Making sense of noise: introducing students to stochastic processes in order to better understand biological behaviors (and even free will).

 Biological systems are characterized by the ubiquitous roles of weak, that is, non-covalent molecular interactions, small, often very small, numbers of specific molecules per cell, and Brownian motion. These combine to produce stochastic behaviors at all levels from the molecular and cellular to the behavioral. That said, students are rarely introduced to the ubiquitous role of stochastic processes in biological systems, and how they produce unpredictable behaviors. Here I present the case that they need to be and provide some suggestions as to how it might be approached.  

Background: Three recent events combined to spur this reflection on stochasticity in biological systems, how it is taught, and why it matters. The first was an article describing an approach to introducing students to homeostatic processes in the context of the bacterial lac operon (Booth et al., 2022), an adaptive gene regulatory system controlled in part by stochastic events. The second were in-class student responses to the question, why do interacting molecules “come back apart” (dissociate).  Finally, there is the increasing attention paid to what are presented as deterministic genetic factors, as illustrated by talk by Kathryn Harden, author of the “The Genetic Lottery: Why DNA matters for social equality” (Harden, 2021).  Previous work has suggested that students, and perhaps some instructors, find the ubiquity, functional roles, and implications of stochastic, that is inherently unpredictable processes, difficult to recognize and apply. Given their practical and philosophical implications, it seems essential to introduce students to stochasticity early in their educational journey.  

What is stochasticity and why is it important for understanding biological systems? Stochasticity results when intrinsically unpredictable events, e.g. molecular collisions, impact the behavior of a system. There are a number of drivers of stochastic behaviors. Perhaps the most obvious, and certainly the most ubiquitous in biological systems is thermal motion. The many molecules within a solution (or a cell) are moving, they have kinetic energy – the energy of motion and mass. The exact momentum of each molecule cannot, however, be accurately and completely characterized without perturbing the system (echos of Heisenberg). Given the impossibility of completely characterizing the system, we are left uncertain as to the state of the system’s components, who is bound to whom, going forward. 

Through collisions energy is exchanged between molecules.  A number of chemical processes are driven by the energy delivered through such collisions. Think about a typical chemical reaction. In the course of the reaction, atoms are rearranged – bonds are broken (a process that requires energy) and bonds are formed (a process that releases energy). Many (most) of the chemical reactions that occur in biological systems require catalysts to bring their required activation energies into the range available within the cell.   [1]  

What makes the impact of thermal motion even more critical for biological systems is that many (most) regulatory interactions and macromolecular complexes, the molecular machines discussed by Alberts (1998) are based on relatively weak, non-covalent surface-surface interactions between or within molecules. Such interactions are central to most regulatory processes, from the activation of signaling pathways to the control of gene expression. The specificity and stability of these non-covalent interactions, which include those involved in determining the three-dimensional structure of macromolecules, are directly impacted by thermal motion, and so by temperature – one reason controlling body temperature is important.  

So why are these interactions stochastic and why does it matter?  A signature property of a stochastic process is that while it may be predictable when large numbers of atoms, molecules, or interactions are involved, the behaviors of individual atoms, molecules, and interactions are not. A classic example, arising from factors intrinsic to the atom, is the decay of radioactive isotopes. While the half-life of a large enough population of a radioactive isotope is well defined, when any particular atom will decay is, in current theory, unknowable, a concept difficult for students (see Hull and Hopf, 2020). This is the reason we cannot accurately predict whether Schrȍdinger’s cat is alive or dead. The same behavior applies to the binding of a regulatory protein to a specific site on a DNA molecule and its subsequent dissociation: predictable in large populations, not-predictable for individual molecules. The situation is exacerbated by the fact that biological systems are composed of cells and cells are, typically, small, and so contain relatively few molecules of each type (Milo and Phillips, 2015). There are typically one or two copies of each gene in a cell, and these may be different from one another (when heterozygous). The expression of any one gene depends upon the binding of specific proteins, transcription factors, that act to activate or repress gene expression. In contrast to a number of other cellular proteins, “as a rule of thumb, the concentrations of such transcription factors are in the nM range, corresponding to only 1-1000 copies per cell in bacteria or 103-106 in mammalian cells” (Milo and Phillips, 2015). Moreover, while DNA binding proteins bind to specific DNA sequences with high affinity, they also bind to DNA “non-specifically” in a largely sequence independent manner with low affinity. Given that there are many more non-specific (non-functional) binding sites in the DNA than functional ones, the effective concentration of a particular transcription factor can be significantly lower than its total cellular concentration would suggest. For example, in the case of the lac repressor of the bacterium Escherichia coli (discussed further below), there are estimated to be ~10 molecules of the tetrameric lac repressor per cell, but “non-specific affinity to the DNA causes >90% of LacI copies to be bound to the DNA at locations that are not the cognate promoter site” (Milo and Phillips, 2015); at most only a few molecules are free in the cytoplasm and available to bind to specific regulatory sites.  Such low affinity binding to DNA allows proteins to undergo one-dimensional diffusion, a process that can greatly speed up the time it takes for a DNA binding protein to “find” high affinity binding sites (Stanford et al., 2000; von Hippel and Berg, 1989). Most transcription factors bind in a functionally significant manner to hundreds to thousands of gene regulatory sites per cell, often with distinct binding affinities. The effective binding affinity can also be influenced by positive and negative interactions with other transcription and accessory factors, chromatin structure, and DNA modifications. Functional complexes can take time to assemble, and once assembled can initiate multiple rounds of polymerase binding and activation, leading to a stochastic phenomena known as transcriptional bursting. An analogous process occurs with RNA-dependent polypeptide synthesis (translation). The result, particularly for genes expressed at lower levels, is that stochastic (unpredictable) bursts of transcription/translation can lead to functionally significant changes in protein levels (Raj et al., 2010; Raj and van Oudenaarden, 2008).

Figure adapted from Elowitz et al 2002

There are many examples of stochastic behaviors in biological systems. Originally noted by Novick and Weiner (1957) in their studies of the lac operon, it was clear that gene expression occurred in an all or none manner. This effect was revealed in a particularly compelling manner by Elowitz et al (2002) who used lac operon promoter elements to drive expression of transgenes encoding cyan and yellow fluorescent proteins (on a single plasmid) in E. coli.  The observed behaviors were dramatic; genetically identical cells were found to express, stochastically, one, the other, both, or neither transgenes. The stochastic expression of genes and downstream effects appear to be the source of much of the variance found in organisms with the same genotype in the same environmental conditions (Honegger and de Bivort, 2018).

Beyond gene expression, the unpredictable effects of stochastic processes can be seen at all levels of biological organization, from the biased random walk behaviors that underlie various forms of chemotaxis (e.g. Spudich and Koshland, 1976) and the search behaviors in C. elegans (Roberts et al., 2016) and other animals (Smouse et al., 2010), the noisiness in the opening of individual neuronal voltage-gated ion channels (Braun, 2021; Neher and Sakmann, 1976), and various processes within the immune system (Hodgkin et al., 2014), to variations in the behavior of individual organisms (e.g. the leafhopper example cited by Honegger and de Bivort, 2018). Stochastic events are involved in a range of “social” processes in bacteria (Bassler and Losick, 2006). Their impact serves as a form of “bet-hedging” in populations that generate phenotypic variation in a homogeneous environment (see Symmons and Raj, 2016). Stochastic events can regulate the efficiency of replication-associated error-prone mutation repair (Uphoff et al., 2016) leading to increased variation in a population, particularly in response to environmental stresses. Stochastic “choices” made by cells can be seen as questions asked of the environment, the system’s response provides information that informs subsequent regulatory decisions (see Lyon, 2015) and the selective pressures on individuals in a population (Jablonka and Lamb, 2005). Together stochastic processes introduce a non-deterministic (i.e. unpredictable) element into higher order behaviors (Murakami et al., 2017; Roberts et al., 2016).

Controlling stochasticity: While stochasticity can be useful, it also needs to be controlled. Not surprisingly then there are a number of strategies for “noise-suppression”, ranging from altering regulatory factor concentrations, the formation of covalent disulfide bonds between or within polypeptides, and regulating the activity of repair systems associated with DNA replication, polypeptide folding, and protein assembly via molecular chaperones and targeted degradation. For example, the identification of “cellular competition” effects has revealed that “eccentric cells” (sometimes, and perhaps unfortunately referred to as of “losers”) can be induced to undergo apoptosis (die) or migration in response to their “normal” neighbors (Akieda et al., 2019; Di Gregorio et al., 2016; Ellis et al., 2019; Hashimoto and Sasaki, 2020; Lima et al., 2021).

Student understanding of stochastic processes: There is ample evidence that students (and perhaps some instructors as well) are confused by or uncertain about the role of thermal motion, that is the transfer of kinetic energy via collisions, and the resulting stochastic behaviors in biological systems. As an example, Champagne-Queloz et al (2016; 2017) found that few students, even after instruction through molecular biology courses, recognize that collisions with other molecules were  responsible for the disassembly of molecular complexes. In fact, many adopt a more “deterministic” model for molecular disassembly after instruction (see part A panel figure on next page). In earlier studies, we found evidence for a similar confusion among instructors (part B of figure on the next page)(Klymkowsky et al., 2010). 

Introducing stochasticity to students: Given that understanding stochastic (random) processes can be difficult for many (e.g. Garvin-Doxas and Klymkowsky, 2008; Taleb, 2005), the question facing course designers and instructors is when and how best to help students develop an appreciation for the ubiquity, specific roles, and implications of stochasticity-dependent processes at all levels in biological systems. I would suggest that  introducing students to the dynamics of non-covalent molecular interactions, prevalent in biological systems in the context of stochastic interactions (i.e. kinetic theory) rather than a ∆G-based approach may be useful. We can use the probability of garnering the energy needed to disrupt an interaction to present concepts of binding specificity (selectivity) and stability. Developing an understanding of the formation and  disassembly of molecular interactions builds on the same logic that Albert Einstein and Ludwig Böltzman used to demonstrate the existence of atoms and molecules and the reversibility of molecular reactions (Bernstein, 2006). Moreover, as noted by Samoilov et al (2006) “stochastic mechanisms open novel classes of regulatory, signaling, and organizational choices that can serve as efficient and effective biological solutions to problems that are more complex, less robust, or otherwise suboptimal to deal with in the context of purely deterministic systems.”

The selectivity (specificity) and stability of molecular interactions can be understood from an energetic perspective – comparing the enthalpic and entropic differences between bound and unbound states. What is often missing from such discussions, aside from the fact of their inherent complexity, particularly in terms of calculating changes in entropy and exactly what is meant by energy (Cooper and Klymkowsky, 2013) is that many students enter biology classes without a robust understanding of enthalpy, entropy, or free energy (Carson and Watson, 2002).  Presenting students with a molecular  collision, kinetic theory-based mechanism for the dissociation of molecular interactions, may help them better understand (and apply) both the dynamics and specificity of molecular interactions. We can gage the strength of an interaction (the sum of the forces stabilizing an interaction) based on the amount of energy (derived from collisions with other molecules) needed to disrupt it.  The implication of student responses to relevant Biology Concepts Instrument (BCI) questions and beSocratic activities (data not shown), as well as a number of studies in chemistry, is that few students consider the kinetic/vibrational energy delivered through collisions with other molecules (a function of temperature), as key to explaining why interactions break (see Carson and Watson, 2002 and references therein).  Although this paper is 20 years old, there is little or no evidence that the situation has improved. Moreover, there is evidence that the conventional focus on mathematics-centered, free energy calculations in the absence of conceptual understanding may serve as an unnecessary barrier to the inclusion of a more socioeconomically diverse, and under-served populations of students (Ralph et al., 2022; Stowe and Cooper, 2019). 

The lac operon as a context for introducing stochasticity: Studies of the E. coli  lac operon hold an iconic place in the history of molecular biology and are often found in introductory courses, although typically presented in a deterministic context. The mutational analysis of the lac operon helped define key elements involved in gene regulation (Jacob and Monod, 1961; Monod et al., 1963). Booth et al (2022) used the lac operon as the context for their “modeling and simulation lesson”, Advanced Concepts in Regulation of the Lac Operon. Given its inherently stochastic regulation (Choi et al., 2008; Elowitz et al., 2002; Novick and Weiner, 1957; Vilar et al., 2003), the lac operon is a good place to start introducing students to stochastic processes. In this light, it is worth noting that Booth et al describes the behavior of the lac operon as “leaky”, which would seem to imply a low, but continuous level of expression, much as a leaky faucet continues to drip. As this is a peer-reviewed lesson, it seems likely that it reflects widely held mis-understandings of how stochastic processes are introduced to, and understood by students and instructors.

E. coli cells respond to the presence of lactose in growth media in a biphasic manner, termed diauxie, due to “the inhibitory action of certain sugars, such as glucose, on adaptive enzymes (meaning an enzyme that appears only in the presence of its substrate)” (Blaiseau and Holmes, 2021). When these (preferred) sugars are depleted from the media, growth slows. If lactose is present, however, growth will resume following a delay associated with the expression of the proteins encoded by the operon that enables the cell to import and metabolize lactose. Although the term homeostatic is used repeatedly by Booth et al, the lac operon is part of an adaptive, rather than a homeostatic, system. In the absence of glucose, cyclic AMP (cAMP) levels in the cell rise. cAMP binds to and activates the catabolite activator protein (CAP), encoded for by the crp gene. Activation of CAP leads to the altered expression of a number of target genes, whose products are involved in adaption to the stress associated with the absence of common and preferred metabolites. cAMP-activated CAP acts as both a transcriptional repressor and activator, “and has been shown to regulate hundreds of genes in the E. coli genome, earning it the status of “global” or “master” regulator” (Frendorf et al., 2019). It is involved in the adaptation to environmental factors, rather than maintaining the cell in a particular state (homeostasis). 

The lac operon is a classic polycistronic bacterial gene, encoding three distinct polypeptides: lacZ (β-galactosidase), lacY (β-galactoside permease), and lacA (galactoside acetyltransferase). When glucose or other preferred energy sources are present, expression of the lac operon is blocked by the inactivity of CAP. The CAP protein is a homodimer and its binding to DNA is regulated by the binding of the allosteric effector cAMP.  cAMP is generated from ATP by the enzyme adenylate cyclase, encoded by the cya gene. In the absence of glucose the enyzme encoded by the crr gene is phosphorylated and acts to activate adenylate cyclase (Krin et al., 2002).  As cAMP levels increase, cAMP binds to the CAP protein, leading to a dramatic change in its structure (↑), such that the protein’s  DNA binding domain becomes available to interact with promoter sequences (figure from Sharma et al., 2009).

Binding of activated (cAMP-bound) CAP is not, by itself sufficient to activate expression of the lac operon because of the presence of the constitutively expressed lac repressor protein, encoded for by the lacI gene. The active repressor is a tetramer, present at very low levels (~10 molecules) per cell. The lac operon contains three repressor (“operator”) binding sites; the tetrameric repressor can bind two operator sites simultaneously (upper figure → from Palanthandalam-Madapusi and Goyal, 2011). In the absence of lactose, but in the presence of cAMP-activated CAP, the operon is expressed in discrete “bursts” (Novick and Weiner, 1957; Vilar et al., 2003). Choi et al (2008) found that these burst come in two types, short and long, with the size of the burst referring to the number of mRNA molecules synthesized (bottm figure adapted from Choi et al ↑). The difference between burst sizes arises from the length of time that the operon’s repressor binding sites are unoccupied by repressor. As noted above, the tetravalent repressor protein can bind to two operator sites at the same time. When released from one site, polymerase binding and initiation produces a small number of mRNA molecules. Persistent binding to the second site means that the repressor concentration remains locally high, favoring rapid rebinding to the operator and the cessation of transcription (RNA synthesis). When the repressor releases from both operator sites, a rarer event, it is free to diffuse away and interact (non-specifically, i.e. with low affinity) with other DNA sites in the cell, leaving the lac operator sites unoccupied for a longer period of time. The number of such non-specific binding sites greatly exceeds the number (three) of specific binding sites in the operon. The result is the synthesis of a larger “burst” (number) of mRNA molecules. The average length of time that the operator  sites remain unoccupied is a function of the small number of repressor molecules present and the repressor’s low but measurable non-sequence specific binding to DNA. 

The expression of the lac operon leads to the appearance of β-galactosidase and β-galactoside permease. An integral membrane protein, β-galactoside permease enables extracellular lactose to enter the cell while cytoplasmic β-galactosidase catalyzes its breakdown and the generation of allolactone, which binds to the lac repressor protein, inhibiting its binding to operator sites, and so removing repression of transcription. In the absence of lactose, there are few if any of the proteins (β-galactosidase and β-galactoside permease) needed to activate the expression of the lac operon, so the obvious question is how, when lactose does appear in the extracellular media, does the lac operon turn on? Booth et al and the Wikipedia entry on the lac operon (accessed 29 June 2022) describe the turn on of the lac operon as “leaky” (see above). The molecular modeling studies of Vilar et al and Choi et al (which, together with Novick and Weiner, are not cited by Booth et al) indicate that the system displays distinct threshold and maintenance concentrations of lactose needed for stable lac gene expression. The term “threshold” does not occur in the Booth et al article. More importantly, when cultures are examined at the single cell level, what is observed is not a uniform increase in lac expression in all cells, as might be expected in the context of leaky expression, but more sporadic (noisy) behaviors. Increasing numbers of cells are “full on” in terms of lac operon expression over time when cultured in lactose concentrations above the operon’s activation threshold. This illustrates the distinctly different implications of a leaky versus a stochastic process in terms of their impacts on gene expression. While a leak is a macroscopic metaphor that produces a continuous, dependable, regular flow (drips), the occurrence of “bursts” of gene expression implies a stochastic (unpredictable) process ( figure from Vilar et al ↓). 

As the ubiquity and functionally significant roles of stochastic processes in biological systems becomes increasingly apparent, e.g. in the prediction of phenotypes from genotypes (Karavani et al., 2019; Mostafavi et al., 2020), helping students appreciate and understand the un-predictable, that is stochastic, aspects of biological systems becomes increasingly important. As an example, revealed dramatically through the application of single cell RNA sequencing studies, variations in gene expression between cells of the same “type” impacts organismic development and a range of behaviors. For example, in diploid eukaryotic cells is now apparent that in many cells, and for many genes, only one of the two alleles present is expressed; such “monoallelic” expression can impact a range of processes (Gendrel et al., 2014). Given that stochastic processes are often not well conveyed through conventional chemistry courses (Williams et al., 2015) or effectively integrated into, and built upon in molecular (and other) biology curricula; presenting them explicitly in introductory biology courses seems necessary and appropriate.

It may also help make sense of discussions of whether humans (and other organisms) have “free will”.  Clearly the situation is complex. From a scientific perspective we are analyzing systems without recourse to non-natural processes. At the same time, “Humans typically experience freely selecting between alternative courses of action” (Maoz et al., 2019)(Maoz et al., 2019a; see also Maoz et al., 2019b)It seems possible that recognizing the intrinsically unpredictable nature of many biological processes (including those of the central nervous system) may lead us to conclude that whether or not free will exists is in fact a non-scientific, unanswerable (and perhaps largely meaningless) question. 

footnotes

[1]  For this discussion I will ignore entropy, a factor that figures in whether a particular reaction in favorable or unfavorable, that is whether, and the extent to which it occurs.  

Acknowledgements: Thanks to Melanie Cooper and Nick Galati for taking a look and Chhavinder Singh for getting it started. Updated 6 January 2023.

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Is it possible to teach evolutionary biology “sensitively”?

Michael Reiss, a professor of science education at University College London and an Anglican Priest, suggests that “we need to rethink the way we teach evolution” largely because conventional approaches can be unduly confrontational and “force religious children to choose between their faith and evolution” or to result in students who”refuse to engage with a lesson.” He suggests that a better strategy would be akin to those use to teach a range of “sensitive” subjects “such as sex, pornography, ethnicity, religion, death studies, terrorism, and others” and could “help some students to consider evolution as a possibility who would otherwise not do so.” [link to his original essay and a previous post on teaching evolution: Go ahead and teach the controversy].

There is no doubt that an effective teacher attempts to present materials sensitively; it is the rare person who will listen to someone who “teaches” ideas in a hostile, alienating, or condescending manner. That said, it can be difficult to avoid the disturbing implications of scientific ideas, implications that can be a barrier to their acceptance. The scientific conclusion that males and females are different but basically the same can upset people on various sides of the theo-political spectrum. 

In point of fact an effective teacher, a teacher who encourages students to question their long held, or perhaps better put, familial or community beliefs, can cause serious social push-back  – Trouble with a capital T.  It is difficult to imagine a more effective teacher than Socrates (~470-399 BCE). Socrates “was found guilty of ‘impiety’ and ‘corrupting the young’, sentenced to death” in part because he was an effective teacher (see Socrates was guilty as charged).  In a religious and political context, challenging accepted Truths (again with a capital T) can be a crime.  In Socrates’ case”Athenians probably genuinely felt that undesirables in their midst had offended Zeus and his fellow deities,” and that, “Socrates, an unconventional thinker who questioned the legitimacy and authority of many of the accepted gods, fitted that bill.”  

So we need to ask of scientists and science instructors, does the presentation of a scientific, that is, a naturalistic and non-supernatural, perspective in and of itself represent an insensitivity to those with a super-natural belief system. Here it is worth noting a point made by the philosopher John Gray, that such systems extend beyond those based on a belief in god(s); they include those who believe, with apocalyptic certainty, in any of a number of Truths, ranging from the triumph of a master race, the forced sterilization of the unfit, the dictatorship of the proletariat, to history’s end in a glorious capitalist and technological utopia. Is a science or science instruction that is “sensitive” to, that is, uncritical of or upsetting to those who hold such beliefs, possible? 

My original impression is that one’s answer to this question is likely to be determined by whether one considers science a path to Truth, with a purposeful capital T, or rather that the goal of scientists is to build a working understanding of the world around and within us.  Working scientists, and particularly biologists who must daily confront the implications of apparently un-intelligent designed organisms (due to ways evolution works) are well aware that absolute certainty is counterproductive. Nevertheless, the proven explanatory and technological power of the scientific enterprise cannot help but reinforce the strong impression that there is some deep link between scientific ideas and the way the world really works.  And while some scientists have advocated unscientific speculations (think multiverses and cosmic consciousness), the truth, with a small t, of scientific thinking is all around us.  

Photograph of the Milky Way by Tim Carl photography, used by permission 

 A science-based appreciation of the unimaginable size and age of the universe, taken together with compelling evidence for the relatively recent appearance of humans (Homo sapiens from their metazoan, vertebrate, tetrapod, mammalian, and primate ancestors) cannot help but impact our thinking as to our significance in the grand scheme of things (assuming that there is such a, possibly ineffable, plan)(1). The demonstrably random processes of mutation and the generally ruthless logic by which organisms survive, reproduce, and evolve, can lead even the most optimistic to question whether existence has any real meaning.  

Consider, as an example, the potential implications of the progress being made in terms of computer-based artificial intelligence, together with advances in our understanding of the molecular and cellular connection networks that underlie human consciousness and self-consciousness. It is a small step to conclude, implicitly or explicitly, that humans (and all other organisms with a nervous system) are “just” wet machines that can (and perhaps should) be controlled and manipulated. The premise, the “self-evident truth”, that humans should be valued in and of themselves, and that their rights should be respected (2) is eroded by the ability of machines to perform what were previously thought to be exclusively human behaviors. 

Humans and their societies have, after all, been around for only a few tens of thousands of years.  During this time, human social organizations have passed from small wandering bands influenced by evolutionary kin and group selection processes to produce various social systems, ranging from more or less functional democracies, pseudo-democracies (including our own growing plutocracy), dictatorships, some religion-based, and totalitarian police states.  Whether humans have a long term future (compared to the millions of years that dinosaurs dominated life on Earth) remains to be seen – although we can be reasonably sure that the Earth, and many of its non-human inhabitants, will continue to exist and evolve for millions to billions of years, at least until the Sun explodes. 

So how do we teach scientific conclusions and their empirical foundations, which combine to argue that science represents how the world really works, without upsetting the most religiously and politically fanatical among us?  Those who most vehemently reject scientific thinking because they are the most threatened by its apparently unavoidable implications. The answer is open to debate, but to my mind it involves teaching students (and encouraging the public) to distinguish empirically-based, and so inherently limited observations and the logical, coherent, and testable scientific models they give rise to from unquestionable TRUTH- and revelation-based belief systems. Perhaps we need to focus explicitly on the value of science rather than its “Truth”. To reinforce what science is ultimately for; what justifies society’s support for it, namely to help reduce human suffering and (where it makes sense) to enhance the human experience, goals anchored in the perhaps logically unjustifiable, but nevertheless essential acceptance of the inherent value of each person.   

  1. Apologies to “Good Omens”
  2. For example, “We hold these truths to be self-evident, that all men are created equal, that they are endowed by their creator with certain unalienable rights, that among these are life, liberty and the pursuit of happiness.” 

Genes – way weirder than you thought

Pretty much everyone, at least in societies with access to public education or exposure to media in its various forms, has been introduced to the idea of the gene, but “exposure does not equate to understanding” (see Lanie et al., 2004).  Here I will argue that part of the problem is that instruction in genetics (or in more modern terms, the molecular biology of the gene and its role in biological processes) has not kept up with the advances in our understanding of the molecular mechanisms underlying biological processes (Gayon, 2016). spacer bar

Let us reflect (for a moment) on the development of the concept of a gene: Over the course of human history, those who have been paying attention to such things have noticed that organisms appear to come in “types”, what biologists refer to as species. At the same time, individual organisms of the same type are not identical to one  another, they vary in various ways. Moreover, these differences can be passed from generation to generation, and by controlling  which organisms were bred together; some of the resulting offspring often displayed more extreme versions of the “selected” traits.  By strictly controlling which individuals were breddogs
together, over a number of generations, people were able to select for the specific traits they desired (→).  As an interesting aside, as people domesticated animals, such as cows and goats, the availability of associated resources (e.g. milk) led to reciprocal effects – resulting in traits such as adult lactose tolerance (see Evolution of (adult) lactose tolerance & Gerbault et al., 2011).  Overall, the process of plant and animal breeding is generally rather harsh (something that the fanciers of strange breeds who object to GMOs might reflect upon), in that individuals that did not display the desired trait(s) were generally destroyed (or at best, not allowed to breed). spacer bar

Charles Darwin took inspiration from this process, substituting “natural” for artificial (human-determined) selection to shape populations, eventually generating new species (Darwin, 1859).  Underlying such evolutionary processes was the presumption that traits, and their variation, was “encoded” in some type of “factors”, eventually known as genes and their variants, alleles.  Genes influenced the organism’s molecular, cellular, and developmental systems, but the nature of these inheritable factors and the molecular trait building machines active in living systems was more or less completely obscure. 

Through his studies on peas, Gregor Mendel was the first to clearly identify some of the rules for the behavior of these inheritable factors using highly stereotyped, and essentially discontinuous traits – a pea was either yellow or green, wrinkled or smooth.  Such traits, while they exist in other organisms, are in fact rare – an example of how the scientific exploration of exceptional situations can help understand general processes, but the downside is the promulgation of the idea that genes and traits are somehow discontinuous – that a trait is yes/no, displayed by an organism or not – in contrast to the realities that the link between the two is complex, a reality rarely directly addressed (apparently) in most introductory genetics courses.  Understanding such processes is critical to appreciating the fact that genetics is often not destiny, but rather alterations in probabilities (see Cooper et al., 2013).  Without such an more nuanced and realistic understanding, it can be difficult to make sense of genetic information.     spacer bar

A gene is part of a molecular machine:  A number of observations transformed the abstraction of Darwin’s and Mendel’s hereditary factors into physical entities and molecular mechanisms (1).  In 1928 Fred Griffith demonstrated that a genetic trait could be transferred from dead to living organisms – implying a degree of physical / chemical stability; subsequent observations implied that the genetic information transferred involved DNA molecules. The determination of the structure of double-stranded DNA immediately suggested how information could be stored in DNA (in variations of bases along the length of the molecule) and how this information could be duplicated (based on the specificity of base pairing).  Mutations could be understood as changes in the sequence of bases along a DNA molecule (introduced by chemicals, radiation, mistakes during replication, or molecular reorganizations associated with DNA repair mechanisms and selfish genetic elements.  

But on their own, DNA molecules are inert – they have functions only within the context of a living organism (or highly artificial, that is man made, experimental systems).  The next critical step was to understand how a gene works within a biological system, that is, within an organism.  This involve appreciating the molecular mechanisms (primarily proteins) involved in identifying which stretches of a particular DNA molecule were used as templates for the synthesis of RNA molecules, which in turn could be used to direct the synthesis of polypeptides (see previous post on polypeptides and proteins).  In the context of the introductory biology courses I am familiar with (please let me know if I am wrong), these processes are based on a rather deterministic context; a gene is either on or off in a particular cell type, leading to the presence or absence of a trait. Such a deterministic presentation ignores the stochastic nature of molecular level processes (see past post: Biology education in the light of single cell/molecule studies) and the dynamic interaction networks that underlie cellular behaviors.  spacer bar

But our level of resolution is changing rapidly (2).  For a number of practical reasons, when the human genome was first sequence, the identification of polypeptide-encoding genes was based on recognizing “open-reading frames” (ORFs) encoding polypeptides of > 100 amino acids in length (> 300 base long coding sequence).  The increasing sensitivity of mass spectrometry-based proteomic studies reveals that smaller ORFs (smORFs) are present and can lead to the synthesis of short (< 50 amino acid long) polypeptides (Chugunova et al., 2017; Couso, 2015).  Typically an ORF was considered a single entity – basically one gene one ORF one polypeptide (3).  A recent, rather surprising discovery is what are known as “alternative ORFs” or altORFs; these RNA molecules that use alternative reading frames to encode small polypeptides.  Such altORFs can be located upstream, downstream, or within the previously identified conventional ORFalternative orfs
(figure →)(see Samandi et al., 2017).  The implication, particularly for the analysis of how variations in genes link to traits, is that a change, a mutation or even the  experimental  deletion of a gene, a common approach in a range of experimental studies, can do much more than previously presumed – not only is the targeted ORF effected, but various altORFs can also be modified.  

The situation is further complicated when the established rules of using RNAs to direct polypeptide synthesis via the process of translation, are violated, as occurs in what is known as “repeat-associated non-ATG (RAN)” polypeptide synthesis (see Cleary and Ranum, 2017).  In this situation, the normal signal for the start of RNA-directed polypeptide synthesis, an AUG codon, is subverted – other RNA synthesis start sites are used leading to underlying or imbedded gene expression.  This process has been found associated with a class of human genetic diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) characterized by the expansion of simple (repeated) DNA sequences  (see Pattamatta et al., 2018).  Once they exceed a certain length, such“repeat” regions have been found to be associated with the (apparently) inappropriarepeat region RAN process
te transcription of RNA in both directions, that is using both DNA strands as templates (← A: normal situation, B: upon expansion of the repeat domain).  These abnormal repeat region RNAs are translated via the RAN process to generate six different types of toxic polypeptides. spacer bar

So what are the molecular factors that control the various types of altORF transcription and translation?  In the case of ALS and FTD, it appears that other genes, and the polypeptides and proteins they encode, are involved in regulating the expression of repeat associated RNAs (Kramer et al., 2016)(Cheng et al., 2018).  Similar or distinct mechanisms may be involved in other  neurodegenerative diseases  (Cavallieri et al., 2017).  

So how should all of these molecular details (and it is likely that there are more to be discovered) influence how genes are presented to students?  I would argue that DNA should be presented as a substrate upon which various molecular mechanisms occur; these include transcription in its various forms (directed and noisy), as well as DNA synthesis, modification, and repair mechanisms occur.   Genes are not static objects, but key parts of dynamic systems.  This may be one reason that classical genetics, that is genes presented within a simple Mendelian (gene to trait) framework, should be moved deeper into the curriculum, where students have the background in molecular mechanisms needed to appreciate its complexities, complexities that arise from the multiple molecular machines acting to access, modify, and use the information captured in DNA (through evolutionary processes), thereby placing the gene in a more realistic cellular perspective (4). 

Footnotes:

1. Described greater detail in biofundamentals™

2. For this discussion, I am completely ignoring the roles of genes that encode RNAs that, as far as is currently know, do not encode polypeptides.  That said, as we go on, you will see that it is possible that some such non-coding RNA may encode small polypeptides.  

3. I am ignoring the complexities associated with alternative promoter elements, introns, and the alternative and often cell-type specific regulated splicing of RNAs, to create multiple ORFs from a single gene.  

4. With respects to Norm Pace – assuming that I have the handedness of the DNA molecules wrong or have exchanged Z for A or B. 

literature cited: 

  • Cavallieri et al, 2017. C9ORF72 and parkinsonism: Weak link, innocent bystander, or central player in neurodegeneration? Journal of the neurological sciences 378, 49.
  • Cheng et al, 2018. C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2α phosphorylation. Nature communications 9, 51.
  • Chugunova et al, 2017. Mining for small translated ORFs. Journal of proteome research 17, 1-11.
  • Cleary & Ranum, 2017. New developments in RAN translation: insights from multiple diseases. Current opinion in genetics & development 44, 125-134.
  • Cooper et al, 2013. Where genotype is not predictive of phenotype: towards an understanding of the molecular basis of reduced penetrance in human inherited disease. Human genetics 132, 1077-1130.
  • Couso, 2015. Finding smORFs: getting closer. Genome biology 16, 189.
  • Darwin, 1859. On the origin of species. London: John Murray.
  • Gayon, 2016. From Mendel to epigenetics: History of genetics. Comptes rendus biologies 339, 225-230.
  • Gerbault et al, 2011. Evolution of lactase persistence: an example of human niche construction. Philosophical Transactions of the Royal Society of London B: Biological Sciences 366, 863-877.
  • Kramer et al, 2016. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science 353, 708-712.
  • Lanie et al, 2004. Exploring the public understanding of basic genetic concepts. Journal of genetic counseling 13, 305-320.
  • Pattamatta et al, 2018. All in the Family: Repeats and ALS/FTD. Trends in neurosciences 41, 247-250.
  • Samandi et al, 2017. Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins. Elife 6.