first A, yielding a piece ending in G and another starting with A. 

Of course, the idea of DNA being composed of letters and words that are read by proteins is metaphorical, too. What is meant is that the molecular shape and chemical nature of the EcoRI restriction enzyme precisely matches the shape and the chemistry of a region of the double helix containing GAATTC, and they thereby may grasp one another in an atomic-level embrace. That interaction between enzyme and DNA allows the bond holding the double helix together to be broken. (And this happens twice: EcoRI cuts between the G and the A on both sides of the double helix, which is symmetric at this site.) 

Colorful drawings produced by world-leading research institutes aside, how do we know this truly is the function of EcoRI? Some molecular biologists, some time ago (Hedgpeth 1972) cut DNA with EcoRI, attached a tag to the beginning of the fragment that had just been lopped off, and then sliced and diced the fragment to bits, to individual letters. (In other words, they deconstructed the larger DNA molecule into individual nucleotides.) They found the smallest tagged bit was an adenine nucleotide: an A. When the piece was chopped to not-quite-so-fine bits, they found a tagged piece that was AA, another AAT, AATT, AATTC.

How did they know how to tag the fragment of DNA, or how to mince it, or identify which letters were in which bits? How did they know a G nucleotide preceded the cut? Read (with skepticism5) their paper, and the papers they reference, and the papers referenced therein. 

And so on, for every assertion made—about the cutting of the DNA, and also how DNA may be stained by a chemical that fluoresces under ultraviolet light when it inserts between nucleotides in a double helix, how DNA is electrically charged and therefore moves in an electric field, through a porous gel, with smaller fragments of DNA moving faster through the holes in the gel, so that with such an electrophoresis procedure, a scientist can separate and analyze the relative amounts and sizes of DNA fragments in a mixture.

The students add one tiny drop of clear liquid taken from a tube labeled enzyme to another drop of clear liquid from a tube labeled DNA. The DNA given to the students contains a single cutting site, just past the halfway point, so each DNA molecule should be snipped into two pieces of slightly different length. The students transfer the mixture—and, separately, a drop containing only the DNA, untouched by enzyme—to a porous gel containing the chemical that fluoresces when it inserts between nucleotides. They submerge the gel in a tank filled with a conductive solution, connect electrical leads at either end of the tank, and flip the switch. After thirty minutes, they turn off the power, disconnect the leads, remove the gel from the tank, and place it under an ultraviolet lamp. Where there is DNA in the gel, it glows.

The electrical current has forced the DNA along a path from the top of the gel to the bottom. In the lane leading from where the uncut DNA, not mixed with enzyme, was put into the gel, there is a single fluorescent band. On a parallel track, farther along from where the cut DNA was placed, there are two.

The enzyme appears to have cut the DNA. At least, the observations are consistent with the students’ (the laboratory manual’s) expectations: the restriction enzyme, when mixed with the DNA sample, catalyzed the chemical reaction that split each DNA molecule at the precise location mandated by the physical interaction of the enzyme with those particular nucleotides, and the two resulting fragments of DNA were shorter than the original DNA molecule and thus moved faster through the gel during the electrophoresis, as demonstrated by their relative locations at the end of the experiment.

One student points out that the lane containing his cut DNA has three glowing bands rather than two. How is he to interpret the difference? Another student observes that the third band is at the same relative position as the band for the uncut DNA. They hypothesize that some, but not all, molecules of DNA were cut. When the first student repeats the exercise, this time ensuring the drop of enzyme is fully mixed with the drop of DNA, he obtains the same results as the rest of the class. He repeats the exercise and confirms the outcome of his second trial.

Another student asks how we know the enzyme is cutting at the exact site the manual claims it cuts at. From an internet database run by the National Center for Biotechnology Information, she downloads the full sequence of the DNA she’s been given. She scans the letters, finds the alleged recognition site for the enzyme, and calculates that the original 649-nucleotide-long DNA should be cut into fragments that are 298 and 351 nucleotides, respectively. She repeats the experiment, but this time also includes a ladder—a sample of DNA with fragments of known sizes—in another lane of the electrophoresis gel. The patterns of bands match what she anticipated: uncut DNA moves at a rate between that of fragments that are 600 and 700 nucleotides long, while one of the bands in the cut DNA is between 300 and 400, and the second is just about 300. From the literature, she learns that the length of DNA and its migration in gel electrophoresis should share a logarithmic relationship. She measures the migration of each band with a ruler and constructs a semilog plot using the given fragment lengths of the bands in the ladder. The data points coincide with the equation of the calculated line of best fit with a coefficient of determination greater than 0.99. The calculated sizes of the cut DNA fragments, according to the observed migration of the two bands and the graph she’s drawn, are 290 and 345 nucleotides. She begins thinking about error estimation.

Another student puzzles over the metaphor of the DNA being read by the enzyme. If the DNA is composed of letters and words, he reasons, changing the letters—the spelling of the words—should change the meaning of the DNA. Put another way, if the restriction enzyme truly cuts at the site claimed by the manual, the textbooks, and the literature, then altering the nucleotides at that site, and that site only, should prevent the enzyme from cutting the DNA. The working model of how the EcoRI restriction enzyme recognizes its GAATTC site—a protein reading the language of DNA—has accumulated sufficient meaning to enable additional inquiry.

The student learns how to generate and purify more DNA; to mutate it, replacing one of the A nucleotides in the recognition site with a G; to verify the sequence of the DNA has indeed been changed. This takes him most of the next term, as he works over ten hours each week under the supervision of an emeritus professor who lets him share her bench, equipment, and supplies; points him to papers with the relevant theory, prior results, and protocols; advises him to apply for the $500 in funding he needs to purchase the reagents; rejoices alongside him after his gel shows that the mutated DNA, when exposed to the enzyme, still migrates as a single fragment: DNA with an altered recognition sequence is not cut.

Later, when he’s presenting the data in an undergraduate research symposium, a snarky chemist asks him how he can be certain the lack of cutting is due to mutation of the recognition site rather than, say, some contaminant in the preparation of mutated DNA preventing the enzyme from working.

That night, the student begins planning experiments in which he’ll mix equal quantities of the original and the mutated DNA, expose the mixture to the enzyme, and demonstrate (he hopes) that exactly half of the DNA is cut. He will purify the cut pieces away from those that remain uncut and glue them back together, ligate them. He will sequence the ligated DNA, and if his prediction is correct, show that the ligation has reconstructed the original, but not the mutated, recognition site.

The success of any experiment in molecular biology, even a student’s classroom laboratory exercise, requires a series of coordinated physicochemical events, along with an accurate understanding of those phenomena and their connections.6 The tenets that underlie the experiment must be sound and in alignment, like the parts of an intricate machine. The workings of that machine—the operation of every individual component and the proper linkages among them—represents the small, perhaps infinitesimal, aspect of nature that has been, if not established, then more closely approached. That the machine works at all comprises the argument.

Afterwords

“That’s wonderful,” he said, not the least bit miffed. “That’s exactly what it’s like when you’re out in the field and you’re first encountering some of those marvelously strange natural adaptations. At first all you’ve got is a few disconnected pieces of raw information, the sheerest glimpses, but you let your mind go, fantasizing the possible connections, projecting the most fanciful life cycles. In a way it’s my favorite part of being a scientist—later on, sure, you have to batten things down, contrive more rigorous hypotheses and the experiments through which to check them out, everything all clean and careful. But that first take—those first fantasies. Those are the best.”

-Lawrence Weschler, Mr. Wilson’s Cabinet of Wonder, 1997, p. 66

“poetry = the best words in the best order”

-Samuel Taylor Coleridge, posthumously quoted in Table Talk, 1835

Notes

1. This argument would serve as the basis of my first scholarly publication, with a twist, for rather than focusing on the men, I focused on the women (at least in the title): “Whatever Happened to the Women in Folktale?” Women’s Studies International Forum 9 (1986): 251-56.

2. Cf. “thigh” in Jessie L. Weston’s verse translation of Wolfram von Eschenbach’s Parzival, vol 1 of 2 (New York: G. E. Stechert & Co., 1912. Rpt. Of London, 1894 ed.), Bk IX, line 799, https://www.gutenberg.org/files/47297/47297-h/47297-h.htm and “genitals” in Cyril Edwards’ prose translation of the same lines in The Romance of Arthur: an Anthology of Medieval Texts in Translation, ed. Norris J. Lacy and James J. Wilhelm (London and New York: Routledge, 2013), p. 198. Still more explicit is the translation “testicles” in the translation by Helen M. Mustard and Charles E. Passage, Parzival (New York: Vintage, 1961), p. 256. Similarly, the question Parzival is expected to ask is also variously translated, in Weston, IX.885, “What aileth thee?”; in Edwards, 199, “What is the nature of your distress?” 

3. All quotations are from the Mustard and Passage Parzival and will be cited in text by page number, unless otherwise noted.

4. Michael Susmoy Sanatani, “The Anfortas Question,” SMAJ 182.11 (10 Aug 2010): 1268. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917944/

5. The Nobel-prize winning physicist Richard Feynman, speaking at the annual meeting of the National Science Teachers Association in 1966 (in a speech republished in The Physics Teacher, 1969), explained that “you have as much right as anyone else, upon hearing about the experiments—but be patient and listen to all the evidence—to judge whether a sensible conclusion has been arrived at.”

6. And, of course, the absence of technical blunders on the part of the experimentalist.

About the Authors

Sandy Feinstein, Professor of English and Honors Program Coordinator at Penn State Berks, has published on medieval literature, including Chaucer, Malory, Chrétien, The Second Shepherd’s Play, and Sir Gawain and the Green Knight, among others. She has also published creative writing—experimental and otherwise—in a range of print and online journals.

Bryan Shawn Wang is Associate Teaching Professor in Biology at Penn State Berks. Originally trained as a molecular biologist, he has published and patented research in protein design and engineering. He also has published creative work in numerous literary journals.

Sandy and Bryan’s writing on interdisciplinary teaching and research has appeared in Angles: New Perspectives on the Anglophone World, Comparative Media Arts Journal, CEA: The Critic, and New Chaucer Studies: Pedagogy & Professions.

Works Cited

Eschenbach, Wolfram von. Parzival. Trans. Helen M. Mustard and Charles E. Passage, New York: Vintage, 1961.

---. Parzival. Trans. Jessie Weston. New York: G. E. Stechert & Co., 1912. Rpt. of London, 1894 ed. https://www.gutenberg.org/files/47297/47297-h/47297-h.htm

---. “Parzival.” Trans. Cyril Edwards. In The Romance of Arthur: an Anthology of Medieval Texts in Translation. Ed. Norris J. Lacy and James J. Wilhelm. London and New York: Routledge, 2013.

Feynman, Richard. “What Is Science.” The Physics Teacher. 7. 1969: 313.

Hedgpeth, Joe, Howard M. Goodman, and Herbert W. Boyer. “DNA Nucleotide Sequence Restricted by the RI Endonuclease.” Proceedings of the National Academy of Sciences. 69.11. 1972: 3448-3452.

Kim, Youngchang, John C. Grable, Robert Love, Patricia J. Greene, and John M. Rosenberg. “Refinement of Eco RI Endonuclease Crystal Structure: a Revised Protein Chain Tracing.” Science. 249.4974. 1990: 1307-1309.

Sanatani, Michael Susmoy. “The Anfortas Question.” Canadian Medical Association Journal.  182.11. 2010: 1268. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917944/