Nobel Laureates: Richard Roberts and Phillip Sharp

 
The Nobel Prize in Physiology or Medicine 1993.

"for their discoveries of split genes"

Richard J. Roberts (1943 - ) and Phillip A. Sharp (1944 - ) received the Nobel Prize in Physiology or Medicine for their discovery of interrupted genes and splicing in eukaryotes [see RNA Splicing: Introns and Exons and Monday's Molecule #31].

Roberts and Sharp discovered that the genes in adenovirus were split into various segments that were combined during RNA processing. The results started to become widely known in 1975-76 and the key papers were published in 1977. Later this gene organization was found to be common in chromosomal eukaryotic genes. Unlike many Nobel Prize discoveries, this one really was revolutionary. Here's the presentation speech by Professor Bertil Daneholt of the Nobel Assembly of the Karolinska Institute.

Your Majesties, Your Royal Highnesses, Ladies and Gentlemen,

Why do children resemble their parents? This question has probably always fascinated humans, but not until the advent of natural science have we arrived at an increasingly satisfactory answer.

In the middle of the last century, the Austrian monk Gregor Mendel conducted his famous breeding experiments with the garden pea. He concluded that every trait of an individual plant is determined by a set of two genes, one obtained from each parental plant. To Mendel a gene was an abstract concept, which he used to interpret his breeding experiments. He had no idea of the physical properties of genes.

Only in the mid-1940s could it be established that in terms of chemistry, genetic material is composed of the nucleic acid DNA. About ten years later the double helical structure of DNA was revealed. Ever since then, progress within the field of molecular biology has been very rapid, and several Nobel prizes have been awarded in this area of research.

Initially, genetic material was studied mainly in simple organisms, particularly in bacteria and bacterial viruses. It was shown that a gene occurs in the form of a single continuous segment of the long, thread-like DNA, and it was generally assumed that the genes in all organisms looked this way. Therefore, it was a scientific sensation when this year's Nobel Laureates, Richard Roberts and Phillip Sharp, in 1977, independently of each other, observed that a gene in higher organisms could be present in the genetic material as several distinct and separate segments. Such a gene resembles a mosaic. Both Roberts and Sharp analyzed an upper respiratory virus, which is particularly suitable for studies of the genetic material in complex organisms. It soon became apparent that most genes in higher organisms, including ourselves, exhibited this mosaic structure.

Roberts' and Sharp's discovery opened up a new perspective on evolution, that is, on how simple organisms develop into more complex ones. Earlier it was believed that genes evolve mainly through the accumulation of small discrete changes in the genetic material. But their mosaic gene structure also permits higher organisms to restructure genes in another, more efficient way. This is because during the course of evolution, gene segments - the individual pieces of the mosaic - are regrouped in the genetic material, which creates new mosaic patterns and hence new genes. This reshuffling process presumably explains the rapid evolution of higher organisms.

Roberts and Sharp also predicted that a specific genetic mechanism is required to enable split genes to direct the synthesis of proteins and thereby to determine the properties of the cell. Researchers had known for many years that a gene contains detailed instructions on how to build a protein. This instruction is first copied from DNA to another type of nucleic acid, known as messenger RNA. Subsequently, the RNA instruction is read, and the protein is synthesized. What Roberts and Sharp were now stating was that the messenger RNA in higher organisms has to be edited. The required process, called splicing, resembles the work that a film editor performs: the unedited film is scrutinized, the superfluous parts are cut out and the remaining ones are joined to form the completed film. Messenger RNA treated in this manner contains only those parts that match the gene segments. It later turned out that the same parts of the original messenger RNA are not always saved during the editing- there are choices. This implies that splicing can regulate the function of the genetic material in a previously unknown way.

Roberts' and Sharp's discovery also helps us understand how diseases arise. One example is a form of anemia called thalassemia, which is due to inherited defects in the genetic material. Several of these defects cause errors in the editing process during splicing; thus, an abnormal messenger RNA is formed and subsequently also a protein that functions poorly or not at all.

The discovery of split genes was revolutionary, triggering an explosion of new scientific contributions. Today this discovery is of fundamental importance for research in biology as well as in medicine.

Dr. Richard Roberts and Dr. Phillip Sharp,

Your discovery of split genes led to the prediction of a new genetic process, that of RNA splicing. The discovery also changed our view of how genes in higher organisms develop during evolution. On behalf of the Nobel Assembly of the Karolinska Institute I wish to convey to you our warmest congratulations, and I now ask you to step forward to receive the Nobel Prize from the hands of His Majesty the King.

What Does the "Support Our Troops" Ribbon mean to You?

 
Since last October emergency vehicles in Toronto have been displaying a decal in support of our troops in Afghanistan. The decals were placed on the vehicles at the request of firefighters and paramedics, whose unions are strong supporters of the soldiers. The original deal was that the decals would stay on for one year and then be removed when the vehicles came in for routine maintenance this Fall.

The issue has turned into a hot political fight that will be decided today at a City Council meeting [Time limit for 'Support Our Troops' ribbons is up].

As you might imagine, there are some city councilors who want the decals to stay on the ambulances and fire trucks.

Some councilors believe the decision to remove the decals is a black mark on the city.

"I was stunned this morning to hear on the radio that some official at the city had ordered emergency services, particularly ambulances, to take off the decal that supports our troops in Afghanistan," city councilor Brian Aston told CTV News on Tuesday.

"These decals are on there and it makes a very strong statement. To take them off, Toronto is the largest city, would just be an outrage. It would be a black eye on the reputation of our city," Ashton said.

It should also come as no surprise that some councilors want to stick to the original agreement and remove the decals in September.

Coun. Janet Davis said just as many councillors want to see the decals removed as those who support their presence on emergency vehicles.

Mayor David Miller said while emergency crews should continue to support Canadian troops, the one-year time limit for the decals was enough time.

"It's controversial on both sides. There are people who see it as support for the troops and there are people who see it as support for war," Miller said.

I'm one of those who believe that the "Support the Troops" ribbon is a political statement. I don't know very many people who are opposed to the war but have this sticker on their car. On the surface it seems like a no-brainer to offer support to our troops while opposing the mission. But, in fact, the term "no-brainer" is quite appropriate in this case. By blindly advertising support for the military you obscure the true difficulty in making rational decisions about how to deploy our army. It's no secret that most people who "support our troops" are also conservatives who are in favour of the war.

The idea that the "Support Our Troops" yellow ribbons are politically neutral is something that only a supporter of the war would say. It's ridiculous. It would be like putting peace symbols on the trucks on the grounds that surely everyone supports peace.

I am very supportive of individual soldiers who are posted to Afghanistan. It's not their fault that our government is insane. They have to follow orders. But that does not mean that I "support our troops" in the way that the decal signifies. As a matter of fact, I do not support our mission in Afghanistan and I would withdraw the troops tomorrow if I could. Every soldier who dies in Afghanistan will have died in vain. That's hardly a way to offer support to our troops.

Having those decals on city vehicles sends the wrong message. For those of us who oppose the war it signifies that the fire fighters and paramedics are on the other side of the issue. That makes me uncomfortable since these are people who deserve my respect and admiration but they're not going to get it if they push a political agenda through advertising on their vehicles.

Take the decals off. It's no place for politics.

RNA Splicing: Introns and Exons

Most eukaryotic protein-encoding genes are interrupted. The coding regions are divided into numerous blocks called "exons" and the exons are separated by "introns."

An example is shown below. The triose phosphate isomerase (TPI) gene from maize is composed of 9 exons and 8 introns. (Triose phosphate isomerase is one of the enzymes in the glycolysis/gluconeogenesis pathway.)


The top line is a cartoon representation of the TPI gene with each exon in a different color. The thick gray lines between them represent the introns. The gene is transcribed from left (5′) to right (3′) beginning at the promoter (P). The long primary RNA transcript contains both intron and exon sequences. Subsequent processing of this primary transcript results in modification of the 5′ end by addition of an m⁷ GTP cap and modification of the 3′ end by addition of adenylate (A) residues to form the poly A tail. More importantly, the introns are spliced out and the exon sequences are fused to form the mature mRNA. This mRNA is then transported to the cytoplasm where it is translated into protein.

Note that all the coding regions in the exons (hatched) are contiguous in the mature mRNA. The relationship between the exons and the structure of the protein is shown on the right where the color of each segment of the protein corresponds to the color of the exons in the upper figure. There is no correlation between the exons and any protein domains or motifs. (It used to be thought that exons corresponded to domains in the protein.)

The splicing reaction is complicated. The cell must cleave the primary transcript at each end of the intron while holding on to the flanking exons so the chopped RNA transcript does not come apart. Then the two exons have to be joined together. For protein-encoding genes the splicing reactions are catalyzed by an RNA/protein complex called a spliceosome. In some cases, the introns can be thousands of nucleotides long—much longer than the exons.

Let's look at a simplified version of this reaction. The various components of the spliceosome have to assemble at the 5′ (left) end of an intron and at the 3′ end. There's a third site in the middle called the branch site. All three sites are identified by specific short sequences in the primary transcript as shown below.


These are the consensus sequences for vertebrates, including us. The splice site and branch site sequences in other species are similar but not identical.

In the first step of the splicing reaction, the various components of the spliceosome bind to the 5′ splice site, the 3′ splice site, and the branch site. Then the three complexes interact with each other to draw together the ends of the intron and position them near the branch site. This forms the spliceosome.

The first reaction involves an attack of the 2′ -OH group of the branch point adenylate residue on the 5′ splice site. This forms an intermediate where the branch site A residue is attached to three different ends of the primary transcript. The structure resembles a lariat or lasso. This is the structure depicted in Monday's Molecule #31.

Meanwhile, the 5′ end of the transcript is still bound to the spliceosome. This is important because it's about to be joined to the next exon and the reaction wouldn't work if the 5′ end were released following the first cleavage reaction.

In the next step, the spliceosome catalyzes the attack of the -OH group at the end of the 5′ exon on the 3′ splice site. This results in cleavage of the 3′ intron/exon junction and joining of the 5′ exon to the 3′ exon. The intron sequence (dark brown) is released as a lariat (looped) structure.

The two reactions are known as transesterification reactions because they require the breaking of one strand of RNA and formation of a new ester linkage. The details are not very important. What's important is to recognize that splicing depends on the correct interaction between the components of the spliceosome and the 5′ and 3′ splice site sequences (and the branch site).

These interactions are mediated by small RNAs that are bound to the spliceosome proteins. These RNAs are called small nuclear RNAs (snRNAs) and they're one example of a host of small RNAs produced by non-protein encoding genes. The snRNA/protein complexes are called small nuclear ribonuclear proteins or snRNPs (snurps).

The snRNAs are complimentary to the splice sites and branch sites and that's how the various snRNPs recognize them. This interaction is very weak since it depends on only three or four base pairs. It can be even less since there are many slice sites that are not perfect matches to the consensus sequences shown above. The relative lack of significant sequence similarity makes splicing a very error-prone reaction.

U1 snRNP recognizes 5′ splice sites, U2 snRNP binds to the branch site, and U5 snRNP binds to the 3′ splice site. A more detailed description of the formation of the splicesome is shown below.

What is a gene, post-ENCODE?

Back in January we had a discussion about the definition of a gene [What is a gene?]. At that time I presented my personal preference for the best definition of a gene.

A gene is a DNA sequence that is transcribed to produce a functional product.

This is a definition that's widely shared among biochemists and molecular biologists but there are competing definitions.

Now, there's a new kid on the block. The recent publication of a slew of papers from the ENCODE project has prompted many of the people involved to proclaim that a revolution is under way. Part of the revolution includes redefining a gene. I'd like to discuss the paper by Mark Gerstein et al. (2007) [What is a gene, post-ENCODE? History and updated definition] to see what this revolution is all about.

The ENCODE project is a large scale attempt to analyze and annotate the human genome. The first results focus on about 1% of the genome spread out over 44 segments. These results have been summarized in an extraordinarily complex Nature paper with massive amounts of supplementary material (The Encode Project Consortium, 2007). The Nature paper is supported by dozens of other papers in various journals. Ryan Gregory has a list of blog references to these papers at ENCODE links.

I haven't yet digested the published results. I suspect that like most bloggers there's just too much there to comment on without investing a great deal of time and effort. I'm going to give it a try but it will require a lot of introductory material, beginning with the concept of alternative splicing, which is this week's theme.

The most widely publicized result is that most of the human genome is transcribed. It might be more correct to say that the ENCODE Project detected RNA's that are either complimentary to much of the human genome or lead to the inference that much of it is transcribed.

This is not news. We've known about this kind of data for 15 years and it's one of the reasons why many scientists over-estimated the number of humans genes in the decade leading up to the publication of the human genome sequence. The importance of the ENCODE project is that a significant fraction of the human genome has been analyzed in detail (1%) and that the group made some serious attempts to find out whether the transcripts really represent functional RNAs.

My initial impression is that they have failed to demonstrate that the rare transcripts of junk DNA are anything other than artifacts or accidents. It's still an open question as far as I'm concerned.

It's not an open question as far as the members of the ENCODE Project are concerned and that brings us to the new definition of a gene. Here's how Gerstein et al. (2007) define the problem.

The ENCODE consortium recently completed its characterization of 1% of the human genome by various high-throughput experimental and computational techniques designed to characterize functional elements (The ENCODE Project Consortium 2007). This project represents a major milestone in the characterization of the human genome, and the current findings show a striking picture of complex molecular activity. While the landmark human genome sequencing surprised many with the small number (relative to simpler organisms) of protein-coding genes that sequence annotators could identify (~21,000, according to the latest estimate [see www.ensembl.org]), ENCODE highlighted the number and complexity of the RNA transcripts that the genome produces. In this regard, ENCODE has changed our view of "what is a gene" considerably more than the sequencing of the Haemophilus influenza and human genomes did (Fleischmann et al. 1995; Lander et al. 2001; Venter et al. 2001). The discrepancy between our previous protein-centric view of the gene and one that is revealed by the extensive transcriptional activity of the genome prompts us to reconsider now what a gene is.

Keep in mind that I personally reject the premise and I don't think I'm alone. As far as I'm concerned, the "extensive transcriptional activity" could be artifact and I haven't had a "protein-centric" view of a gene since I learned about tRNA and ribosomal RNA genes as an undergraduate in 1967. Even if the ENCODE results are correct my preferred definition of a gene is not threatened. So, what's the fuss all about?

Regulatory Sequences

Gerstein et al. are worried because many definitions of a gene include regulatory sequences. Their results suggest that many genes have multiple large regions that control transcription and these may be located at some distance from the transcription start site. This isn't a problem if regulatory sequences are not part of the gene, as in the definition quoted above (a gene is a transcribed region). As a mater of fact, the fuzziness of control regions is one reason why most modern definitions of a gene don't include them.

Overlapping Genes

According to Gerstein et al.

As genes, mRNAs, and eventually complete genomes were sequenced, the simple operon model turned out to be applicable only to genes of prokaryotes and their phages. Eukaryotes were different in many respects, including genetic organization and information flow. The model of genes as hereditary units that are nonoverlapping and continuous was shown to be incorrect by the precise mapping of the coding sequences of genes. In fact, some genes have been found to overlap one another, sharing the same DNA sequence in a different reading frame or on the opposite strand. The discontinuous structure of genes potentially allows one gene to be completely contained inside another one’s intron, or one gene to overlap with another on the same strand without sharing any exons or regulatory elements.

We've known about overlapping genes ever since the sequences of the first bacterial operons and the first phage genomes were published. We've known about all the other problems for 20 years. There's nothing new here. No definition of a gene is perfect—all of them have exceptions that are difficult to squeeze into a one-size-fits-all definition of a gene. The problem with the ENCODE data is not that they've just discovered overlapping genes, it's that their data suggests that overlapping genes in the human genome are more the rule than the exception. We need more information before accepting this conclusion and redefining the concept of a gene based on analysis of the human genome.

Splicing

Splicing was discovered in 1977 (Berget et al. 1977; Chow et al. 1977; Gelinas and Roberts 1977). It soon became clear that the gene was not a simple unit of heredity or function, but rather a series of exons, coding for, in some cases, discrete protein domains, and separated by long noncoding stretches called introns. With alternative splicing, one genetic locus could code for multiple different mRNA transcripts. This discovery complicated the concept of the gene radically.

Perhaps back in 1978 the discovery of splicing prompted a re-evaluation of the concept of a gene. That was almost 30 years ago and we've moved on. Now, many of us think of a gene as a region of DNA that's transcribed and this includes exons and introns. In fact, the modern definition doesn't have anything to do with proteins.

Alternative splicing does present a problem if you want a rigorous definition with no fuzziness. But biology isn't like that. It's messy and you can't get rid of fuzziness. I think of a gene as the region of DNA that includes the longest transcript. Genes can produce multiple protein products by alternative splicing. (The fact that the definition above says "a" functional product shouldn't mislead anyone. That was not meant to exclude multiple products.)

The real problem here is that the ENCODE project predicts that alternative splicing is abundant and complex. They claim to have discovered many examples of splice variants that include exons from adjacent genes as shown in the figure from their paper. Each of the lines below the genome represents a different kind of transcript. You can see that there are many transcripts that include exons from "gene 1" and "gene 2" and another that include exons from "gene 1" and "gene 4." The combinations and permutations are extraordinarily complex.

If this represents the true picture of gene expression in the human genome, then it would require a radical rethinking of what we know about molecular biology and evolution. On the other hand, if it's mostly artifact then there's no revolution under way. The issue has been fought out in the scientific literature over the past 20 years and it hasn't been resolved to anyone's satisfaction. As far as I'm concerned the data overwhelmingly suggests that very little of that complexity is real. Alternative splicing exists but not the kind of alternative splicing shown in the figure. In my opinion, that kind of complexity is mostly an artifact due to spurious transcription and splicing errors.

Trans-splicing

Trans-splicing refers to a phenomenon where the transcript from one part of the genome is attached to the transcript from another part of the genome. The phenomenon has been known for over 20 years—it's especially common in C. elegans. It's another exception to the rule. No simple definition of a gene can handle it.

Parasitic and mobile genes

This refers mostly to transposons. Gerstein et al say, "Transposons have altered our view of the gene by demonstrating that a gene is not fixed in its location." This isn't true. Nobody has claimed that the location of genes is fixed.

The large amount of "junk DNA" under selection

If a large amount of what we now think of as junk DNA turns out to be transcribed to produce functional RNA (or proteins) then that will be a genuine surprise to some of us. It won't change the definition of a gene as far as I can see.

The paper goes on for many more pages but the essential points are covered above. What's the bottom line? The new definition of an ENCODE gene is:

There are three aspects to the definition that we will list below, before providing the succinct definition:

1. A gene is a genomic sequence (DNA or RNA) directly encoding functional product molecules, either RNA or protein.
2. In the case that there are several functional products sharing overlapping regions, one takes the union of all overlapping genomic sequences coding for them.
3. This union must be coherent—i.e., done separately for final protein and RNA products—but does not require that all products necessarily share a common subsequence.

This can be concisely summarized as:

The gene is a union of genomic sequences encoding a coherent set of potentially overlapping functional products.

On the surface this doesn't seem to be much different from the definition of a gene as a transcribed region but there are subtle differences. The authors describe how their new definition works using a hypothetical example.

How the proposed definition of the gene can be applied to a sample case. A genomic region produces three primary transcripts. After alternative splicing, products of two of these encode five protein products, while the third encodes for a noncoding RNA (ncRNA) product. The protein products are encoded by three clusters of DNA sequence segments (A, B, and C; D; and E). In the case of the three-segment cluster (A, B, C), each DNA sequence segment is shared by at least two of the products. Two primary transcripts share a 5' untranslated region, but their translated regions D and E do not overlap. There is also one noncoding RNA product, and because its sequence is of RNA, not protein, the fact that it shares its genomic sequences (X and Y) with the protein-coding genomic segments A and E does not make it a co-product of these protein-coding genes. In summary, there are four genes in this region, and they are the sets of sequences shown inside the orange dashed lines: Gene 1 consists of the sequence segments A, B, and C; gene 2 consists of D; gene 3 of E; and gene 4 of X and Y. In the diagram, for clarity, the exonic and protein sequences A and E have been lined up vertically, so the dashed lines for the spliced transcripts and functional products indicate connectivity between the proteins sequences (ovals) and RNA sequences (boxes). (Solid boxes on transcripts) Untranslated sequences, (open boxes) translated sequences.

This isn't much different from my preferred definition except that I would have called the region containing exons C and D a single gene with two different protein products. Gerstein et al (2007) split it into two different genes.

The bottom line is that in spite of all the rhetoric the "new" definition of a gene isn't much different from the old one that some of us have been using for a couple of decades. It's different from some old definitions that other scientists still prefer but this isn't revolutionary. That discussion has already been going on since 1980.

Let me close by making one further point. The "data" produced by the ENCODE consortium is intriguing but it would be a big mistake to conclude that everything they say is a proven fact. Skepticism about the relevance of those extra transcripts is quite justified as is skepticism about the frequency of alternative splicing.

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Gerstein, M.B., Bruce, C., Rozowsky, J.S., Zheng, D., Du, J., Korbel, J.O., Emanuelsson, O., Zhang, Z.D., Weissman, S. and Snyder, M. (2007) What is a gene, post-ENCODE? History and updated definition. Genome Res. 17:669-681.

The ENCODE Project Consortium (2007) Nature 447:799-816. [PDF]

[Hat Tip: Michael White at Adaptive Complexity]

Gene Genie #9

 
The latest issue of the carnival Gene Genie has just been posted on DNAdirect talk.

Skepticism About "Out-of-Africa"

 
Alan R. Templeton has long been a critic of those who would over-interpret the genetic data on human origins. He's not alone. There are a surprisingly large number of biologists who refuse to jump on the "Out-of-Africa" bandwagon. This group does not get the same amount of publicity as the advocates of a recent (<100,000 years) wave of migration out of Africa. I think it's because skepticism of a new theory is seen as sour grapes. That, plus the fact that it's hard to publish criticisms of work that's already in the scientific literature.

An upcoming issue of the journal Evolution will contain a review by Templeton on human origins and the Out-of-Africa theory. Right now it's only available online [GENETICS AND RECENT HUMAN EVOLUTION]. Here's the abstract,

Starting with "mitochondrial Eve" in 1987, genetics has played an increasingly important role in studies of the last two million years of human evolution. It initially appeared that genetic data resolved the basic models of recent human evolution in favor of the "out-of-Africa replacement" hypothesis in which anatomically modern humans evolved in Africa about 150,000 years ago, started to spread throughout the world about 100,000 years ago, and subsequently drove to complete genetic extinction (replacement) all other human populations in Eurasia. Unfortunately, many of the genetic studies on recent human evolution have suffered from scientific flaws, including misrepresenting the models of recent human evolution, focusing upon hypothesis compatibility rather than hypothesis testing, committing the ecological fallacy, and failing to consider a broader array of alternative hypotheses. Once these flaws are corrected, there is actually little genetic support for the out-of-Africa replacement hypothesis. Indeed, when genetic data are used in a hypothesis-testing framework, the out-of-Africa replacement hypothesis is strongly rejected. The model of recent human evolution that emerges from a statistical hypothesis-testing framework does not correspond to any of the traditional models of human evolution, but it is compatible with fossil and archaeological data. These studies also reveal that any one gene or DNA region captures only a small part of human evolutionary history, so multilocus studies are essential. As more and more loci became available, genetics will undoubtedly offer additional insights and resolutions of human evolution.

[Hat Tip: Gene Expression]

Skepticism About Evo-Devo

 
The May issue of Evolution contains an article by Hoekstra and Coyne on Evolutionary-Developmental Biology or Evo-Devo. There are many evolutionary biologists who have serious doubts about the claims of evo-devo but these doubts don't often make it into the scientific literature because it's very hard to publish critiques. The Hoekstra and Coyne (2007) article is a welcome contribution to the debate.

Here's the abstract,

An important tenet of evolutionary developmental biology (“evo devo”) is that adaptive mutations affecting morphology are more likely to occur in the cis-regulatory regions than in the protein-coding regions of genes. This argument rests on two claims: (1) the modular nature of cis-regulatory elements largely frees them from deleterious pleiotropic effects, and (2) a growing body of empirical evidence appears to support the predominant role of gene regulatory change in adaptation, especially morphological adaptation. Here we discuss and critique these assertions. We first show that there is no theoretical or empirical basis for the evo devo contention that adaptations involving morphology evolve by genetic mechanisms different from those involving physiology and other traits. In addition, some forms of protein evolution can avoid the negative consequences of pleiotropy, most notably via gene duplication. In light of evo devo claims, we then examine the substantial data on the genetic basis of adaptation from both genome-wide surveys and single-locus studies. Genomic studies lend little support to the cis-regulatory theory: many of these have detected adaptation in protein-coding regions, including transcription factors, whereas few have examined regulatory regions. Turning to single-locus studies, we note that the most widely cited examples of adaptive cis-regulatory mutations focus on trait loss rather than gain, and none have yet pinpointed an evolved regulatory site. In contrast, there are many studies that have both identified structural mutations and functionally verified their contribution to adaptation and speciation. Neither the theoretical arguments nor the data from nature, then, support the claim for a predominance of cis-regulatory mutations in evolution. Although this claim may be true, it is at best premature. Adaptation and speciation probably proceed through a combination of cis-regulatory and structural mutations, with a substantial contribution of the latter.

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Hoekstra, Hopi, E. and Coyne, Jerry, A. (2007) THE LOCUS OF EVOLUTION: EVO DEVO AND THE GENETICS OF ADAPTATION. Evolution 65:995–1016.

Monday's Molecule #31

 
Today's molecule is complicated but it makes a lot of sense if you know your basic biochemistry. We don't need a long complicated name this time. It's sufficient to simply describe what you're looking at and why it's significant. You have to identify the key residue to get credit for the answer.

As usual, there's a connection between Monday's molecule and this Wednesday's Nobel Laureate(s). This one is an obvious direct connection. Once you have identified the molecule you should be able to name the Nobel Laureate(s).

The reward (free lunch) goes to the person who correctly identifies the molecule and the reaction and the Nobel Laureate(s). Previous free lunch winners are ineligible for one month from the time they first collected the prize. There are no ineligible candidates for this Wednesday's reward since recent winners (including last week's winner, "Kyo") have declined the prize on the grounds that they live in another country and can't make it for lunch on Thursday (a feeble excuse, in my opinion, haven't you heard of airplanes?).

Comments will be blocked for 24 hours. Comments are now open.

UPDATE: The molecule is the lariat structure of the RNA splicing intermediate. The key residue is the adenylate residue that's joined through its 2′ hydroxyl group to the 5′ end of the intron [see RNA Splicing: Introns and Exons]. The Nobel Laureates are Rich Roberts and Phil Sharp. (See the comments for an interesting anecdote concerning the discovery of this molecule.)

Cellular Respiration Ninja Enzymes

 
Here's a student video presentation of glycolysis and respiration. It's much better than most [e.g., An Example of High School Biochemistry]. However, there are two errors in the video. The first one is fairly serious. The second one is less serious but it's something we cover in my class and it helps illustrate a fundamental concept about how certain reactions work. The second error was very common in most biochemistry textbooks in the past but it's been eliminated from the majority of 21st century textbooks. Can you spot both errors?



[Hat Tip: Greg Laden]
[Hint: What are the products produced by glycolysis and by the Krebs cycle?]

Penicillin Resistance in Bacteria: After 1960

 
The widespread appearance of penicillin-resistant bacteria by 1960 prompted the introduction of new drugs that could not be degraded by newly evolved β-lactamases [see Penicillin Resistance in Bacteria: Before 1960].

The most important of these new drugs are the cephalosporins, modified β-lactams with bulky side chains at two different positions. These drugs still inhibit the transpeptidases and prevent cell wall formation but because of the bulky side chains they cannot be hydrolyzed by β-lactamases. Thus, they are effective against most of the penicillin-resistant strains that arose before 1960.

Other drugs, such as methicillin, were modified penicillins. They also had modified side chains that prevented degradation by the β-lactamases.

It wasn't long before cephalosporin- and methicillin-resistant strains began to appear in hospitals. As a general rule, these strains were not completely resistant to high doses of the new class of drugs but as time went on the resistant strains became more and more immune to the drugs.

The new version of drug resistance also involves the transpeptidase target but instead of developing into β-lactamases they evolve into enzymes that can no longer bind the cephalosporins. Usually the development of resistance takes place in several stages.

There are many different transpeptidases in most species of bacteria. The are usually referred to as penicillin-binding proteins or PBP's. Often the first sign of non-lactamase drug resistance is a mutant version of one PDP (e.g., PDP1a) and subsequent development of greater resistance requires the evolution of other PDB's that don't bind the drug. In the most resistant strains there will be one particular PDB (e.g., PDB2a) that is still active at high drug concentrations while the other transpeptidases will be inhibited.

Resistant enzymes have multiple mutations, which explains the slow, stepwise acquisition of drug resistance. An example is shown in the figure. This is PDP1a from Streptococcus pneumoniae (Contreras, et al. 2006) and the mutant amino acids are displayed as gold spheres. Most of the mutations do not affect the binding of the drug but those surrounding the entry to the active site are crucial. The necessary amino acid substitutions are numbered in the figure. You can see that they line the groove where the cephalosporin drug (purple) is bound. The effect of the mutations is to prevent the bulky β-lactam from inhibiting the enzyme. This is a very different form of drug resistance than the evolution of degradation enzymes that characterized the first stage of penicillin resistant bacteria.

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Chambers, H.F. (2003) Solving staphylococcal resistance to beta-lactams. Trends Microbiol. 11:145-148.

Contreras-Martel, C., Job, V., Di Guilmi, A.M., Vernet, T., Dideberg, O. and Dessen, A. (2006) Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae. J. Mol. Biol. 355:684-696.

Livermore, D.M. (2000) Antibiotic resistance in staphylococci. Int. J. Antimicrob. Agents 16:s3-s10.

Penicillin Resistance in Bacteria: Before 1960

 
The Nobel Prize for the discovery and analysis of penicillin was awarded in 1945 [Nobel Laureates: Sir Alexander Fleming, Ernst Boris Chain, Sir Howard Walter Florey]. It was about this time that penicillin became widely available in Europe and North America.

By 1946 6% of Staphylococcus aureus strains were resistant to penicillin. Resistance in other species of bacteria was also detected in the 1940's. By 1960 up to 60% of Staphylococcus aureus strains were resistant with similar levels of resistance reported in other clinically relevant strains causing a wide variety of diseases (Livermore, 2000).

Penicillins are a class of antibiotics with a core structure called a β-lactam. The different types of penicillin have different R groups on one end of the core structure. A typical examples of a penicillin is penicillin G [Monday's Molecule #30]. Others common derivatives are ampicillin and amoxicillin.

The original resistance to this entire class of drugs was caused mostly by the evolution of bacterial enzymes that could degrade them before they could block cell wall synthesis. (Recall that bacteria have cell walls and penicillin blocks cell wall synthesis [How Penicillin Works to Kill Bacteria].)
It seems strange that the evolution of penicillin resistance would require a totally new enzyme for degrading the drug. Where did this enzyme come from? And how did it arise so quickly in so many different species?

The degrading enzyme is called penicillinase, β-lactamase, or oxacillinase. They all refer to the same class of enzyme that binds penicillins and then cleaves the β-lactam unit releasing fragments that are inactive. The enzymes are related to the cell wall transpeptidase that is the target of the drug. The inhibition of the transpeptidase is effective because penicillin resembles the natural substrate of the reaction: the dipeptide, D-alanine-D-alanine.

In the normal reaction, D-Ala-D-Ala binds to the enzyme and the peptide bond is cleaved causing release one of the D-Ala residues. The other one, which is part of the cell wall peptidoglycan, remains bound to the enzyme. In the second part of the reaction, the peptidoglycan product is transferred from the enzyme to a cell wall crosslinking molecule. This frees the enzyme for further reactions (see How Penicillin Works to Kill Bacteria for more information).

Penicillin binds to the peptidase as well and the β-lactam bond is cleaved resulting in the covalent attachment of the drug to the enzyme. However, unlike the normal substrate, the drug moiety cannot be released from the transpeptidase so the enzyme is permanently inactivated. This leads to disruption of cell wall synthesis and death.

Resistant strains have acquired mutations in the transpeptidase gene that allow the release of the cleaved drug. Thus, the mutant enzyme acts like a β-lactamase by binding penicillins, cleaving them, and releasing the products. Although the β-lactamases evolved from the transpeptidase target enzymes, the sequence similarity between them is often quite low in any given species. This is one of the cases where structural similarity reveals the common ancestry [see the SCOP Family beta-Lactamase/D-ala carboxypeptidase]. It's clear that several different β-lactamases have evolved independently but, in many cases, a particular species of bacteria seems to have licked picked up a β-lactamase gene by horizontal transfer from another species. The transfer can be mediated by bacteriophage or plasmids.

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Livermore, D.M. (2000) Antibiotic resistance in staphylococci. Int. J. Antimicrob. Agents 16:s3-s10.

Catherine Shaffer Responds to My Comments About Her WIRED Article

 
Over on the WIRED website there's a discussion about the article on junk DNA [One Scientist's Junk Is a Creationist's Treasure]. In the comments section, the author Catherine Shaffer responds to my recent posting about her qualifications [see WIRED on Junk DNA]. She says,

You might be interested to learn that I contacted Larry Moran while working on this article and after reading the archives of his blog. I wanted to ask him to expand upon his assertion that junk DNA disproves intelligent design. His response was fairly brief, did not provide any references, and did not invite further discussion. It's interesting that he's now willing to write a thousand words or so about how wrong I am publicly, but was not able to engage this subject privately with me.

Catherine Shaffer sent me a brief email message where she mentioned that she had read my article on Junk DNA Disproves Intelligent Design Creationism. She wanted to know more about this argument and she wanted references to those scientists who were making this argument. Ms. Shaffer mentioned that she was working on an article about intelligent design creationism and junk DNA.

I responded by saying that the presence of junk DNA was expected according to evolution and that it was not consistent with intelligent design. I also said that, "The presence of large amounts of junk DNA in our genome is a well established fact in spite of anything you might have heard in the popular press, which includes press releases." She did not follow up on my response.

His blog post is inaccurate in a couple of ways. First, I did not make the claim, and was very careful to avoid doing so, that “most” DNA is not junk. No one knows how much is functional and how much is not, and none of my sources would even venture to speculate upon this, not even to the extent of “some” or “most.”

Her article says, "Since the early '70s, many scientists have believed that a large amount of many organisms' DNA is useless junk. But recently, genome researchers are finding that these "noncoding" genome regions are responsible for important biological functions." Technically she did not say that most DNA is not junk. She just strongly implied it.

I find it difficult to believe that Ryan Gregory would not venture to speculate on the amount of junk DNA but I'll let him address the validity of Ms. Shaffer's statement.

Moran also mistakenly attributed a statement to Steven Meyer that Meyer did not make.

I can see why someone might have "misunderstood" my reference to what Myer said so I've edited my posting to make it clear.

Judmarc and RickRadditz—Here is a link to the full text of the genome biology article on the opossum genome: Regulatory conservation of protein coding and microRNA genes in vertebrates: lessons from the opossum genome. We didn't have space to cover this in detail, but in essence what the researchers found was that upstream intergenic regions were more highly conserved in the possum compared to coding regions, but also represented a greater area of difference between possums and humans.

This appears to be a reference to the paper she was discussing in her article. It wasn't at all clear to me that this was the article she was thinking about in the first few paragraphs of her WIRED article.

Interested readers might want to read the comment by "Andrea" over on the WIRED site. She He doesn't pull any punches in demonstrating that Catherine Shaffer failed to understand what the scientific paper was saying. Why am I not surprised? (Recall that this is a science writer who prides herself on being accurate.)

So, yes, this does run counter to the received wisdom, which makes it fascinating. You are right that the discussion of junk vs. nonjunk and conserved vs. nonconserved is much more nuanced, and we really couldn't do it justice in this space. Here is another reference you might enjoy that begins to deconstruct even our idea of what conservation means: “Conservation of RET regulatory function from human to zebrafish without sequence similarity.” Science. 2006 Apr 14;312(5771):276-9. Epub 2006 Mar 23. Revjim—If you have found typographical errors in the copy, please do point them out to us. The advantage of online publication is that we do get a chance to correct these after publication.

Sounds to me like Catherine Shaffer is grasping at straws (or strawmen).

For Katharos and others—I interviewed five scientists for this article. Dr. Francis Collins, Dr. Michael Behe, Dr. Steve Meyers, Dr. T. Ryan Gregory, and Dr. Gill Bejerano. Each one is a gentleman and a credentialed expert either in biology or genetics. I am grateful to all of them for their time and kindness.

I think we all know just how "credentialed" Stephen Meyer is. He has a Ph.D. in the history and philosophy of science. Most of us are familiar with the main areas of expertise of Michael Behe and none of them appear to be science.

WIRED on Junk DNA

Junk DNA is the DNA in your genome that has no function. Much of it accumulates mutations in a pattern that's consistent with random genetic drift implying strongly that the sequences in junk DNA are unimportant. In fact, the high frequency of sequence change (mutation plus fixation) is one of the most powerful bits of evidence for lack of function.

Catherine Shaffer is a science writer who describes herself like this on her website,

I am a writer specializing in biotechnology, genetics, genomics, and other molecular, biological sciences. I have experience with news and features. My strengths include a meticulous attention to detail, an absolutely fanatical devotion to scientific accuracy, and enthusiasm. Readers appreciate my clean, uncluttered prose; my crisp, novelistic style; and (sometimes) my zany sense of humor. I am a writer who always meets deadlines and is organized and dependable.

I studied biochemistry at the graduate level at the University of Michigan, and worked in the pharmaceutical industry for several years. I am especially knowledgeable about genomics, proteomics, biotechnology, drug discovery, and chromatographic separations.

She has written an article for WIRED on junk DNA [One Scientist's Junk Is a Creationist's Treasure]. Here's how the article begins,

Without your "junk DNA" you might be reading this article while hanging upside down by your tail.

That's one of the key findings of the opossum genome-sequencing project, and a surprising group is embracing the results: intelligent-design advocates. Since the early '70s, many scientists have believed that a large amount of many organisms' DNA is useless junk. But recently, genome researchers are finding that these "noncoding" genome regions are responsible for important biological functions.

The opossum data revealed that more than 95 percent of the evolutionary genetic changes in humans since the split with a common human-possum ancestor occurred in the "junk" regions of the genome. Creationists say it's also evidence that God created all life, because God does not create junk. Nothing in creation, they say, was left to chance.

"It is a confirmation of a natural empirical prediction or expectation of the theory of intelligent design, and it disconfirms the neo-Darwinian hypothesis," said Stephen Meyer, director of the Center for Science and Culture at the Discovery Institute in Seattle.

Advocates like Meyer are increasingly latching onto scientific evidence to support the theory of intelligent design, a modern arm of creationism that claims life is not the result of natural selection but of an intelligent creator. Most scientists believe that intelligent design is not science. But Meyer says the opossum data supports intelligent design's prediction that junk DNA sequences aren't random, but important genetic material. It's an argument Meyer makes in his yet-to-be-published manuscript, The DNA Enigma.

Hmmmm ... This is so confused that it's difficult to know where to begin. First, the connection between my junk DNA and whether I am an opossum completely escapes me. I don't know of any credible scientist who claims that it's changes in junk DNA that makes us so different from the common ancestor of opossums. (And none who claim that we are descended from opossums.)

Second, the implication that most junk DNA is turning out to have a function is completely false and the confusion about the difference between junk DNA and noncoding DNA is inexcusable from someone who claims to be an expert on genomics [see Noncoding DNA and Junk DNA, The Deflated Ego Problem].

Third, the idea that large amounts of evolution in junk DNA supports Intelligent Design Creationism is crazy. But, in fairness, I don't think Shaffer is making the connection between the sequence variation and Intelligent Design Creationism; instead, she's making the (factually incorrect) connection between the discovery of some functions in noncoding, nonjunk, DNA and Intelligent Design Creationism (IDC). I think Steve Meyer is suggesting that IDC predicts that junk DNA will have a function and that's why he's being quoted here in the article (see above).

Scientists have made several discoveries about what some call the "dark matter of the genome" in recent years, but they say the research holds up the theory of natural selection rather than creationism.

When sequences in noncoding DNA are conserved, this is taken as evidence of negative selection. In that sense, it supports the theory of natural selection. However, most of the sequence comparisons show that junk DNA is not conserved. This does not support the theory of natural selection. It supports Neutral Theory and the mechanism of evolution by random genetic drift.

The article then describes one recent study suggesting that some noncoding DNA is not junk (Lowe et al. 2007). It appears to be the justification for writing the article since it compares short stretches of sequences in the human and opossum genomes. This is not news so I won't bother commenting.

With scientists increasingly believing that so-called junk DNA regulates other genes, among other functions, creationists like Michael Behe, a biochemistry professor at Lehigh University in Pennsylvania and author of the controversial new book on intelligent design, The Edge of Evolution, are more than happy to point out their errors.

"From the very beginning Darwinism thought whatever it didn't understand must be simple, must be nonfunctional," Behe said. "It's only in retrospect that Darwinists try to fit that into their theory."

The concept of junk DNA is not based on ignorance in spite of what the IDiots say. It's based on good scientific evidence and deduction. Of course most IDiots wouldn't recognize scientific evidence even if it bit them on the ...

Is this just a way of getting in another quote from a prominent advocate of Intelligent Design Creationism? Why is Shaffer so interested in the IDiots? This seems to be more than just seeking out controversy since the proper way to do that would be to interview real scientists who can put the work into perspective and comment on it's significance (see below).

Part of the difficulty in studying junk DNA is that it's impossible to prove a negative, i.e., that any particular DNA does not have a function.

That's why T. Ryan Gregory, an assistant professor in biology at the University of Guelph, believes that nonfunctional should be the default assumption. "Function at the organism level is something that requires evidence," he said.

That's how a real scientist speaks [see A word about "junk DNA" and Comments on "Noncoding DNA and Junk DNA"].

This is getting to be a familiar pattern among science writers. Many of them seem to be incapable of sorting out the actual science from the rhetoric. In this case the problem is exacerbated by introducing IDiots as though their opinion had a bearing on the subject. Not only that, the poor science writing stands in sharp contrast to the claim that, "My strengths include a meticulous attention to detail, an absolutely fanatical devotion to scientific accuracy, and enthusiasm."

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Lowe, C.B., Bejerano, G. and Haussler, D. (2007) Thousands of human mobile element fragments undergo strong purifying selection near developmental genes. Proc. Natl. Acad. Sci. (USA) 104:8005-8010. [PubMed]

University College London Restores Professor Colquhoun's Website

 
David Colquhoun has a website at University College London where he regularly debunks the claims of "medical" quacks. Recently a herbal medicine practitioner took offense at this debunking and threatened legal action against the university. The university responded by removing the website.

Today the website has been restored [DC's Improbable Science] and University College London has published a press release explaining why [Joint statement by Professor Colquhoun and UCL].

While it's encouraging that the university decided to restore the website, the fact that it buckled to pressure in the first place is disturbing. What's the point of academic freedom if you abandon it whenever you're threatened with a lawsuit?

UCL has a long and outstanding liberal tradition and is committed to encouraging free and frank academic debate. The evidence (or lack thereof) for the claims made for health supplements is a matter of great public interest, and UCL supports all contributions to that debate. The only restriction it places on the use of its facilities is that its staff should use their academic freedom responsibly within the law.

To this end, the Provost and Professor Colquhoun have taken advice from a senior defamation Queen’s Counsel, and we are pleased to announce that Professor Colquhoun’s website – with some modifications effected by him on counsel’s advice - will shortly be restored to UCL’s servers. UCL will not allow staff to use its website for the making of personal attacks on individuals, but continues strongly to support and uphold Professor Colquhoun’s expression of uncompromising opinions as to the claims made for the effectiveness of treatments by the health supplements industry or other similar bodies.

I'm curious about the "minor modifications" and I'm troubled by the prohibition against "the making of personal attacks on individuals." It seems to me that such a prohibition could be used in a way that inhibits academic freedom. For example, would it prohibit a university Professor from criticizing Tony Blair for the war in Iraq? Would it block any negative comments about Prince Charles (pictured at left)? Does it mean that the UCL website is completely devoid of any negative comments about Richard Dawkins?

Perhaps more importantly, does this mean that university Professors cannot point out on their websites the stupidity of administration officials such as UCL President and Provost Malcolm Grant?

Nobel Laureates: Sir Alexander Fleming, Ernst Boris Chain, Sir Howard Walter Florey

 
The Nobel Prize in Physiology or Medicine 1945.

"for the discovery of penicillin and its curative effect in various infectious diseases"


Sir Alexander Fleming (1881-1955), Ernst Boris Chain (1906-1979) and Sir Howard Walter Florey (1898-1968) received the Nobel Prize in Physiology or Medicine for their work on penicillin [see Monday's Molecule #30 and How Penicillin Works to Kill Bacteria]. The Presentation Speech was delivered by by Professor G. Liljestrand of the Royal Caroline Institute (Karolinska Institutet), on December 10, 1945.

Attempts have been made to reach the goal of medical art - the prevention and cure of disease - by many different paths. New and reliable ones have become practicable as our knowledge of the nature of the different diseases has widened. Thus the successful combating of certain disturbances in the activities of the organs of internal secretion, as also of the deficiency diseases, or avitaminoses, has been a direct result of the increase in our knowledge of the nature of these afflictions. When, thanks to the research work of Louis Pasteur and Robert Koch, the nature of the infectious diseases was laid bare, and the connection between them and the invasion of the body by bacteria and other micro-organisms was elucidated, fully a generation ago, this was an enormous advance, both for the prevention and the treatment of this important group of diseases. This was so much the more important as the group included a number of the worst scourges of humanity, which had slain whole peoples, and at times had laid waste wide areas. But now possibilities were revealed which have not yet been by any means fully utilized. In rapid succession, different forms of vaccination were evolved, and subsequently also serum treatment, for the introduction of which the first Nobel Prize for Physiology or Medicine was given 44 years ago today. In these cases advantage was taken of the capacity of the human and animal bodies themselves to produce protective substances in the fight against the invaders, and to do so in great abundance. But it is by no means the higher organisms only that are able to produce such substances. In cooperation with Joubert (1877), Pasteur himself observed that anthrax bacilli cultivated outside the body were destroyed if bacteria from the air were admitted, and with prophetic acumen he realized that it was justifiable to attach great hopes to this observation in the treatment of infectious diseases. Nevertheless more than two decades passed before an attempt was made to profit by the struggle for existence which goes on between different species of micro-organisms. Experiments carried out by Emmerich and Loew (1899) did not give such favourable results, however, that any great interest was aroused, nor did success attend the later efforts of Gratia and Dath and others. It was reserved to this year's Nobel Prize winners to realize Pasteur's idea.

The observation made by Professor Alexander Fleming which led to the discovery of penicillin, is now almost classical. In 1928, in the course of experiments with pyogenic bacteria of the staphylococcus group, he noticed that, around a spot of mould which had chanced to contaminate one of his cultures, the colonies of bacteria had been killed and had dissolved away. Fleming had earlier made a study of different substances which prevent the growth of bacteria and, inter alia, had come upon one in lacrimal fluid and saliva, the so-called lysozyme. As he points out himself, he was therefore always on the look-out for fresh substances which checked bacteria, and he became sufficiently interested in his latest find to make a closer investigation of the phenomenon. The mould was therefore cultivated and subsequently transferred to broth, where it grew on the surface in the form of a felted green mass. When the latter was filtered off a week later, it was found that the broth had such a strongly checking effect on bacteria that even when diluted 500-800 times it completely prevented the growth of staphylococci; consequently an extremely active substance had passed to the broth from the mould. This proved to belong to the Penicillium group or brush moulds, and therefore first the broth, and later the substance itself, was called «penicillin». It was soon realized that most of the species of Penicillium did not form it at all, and a closer scrutiny showed that the species which polluted Fleming's culture was Penicillium notatum. It had been described for the first time by Richard Westling, in the thesis which he defended in the autumn of 1911 at the University of Stockholm for the degree of Doctor of Philosophy - an illustration of the international nature of science, but also of the suddenly increased importance which sometimes accrues to sound work as a result of further developments. Fleming also showed that penicillin was extremely effective against cultures of many different kinds of bacteria, above all against those belonging to the coccus group, among them those that usually give rise to suppuration, pneumonia and cerebral meningitis, but also against certain other types, such as diphtheria, anthrax, and gas gangrene bacteria. But as numerous other species, among them the influenza, coli, typhoid and tuberculosis bacilli, grew even if they were exposed to moderate quantities of penicillin, Fleming was able to work out a method for isolating out from a mixture of bacteria those which were insensitive to penicillin. He found, further, that the white blood corpuscles, which are usually so sensitive, were not affected by penicillin. When injected into mice, too, it was fairly harmless. In this respect penicillin differs decisively from other substances which had been produced earlier from micro-organisms, and which were certainly found to be noxious to bacteria, but at the same time at least equally noxious to the cells of the higher animals. The possibility that penicillin might be used as a remedy was therefore within reach, and Fleming tested its effect on infected wounds, in some cases with moderate success.

Three years after Fleming's discovery, the English biochemists Clutterbuck, Lovell, and Raistrick, endeavoured to obtain penicillin in the pure form, but without success. They established, inter alia, that it was a sensitive substance which easily lost its antibacterial effect during the purifying process, and this was soon confirmed in other quarters.

Penicillin would undoubtedly still have remained a fairly unknown substance, interesting to the bacteriologist but of no great practical importance, if it had not been taken up at the Pathological Institute at the venerable University of Oxford. This time a start was again made from what is usually called basic research. Professor Howard Florey, who devoted his attention to the body's own natural protective powers against infectious diseases, together with his co-workers, had studied the lysozyme referred to above, the nature of which they succeeded in elucidating. Dr. Ernst Boris Chain, a chemist, took part in the final stage of these investigations, and during 1938 the two researchers jointly decided to investigate other antibacterial substances which are formed by micro-organisms, and in that connection they fortunately thought first of penicillin. It was certainly obvious that the preparation of the substance in a pure form must involve great difficulties, but on the other hand its powerful effect against many bacteria gave some promise of success. The work was planned by Chain and Florey, who, however, owing to the vastness of the task, associated with themselves a number of enthusiastic co-workers, among whom mention should be made especially of Abraham, Fletcher, Gardner, Heatley, Jennings, Orr-Ewing, Sanders and Lady Florey. Heatley worked out a convenient method of determining the relative strength of a fluid with a penicillin content, by means of a comparison under standard conditions of its antibacterial effect with that of a penicillin solution prepared at the laboratory. The amount of penicillin found in one cc. of the latter was called an Oxford unit.

In the purifying experiments then made, the mould was cultivated in a special nutritive fluid in vessels, to which air could only gain access after it had been filtered through cotton wool. After about a week the penicillin content reached its highest value, and extraction followed. In this connection advantage was taken of the observation that the free penicillin is an acid which is more easily dissolved in certain organic solvents than in water, while its salts with alkali are more readily dissolved in water. The culture fluid was therefore shaken with acidified ether or amyl acetate. As, however, the penicillin was easily broken up in water solution, the operation was performed at a low temperature. Thus the penicillin could be returned to the water solution after the degree of acidity had been reduced to almost neutral reaction. In this way numerous impurities could be removed, and after the solution had been evaporated at a low temperature it was possible to obtain a stable dry preparation. The strength of this was up to 40-50 units per mg and it prevented the growth of staphylococci in a dilution of at least 1 per 1 million - thus the active substance had been successfully concentrated very considerably. It was therefore quite reasonable that it was thought that almost pure penicillin had been obtained - in a similar manner, in their work with strongly biologically active substances, many earlier researchers had thought that they were near to producing the pure substance. The further experiments, which were made subsequently with the help of the magnificent resources of modern biochemistry proved, however, that such was not the case. In reality the preparation just mentioned contained only a small percentage of penicillin. Now when it has become possible to produce pure penicillin in a crystalline form, it has been found that one mg contains about 1,650 Oxford units. It is also known that penicillin is met with in some different forms, which possibly have somewhat different effects. The chemical composition of penicillin has also been elucidated in recent years, and in this work Chain and Abraham have successfully taken part.

The Oxford school was able to confirm Fleming's observation that penicillin was only slightly toxic, and they found that its effect was not weakened to any extent worth mentioning in the presence of blood or pus. It is readily destroyed in the digestive apparatus, but after injection under the skin or into the muscles, it is quickly absorbed into the body, to be rapidly excreted again by way of the kidneys. If it is to have an effect on sick persons or animals, it should therefore be supplied uninterruptedly or by means of closely repeated injections - some more recent experiments indicate that gradually perhaps it will be possible to overcome the difficulties in connection with taking the preparation by mouth. Experiments on mice infected with large doses of pyogenic or gas gangrene bacteria, which are sensitive to penicillin, proved convincingly that it had a favourable effect. While over 90% of the animals treated with penicillin recovered, all the untreated control animals died.

Experiments on animals play an immense role for modern medicine; indeed it would certainly be catastrophic if we ventured to test remedies on healthy or sick persons, without having first convinced ourselves by experiments on animals that the toxic effect is not too great, and that at the same time there is reason to anticipate a beneficial result. Tests on human beings may, however, involve many disappointments, even if the results of experiments on animals appear to be clear. At first this seemed to be the case with penicillin, in that the preparation gave rise to fever. Fortunately this was only due to an impurity, and with better preparations it has subsequently been possible to avoid this unpleasant effect.

The first experiments in which penicillin was given to sick persons were published in August 1941 and appeared promising, but owing to the insufficient supplies of the drug, the treatment in some cases had to be discontinued prematurely. However, Florey succeeded in arousing the interest of the authorities in the United States in the new substance, and with the cooperation of numerous research workers it was soon possible, by means of intensive work, to obtain materially improved results there and to carry on the preparation in pure form to the crystallization stage just mentioned. Large quantities of penicillin could be made available, and numerous tests were made above all in the field, but to a certain extent also in the treatment of civilians. Many cases were reported of patients who had been considered doomed or had suffered from illness for a long period without improvement, although all the resources of modern medicine had been tried, but in which the penicillin treatment had led to recoveries which not infrequently seemed miraculous. Naturally such testimony from experienced doctors must not be underestimated, but on the other hand we must bear in mind the great difficulties in judging the course of a disease. «Experience is deceptive, judgment difficult», is one of Hippocrates' famous aphorisms. Therefore it is important that a remedy should be tested on a large material and in such a way that comparison can be made with cases which have not been given the remedy but had otherwise received exactly the same treatment. There are now many reports of such investigations. The extraordinarily good effects of penicillin have been established in a number of important infectious illnesses, such as general blood poisoning, cerebral meningitis, gas gangrene, pneumonia, syphilis, gonorrhea and many others. It is of special importance that even sick persons who are not favourably affected by the modern sulfa drugs are not infrequently cured with penicillin. The effect naturally depends on the remedy being given in a suitable manner and in sufficient doses. On the other hand, experience has confirmed what might have been surmised, namely that penicillin is not effective in cases of, e.g. tuberculosis, typhoid fever, poliomyelitis, and a number of other infectious diseases. Consequently penicillin is not a universal remedy, but it is of the highest value for certain diseases. And it appears not improbable that, with the guidance of experience with penicillin, it will be possible to produce new remedies which can compete with or perhaps surpass it in certain respects.

Four years is a short time in which to arrive at definite conclusions as to the value of a remedy. But during these last few years experiences of penicillin have been assembled which, under ordinary conditions, would have required decades. And therefore there is no doubt at the present time that the discovery of penicillin and its curative properties in the case of various infection diseases for which this year's Nobel Prize is awarded, is of the greatest importance for medical science.

Sir Alexander Fleming, Doctor Chain, and Sir Howard Florey. The story of penicillin is well-known throughout the world. It affords a splendid example of different scientific methods cooperating for a great common purpose. Once again it has shown us the fundamental importance of basic research. The starting-point was a purely academic investigation, which led to a so-called accidental observation. This gave the nucleus, around which one of the most efficient remedies ever known could be crystallized. This difficult process was made possible with the aid of modern biochemistry, bacteriology, and clinical research. To overcome the numerous obstacles, all this work demanded not only assistance from many different quarters, but also an unusual amount of scientific enthusiasm, and a firm belief in an idea. In a time when annihilation and destruction through the inventions of man have been greater than ever before in history, the introduction of penicillin is a brilliant demonstration that human genius is just as well able to save life and combat disease.

In the name of the Caroline Institute I extend to you hearty congratulations on one of the most valuable contributions to modern medicine. And now I have the honour of calling on you to accept the Nobel Prize for Physiology or Medicine for the year 1945 from the hands of His Majesty the King.