How to read the scientific literature?

Science addressed the problem of How to (seriously) read a scientific paper by asking a group of Ph.D. students, post-docs, and scientists how they read the scientific literature. None of the answers will surprise you. The general theme is that you read the abstract to see if the work is relevant then skim the figures and the conclusions before buckling down to slog through the entire paper.

None of the respondents address the most serious problems such as trying to figure out what the researchers actually did while not having a clue how they did it. Nor do they address the serious issue of misleading conclusions and faulty logic.

I asked on Facebook whether we could teach undergraduates to read the primary scientific literature. I'm skeptical since I believe it takes a great deal of experience to be able to profitably read recent scientific papers and it takes a great deal of knowledge of fundamental concepts and principles. We know from experience that many professional scientists can be taken in by papers that are published in the scientific literature. Arseniclife is one example and the ENCODE papers published in September 2012 are another. If professional scientists can be fooled, how are we going to teach undergraduates to be skeptical?

Proponents of the Extended Evolutionary Synthesis (EES) explain their logic using the Central Dogma as an example

There's a group of biologists who think that the current version of evolutionary theory is insufficient. They want to create an extended evolutionary synthesis that incorporates evo-devo, plasticity, niche construction, evolvability, epigenetics, and other things.

You might be wondering how these things could be incorporated and what that would do to "classic" evolutionary theory. Fortunately, we have a road map provided by Massimo Pigliucci and Gerd Müller in chapter one of Evolution: The Extened Synthesis. They help us out by providing an analogy.

As we will see in the rest of this volume, several of these tenets [of the Modern Synthesis] are being challenged as either inaccurate or incomplete. It is important, however, to understand the kind of challenge being posed here, in order to avoid wasting time on unproductive discussions that missed the point of an extended evolutionary synthesis. Perhaps a parallel with another branch of biology will be helpful. After Watson and Crick discovered the double-helix structure of DNA, and the molecular revolution got started in earnest, one of the first principles to emerge from the new discipline was the unfortunately named "central dogma" of molecular biology. The dogma (a word that arguably should never be used in science) stated that the flow of information in biological systems is always one way, from DNA to RNA to proteins. Later on, however, it was discovered that the DNA > RNA flow can be reversed by the appropriately named process of reverse transcription, which takes place in a variety of organisms, including some viruses and eukaryotes (through retrotransposons). Moreover, we now know that some viruses replicate their RNA directly by means of RNA dependent RNA polymerases, enzymes also found in eukaryotes, where they mediate RNA silencing. Prions have shown us how some proteins can catalyze conformational changes in similar proteins, a phenomenon that is not a case of replication, but certainly qualifies as information transfer. Finally, we also have examples of direct DNA translation to protein in cell-free experimental systems in the presence of ribosomes but not of mRNA. All of these molecular processes clearly demolish the alleged central dogma, and yet do not call for the rejection of any of the empirical discoveries or conceptual advances made in molecular biology since the 1950s. Similarly, we argue, individual tenets of the Modern Synthesis can be modified, or even rejected, without generating a fundamental crisis in the structure of evolutionary theory—just as the Modern Synthesis itself improved upon but did not cause rejection of either Darwinism or neo-Darwinism.

I thank Pigliucci and Müller for giving us a clear idea of the logic behind their attack on the Modern Synthesis.

... I must correct a wrong idea that has been spreading for the past three or four years. It was discovered some years ago that in some cases, the transcription step from DNA to RNA works in the reverse direction. That is nothing surprising. ... it could be predicted that such events could occur. They do occur, indeed, but this must not be taken to mean that information from protein could possibly go back to the genome. ... I am ready to take any bet you like that this is never going to turn out to be the case.

Jacques Monod (1974) p.394The original, and correct, version of the Central Dogma of Molecular Biology was stated clearly by Francis Crick in 1958. Crick restated the Central Dogma of Molecular Biology in a famous paper published in 1970 at a time when the premature slaying of the Central Dogma by reverse transcriptase was being announced (Crick, 1970). According to Crick, the correct, concise version of the Central Dogma is ...

... once (sequential) information has passed into protein it cannot get out again (F.H.C. Crick, 1958)

The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred from protein to either protein or nucleic acid. (F.H.C. Crick, 1970)

Jim Watson published the well-known, but incorrect, version in his 1960s textbook but anyone who does even a little bit of research will discover that the Crick version is the original [see: The Central Dogma of Molecular Biology].

Here's the summary provided by Francis Crick in his 1970 Nature paper.

Fig. 1. Information flow and the sequence hypothesis. These diagrams of potential information flow were used by Crick (1958) to illustrate all possible transfers of information (left) and those that are permitted (right). The sequence hypothesis refers to the idea that information encoded in the sequence of nucleotides specifies the sequence of amino acids in the protein.

This is important because whenever someone attacks the Central Dogma you can get a good idea of their academic ability by seeing if they understand the concept they attack. In this case, there's a question about proponents of the extended evolutionary synthesis and whether they have a sufficient grasp of evolutionary theory to be challenging it. Pigliucci and Müller have tried to convince us that they know what they are talking about by giving us an analogy; namely, the "demolition" of the Central Dogma of Molecular Biology.

They didn't do their homework. That doesn't inspire confidence in their ability to overthrow modern evolutionary theory.

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Crick, F.H.C. (1958) On protein synthesis. Symp. Soc. Exp. Biol. XII:138-163. [PDF]
Crick, F. (1970) Central Dogma of Molecular Biology. Nature 227, 561-563. [PDF file]
Monod, J. (1974) "On the Molecular Theory of Evolution" reprinted in Mark Ridley (editor) Evolution (1997) p. 389

How do you characterize these scientists?

We've been having a discussion on another thread about ID proponents. Are some of them acting in good faith or are they all lying and deceiving their followers?

I have similar problems about many scientists. I've been reading up on pervasive transcription and the potential number of genes for noncoding, functional, RNAs in the human genome. As far as I can tell, there are only a few hundred examples that have any supporting evidence. There are good scientific reasons to believe that most of the detected transcripts are junk RNA produced as the result of accidental, spurious, transcription.

There are about 20,000 protein-coding genes in the human genome. I think it's unlikely that there are more than a few thousand genes for functional RNAs for a total of less than 25,000 genes.

Here's one of the papers I found.

Guil, S. and Esteller, M. (2015) RNA–RNA interactions in gene regulation: the coding and noncoding players. Trends in Biochemical Sciences 40:248-256. [doi: 10.1016/j.tibs.2015.03.001]

Trends in Biochemical Sciences is a good journal and this is a review of the field by supposed experts. The authors are from the Department of Physiological Sciences II at the University of Barcelona School of Medicine in Barcelona, Catalonia, Spain. The senior author, Manel Esteller, has a Wikipedia entry [Manel Esteller].

Here's the first paragraph of the introduction.

There are more genes encoding regulatory RNAs than encoding proteins. This evidence, obtained in recent years from the sum of numerous post-genomic deep-sequencing studies, give a good clue of the gigantic step we have taken from the years of the central dogma: one gene gives rise to one RNA to produce one protein.

The first sentence is not true by any stretch of the imagination. The best that could be said is that there "may" be more genes for regulatory RNAs (> 20,000) but there's no strong consensus yet. Since the first sentence is an untruth, it follows that it is incorrect to say that the evidence supports such a claim.

It's also untrue to distort the real meaning of the Central Dogma of Molecular Biology, which never said that all genes have to encode proteins. The authors don't understand the history of their field in spite of the fact they are writing a review of that field.

Here's the problem. Are these scientists acting in good faith when they say such nonsense? Does acting in "good faith" require healthy criticism and critical thinking or is "honesty" the only criterion? The authors are clearly deluded about the controversy since they assume that it has been resolved in favor of their personal biases but they aren't lying. Can we distinguish between competent science and bad science based on such statements? Can we say that these scientists are incompetent or is that too harsh?

Furthermore, what ever happened to peer review? Isn't the system supposed to prevent such mistakes?

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Junk DNA doesn't exist according to "Conceptual Revolutions in Science"

The blog "Conceptual Revolutions in Science" only publishes "evidence-based, paradigm-shifting scientific news" according to their home page.

The man behind the website is Adam B. Dorfman (@DorfmanAdam). He has an MBA from my university and he currently works at a software company. Here's how he describes himself on the website.

Methodological naturalism at Dover

I'm one of those scientists who don't think that science as a way of knowing is restricted to investigating natural causes [John Wilkins Revisits Methodological Naturalism ]. I think that science can easily investigate supernatural claims and show that they are wrong. In theory, science might even show that the supernatural exists. Some (most?) philosophers agree. Maarten Boudry is the best known [Is Science Restricted to Methodologial Naturalism?].

This year is the tenth anniversary of Kitzmiller v. Dover Area School District. At that trial, the plaintiffs successfully convinced Judge Jones that intelligent design isn't a science because it invokes supernatural causes. The expert witnesses testified that, by definition, science is limited by methodological naturalism. I disagree with the expert witnesses at the trial and I agree with many leading philosophers that science is not restricted to methodological naturalism [Can Science Test Supernatural Worldviews? ].

Best blog post in the past year

3 Quarks Daily is running their annual contest to pick the best blog posts in the past year. The finalists will be picked by popular vote and the winner will be selected from the finalists by Nick Lane. You can review the rules at: Nick Lane to Judge 6th Annual 3QD Science Prize.

The formal description of the prize is "6th annual prize for the best blog and online-only writing in the category of science." This is important because although the rules refer to "blog posts" and "blog entries" it's clear that most of the nominees are more like online poplar science articles than typical blog posts.

Here's a list of the current nominees ...

The value of critique in science education

One of the most difficult concepts to get across to science educators (e.g. professors in a biochemistry department ) is the idea that students need to be exposed to ideas that you think are incorrect and they need to be given the opportunity to make a choice. It's part of critical thinking and it's part of a good science education. Part of the problem is that there's a general reluctance to even teach "ideas" as opposed to facts and techniques.

There's an extensive pedagogical literature on this but university professors are reluctant to admit that there might be better ways to teach. While browsing this literature, I came across a recent article by Henderson et al. (2015) that makes a good case.

Here's why you can ignore Günther Witzany

Günther Witzany is one of those people who think the Modern Synthesis needs to be overthrown but he missed the real revolution that took place in the late 1960s. He's part of The Third Way crowd that includes Denis Noble and Jim Shapiro [see Physiologists fall for the Third Way and The Third Fourth Way].

Susan Mazur interviews him for the Huffington Post [Günther Witzany: Modern Synthesis "Must Be Replaced," Communication Key to Evolution]. Recall that Susan Mazur is fixated on the Altenburg 16 and their attempts to radically revise evolutionary theory without understanding anything about Neutral Theory and random genetic drift. Günther Witzany is a philosopher. He was not one of the Altenberg 16 but he clearly wants to be part of the outer circle. It's not clear why anyone should consider him an expert on evolutionary biology.

Susan Mazur did us a great favor when she asked him if he would like to make a final point. His answer shows us why we can ignore him.

The older concepts we have now for a half century cannot sufficiently explain the complex tendency of the genetic code. They can't explain the functions of mobile genetic elements and the endogenous retroviruses and non-coding RNAs. Also, the central dogma of molecular biology has been falsified -- that is, the way is always from DNA to RNA to proteins to anything else, or the other "dogmas," e.g., replication errors drive evolutionary genetic variation, that one gene codes for one protein and that non-coding DNA is junk. All these concepts that dominated science for half a century are falsified now. ...

Thank-you Susan. Keep up the good work. Fools need to be exposed.

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Readings from Trends in Biochemical Sciences on the Central Dogma

I'm re-reading The Inside Story edited by Jan Witkowski, the former editor-in-chief of Trends in Biochemical Sciences (TIBS). The book is a collection of essays that appeared in the journal. The collection centers around "the theme of the Central Dogma of molecular biology." Here's how Jan Witkowski describes the collection in the preface (page xii)...

When I came to look more closely, it was clear that the area the articles covered most comprehensively, where the most interesting selection could be made, was the Central Dogma, that is DNA, RNA, and protein synthesis. And the number of relevant articles was just right for the size of book we had in mind.

This explains the subtitle of the book, "DNA to RNA to Protein."

This is not going to be another complaint about misinterpretations of the Central Dogma. Quite the contrary, as we shall see.

The Forward was written by Tim Hunt who was the editor-in-chief from 1992-2000. He refers to "The General Idea."

"Jim, you might say, had it first. DNA makes RNA makes protein. That became the general idea." Thus did Francis Crick explain to Horace Judson years later, long after he had written with such clarity and force on the subject of protein synthesis in the 1958 Symposium on "The Biological Replication of Macromolecules" [see Crick, 1959). This article is celebrated for its prediction of the existence of tRNA (although by the time the article appeared in print, tRNA had been discovered), but it is chiefly worth reading and rereading, even today, for its enunciation of the two principles that together constitute the "General Idea." The first principle is the Sequence Hypothesis; the idea that the sequence of amino acids in proteins is specified by the sequence of bases in DNA and RNA. The second principle is the famous "Central Dogma"; not DNA makes RNA makes Protein, but the assertion that "Once information has passed into protein it cannot get out again." It isn't completely clear why one is a hypothesis and the other a dogma and the two together an idea. The Dogma stuck in some throats, mainly because it was called a dogma, with heavy religious overtones.

I quote Tim Hunt to show that there are some knowledgeable scientists who understand the Central Dogma [see The Central Dogma of Molecular Biology].

Hunt continues ...

Crick explains that calling it a dogma was a misunderstanding on his part: he thought the word stood for "an idea for which there was no reasonable evidence," blaming his "curious religious upbringing" for the error. But it probably wasn't that much of a mistake after all, for the Oxford Dictionary allows dogma to mean simply a principle, although the alternative "Arrogant declaration of opinion" is probably how most people who were not molecular biologists took it, considering its never modest author. That is probably how they were meant to take it, too. It was the most important article of faith among the circle of biologists centered on Watson and Crick and remained so for quite a long time until the mechanism of protein synthesis became clear. Crick said that if you did not subscribe to the sequence hypothesis and the central dogma "you generally ended up in the wilderness," although he did not offer alternative scenarios for public consumption, even though they probably played an important part in convincing him of the dogmatic status of the General Idea's second component.

This is the concept that I "grew up" with as a graduate student in the late 1960s. We saw the "General Idea" as an important concept and a way of understanding the data that was coming out of many labs working on DNA replication, transcription, and protein synthesis. We knew, especially after 1970 (Crick, 1970), that RNA could be used as a template to make DNA and that there were many types of RNA other than messenger RNA. We also knew that Francis Crick was a very smart man and it was unwise to disagree with him because he was usually right about big ideas.

Fig. 1. Information flow and the sequence hypothesis. These diagrams of potential information flow were used by Crick (1958) to illustrate all possible transfers of information (left) and those that are permitted (right). The sequence hypothesis refers to the idea that information encoded in the sequence of nucleotides specifies the sequence of amino acids in the protein.

At some point in the last 40 year the "General Idea" has been subverted in two ways.

1. The Sequence Hypothesis has come to be interpreted as the Central Dogma. This is mostly due to Jim Watson who propagated this misinterpretation in his Molecular Biology of the Gene textbook.
2. The Central Dogma is taken to mean that the ONLY important information in the genome is that which encodes proteins. It's assumed, incorrectly, that Crick meant to say that the role of all genes is to encode proteins.

One of the essays in The Inside Story is "Forty Years under the Central Dogma," published in 1998. The authors are Denis Thieffry and Sahotra Sarkar (Thieffry and Sarkar, 1998).

Here's how they explain some of the confusion about the Central Dogma ...

The most obvious interpretation of Crick’s original (1958) formulation of the Central Dogma is in negative terms. The Central Dogma only forbids a few types of information transfer, namely, from proteins to proteins and from proteins to nucleic acids. However, after its rapid adoption by most of the biologists interested in protein synthesis, it was most often interpreted or reformulated in a more restrictive way, constricting the flow of information from DNA to RNA and from RNA to protein (Fig. 1).

Figure 1 The Central Dogma as envisioned by Watson in 1965. ‘We should first look at the evidence that DNA itself is not the direct template that orders amino acid sequences. Instead, the genetic information of DNA is transferred to another class of molecules, which then serve as the protein templates. These intermediate templates are molecules of ribonucleic acid (RNA)...Their relation to DNA and protein is usually summarised by the formula (often called the central dogma).'

According to Watson’s autobiography, he had already derived this ‘formula’ (Fig. 1) in 1952. In fact, such schemes were commonly entertained during the early 1950s, at least among the biologists interested in protein synthesis. ... Much more restrictive than Crick’s original statement, Watson’s formula was immediately confronted with a series of possible exceptions, some of which are mentioned below. Crick, meanwhile, remained rather cautious in his interpretation of the Central Dogma. On several occasions, he felt it necessary to come back to his original idea and explicate what he thought to be its correct interpretation. For example, in 1970, Crick devoted a paper specifically to the Central Dogma, including a diagram reportedly conceived (but not published) in 1958.[see the figure at the top of this page]

The authors recognize several challenges to the Central Dogma, at least to the version preferred by Watson. There were two discoveries in the 1960s that seemed to threaten the Central Dogma. The first was the discovery that the genetic material of some viruses (e.g. TMV) was RNA, not DNA. The second was the discovery that RNA could be copied into DNA by reverse transcriptase. This was not a problem for Crick ....

These findings prompted Crick to write his 1970 piece for Nature, in which he explicitly showed how the new facts fitted into his scheme.

It's difficult to evaluate the importance of the Central Dogma in the 21st century because so many scientists don't understand it. The incorrect version seems to mostly serve as a whipping boy to promote "new" ideas that overthrow the strawman version of the Central Dogma.

Back in 1998, the authors of this article asked Crick what he thought of the Central Dogma ...

In a recent answer to a question addressing the relevance of these challenges, Crick stated that he still believes in the value of the Central Dogma today (F.H.C. Crick, pers. commun.). However, he also acknowledges the existence of various exceptions, most of which he regards as minor. For him, the most significant exception is RNA editing. Still, according to Crick, simplifications of the Central Dogma in terms such as ‘DNA makes RNA and RNA makes protein’ were clearly inadequate from the beginning.

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Crick, F.H.C. (1958) On protein synthesis. Symp. Soc. Exp. Biol. XII:138-163. [PDF]

Crick, F. (1970) Central Dogma of Molecular Biology. Nature 227, 561-563. [PDF file]

Thieffry, D. and Sarkar, S. (1998) "Forty years under the central dogma." Trends in Biochemical Sciences 23:312–316. [doi: 10.1016/S0968-0004(98)01244-4}

More confusion about the central dogma of molecular biology

I was doing some reading on lncRNAs (long non-coding RNAs) in order to find out how many of them had been assigned real biological functions. My reading was prompted by the one of the latest updates to the human genome sequence; namely, assembly GRCh38.p3 from June 2015. The Ensembl website lists 14,889 lncRNA genes but I'm sure that most of these are just speculative [Ensembl Whole Genome].

The latest review by my colleagues here in the biochemistry department at the University of Toronto (Toronto, Canada), concludes that only a small fraction of these putative lncRNAs have a function (Palazzo and Lee, 2015). They point out that in the absence of evidence for function, the null hypothesis is that these RNAs are junk and the genes don't exist. That's not the view that annotators at Ensembl take.

I stumbled across a paper by Ling et al. (2015) that tries to make a case for function. I don't think their case is convincing but that's not what I want to discuss. I want to discuss their view of the Central Dogma of Molecular Biology. Here's the abstract ...

The central dogma of molecular biology states that the flow of genetic information moves from DNA to RNA to protein. However, in the last decade this dogma has been challenged by new findings on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). More recently, long non-coding RNAs (lncRNAs) have attracted much attention due to their large number and biological significance. Many lncRNAs have been identified as mapping to regulatory elements including gene promoters and enhancers, ultraconserved regions and intergenic regions of protein-coding genes. Yet, the biological function and molecular mechanisms of lncRNA in human diseases in general and cancer in particular remain largely unknown. Data from the literature suggest that lncRNA, often via interaction with proteins, functions in specific genomic loci or use their own transcription loci for regulatory activity. In this review, we summarize recent findings supporting the importance of DNA loci in lncRNA function and the underlying molecular mechanisms via cis or trans regulation, and discuss their implications in cancer. In addition, we use the 8q24 genomic locus, a region containing interactive SNPs, DNA regulatory elements and lncRNAs, as an example to illustrate how single nucleotide polymorphism (SNP) located within lncRNAs may be functionally associated with the individual’s susceptibility to cancer.

This is getting to be a familiar refrain. I understand how modern scientists might be confused about the difference between the Watson and the Crick versions of the Central Dogma [see The Central Dogma of Molecular Biology]. Many textbooks perpetuate the myth that Crick's sequence hypothesis is actually the Central Dogma. That's bad enough but lots of researchers seem to think that their false view of the Central Dogma goes even further. They think it means that the ONLY kind of genes in your genome are those that produce mRNA and protein.

I don't understand how such a ridiculous notion could arise but it must be a common misconception, otherwise why would these authors think that non-coding RNAs are a challenge to the Central Dogma? And why would the reviewers and editors think this was okay?

I'm pretty sure that I've interpreted their meaning correctly. Here's the opening sentences of the introduction to their paper ...

The Encyclopedia of DNA Elements (ENCODE) project has revealed that at least 75% of the human genome is transcribed into RNAs, while protein-coding genes comprise only 3% of the human genome. Because of a long-held protein-centered bias, many of the genomic regions that are transcribed into non-coding RNAs (ncRNAs) had been viewed as ‘junk’ in the genome, and the associated transcription had been regarded as transcriptional ‘noise’ lacking biological meaning.

They think that the Central Dogma is a "protein-centered bias." They think the Central Dogma rules out genes that specify noncoding RNAs. (Like tRNA and ribosomal RNA?)

Later on they say ....

The protein-centered dogma had viewed genomic regions not coding for proteins as ‘junk’ DNA. We now understand that many lncRNAs are transcribed from ‘junk’ regions, and even those encompassing transposons, pseudogenes and simple repeats represent important functional regulators with biological relevance.

It's simply not true that scientists in the past viewed all noncoding DNA as junk, at least not knowledgeable scientists [What's in Your Genome?]. Furthermore, no knowledgeable scientists ever interpreted the Central Dogma of Molecular Biology to mean that the only functional genes in a genome were those that encoded proteins.

Apparently Lee, Vincent, Picler, Fodde, Berindan-Neagoe, Slack, and Calin knew scientists who DID believe such nonsense. Maybe they even believed it themselves.

Judging by the frequency with with such statements appear in the scientific literature, I can only assume that this belief is widespread among biochemists and molecular biologists. How in the world did this happen? How many Sandwalk readers were taught that the Central Dogma rules out all genes for noncoding RNAs? Did you have such a protein-centered bias about the role of genes? Who were your teachers?

Didn't anyone teach you who won the Nobel Prize in 1989? Didn't you learn about snRNAs? What did you think RNA polymerases I and III were doing in the cell?

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Ling, H., Vincent, K., Pichler, M., Fodde, R., Berindan-Neagoe, I., Slack, F.J., and Calin, G.A. (2015) Junk DNA and the long non-coding RNA twist in cancer genetics. Oncogene (published online January 26, 2015) [PDF]

Palazzo, A.F. and Lee, E.S. (2015) Non-coding RNA: what is functional and what is junk? Frontiers in genetics 6: 2 (published online January 26, 2015 [Abstract]

John Parrington and modern evolutionary theory

We are continuing our discussion of John Parrington's book The Deeper Genome: Why there is more to the human genome than meets the eye. This is the third of five posts on: Five Things You Should Know if You Want to Participate in the Junk DNA Debate

1. Genetic load

John Parrington and the genetic load argument

2. C-Value paradox

John Parrington and the c-value paradox

3. Modern evolutionary theory (this post)

John Parrington and modern evolutionary theory

4. Pseudogenes and broken genes are junk

John Parrington discusses pseudogenes and broken genes

5. Most of the genome is not conserved

John Parrington discusses genome sequence conservation

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3. Modern evolutionary theory

You can't understand the junk DNA debate unless you've read Michael Lynch's book The Origins of Genome Architecture. That means you have to understand modern population genetics and the role of random genetic drift in the evolution of genomes. There's no evidence in Parrington's book that he has read The Origins of Genome Architecture and no evidence that he understands modern evolutionary theory. The only evolution he talks about is natural selection (Chapter 1).

Here's an example where he demonstrates adaptationist thinking and the fact that he hasn't read Lynch's book ...

At first glance, the existence of junk DNA seems to pose another problem for Crick's central dogma. If information flows in a one-way direction from DNA to RNA to protein, then there would appear to be no function for such noncoding DNA. But if 'junk DNA' really is useless, then isn't it incredibly wasteful to carry it around in our genome? After all, the reproduction of the genome that takes place during each cell division uses valuable cellular energy. And there is also the issue of packaging the approximately 3 billion base pairs of the human genome into the tiny cell nucleus. So surely natural selection would favor a situation where both genomic energy requirements and packaging needs are reduced fiftyfold?¹

Nobody who understands modern evolutionary theory would ask such a question. They would have read all the published work on the issue and they would know about the limits of natural selection and why species can't necessarily get rid of junk DNA even if it seems harmful.

People like that would also understand the central dogma of molecular biology.

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1. He goes on to propose a solution to this adaptationist paradox. Apparently, most of our genome consists of parasites (transposons), an idea he mistakenly attributes to Richard Dawkins' concept of The Selfish Gene. Parrington seems to have forgotten that most of the sequence of active transposons consists of protein-coding genes so it doesn't work very well as an explanation for excess noncoding DNA.

John Parrington and the genetic load argument

We are discussing John Parrington's book The Deeper Genome: Why there is more to the human genome than meets the eye. This is the first of five posts on: Five Things You Should Know if You Want to Participate in the Junk DNA Debate

1. Genetic load (this post)

John Parrington and the genetic load argument

2. C-Value paradox

John Parrington and the c-value paradox

3. Modern evolutionary theory

John Parrington and modern evolutionary theory

4. Pseudogenes and broken genes are junk

John Parrington discusses pseudogenes and broken genes

5. Most of the genome is not conserved

John Parrington discusses genome sequence conservation

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1. Genetic load

The genetic load argument has been around for 50 years. It's why experts did not expect a huge number of genes when the genome sequence was published. It's why the sequence of most of our genome must be irrelevant from an evolutionary perspective.

This argument does not rule out bulk DNA hypotheses but it does rule out all those functions that require specific sequences in order to confer biological function. This includes the speculation that most transcripts have a function and it includes the speculation that there's a vast amount of regulatory sequence in our genome. Chapter 5 of The Deeper Genome is all about the importance of regulatory RNAs.

So, starting from a failed attempt top turn a petunia purple, the discovery of RNA interference has revealed a whole new network of gene regulation mediated by RNAs and operating in parallel to the more established one of protein regulatory factors. ... Studies have revealed that a surprising 60 per cent of miRNAs turn out to be recycled introns, with the remainder being generated from the regions between genes. Yet these were parts of the genome formerly viewed as junk. Does this mean we need a reconsideration of this question? This is an issue we will discuss in Chapter 6, in particular with regard to the ENCODE project ...

The implication here is that a substantial part of the genome is devoted to the production of regulatory RNAs. Presumably, the sequences of those RNAs are important. But this conflicts with the genetic load argument unless we're only talking about an insignificant fraction of the genome.

But that's only one part of Parrington's argument against junk DNA. Here's the summary from the last Chapter ("Conclusion") ...

As we've discussed in this book, a major part of the debate about the ENCODE findings has focused on the question of what proportion of the genome is functional. Given that the two sides of this debate use quite different criteria to assess functionality it is likely that it will be some time before we have a clearer idea about who is the most correct in this debate. Yet, in framing the debate in this quantitative way, there is a danger that we might lose sight of an exciting qualitative shift that has been taking place in biology over the past decade or so. So a previous emphasis on a linear flow of information, from DNA to RNA to protein through a genetic code, is now giving way to a much more complex picture in which multiple codes are superimposed on one another. Such a viewpoint sees the gene as more than just a protein-coding unit; instead it can equally be seen as an accumulation of chemical modifications in the DNA or its associated histones, a site for non-coding RNA synthesis, or a nexus in a 3D network. Moreover, since we now know that multiple sites in the genome outside the protein-coding regions can produce RNAs, and that even many pseudo-genes are turning out to be functional, the very question of what constitutes a gene is now being challenged. Or, as Ed Weiss at the University of Pennsylvania recently put it, 'the concept of a gene is shredding.' Such is the nature of the shift that now we face the challenge of not just recognizing the true scale of this complexity, but explaining how it all comes together to make a living, functioning, human being.

I've already addressed some of the fuzzy thinking in this paragraph [The fuzzy thinking of John Parrington: The Central Dogma and The fuzzy thinking of John Parrington: pervasive transcription]. The point I want to make here is that Parrington's arguments for function in the genome require a great deal of sequence information. They all conflict with the genetic load argument.

Parrington doesn't cover the genetic load argument at all in his book. I don't know why since it seems very relevant. We could not survive as a species if the sequence of most of our genome was important for biological function.

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The fuzzy thinking of John Parrington: pervasive transcription

Opponents of junk DNA usually emphasize the point that they were surprised when the draft human genome sequence was published in 2001. They expected about 100,000 genes but the initial results suggested less than 30,000 (the final number is about 25,000¹. The reason they were surprised was because they had not kept up with the literature on the subject and they had not been paying attention when the sequence of chromosome 22 was published in 1999 [see Facts and Myths Concerning the Historical Estimates of the Number of Genes in the Human Genome].

The experts were expecting about 30,000 genes and that's what the genome sequence showed. Normally this wouldn't be such a big deal. Those who were expecting a large number of genes would just admit that they were wrong and they hadn't kept up with the literature over the past 30 years. They should have realized that discoveries in other species and advances in developmental biology had reinforced the idea that mammals only needed about the same number of genes as other multicellular organisms. Most of the differences are due to regulation. There was no good reason to expect that humans would need a huge number of extra genes.

That's not what happened. Instead, opponents of junk DNA insist that the complexity of the human genome cannot be explained by such a low number of genes. There must be some other explanation to account for the the missing genes. This sets the stage for at least seven different hypotheses that might resolve The Deflated Ego Problem. One of them is the idea that the human genome contains thousands and thousands of nonconserved genes for various regulatory RNAs. These are the missing genes and they account for a lot of the "dark matter" of the genome—sequences that were thought to be junk.

Here's how John Parrington describes it on page 91 of his book.

The study [ENCODE] also found that 80 per cent of the genome was generating RNA transcripts having importance, many were found only in specific cellular compartments, indicating that they have fixed addresses where they operate. Surely there could hardly be a greater divergence from Crick's central dogma than this demonstration that RNAs were produced in far greater numbers across the genome than could be expected if they were simply intermediates between DNA and protein. Indeed, some ENCODE researchers argued that the basic unit of transcription should now be considered as the transcript. So Stamatoyannopoulos claimed that 'the project has played an important role in changing our concept of the gene.'

This passage illustrates my difficulty in coming to grips with Parrington's logic in The Deeper genome. Just about every page contains statements that are either wrong or misleading and when he strings them together they lead to a fundamentally flawed conclusion. In order to critique the main point, you have to correct each of the so-called "facts" that he gets wrong. This is very tedious.

I've already explained why Parrington is wrong about the Central Dogma of Molecular Biology [John Avise doesn't understand the Central Dogma of Molecular Biology]. His readers don't know that he's wrong so they think that the discovery of noncoding RNAs is a revolution in our understanding of biochemisty—a revolution led by the likes of John A. Stamatoyannopoulos in 2012.

The reference in the book to the statement by Stamatoyannopoulos is from the infamous Elizabeth Pennisi article on ENCODE Project Writes Eulogy for Junk DNA (Pennisi, 2012). Here's what she said in that article ...

As a result of ENCODE, Gingeras and others argue that the fundamental unit of the genome and the basic unit of heredity should be the transcript—the piece of RNA decoded from DNA—and not the gene. “The project has played an important role in changing our concept of the gene,” Stamatoyannopoulos says.

I'm not sure what concept of a gene these people had before 2012. It appears that John Parrington is under the impression that genes are units that encode proteins and maybe that's what Pennisi and Stamatoyannopoulos thought as well.

If so, then perhaps the publicity surrounding ENCODE really did change their concept of a gene but all that proves is that they were remarkably uniformed before 2012. Intelligent biochemists have known for decades that the best definition of a gene is "a DNA sequence that is transcribed to produce a functional product."² In other words, we have been defining a gene in terms of transcripts for 45 years [What Is a Gene?].

This is just another example of wrong and misleading statements that will confuse readers. If I were writing a book I would say, "The human genome sequence confirmed the predictions of the experts that there would be no more than 30,000 genes. There's nothing in the genome sequence or the ENCODE results that has any bearing on the correct understanding of the Central Dogma and there's nothing that changes the correct definition of a gene."

You can see where John Parrington's thinking is headed. Apparently, Parrington is one of those scientists who were completely unaware of the fact that genes could specify functional RNAs and completely unaware of the fact that Crick knew this back in 1970 when he tried to correct people like Parrington. Thus, Parrington and his colleagues were shocked to learn that the human genome only had only 25,000 genes and many of them didn't encode proteins. Instead of realizing that his view was wrong, he thinks that the ENCODE results overthrew those old definitions and changed the way we think about genes. He tries to convince his readers that there was a revolution in 2012.

Parrington seems to be vaguely aware of the idea that most pervasive transcription is due to noise or junk RNA. However, he gives his readers no explanation of the reasoning behind such a claim. Spurious transcription is predicted because we understand the basic concept of transcription initiation. We know that promoter sequences and transcription binding sites are short sequences and we know that they HAVE to occur a high frequency in large genomes just by chance. This is not just speculation. [see The "duon" delusion and why transcription factors MUST bind non-functionally to exon sequences and How RNA Polymerase Binds to DNA]

If our understanding of transcription initiation is correct then all you need is a activator transcription factor binding site near something that's compatible with a promoter sequence. Any given cell type will contain a number of such factors and they must bind to a large number of nonfunctional sites in a large genome. Many of these will cause occasional transcription giving rise to low abundance junk RNA. (Most of the ENCODE transcripts are present at less than one copy per cell.)

Different tissues will have different transcription factors. Thus, the low abundance junk RNAs must exhibit tissue specificity if our prediction is correct. Parrington and the ENCODE workers seem to think that the cell specificity of these low abundance transcripts is evidence of function. It isn't—it's exactly what you expect of spurious transcription. Parrington and the ENCODE leaders don't understand the scientific literature on transription initiation and transcription factors binding sites.

It takes me an entire blog post to explain the flaws in just one paragraph of Parrington's book. The whole book is like this. The only thing it has going for it is that it's better than Nessa Carey's book [Nessa Carey doesn't understand junk DNA].

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1. There are about 20,000 protein-encoding genes and an unknown number of genes specifying functional RNAs. I'm estimating that there are about 5,000 but some people think there are many more.

2. No definition is perfect. My point is that defining a gene as a DNA sequence that encodes a protein is something that should have been purged from textbooks decades ago. Any biochemist who ever thought seriously enough about the definition to bring it up in a scientific paper should be embarrassed to admit that they ever believed such a ridiculous definition.

Pennisi, E. (2012) "ENCODE Project Writes Eulogy for Junk DNA." Science 337: 1159-1161. [doi:10.1126/science.337.6099.1159"]

John Avise doesn't understand the Central Dogma of Molecular Biology

I've just read Conceptual Breakthroughs in Evolutionary Genetics by John Avise. Avise is a Distinguished Professor of Ecology & Evolutionary Biology in the School of Biological Sciences at the University of Califonia at Davis (Davis, California, USA). He has written a number of excellent books including, Inside the Human Genome: A Case for Non-Intelligent Design.

His latest book consists of 70 idiosyncratic "breakthroughs" that have changed the way we think about biology. Each one is introduced with a short paragraph outlining "The Standard Paradigm" followed by another paragraph on "The Conceptual Revolution." There are 70 chapters, one for each "breakthrough," and all of them are two pages in length.

Chapter 42 is entitled: "1970 The Flow of Information."

Here's the "standard paradigm" according to John Avise.

In biochemical genetics, the molecular direction of information flow is invariably from DNA → RNA → protein. In other words, DNA is first transcribed into RNA, which then may be translated into polypeptides that make up proteins. This view was so ensconced in the field that it had become known as the "central dogma" (Crick, 1970) of molecular biology.

It's true that the Watson version of the Central Dogma was "ensconced" by 1970 and it's true that the incorrect Watson version is still "ensconced" in the textbooks.

It is NOT TRUE that this is the version that Crick described in 1970 or in his 1958 paper [see Basic Concepts: The Central Dogma of Molecular Biology]. Here's how Crick actually described the Central Dogma.

... once (sequential) information has passed into protein it cannot get out again (F.H.C. Crick, 1958)

The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred from protein to either protein or nucleic acid. (F.H.C. Crick, 1970)

The version that John Avise refers to is the incorrect version promoted by Jim Watson.

I understand that many biologists have been taught an incorrect version of the Central Dogma but if you are going to write about it you are wise to read the original papers. In this case, Avise quotes the correct paper but he clearly has not read it.

Now let's look at the "conceptual revolution" according to John Avise.

Researchers showed that biochemical information could also flow from RNA → DNA. The key discovery came when Howard Temin and David Baltimore, working independently and on different viral systems, identified an enzyme (reverse transcriptase) that catalyzes the conversion of RNA into DNA, thus enabling the passage of genetic information in a direction contrary to the central dogma.

How do I know that John Avise has not read Crick's 1970 paper? Because here's what Crick says in that paper ...

"The central dogma, enunciated by Crick in 1958 and the keystone of molecular biology ever since, is likely to prove a considerable over-simplification."

This quotation is taken from the beginning of an unsigned article headed "Central dogma reversed", recounting the very important work of Dr Howard Temin and others showing that an RNA tumor virus can use viral RNA as a template for DNA synthesis. This is not the first time that the idea of the central dogma has been misunderstood, in one way or another. In this article I explain why the term was originally introduced, its true meaning, and state why I think that, properly understood, it is still an idea of fundamental importance.

Crick tells us that the discovery of reverse transcriptase did NOT conflict with the central dogma. Thus, John Avise's conceptual revolution never happened. What happened instead, at least for some biologists, is that the discovery of reverse transcriptase taught them that their view of the central dogma was wrong. Most biologists still haven't experienced that particular conceptual revolution.

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Crick, F.H.C. (1958) On protein synthesis. Symp. Soc. Exp. Biol. XII:138-163.

Crick, F. (1970) Central Dogma of Molecular Biology. Nature 227, 561-563. [PDF file]

The fuzzy thinking of John Parrington: The Central Dogma

My copy of The Deeper Genome: Why there's more to the human genome than meets the eye has arrived and I've finished reading it. It's a huge disappointment. Parrington makes no attempt to describe what's in your genome in more than general hand-waving terms. His main theme is that the genome is really complicated and so are we. Gosh, golly, gee whiz! Re-write the textbooks!

You will look in vain for any hard numbers such as the total number of genes or the amount of the genome devoted to centromeres, regulatory sequences etc. etc. [see What's in your genome?]. Instead, you will find a wishy-washy defense of ENCODE results and tributes to the views of John Mattick.

John Parrington is an Associate Professor of Cellular & Molecular Pharmacology at the University of Oxford (Oxford, UK). He works on the physiology of calcium signalling in mammals. This should make him well-qualified to write a book about biochemistry, molecular biology, and genomes. Unfortunately, his writing leaves a great deal to be desired. He seems to be part of a younger generation of scientists who were poorly trained as graduate students (he got his Ph.D. in 1992). He exhibits the same kind of fuzzy thinking as many of the ENCODE leaders.

Let me give you just one example.

Physiologists fall for the Third Way

I looked forward to this "conversation" because I was already familiar with Denis Noble and his strange views of evolution [A physiologist thinks about evolution]. Noble reiterated his view of modern evolutionary theory at the meeting. He thinks that modern evolutionary theory (The Modern Synthesis or Neo-Darwinism) is all about random mutation and natural selection. He thinks it is based on the views of Richard Dawkins in The Selfish Gene. Neither he nor Michael Joyner (an anaethesiologist at the Mayo Clinic) have learned about random genetic drift or Neutral Theory and neither of them have much knowledge of population genetics. In other words, they are pretty ignorant about evolution even though they feel entitled to attack it.

Quantifying the "central dogma"

There was a short article in a recent issue of Science that caught my eye. The title was "Statistics requantitates the central dogma."

As most Sandwalk readers know, The Central Dogma of Molecular Biology says,

... once (sequential) information has passed into protein it cannot get out again (F.H.C. Crick, 1958)

The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred from protein to either protein or nucleic acid. (F.H.C. Crick, 1970)

You might wonder how you can quantify the idea that once information gets into protein it can't flow back to nucleic acids. You can't, of course.

The authors are referring to the standard scheme of information flow from DNA to RNA to protein. This is often mistakenly referred to as the Central Dogma by those scientists who haven't read the original papers. In this case, the authors of the Science article are asking whether the levels of protein in different cells are mostly controlled at the level of transcription, translation, mRNA degradation, or protein degradation.

A physicist tries to understand junk DNA: Part II

Yesterday I posted some comments on a blog post by physicist Rob Sheldon [A physicist tries to understand junk DNA ]. My comments were based on what I had seen on Uncommon Descent but it turns out that was only a summary of a longer post that appeared on Evolution News & Views (sic): More on Junk DNA and the "Onion Test".

The longer post doesn't add very much to the argument but it does have something interesting at the bottom. Here's what Rob Sheldon says about the Onion Test and junk DNA.

Functional RNAs?

One of the most important problems in biochemistry & molecular biology is the role (if any) of pervasive transcription. We've known for decades that most of the genome is transcribed at some time or other. In the case of organisms with large genomes, this means that tens of thousand of RNA molecules are produced from regions of the genome that are not (yet?) recognized as functional genes.

Do these RNAs have a function?

Most knowledgeable biochemists are aware of the fact that transcription factors and RNA polymerase can bind at many sites in the genome that have nothing to do with transcription of a normal gene. This simply has to be the case based on our knowledge of DNA binding proteins [see The "duon" delusion and why transcription factors MUST bind non-functionally to exon sequences and How RNA Polymerase Binds to DNA].

If you have a genome containing large amounts of junk DNA then it follows, as night follows day, that there will be a great deal of spurious transcription. The RNAs produced by these accidental events will not have a biological function.

The textbooks are wrong about protein synthesis according to a press release from the University of Utah

A recent paper in Science provides evidence that when protein synthesis is stalled a protein called Rqc2 ("conserved from yeast to man") catalyzes the addition of random amounts of alanine and threonine the the C-terminus of the proteins that's about to be destroyed (Shen et al., 2015).

Here's the editorial summary of the work ...

During the translation of a messenger RNA (mRNA) into protein, ribosomes can sometimes stall. Truncated proteins thus formed can be toxic to the cell and must be destroyed. Shen et al. show that the proteins Ltn1p and Rqc2p, subunits of the ribosome quality control complex, bind to the stalled and partially disassembled ribosome. Ltn1p, a ubiquitin ligase, binds near the nascent polypeptide exit tunnel on the ribosome, well placed to tag the truncated protein for destruction. The Rqc2p protein interacts with the transfer RNA binding sites on the partial ribosome and recruits alanine- and threonine-bearing tRNAs. Rqc2p then catalyzes the addition of these amino acids onto the unfinished protein, in the absence of both the fully assembled ribosome and mRNA. These so-called CAT tails may promote the heat shock response, which helps buffer against malformed proteins

This is mildly interesting. We've known about ubiquitin ligase for decades but this is a different way of tagging proteins for destruction.

We'll have to see if this work stands up to verification but even if it does, it's not going to make it into the textbooks.

Let's see what the University of Utah Press Office has to say ...

Defying Textbook Science, Study Finds New Role for Proteins

Open any introductory biology textbook and one of the first things you’ll learn is that our DNA spells out the instructions for making proteins, tiny machines that do much of the work in our body’s cells. Results from a study published on Jan. 2 in Science defy textbook science, showing for the first time that the building blocks of a protein, called amino acids, can be assembled without blueprints – DNA and an intermediate template called messenger RNA (mRNA). A team of researchers has observed a case in which another protein specifies which amino acids are added.

"This surprising discovery reflects how incomplete our understanding of biology is,” says first author Peter Shen, Ph.D., a postdoctoral fellow in biochemistry at the University of Utah. “Nature is capable of more than we realize." ...

Mathew Cobb, writing on Jerry Coynes blog, explains why this isn't really a big deal [CAT tails weaken the central dogma – why it matters and why it doesn’t]. Let me just add that the synthesis of peptides with defined sequences in the absence of mRNA and ribosomes has been described in most textbooks since the 1980s. The best examples are the peptides involved in pepditogylcan synthesis (cell walls) and peptide antibiotics.

Here's a figure from my book.

What this means is that the statement, "... showing for the first time that the building blocks of a protein, called amino acids, can be assembled without blueprints – DNA and an intermediate template called messenger RNA (mRNA)" is simply not true.

We really, really, need to do something about university press releases.

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Shen, P.S., Park, J., Qin, Y., Li, X., Parsawar, K., Larson, M.H., Cox, J., Cheng, Y., Lambowitz, A.M., Weissman, J.S., Brandman, O., and Frost, A. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347:75-78. [doi: 10.1126/science.1259724 ]