3,000 new genes discovered in the human genome - dark matter revealed

Let's look a a recent paper published by a large group of medical researchers at the University of California, Los Angeles (USA). The paper was published online a few days ago (Oct. 26, 2015) in Nature Immunology.

The authors clam to have discoverd 3,000 previously unknown genes in the human genome.

The complete reference is ...

Nature publishes a misleading history of the discovery of DNA repair

The history of DNA repair is well-known. Here's a quote from "Early days of DNA repair: discovery of nucleotide excision repair and homology-dependent recombinational repair" by W.D. Rupp in 2013 (Rupp, 2013).

This article describes events related to the first papers published in the 1960s describing nucleotide excision repair (NER) and homology-dependent recombinational repair.

Here's are the relevant papers.

Setlow, R.B., and Carrier, W.L. (1964) The disappearance of thymine dimers from DNA: An error-correcting mechanism. Proc. Natl. Acad. Sci. (USA) 51:226–231. [Full Text]

Boyce, R.P., Howard-Flanders, P. (1964) Release of ultraviolet light-induced thymine dimers from DNA in E. coli K-12. Proc. Natl. Acad. Sci. (USA) 51:293–300. [Full Text]

Pettijohn, D, and Hanawalt, P. (1964) Evidence for repair-replication of ultraviolet damaged DNA in bacteria. J. Mol. Biol. 9:395–410. [PubMed]

Human mutation rates

I was excited when I saw the cover of the Sept. 25th (2015) issue of Science because I'm very interested in human mutation rates. I figured there would have to be an article that discussed current views on the number of new mutations per generation even though I was certain that the focus would be on the medical relevance of mutations. I was right. There was one article that discussed germline mutations and the overall mutation rate.

The article by Shendure and Akay (2015) is the only one that addresses human mutation rates in any meaningful way. They begin their review with ...

Despite the exquisite molecular mechanisms that have evolved to replicate and repair DNA with high fidelity, mutations happen. Each human is estimated to carry on average ~60 de novo point mutations (with considerable variability among individuals) that arose in the germline of their parents (1–4). Consequently, across all seven billion humans, about 10¹¹ germline mutations—well in excess of the number of nucleotides in the human genome—occurred in just the last generation (5). Furthermore, the number of somatic mutations that arise during development and throughout the lifetime of each individual human is potentially staggering, with proliferative tissues such as the intestinal epithelium expected to harbor a mutation at nearly every genomic site in at least one cell by the time an individual reaches the age of 60 (6).

Nobel Prize for DNA repair

Tomas Lindahl, Paul Modrich, and Aziz Sancar shared the 2015 Nobel Prize in Chemistry for "for mechanistic studies of DNA repair" [Nobel Prize, Chemistry 2015].

Here's some of the press release.

In the early 1970s, scientists believed that DNA was an extremely stable molecule, but Tomas Lindahl demonstrated that DNA decays at a rate that ought to have made the development of life on Earth impossible. This insight led him to discover a molecular machinery, base excision repair, which constantly counteracts the collapse of our DNA.

Aziz Sancar has mapped nucleotide excision repair, the mechanism that cells use to repair UV damage to DNA. People born with defects in this repair system will develop skin cancer if they are exposed to sunlight. The cell also utilises nucleotide excision repair to correct defects caused by mutagenic substances, among other things.

Paul Modrich has demonstrated how the cell corrects errors that occur when DNA is replicated during cell division. This mechanism, mismatch repair, reduces the error frequency during DNA replication by about a thousandfold. Congenital defects in mismatch repair are known, for example, to cause a hereditary variant of colon cancer.

What about Phil Hanawalt?

Meanwhile, in other news: Discovery and Characterization of DNA Excision Repair Pathways: the Work of Philip Courtland Hanawalt ...

In 1963, Hanawalt and his first graduate student, David Pettijohn, observed an unusual density distribution of newly synthesized DNA during labeling with 5-bromouracil in UV-irradiated E. coli. These studies, along with the discovery of CPD excision by the Setlow and Paul Howard-Flanders groups, represented the co-discovery of nucleotide excision repair.

And Wikipedia [Philip Hanawalt] says,

Philip C. Hanawalt (born in Akron, Ohio in 1931) is an American biologist who discovered the process of repair replication of damaged DNA in 1963. He is also considered the co-discoverer of the ubiquitous process of DNA excision repair along with his mentor, Richard Setlow, and Paul Howard-Flanders. He holds the Dr. Morris Herzstein Professorship in the Department of Biology at Stanford University,[1] with a joint appointment in the Dermatology Department in Stanford University School of Medicine.

Here's what Hanawalt himself says about discovering DNA excision repair [The Awakening of DNA Repair at Yale] ...

Upon joining the faculty at Stanford University in late 1961 as Research Biophysicist and Lecturer, I returned to the problem of what UV did to DNA replication, now that we knew the principal photoproducts. I wanted to understand the behavior of replication forks upon encountering pyrimidine dimers, and I was hoping to catch a blocked replication fork at a dimer. Using density labeling with 5-bromouracil and radioactive labeling of newly-synthesized DNA, we were able to observe partially replicated DNA fragments in E. coli [13]. However, in samples from UV irradiated bacterial cultures, the density patterns of nascent DNA indicated that much of the observed synthesis was in very short stretches, too short to appreciably shift the density of the DNA fragments containing them [14]. I communicated these results to Setlow by phone and learned that he had just discovered that pyrimidine dimers in wild type cells, but not in Ruth Hill’s UV sensitive mutant, were released from the DNA into an acid soluble fraction. We speculated in discussion that my student, David Pettijohn, and I were detecting a patching step by which a process of repair replication might use the complementary DNA strand as template to fill the single-strand gaps remaining after the pyrimidine dimers had been removed. At about the same time, Paul Howard-Flanders in the Department of Therapeutic Radiology at Yale had isolated a number of UV-sensitive mutants from E. coli K12 strains, and he was able to show that these mutants were also deficient in removing pyrimidine dimers from their DNA. The seminal discovery of dimer excision was published by the Setlow and Howard-Flanders groups, as the first indication of an excision repair pathway [15,16]. Of course, the excision per se is not a repair event but only the first step, since it generates another lesion, the gap in one strand of the DNA. We carried out more controls, to then claim that we had discovered a non-conservative mode of repair replication, constituting the presumed patching step in the postulated excision-repair pathway [17]. I later showed that DNA containing the repair patches could undergo semiconservative replication with no remaining blockage [18].

Richard Boyce and Howard-Flanders at Yale also documented excision of lesions induced by mitomycin C in E. coli K12 strains, indicating some versatility of excision repair [19]. In a collaboration with Robert Haynes, I found a similar pattern of repair replication after nitrogen mustard exposure to that following UV, and we concluded that “it is not the precise nature of the base damage that is recognized, but rather some associated secondary structural alteration …” We speculated that “[s]uch a mechanism might even be able to detect accidental mispairing of bases after normal replication,” thus predicting the existence of a mismatch repair pathway [20]. Mismatch repair was reported by Wagner and Meselson a decade later [21] and yet another excision repair mode, termed base excision repair, was discovered by Tomas Lindahl [22].

One of Hanawalt's students was Jonathan Eisen [Tree of Life]. I'll be interested in hearing what he has to say about this Nobel Prize. It seems unfair to me.

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How many RNA molecules per cell are needed for function?

One of the issues in the junk DNA wars is the importance of all those RNAs that are detected in sensitive assays. About 90% of the human genome is complementary to RNAs that are made at some time in some tissue or other. Does this pervasive transcription mean that most of the genome is functional or are most of these transcripts just background noise due to accidental transcription?

A little learning of biochemistry ...

A little learning is a     dangerous thing;
drink deep, or taste not the     Pierian spring:
there shallow draughts     intoxicate the brain,
and drinking largely     sobers us again.
                  Alexander PopeI've been following Angelo Grasso on Facebook because he posts a lot of biochemistry stuff. His schtick is to post some complicated pathway or structure then marvel at how complex it is and how it had to be designed. For a while I was commenting on his posts in order to show him why his interpretation was wrong or misleading but he just kept posting more examples gleaned from biochemistry textbooks.

This is a classic examples of someone who knows just enough to be dangerous. His latest post is about glycolysis and membrane-associated electron transport in animals. You can see it on the reasonandscience.heavenforum website: Glycolysis. Here's the bottom line ...

Four things that Francis Collins learned from sequencing the human genome

I've been doing a bit of research on the human genome in preparation for a book. This led me to an article published in 2003 by Francis Collins, former head of the Human Genome Consortium (Collins, 2003). It's mostly about how he deals with science and religion but there was an interesting description of what he learned from completing the human genome sequence.

Here's what he said ....

We discovered some pretty surprising things in reading out the human genome sequence. Here are four highlights.

1. Humans have fewer genes than expected. My definition of a gene here—because different people use different terminology—is a stretch of DNA that codes for a particular protein. There are probably stretches of DNA that code for RNAs that do not go on to make proteins. That understanding is only now beginning to emerge and may be fairly complicated. But the standard definition of “a segment of DNA that codes for a protein” gives one a surprisingly small number of about 30,000 for the number of human genes. Considering that we’ve been talking about 100,000 genes for the last fifteen years (that’s what most of the textbooks still say), this was a bit of a shock. In fact, some people took it quite personally. I think they were particularly distressed because the gene count for some other simpler organisms had been previously determined. After all, a roundworm has 19,000 genes, and mustard weed has 25,000 genes, and we only have 30,000? Does that seem fair? Even worse, when they decoded the genome of the rice, it looks as if rice has about 55,000 genes. So you need to have more respect for dinner tonight! What does that mean? Surely, an alien coming from outer space looking at a human being and looking at a rice plant would say the human being is biologically more complex. I don’t think there’s much doubt about that. So gene count must not be the whole story. So what is going on?

2. Human genes make more proteins than those of other critters. One of the things going on is that we begin to realize that one gene does not just make one protein in humans and other mammals. On the average, it makes about three, using the phenomenon of alternative splicing to create proteins with different architectures. One is beginning to recover some sense of pride here in our genome, which was briefly under attack, because now we can say, “Well, we don’t have very many genes but boy are they clever genes. Look what they can do!”

3. The male mutation rate is twice that of females. We also discovered that simply by looking at the Y chromosome and comparing it to the rest of the genome—of course, the Y chromosome only passes from fathers to sons, so it only travels through males—you can get a fix on the mutation rate in males compared to females. This was not particularly good news for the boys in this project because it seems that we make mistakes about twice as often as the women do in passing our DNA to the next generation. That means, guys, we have to take responsibility for the majority of genetic disease. It has to start somewhere; the majority of the time, it starts in us. If you are feeling depressed about that, let me also point out we can take credit for the majority of evolutionary progress, which after all is the same phenomenon.

4. “Junk” DNA may not be junk after all. I have been troubled for a long time about the way in which we dismissed about 95% of the genome as being junk because we didn’t know what its function was. We did not think it had one because we had not discovered one yet. I found it quite gratifying to discover that when you have the whole genome in front of you, it is pretty clear that a lot of the stuff we call “junk” has the fingerprints of being a DNA sequence that is actually doing something, at least, judging by the way evolution has treated it. So I think we should probably remove the term “junk” from the genome. At least most of it looks like it may very well have some kind of function.

Insulators, junk DNA, and more hype and misconceptions

The folks at Evolution News & Views (sic) can serve a very useful purpose. They are constantly scanning the scientific literature for any hint of evidence to support their claim about junk DNA. Recall that Intelligent Design Creationists have declared that if most of our genome is junk then intelligent design is falsified since one of the main predictions of intelligent design is that most of our genome will be functional.

THEME

Genomes & Junk DNA
They must be getting worried because their most recent posts sounds quite desperate. The last one is: The Un-Junk Industry. It quotes a popular press report on a paper published recently in Procedings of the National Academy of Sciences (USA). The creationists concede that the paper itself doesn't even mention junk DNA but the article in EurekAlert does.

How to write about RNA

I find it very frustrating to read reports about RNA these days because the writers almost always misrepresent the history of the field and exaggerate the significance of recent discoveries. An article in the July 23, 2015 issue of Nature illustrates the problem. The article is written by Elie Dolgin (@ElieDolgin), a freelance science journalist based in Massachusetts (USA). He graduated from McGill University (Montreal, Quebec, Canada) with a degree in biology and obtained a Ph.D. in genetics and evolution from the University of Edinburgh (Edinburgh, Scotland, UK).

On the total length of all DNA molecules on the planet

If you were to line up all the DNA molecules from all the individuals in all the species on Earth, how long would it be? This is a kind of "Fermi question" or "Fermi problem." You should be able to estimate an answer based on what you know and reasonable assumptions.

Michael Lynch has a crude estimate in his book The Origins of Genome Architecture. Without reading the book, can you come up with an estimate of your own? Is it larger than the circumference of the Earth? Larger than the distance to Pluto? Longer than the distance to the nearest star (other than the sun) or the the center of the galaxy? Would the string of DNA molecules stretch to the nearest large galaxy (Andromeda)? Or, would it be even longer than that?

In case you've forgotten everything you once knew about the structure of DNA, here's a brief refresher: The Three-Dimensional Structure of DNA.

You may assume that all of the DNA molecules are in the standard B-form with the dimensions shown in the figure.

I will not accept any answers in archaic measurement units like leagues, miles, yojana, or cubits.

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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}

University of Toronto Professor, teaching stream

After years of negotiation between the administration and the Faculty Association, the university has finally allowed full time lecturers to calls themselves "professors" [U of T introduces new teaching stream professorial ranks]. This brings my university into line with some other progressive universities that recognize the value of teaching.

Unfortunately, the news isn't all good. These new professors will have a qualifier attached to their titles. The new positions are: assistant professor (conditional), teaching stream; assistant professor, teaching stream; associate professor, teaching stream; and professor, teaching stream. Research and scholarly activity is an important component of these positions. The fact that the activity is in the field of pedagogy or the discipline in which they teach should not make a difference.

Meanwhile, current professors will not have qualifiers such as "professor: research," or "professor: administration," or "professor: physician," or "professor: mostly teaching."

The next step is to increase the status of these new professors by making searches more rigorous and more competitive, by keeping the salaries competitive with other professors in the university, and by insisting on high quality research and scholarly activity in the field of pedagogy. The new professors will have to establish an national and international reputation in their field just like other professors. They will have to publish in the pedagogical literature. They are not just lecturers. Almost all of them can do this if they are given the chance.

Some departments have to change the way they treat the new professors. The University of Toronto Faculty Association (UTFA) has published a guideline: Teaching Stream Workload. Here's the part on research and scholarly activity ....

• In section 7.2, the WLPP offers the following definition of scholarship: “Scholarship refers to any combination of discipline-based scholarship in relation to or relevant to the field in which the faculty member teaches, the scholarship of teaching and learning, and creative/professional activities. Teaching stream faculty are entitled to reasonable time for pedagogical/professional development in determining workload.”
• It is imperative that teaching stream faculty have enough time in their schedules, that is, enough “space” in their appointments, to allow for the “continued pedagogical/professional development” that the appointments policy (PPAA) calls for. Faculty teaching excessive numbers of courses or with excessive administrative loads will not have the time to engage in scholarly activity. Remember that UTFA fought an Association grievance to win the right for teaching stream faculty to “count” their discipline-based scholarship. That scholarship “counts” in both PTR review and review for promotion to senior lecturer.

And here's a rule that many departments disobey ...

Under 4.1, the WLPP reminds us of a Memorandum of Agreement workload protection: “faculty will not be required to teach in all three terms, nor shall they be pressured to volunteer to do so.” Any faculty member who must teach in all three terms should come to see UTFA.

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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]

Biochemists can be astronauts!

The latest issue of ASBMS Today has an article about the American astronaut Paggy Whitson [see A lab with a view].

Peggy Whitson is a biochemist. She did her Ph.D. with Kathleen Mathews at Rice University in Houston, Texas, USA. I frequently refer to her work on the lac repressor and its interaction with lac operator sequences [see Repression of the lac Operon]. Here are some of her papers. Once you understand this stuff, you are in a better position to judge the ENCODE results and the role of spurious binding sites.

Hsieh, W.T., Whitson, P.A., Matthews, K.S., and Wells, R.D. (1987) Influence of sequence and distance between two operators on interaction with the lac repressor. Journal of Biological Chemistry, 262: 14583-14591.

The influence of additional operator or pseudooperator sequences on the lactose repressor-operator interaction has been investigated. Results of kinetic and equilibrium binding measurements suggest an important in vivo role for the Z-gene pseudooperator in repressor-operator binding; the formation of a ternary, looped complex is indicated by the influence of secondary operator sites on binding parameters. Although the binding affinity of the Z-gene pseudooperator [Oz] is only approximately 1/30 that observed for the primary operator [O], the binding affinity to DNA containing both Oz and O is significantly higher than either sequence alone or the sum of the two. This synergistic effect is enhanced further by replacing the pseudooperator sequence [Oz] with the primary operator sequence and results in an even stronger ternary complex in plasmids with duplicate primary sites. The distance between the center of the two primary operators affects the formation of a ternary complex in the linear DNA molecules. Decreased dissociation rate constants were observed with spacing of operator-like sequences between 300 and 500 base pairs (bp). Minimal influence of the second operator on repressor binding is observed when the operators are separated by approximately 4000 or approximately 100 bp. The significant influence of distance on kinetic and equilibrium parameters was demonstrated by measurements on plasmid pRW1511 [Oi-O-PL-Oz] cleaved with restriction enzymes either in the polylinker region to place Oi-O and Oz on opposite ends of the linear plasmid or outside this region to maintain the sites within 500 bp. These results are consistent with the formation of operator-repressor-pseudooperator ternary complex to generate a looped DNA structure.

Whitson, P.A., Hsieh, W.T., Wells, R.D., and Matthews, K.S. (1987) Supercoiling facilitates lac operator-repressor-pseudooperator interactions. Journal of Biological Chemistry, 262:4943-4946.

The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling. Dissociation rate constants for plasmids containing a single operator site decreased as a function of the negative supercoil density. However, the presence of pseudooperators in the plasmid DNA in addition to the primary operator sequence resulted in a significant decrease in the operator-plasmid dissociation rate at higher negative supercoil densities. Approximately eight ionic interactions were determined for both the supercoiled plasmids and the linear DNAs examined. These results suggest that the stabilization provided by the topology of supercoiled DNA affects the nonionic component of the protein-DNA interaction. The ability to form a ternary complex of protein with two DNA segments is increased by the presence of multiple operator-like sites on the DNA. Furthermore, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate and results in a remarkably stable ternary, presumably looped complex (t1/2 approximately 28 h). These data suggest a critical role in vivo for DNA topology and pseudooperator(s) in transcriptional regulation of the lac operon.

Whitson, P.A., Hsieh, W.T., Wells, R.D., and Matthews, K.S. (1987) Influence of supercoiling and sequence context on operator DNA binding with lac repressor. Journal of Biological Chemistry, 262(30), 14592-14599.

The dissociation of the repressor-operator complex from a series of negatively supercoiled plasmid DNAs was examined as a function of the sequence context, orientation, and spacing. The plasmids were grouped into four classes, each with common sequence context. The highest dissociation rate constants were observed for the plasmids containing only a single operator (or pseudooperator) sequence, while approximately 10-fold lower rate constants were measured for plasmids with the I gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator. Comparison of the behavior of these two classes of plasmids demonstrated the importance of two operator sequences and supported a model of DNA loop formation to stabilize the repressor-operator complex (Whitson, P. A., and Matthews, K. S. (1986) Biochemistry 25, 3845-3852; Whitson, P. A., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 3852-3858; Whitson, P. A., Hsieh, W. T., Wells, R. D., and Matthews, K. S. (1987) J. Biol. Chem. 262, 4943-4946; Krämer, H., Niemöller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491). The third class, with intermediate dissociation rate constants, was comprised of plasmids which contained the primary operator and the higher affinity pseudooperator normally located in the Z gene. Neither the additional presence of the I gene pseudooperator nor the orientation of the primary operator relative to the Z gene pseudooperator significantly affected the dissociation rate constants. The binding characteristics of this group of plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternary complex and suggested an in vivo function for this pseudooperator. Plasmids containing two primary operator sequences were the class with lowest dissociation rate constants from lac repressor, and minimal effects of salt or spacing on dissociation of this class were observed. These data are consistent with formation of an intramolecular complex with a looped DNA segment stabilized by the combination of increased local concentration of binding sites and torsional stresses on the DNA which favor binding in supercoiled DNA.

Whitson, P.A., and Matthews, K.S. (1986) Dissociation of the lactose repressor-operator DNA complex: Effects of size and sequence context of operator-containing DNA. Biochemistry, 25:3845-3852.

The dissociation kinetics for repressor-³²P-labeled operator DNA have been examined by adding unlabeled operator DNA to trap released repressor or by adding a small volume of concentrated salt solution to shift the Kd of repressor-operator interaction. The dissociation rate constant for pLA 322-8, an operator-containing derivative of pBR 322, was 2.4 × 10^-3 s^-1 in 0.15 M KCl. The dissociation rate constant at 0.15 M KC1 for both Xplac and pIQ, each of which contain two pseudooperator sequences, was ~6 × l0^-4 s^-1. Elimination of Elimination flanking nonspecific DNA sequences by use of a 40 base pair operator-containing DNA fragment yielded a dissociation rate constant of 9.3 × 10^-3 s^-l. The size and salt dependences of the rate constants suggest that dissociation occurs as a multistep process. The data for all the DNAs examined are consistent with a sliding mechanism of facilitated diffusion to/from the operator site. The ability to form a ternary complex of two operators per repressor, determined by stoichiometry measurements, and the diminished dissociation rates in the presence of intramolecular nonspecific and pseudooperator DNA sites suggest the formation of an intramolecular ternary complex. The salt dependence of the dissociation rate constant for pLA 322-8 at high salt concentrations converges with that for a 40 base pair operator. The similarity in dissociation rate constants for pLA 322-8 and a 40 base pair operator fragment under these conditions indicates a common dissociation mechanism from a primary operator site on the repressor.

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Are biochemistry instructors going to treat evolution as a core concept or are they going to teach to the MCAT?

The American Society for Biochemistry and Molecular Biology (ASBMB) has recommended that biochemistry courses concentrate on core concepts rather than details. It has defined five categories of core concepts that are essential in understanding biochemistry and molecular biology [see ASBMB Core Concepts in Biochemistry and Molecular Biology: Molecular Structure and Function].

Theme

Better BiochemistryI strongly support the concept of teaching core concepts even though I disagree with many of the actual concepts that are proposed. Here are the five core concepts with links to my discussions.

1. evolution [ASBMB Core Concepts in Biochemistry and Molecular Biology: Evolution ]
2. matter and energy transformation [ASBMB Core Concepts in Biochemistry and Molecular Biology: Matter and Energy Transformation]
3. homeostasis [ASBMB Core Concepts in Biochemistry and Molecular Biology: Homeostasis]
4. biological information [ASBMB Core Concepts in Biochemistry and Molecular Biology: Biological Information]
5. macromolecular structure and function [ASBMB Core Concepts in Biochemistry and Molecular Biology: Molecular Structure and Function]

Nature reviews Nessa Carey's book on junk DNA

Read it at" Genetics: We are the 98%. Here's the important bit ...

Finally, Junk DNA, like the genome, is crammed with repetitious elements and superfluous text. Bite-sized chapters parade gee-whizz moments of genomics. Carey's The Epigenetics Revolution (Columbia University Press, 2012) offered lucid science writing and vivid imagery. Here the metaphors have been deregulated: they metastasize through an otherwise knowledgeable survey of non-coding DNA. At one point, the reader must run a gauntlet of baseball bats, iron discs, Velcro and “pretty fabric flowers” to understand “what happens when women make eggs”. The genome seems to provoke overheated prose, unbridled speculation and Panglossian optimism. Junk DNA produces a lot of DNA junk.

The idea that the many functions of non-coding DNA make the concept of junk DNA obsolete oversells a body of research that is exciting enough. ENCODE's claim of 80% functionality strikes many in the genome community as better marketing than science.

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Nessa Carey doesn't understand junk DNA

Nessa Carey is a science writer with a Ph.D. in virology and she is a former Senior Lecturer in Molecular Biology at Imperial College, London.

She has written a book on junk DNA but it's not available yet (in Canada). Judging by her background, she should be able to sort through the controversy and make a valuable contribution to informing the public but, as we've already noted Nessa Carey and New Scientist don't understand the junk DNA debate.

Casey Luskin has a copy of the book so he wrote a blog post on Evolution News & Views. He's thrilled to find someone else who dismisses junk DNA and "confirms" the predictions of Intelligent Design Creationism. I hope Nessa Carey is happy that the IDiots are pleased with her book [New Book on "Junk DNA" Surveys the Functions of Non-Coding DNA].

The Virtual Cell Animation Collection

I'm interested in science education in general and teaching biochemistry and molecular biology in particular. A recent publication in PLoS Biology caught my eye ...

Reindl, K.M., White, A. R., Johnson, C., Vender, B., Slator, B.M., and McClean, P. (2105) The Virtual Cell Animation Collection: Tools for Teaching Molecular and Cellular Biology. PLoS Biology 13(4): e1002118 DOI: 10.1371/journal.pbio.1002118

The paper focuses on the value of short animations for teaching biochemistry and molecular biology to advanced high school students and college students.

There's nothing in the paper about the scientific accuracy of the presentations or the pedagogical approach and this is unfortunate. The animations only show complex eukaryotic cells in spite of the fact that the American Society for Biochemistry and Molecular Biology recommends an evolutionary approach to teaching. The fact that the videos emphasize eukaryotes leads to some interesting descriptions of fundamental processes.

Look at the video on transcription regulation for example [Regulated Transcription]. The textbooks teach this using simple systems such as E. coli transcription then they move on to more complex prokarotic systems such as the lac operon. Then they cover the eukaryotic examples pointing out how they differ from the simple bacterial systems. This has always been a successful approach to teaching the basic concepts of transcription and transcription regulation. ¹

Is the approach taken by the authors of The Virtual Cell Animation project better? I don't think so. What do you think? Does anyone out there teach transcription without introducing it first in bacteria?

Let's not forget my favorite example of biochemical misconceptions: the Citric Acid Cycle. Did you know that it's sometimes called the "tricarboxylic acid cycle" because three CO₂ molecules are released for every pyruvate molecule? ²

The carboxylate groups on citrate, isocitrate etc. are shown as -COOH instead of COO^- as in the textbooks. I don't know why they did this ... it leads to some extra protons being released in the reactions.

The authors make a very common mistake with succinate dehydrogenase. They show FADH₂ as one of the products of the reaction whereas the IUBMB database shows that the real final product is QH₂ [see Succinate Dehydrogenase]. I don't understand why biochemistry teachers can't check out a leading textbook (or the scientific literature) before producing a video.

Did you know that some of the reactions of glycolysis are irreversible? Check out the video on Glycolysis to find out which reactions have this interesting property. ³ There is no video on gluconeogenesis and that's surprising because the synthesis of glucose is far more important than glycolysis in most species.

I wonder if the editors of PLoS bothered to watch the videos or whether they just assumed that they were scientifically accurate and pedagogically sound? I'm guessing that they didn't see the need to review the videos and simply concentrated on whether all the words in the article were spelled correctly.

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1. There's a separate video on the lac Operon. How many errors, flaws, or missed opportunities, can you spot?

2. Silly me. I always though it had something to do with the fact that two of the key intermediates (citrate and isocitrate) were tricarboxylic acids. Most of the others are dicarboxylic acids.

3. Maybe I'm quibbling. In my textbook I describe these reactions as "metabolically irreversible" because the activities of the enzymes are regulated. That's not the same as saying that the reactions are irreversible.

Mary Lyon (1925 - 2014)

Mary Lyon died on Christmas day last December. She was 89 years old.

She was a famous mouse geneticist who spend most of her working career at the MRC labs in Harwell, United Kingdom (near Oxford). The labs are known as an international center for mouse genetics.

Mary Lyon is famous for discovering the phenomenon of X chromosome inactivation. This is when one the the X chromosomes of female mammals is selectively inactivated so that the products of the X chromosome genes are quantitatively similar to the dosage in males where there's only one X chromosome. The phenomenon used to be referred to as Lyonization.

I never met Mary Lyon but from what people say about her, I'm sure I would have liked her. Here's an excerpt from the obituary in Nature: Mary F. Lyon (1925 - 2014).

Lyon was a central figure in twentieth-century mouse genetics. She laid the intellectual foundations and developed the genetic tools for the use of mice as model organisms in molecular medicine, cell and developmental biology and in deciphering the function of the human genome. Lyon was editor of Mouse News Letter from 1956 to 1970, a publication that had a key role in establishing a mouse-focused research community in the pre-Internet age. She also helped to develop a common language for the field by chairing the Committee on Standardised Genetic Nomenclature for Mice from 1975 to 1990. Her pivotal contribution was recognized by the naming of the Mary Lyon Centre, an international facility for mouse-genetic resources, opened at Harwell in 2004, and by the creation of the Mary Lyon Medal by the UK Genetics Society in 2014.

Because everything Mary said was so carefully thought through, she could be difficult to talk to: on the phone, it was easy to think you had been cut off. She did not suffer fools gladly, but was a great supporter of the bright young scientist, often eschewing authorship of publications to enhance the profile of junior collaborators. She was intellectually rigorous but not dictatorial. When I began my PhD with her in 1977, she gave me a handful of papers, showed me the genetic tools — mice carrying the various mutations and chromosomal rearrangements — and said, “do something on X-inactivation”. That degree of academic freedom was exhilarating, coupled as it was with the safety net of robust critique.

... Her first love was mice, although she always had a cat — a tortoiseshell, of course.

X chromosome inactivation is one of the classic examples of epigenetics, sensu stricto. It was the subject of one of my most popular posts of all time: Calico cats. Calico cats almost always have to be female but there are very rare examples of male calico cats. Can anyone figure out why?

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