Sequence Alignment

Sequence alignment is one of the crucial steps in deciding whether two genes/proteins are homologous. The two sequences are aligned from one end to the other and the number of identical, or similar, residues is counted. If this number reaches a significant percentage of the total length (usually >25%) then the two sequences are homologous—they descend from a common ancestor.

Sequence alignment is not straightforward, even for two sequences, because in addition to substitutions the genes might have undergone insertions or deletions (indels). In order to identify conserved residues, one needs to insert gaps in one sequence or the other to compensate for these indel events.

You can't just willy-nilly stick in gaps to maximize the number of aligned residues because the gaps represent true historical events (insertions and deletions). In theory, you can get high identity scores with any two sequences as long as you insert enough gaps but that isn't allowed. When the alignment is done by computer algorithm, each gap is associated with a gap penalty.

The determination of proper gap penalties is a major challenge in multiple sequence alignment. A crude estimate is that each gap comes with a penalty of 3—that is you have to generate at least three identities in order to make the gap worthwhile. The number of gaps and gap penalties have to be subtracted from the identity/similarity scores when deciding about homology. (This isn't always done.)

Here's an example of a multiple sequence alignment from a region of bacterial HSP70 genes. The letters represent the amino acid residues and the dashes are gaps due to insertions and deletions.


The HSP70 genes are the most highly conserved genes in biology so, in principle, it should be easy to align them. In fact, it is easy in most regions but the one shown above is the most difficult. This is a manual alignment that takes into account the similarities of groups of sequences. Those that are most similar are clustered together and whenever possible the alignment is adjusted so that the positions of the gaps in the most closely related sequences are identical.

This is a procedure known as phylogenetic alignment but it would be better to call it similarity alignment because what we're actually doing is clustering sequences by their overall similarity and not their phylogeny. (The fact that their phylogenetic relatedness closely corresponds to their similarity is a consequence of the the analysis and not a cause.)^1.

The placing of gaps in this region of HSP70 sequences is very difficult. No computer program can come close to achieving the quality of alignments that well trained humans can achieve. That's because the overall alignment has to take into account a number of variables simultaneously and the progressive alignment takes many trial-and-error steps. As a general rule of thumb, if you see a paper where phylogenetic trees are constructed using computer-generated multiple sequence alignments only, then you should assign a low confidence value to that work.

Is this important? Indeed it is. The exact nature and position of the large gap in the above sequences, for example, plays an important role in testing the Three Domain Hypothesis. Different alignments give different trees and the most important variable is the position of gaps.

This brings me to an important paper just published in this week's issue of Science. Löytynoja and Goldman (2008) have developed a new algorithm for multiple sequence alignment. The abstract of their paper describes the problem, and their solution.

Genetic sequence alignment is the basis of many evolutionary and comparative studies, and errors in alignments lead to errors in the interpretation of evolutionary information in genomes. Traditional multiple sequence alignment methods disregard the phylogenetic implications of gap patterns that they create and infer systematically biased alignments with excess deletions and substitutions, too few insertions, and implausible insertion-deletion–event histories. We present a method that prevents these systematic errors by recognizing insertions and deletions as distinct evolutionary events. We show theoretically and practically that this improves the quality of sequence alignments and downstream analyses over a wide range of realistic alignment problems. These results suggest that insertions and sequence turnover are more common than is currently thought and challenge the conventional picture of sequence evolution and mechanisms of functional and structural changes.

The authors test their phylogeny-aware program (PRANK) against several other multiple sequence alignment programs (ClustalW, MAFFT, MUSCLE, and T-COFFEE) using a set of sequences that were "evolved" using a computer program that created substitutions and insertions/deletions. Since the true phylogeny of this artificial set is known, they were able to evaluate the performance of the various programs.

As you might expect, PRANK came out best in this test. I'm not sure that it would work best with real data but that's not really my point. My point is that this is an ongoing problem that has not been fully solved. It is still best to avoid multiple sequence alignments that have not been manually improved by humans with considerable experience in sequence alignment.

I'll close by quoting from the discussion in Löytynoja and Goldman (2008) just to remind everyone how important this is. They argue that even post-alignment human "refinement" of computer generated sequence alignments suffers from systemic bias.

Our analyses show that sequence alignment remains a challenging task, and alignments generated with methods based on the traditional progressive algorithm may lead to seriously incorrect conclusions in evolutionary and comparative studies. The main reason for their systematic error is disregard of the phylogenetic implications of gap patterns created—which is not corrected by considering alignment consistency (13) or using post alignment refinement (14, 15)—and this error is intensified by methods that intentionally force gaps into tight blocks. Affected methods can be positively misleading and become increasingly confident of erroneous solutions as more sequences are included. It is not the progressive algorithm as such that is defective, rather, correct alignment requires that we take account of sequences' phylogeny, irrespective of alignment method used or data type, but the original implementations of the progressive algorithm have a flaw that has gone unnoticed as long as different methods have been consistent in the error they create.

That such a significant error has passed undetected may be explained by the alignment field's historical focus on proteins, where these biases tend to be manifested in less-constrained regions such as loops (compare Fig. 1). Alignments with insertions and deletions squeezed compactly between conserved blocks may suffice for, and even be preferred by, some molecular biologists working with proteins. We have shown, however, that these patterns are, in fact, imposed by systematic biases in alignment algorithms, even in cases where they are incorrect and, indeed, phylogenetically unreasonable. We contend that algorithms that impose gap patterns like those found in structural alignments of proteins are inappropriate for the increasingly widespread analysis of genomic DNA and are likely to cause error when the resulting alignments are used for evolutionary inferences.

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1. In a sense, phylogenetic alignment creates a circular argument. What we're trying to do is to build a phylogenetic tree from the multiple sequence alignments. If we use the presumed phylogeny to generate the alignments then we have a problem. Part of the problem goes away once we recognize that the alignment is driven by clustering similar sequences rather than phylogenetically related sequence.

Löytynoja, A. and Goldman, N. (2008) Phylogeny-Aware Gap Placement Prevents Errors in Sequence Alignment and Evolutionary Analysis. Science 320:1632-1635. [DOI: 10.1126/science.1158395]

A Graduate Student Oath

 
The Institute of Medical Studies (IMS) at the University of Toronto is a large department with many graduate students. Many of them are M.D.s doing clinical research.

The department has instituted a graduate student oath that beginning graduate students recite at their first meeting. The idea is to teach students the value of social and moral responsibilities. Beginning graduate students also have to take a mandatory seminar course on ethics.

The oath is explained and reproduced in this week's issue of science magazine in an article by Davis et al. (2008). Here it is.

"I, [NAME], have entered the serious pursuit of new knowledge as a member of the community of graduate students at the University of Toronto.

"I declare the following:

"Pride: I solemnly declare my pride in belonging to the international community of research scholars.

"Integrity: I promise never to allow financial gain, competitiveness, or ambition cloud my judgment in the conduct of ethical research and scholarship.

"Pursuit: I will pursue knowledge and create knowledge for the greater good, but never to the detriment of colleagues, supervisors, research subjects or the international community of scholars of which I am now a member.

"By pronouncing this Graduate Student Oath, I affirm my commitment to professional conduct and to abide by the principles of ethical conduct and research policies as set out by the University of Toronto."

What do you think? Is this something that all departments should consider?

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Davis, K.D., Seeman, M.V., Chapman, J. and Rotstein, O.D. (2008) A Graduate Student Oath. Science 320:1587-1588. [DOI: 10.1126/science.320.5883.1587b]

Errors in Sequence Databases

Sandra Porter at Discovering Biology in a Digital World brings up an issue that has been bugging me for two decades [Biologists vs. the Age of Information]. The issue is the accuracy of information in biological databases.

Let's begin with GenBank - GenBank is the main database of nucleotide sequences at the NCBI. Sequence data are submitted to GenBank by researchers or sequencing centers. If mistakes are found, the information in the records can be updated by the submitters or by third parties if the corrected versions are published. This correction activity doesn't always happen though, and the requirement for third party annotations to be published makes it pretty unlikely that anyone will submit small corrections to a sequence.

This is why we see these kinds of quotes from Steven Salzberg (3):

So you think that gene you just retrieved from GenBank [1] is correct? Are you certain? If it is a eukaryotic gene, and especially if it is from an unfinished genome, there is a pretty good chance that the amino acid sequence is wrong. And depending on when the genome was sequenced and annotated, there is a chance that the description of its function is wrong too.

This is a serious problem. Most people don't realize that GenBank is full of sequences that are known to be incorrect and/or poorly annotated. In most cases, the errors are relatively minor such as one or two incorrect codons or deletion of a single codon. In other cases, the errors are more important, such as a pseudogene being represented as a real gene, or missing exons. Sometimes the identity of a gene is completely wrong. I've even seen examples where the species is incorrectly identified.

Sandra asks,

So what do we do? Do we care if the database information is up-to-date? If so, who should be responsible for the updates?

I'm sure some people would like the NCBI to be the final authority and just fix everything but I don't think that's very realistic.

Other people have proposed that wikis are the answer. Maybe they're right, but I really wonder if researchers would be any better at updating wikis than they are at updating information in places like the NCBI.

Well, dear readers, what do you think? Does GenBank need to be fixed? Do we just need more alternatives? Does it even matter?

Back in 1992, I spent part of a summer at the GenBank site in Los Alamos (New Mexico, USA). That was before GenBank moved to NCBI in Bethesda. My task was to explore the possibility of curating GenBank to fix all the errors. I worked with the HSP70 sequences since I had already documented most of the errors in those sequences (The HSP70 Sequence Database).

We decided that I could make corrections to any HSP70 sequence as long as I annotated the changes and got permission from the authors by 'phone.¹ This didn't work. Most of the authors were unwilling to allow changes 'cause they weren't aware of the fact that there was a conflict between their sequences and the aligned sequence database. They didn't even know that others had sequenced the same gene and gotten a different sequence.

We discussed this problem. At the time, everyone was aware of the fact that the SwissProt database was curated and that the curators were making decisions on their own about which sequences were correct and which ones were errors. Here's an example of the entry for human HSPA1A showing the conflicts and variations.

Sometimes the SwissProt curators get it wrong and identify the correct sequence as an error and vice versa. Sometimes they really screw up. Here's an example of that mistake [P23931].

Curating a sequence database is incredibly expensive. You need to hire hundreds of competent workers who can analyze every sequence as it comes in. There are some tools that will help identify errors but in order to reach an acceptable level of accuracy you need to build aligned sequence databases for every gene. That can't be done automatically; you need to have real people look at the data and make the best alignment if you are going to use it to make judgements on the accuracy of a submitted sequence.

The final decision at GenBank was to forget about correcting errors and treat the database as an archive of submitted sequences. It would be up to every researcher to become aware of the error-prone nature of the database before drawing any conclusions. I think this was the correct decision—it was the only realistic decision. Unfortunately, the average researcher doesn't realize how may errors are being propagated in the sequence databases.

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1. It was a huge ego-trip to have the power to change records in GenBank. All of the changes I made to other people's sequences have been removed but the ones I made to my own sequences are still there. You can check out [M76613] to see an example of what an annotated sequence could have looked like. Note the references to "old-sequence," "conflict," "variation," and "unsure." These represent differences between the genomic sequence and our older error-prone cDNA sequences.

Kristin Roovers Punished for Falsifying Data

 
Kristin Roovers was a post-doc at the Ottawa Health Research Institute in Ottawa (Canada) until last week. Her job was abruptly terminated when OHRI learned that she had been convicted and punished for falsifying data while she was a graduate student and a post-doc at the University of Pennsylvania. Apparently they first heard that something was wrong from an article in The Chronicles of Higher Education [Journals Find Fakery in Many Images Submitted to Support Research].

Read about it in yesterday's Ottawa Citizen [Researcher's tainted past leads Ottawa health facility to sever ties]. See the fraudulent data on baylab [Kristin-gate at the OHRI].

You can read the July 2007 report from the Office of Research Integrity (USA) at Case Summary - Kristin Roovers.

Here's the question. Why was she hired at OHRI? They probably didn't ask for letters of reference and they certainly didn't Google her name.

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An Unusual Science Conference

 

An unusual science conference was held recently in Azeroth. Many of you, like me, who know where Azeroth is. It's the virtual world of World of Warcraft. There were more than 200 people in attendance.

The three days of meetings were packed full of interesting discussion about science, or so I'm told. What was most exciting were the social events, culminating on the last day when all of the participants died in a mass attack on an enemy fortress. I've never been to a science conference that was quite like that.

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Gunther Stent (1924 - 2008)

 
Gunther Stent was one of the leading figures in the 'phage group, a group of molecular biologists who transformed the science of biology back in the 1940's, '50's, and 60's. He died on June 12th [Gunther Stent, an Early Researcher in Molecular Biology, Is Dead at 84].

Today's Citation Classic from John Dennehy is the book The Molecular Biology of Bacterial Viruses by Gunther Stent. In keeping with his main theme, John often uses the citation classic to highlight the influence of past scientists and not necessarily the significance of a single paper. Stent's name is not associated with any one experiment, or even a series of experiments. His influence extended well beyond his ability to do important experiments.

I first encountered Gunther Stent at the annual 'phage meeting held at Cold Spring Harbor every summer. I learned pretty quickly that he thought on a different plane than the rest of us. I also observed first hand the respect he earned from other famous biologists. At the time I was just a graduate student, I'm certain that Gunther Stent was unaware of my existence.

Later on I began to read Stent's articles on the history and philosophy of biology and I was greatly influenced by his writing.¹ Stent had an amazing ability to sift through the garbage and get to the heart of an argument; especially if that meant going against the perceived wisdom of his intellectual peers. Here are two examples from THE DILEMMA OF SCIENCE AND MORALS published in Zygon in March 1975. Stent is discussing contradictions between modern science and Western moral traditions.

The first example we might consider concerns the teaching of evolution in the public schools, which evidently has come a long way from the days of the Scopes Monkey Trial in Tennessee half a century ago. In 1972 the Curriculum Commission of the California State Board of Education held hearings in response to the demand of some Christian fundamentalist groups that in the officially approved biology textbooks the biblical account of Creation ought to be presented on an equal footing with the Darwinian view as an explanation of the origin of’ life and of the species. Although much of the argument before the Commission pertained to the question of whether the theory of evolution is merely an unproven speculation, as alleged by the fundamentalists, or a solidly documented scientific proposition, as claimed by the biologists, the deeper point at issue was religious freedom.

For the fundamentalists held that a Christian child in a tax supported school has as much right to be protected from the dogmas of atheism as an atheist child has to be protected from prayer. Hence, it would follow that the classroom teaching of Darwinism as the only explanation of biocosmogony is an infringement of the religious freedom of Christian parents to raise their children in the faith of their choice. This argument seems completely justified, whether or not it is true as claimed in pro-Darwinian testimony at the hearings by liberal, apologist clergymen that one can be a good Christian without taking the biblical account of Genesis all that literally. After all, the fundamentalist faith is to take the Bible literally. But the inference that follows from admitting the justice of the fundamentalist claim is not that biology texts should give Genesis equal time with evolution. Rather, it is to be concluded that no public school system can operate effectively in a heterogeneous social setting without having its curriculum prejudice the minds of the pupils against the cherished beliefs of some of the citizens. In other words, in this case the ultimate Christian ethical aim of freedom and individual rights has to give way to the pagan aim of mounting a pedagogically effective society.

The second example is much more controversial, yet the logic is impeccable. This is not the sort of thing that modern liberals (I am one) want to hear, but the very fact that they cover their ears and chant nonsense verses at the top of their lungs is the problem that Stent addresses. Most of us don't realize that the conflict between science and culture is much deeper than the fight between scientist and Biblical literalists would suggest. If you are going to adopt the positions of science and rationalism then there are some implications that may be hard to confront. Sweeping them under the rug, as many try to do, is hypocritical.

We may now consider the ethical conflicts surrounding two applications of human genetics. One of these is the very troublesome matter, at least for present-day American society, of the heritability of intelligence and in particular of the problem whether there exist significant racial differences in intelligence genotype. On the one hand, it seems reasonable to think that if there is a significant variation in the genetic contribution to intelligence between individuals, or between racial groups, then this factor ought to be taken into account in the organization of society. But, on the other hand, the mere acknowledgment of the existence of this factor, let alone taking it into account in social action, seems morally inadmissible, a scientistic underpinning of racist ideology. An excellent exposition of this problem was recently provided by W. Bodmer and L. L. Cavalli-Sforza, who show that the heritability of intelligence, unlike extrasensory perception and telepathy, is a genuine scientific proposition.

First, it is possible to obtain a meaningful measure of intelligence through IQ tests, at least insofar as the concept of intelligence applies to the capacity to succeed in the society in whose contextual setting the tests are given. Second, there do exist significant differences in IQ between individuals and between social and racial subgroups. Third, it is possible, at least in principle, to perform studies that can ascertain the relative contribution of genetic and environmental factors to the observed differences in IQ. Bodmer and Cavalli-Sforza find that there is sufficient evidence at present to make it very likely that within a socioeconomically homogeneous group heredity does make a significant contribution to extant differences in IQ. When it comes to the considerably lower mean IQ of American blacks, however, they conclude not only that the currently available data are inadequate to ascertain whether this fact is attributable mainly to hereditary or mainly to environmental differences, but “that the question of a possible genetic basis for the race IQ differences will be almost impossible to answer satisfactorily before the environmental differences between U.S. blacks and whites have been substantially reduced. . . .” Finally, “[since] for the present at least, no good case can be made for [studies on racial IQ differences], either on scientific or practical grounds, we do not see any point in particularly encouraging the use of public funds for their support. There are many more useful biological problems for the scientist to attack.”

In my opinion, this recommendation, which trivializes the problem scientifically, amounts to taking the easy way out from a serious dilemma. What if, as Bodmer and Cavalli-Sforza admit could be true, there does exist a significant genetic contribution to the mean IQ differences found between blacks and whites? They think that this “should not, in a genuinely democratic society free of race prejudice, make any differene.”’~ But if the races really differed hereditarily in intelligence, then racism would not be a “prejudice” but a true perception ofthe world and one of which a rational society ought to take account. For instance, in this case, the black-white disparities in socioeconomic levels would not reflect discrimination at all but merely an underlying biological reality. And hence the aim of an egalitarian, multiracial society would be just another unattainable, utopian dream. We thus encounter another Machiavellian contradiction between the two incompatible ethical systems of our heritage. The pagan ethics of communal purpose, which science serves, would demand that every effort be made to ascertain whether the member races of a multiracial society do in fact differ hereditarily in their intelligence. But the Christian ethics of ultimate values, which inspire science, holds racism to be an absolute evil in that it is subversive of the fundamental concept of the freedom and responsibility of the human soul. Hence, these ethics demand an uncompromisingly hard line against research on race intelligence. Since there must not be any hereditarily determined racial differences in intelligence, research that entertains the possibility of such differences is a priori evil.

In today's world we need more Gunther Stent's, not fewer.

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1. Incidentally, I'm currently reading Richard Dawkin's anthology of Modern Science Writing. Stent is not in that book but, then again, neither are many other scientists who should be there. It's probably no coincidence that most of those scientists express opinions that differ from those of Richard Dawkins.

[Photo Credit: Left-to-right: Esther Lederberg, Gunther Stent, Sydney Brenner and Joshua Lederberg. From Wikipedia : "The original photo is owned by the Esther M. Zimmer Lederberg Estate. With the permission of that Estate's Trustee, Matthew Simon, I have adapted the photo for free use."

You've Been Left Behind

 

You've Been Left Behind is a very special website.

If you are sure that you've led a good Christian life then you can expect to be raptured. This could happen at any time. What about the friends and relative you leave behind? You've Been Left Behind will automatically send out email messages telling everyone where you've gone. It might give them one last chance.

We all have family and friends who have failed to receive the Good News of the Gospel.

The unsaved will be 'left behind' on earth to go through the "tribulation period" after the "Rapture". You remember how, for a short time, after (9/11/01) people were open to spiritual things and answers. (We are still singing "God Bless America" at baseballs' seventh inning stretch.) Imagine how taken back they will be by the millions of missing Christians and devastation at the rapture. They will know it was true and that they have blown it. There will be a small window of time where they might be reached for the Kingdom of God. We have made it possible for you to send them a letter of love and a plea to receive Christ one last time. You can also send information based on scripture as to what will happen next. Each fulfilled prophecy will cause your letter and plea to be remembered and a decision to be made.

"WHY" is one last chance to bring them to Christ and snatch them from the flames!

How does it work?

We have set up a system to send documents by the email, to the addresses you provide, 6 days after the "Rapture" of the Church. This occurs when 3 of our 5 team members scattered around the U.S fail to log in over a 3 day period. Another 3 days are given to fail safe any false triggering of the system.

We give you 150mb of encrypted storage that can be sent to 12 possible email addresses, in Box #1. You up load any documents and choose which documents go to who. You can edit these documents at any time and change the addresses they will be sent to as needed. Box #1 is for your personal private letters to your closest lost friends and relatives.

We give you another 100mb. of unencrypted storage that can be sent to up to 50 email addresses, in Box #2. You can edit the documents and the addresses any time. Box #2 is for more generic documents to lost family & friends.

The cost is $40 for the first year. Re-subscription will be reduced as the number of subscribers increases. Tell your friends about You've Been left behind.

I can foresee a few problems. What if three of the five Christians don't get raptured in spite of the fact that they've led a good Christian life? Maybe God will punish them for trying to scam their fellow Christians? Wouldn't that be a bummer? We might go for a week or so before noticing that some people were missing and by then it may be too late to recant.

What happens if all the ISP technical people get raptured and the server goes down within 48 hours? Have they thought of that? They should make sure that everyone working for the ISP is a heathen and/or an extremely disreputable person. That should also be a requirement for airline pilots, police, and lawyers doctors. We're gonna need them.

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[Hat Tip: RichardDawkins.net]

[Image credit: The cartoons are from There's a New World Coming by Hal Lindsey. Thanks to Brian Larnder of Primordial Blog for bringing it to my atention (Hallucinogenic Christian Comic). Apparently Brian used to read, and believe, these comics when he was little.]

Nobel Laureates: Gerald Edelman and Rodney Porter

 

The Nobel Prize in Physiology or Medicine 1972.

"for their discoveries concerning the chemical structure of antibodies"

Gerald M. Edelman (1929 - ) and Rodney R. Porter (1917 - 1985) received the Nobel Prize in Physiology or Medicine for elucidating the structure of immunoglobulins (antibodies). They determined that immunoglobulins were composed of two heavy chains and two light chains. There are three domains in the molecule. Two of them form binding sites for antigens and the third one links the two heavy chains together.

Edelman and Porter founded the field of molecular immunology, a field that today encompasses hundreds of labs. If you count all the clinical immunologists and cellular immunologists, there are as many immunology labs in the world as there are biochemistry labs. That was not true in the 1950's when Edelman and Porter began their work.

The presentation speech was in Swedish by Professor Sven Gard of the Karolinska Medico-Chirurgical Instit.

THEME:
Nobel Laureates

Your Royal Highnesses, Ladies and Gentlemen,

Immunebodies or antibodies is the designation of a group of proteins in the blood, that play an important part in the defense against infections and in the development of many different diseases. Their perhaps most characteristic property is the capacity to react and combine with substances, foreign to the organism, so-called antigens and to do so in a highly specific manner. There probably exist more than 50,000 different antibodies in the blood, each of them reactive against one particular antigen. Their main features are similar but they show individual characteristics and constitute, therefore, an extremely heterogeneous group. Since, in addition, they appear as very large molecules of a complex structure, it is understandable that the study of their chemistry for a long time offered great difficulties.

Up to 1959 the knowledge about their nature and mechanism of action was rather incomplete. That same year, however, Edelman and Porter separately and independently published their fundamental studies of the molecular structure of antibodies. Both of them had aimed at splitting the giant molecule into smaller, well defined fragments that might be more easily analysed than would the whole complex.

Porter's aim was to separate those parts of the antibody which are responsible for their specific reactivity. He hoped by this means to obtain a preparation lacking most of the biologic functions of the antibody but, on account of its capacity of combination, capable of competing with the antibody for the binding sites of the antigen. He succeeded in achieving this by means of treatment of the antibody, under strictly controlled conditions, with a protein-splitting enzyme called papain. By this treatment the antibody split into three parts. Two of these could combine specifically with the antigen and they were almost identical in other respects as well. The third fragment differed distinctly from the others, lacked binding capacity but possessed certain other biologic characteristics of the intact molecule.

Edelman for his part assumed the molecule, like those of many other proteins, to be composed of two or more separate chain structures held together by cross links of some kind, most probably so-called sulphide bonds. His assumption turned out to be correct. By means of a fairly rough treatment he was able to sever the cross bonds and release a number of separate chain molecules. Both he and Porter could later show that the antibody was in fact composed of four chains, one pair of identical, "light" chains and one pair of like- wise identical, "heavy" chains.

On the basis of the collected evidence Porter built a model of the molecule which has later, with overwhelming probability, been proven correct.

Accordingly the antibody molecule appears in the shape of the letter Y, with a stem and two angled branches. Each branch is composed of one light and one half of a heavy chain in side by side arrangement. The stem is made up of the remaining halves of the heavy chains. The specific combining capacity is accounted for by the structure of the free tips of the branches and in like measure by the light and the heavy chain; separately they are inactive. Porter's papain treatment attacks the molecule exactly at the point of branching and splits off the branches from the stem.

These discoveries incited an intense activity in laboratories in the four corners of the world. Apparently there existed a latent need for immunochemical research that could not be satisfied until today's prize winners had opened the way and provided the means. During the two decades that have since past our knowledge about the processes of immunity has broadened and deepened to an extent that perhaps has not yet been fully appreciated, even by some specialists in closely related fields. Many novel and fascinating aspects on problems in the fields of molecular biology and genetics have grown out of the immunochemical studies. We have now a new and firmer grasp of the question of the role of immunity as defense against and as cause of disease. Our possibilities to make use of immune reactions for diagnostic and therapeutic purposes have improved. It is, thus, a very important pioneer contribution that has been rewarded with this year's prize in physiology or medicine.

Gerald Edelman, Rodney Porter,

By clarifying the principal chemical structure of immunoglobulins you achieved an extremely important break-through in the field of immunochemistry. You, so to speak, opened the sluice-gates and gave impetus to the flood of research that soon started gushing forth, irrigating previously arid land, making it fertile and producing rich harvests. By awarding you the prize in 'physiology or medicine the Karolinska Institute has recognized the great significance of your accomplishments for biology in general and medicine in particular. On behalf of the Institute I wish to express our admiration and extend to you our heart-felt felicitations.

Now I ask you to proceed to receive your prize from the hands of His Royal Highness the Crown Prince.

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[Image Credit: The cartoon of an immunoglobulin molecule is from the Genetics Home Reference website of the National Institutes of Health (USA).]

How long does it take to synthetize a molecule of leucine anyway?

Bora Zivkovic asked this question on A Blog Around the Clock: How long does it take to synthesize a molecule of leucine anyway?.

A dozen or so years ago, I drove my Biochemistry prof to tears with questions - she had 200 people in front of her and she tried hard to make Biochem interesting enough not to get us all bored to tears, and she was pretty good at that, as much as it is possible not to make people bored to tears with Biochem. But my questions exasperated her mainly because she could not answer them, because, as I learned later, the field of biochemistry was not able to answer those questions yet at the time: questions about dynamics - how fast is a reaction, how long it takes for a pathway to go from beginning to end, how many individual molecules are synthesized per unit of time?, etc.

Bora, I'm sorry your Professor wasn't able to answer your questions. The answers have been known for decades. Perhaps she didn't know the answers, or perhaps there was another reason why she didn't answer.

The rate of enzymatic reactions is part of the field of enzyme kinetics. The material is usually covered in introductory biochemistry. The particular value you were looking for is called the catalytic constant, k_cat. It represents the number of moles of substrate converted to product per second per mole of enzyme under saturating conditions. In other words, it's the maximum speed of an enzyme. This is also called the turnover number.

Typical values for most enzymes are between 10² and 10³. What this means is that a given enzyme can catalyze between 100 and 1000 reactions per second.

A metabolic pathway in cells is a series of reactions where a substrate is converted to a product in several steps. The pathway for leucine biosynthesis is well known. It begins with pyruvate, which is converted in three reactions to α-ketoisovalerate. That intermediate can be converted directly to valine or it can serve as the substrate for a series of four reactions leading to leucine.

All of the enzymes have been studied. I'd have to look up all of the k_cat values to give you a precise answer but it's easy to give a reasonable estimate.¹

Biochemical pathways operate, for the most part, under near-equilibrium conditions. What this means is that there is a steady state concentration of all reactants in the pathway. These concentrations correspond to the equilibrium values for each reaction.

The flux in a pathway depends on how quickly the end product is utilized. Under normal conditions leucine will be used up in protein synthesis at a nearly constant rate but that rate might rise if the cell is growing rapidly and it might fall if the cell is starved for nutrients. When leucine synthesis is required, for whatever reason, it's maximum rate of synthesis will be equal to the turnover number of the slowest enzyme in the pathway.

You can safely assume that this will be between 100 and 1000 molecules per second per enzyme. That's the answer you should have been given. In a big mammalian cell growing in tissue culture there will be lots and lots of enzyme and the flux could be a million molecules per second. It will be much less in smaller cells that are not growing.

The key to understanding metabolic pathways is to appreciate that there is a pool of leucine in the cell and a pool of the last intermediate. These pools of metabolites are at steady-state concentrations and the enzyme is constantly making leucine and converting leucine back to the intermediate because that's what happens under equilibrium conditions. The rates of the forward and reverse reactions are equal, and fast.

As soon as the leucine pool is depleted there will be some net synthesis of leucine made from the pool of the last intermediate to restore the steady-state equilibrium concentrations. The rate of this reaction is very rapid.²

Then the pool of the last intermediate is replenished from the second-last intermediate etc. etc. All of these reactions are rapid. Most students seem to think that there are no intermediates and when leucine is needed the enzymes have to grab a pyruvate molecule and run through the entire pathway to make a new molecule of leucine. Such a pathway is impossible.

Well, the field is starting to catch up with my questions lately - adding the temporal dimension to the understanding of what is going on inside the cell. In today's issue of PLoS Biology, there is a new article that is trying to address exactly this concern: Dynamics and Design Principles of a Basic Regulatory Architecture Controlling Metabolic Pathways:

That's an interesting paper but it doesn't answer any of your questions.

The paper address the induction of enzymes in yeast. When yeast cells grow in the presence of leucine they turn off synthesis of the pathway enzymes because there's no need to synthesize leucine when it's available in the medium. If you then shift the cells to leucine free medium they will begin to make the leucine pathway enzymes. It takes about one hour to make signifcant amounts of enzyme.

Enzyme induction has been studied for over 50 years. The diagram below is from Monod's Nobel Lecture of 1965. The current PLoS paper adds some information to this field but, with all due respect, it is not a breakthrough and it does not answer fundamental questions in biochemistry that were unknown when you were a student. You may not have been aware of kinetic studies when you were a student but that's a reflection on the quality of your education and not on what was known in biochemistry at the time.

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1. Perhaps your biochemistry Professor didn't want to spend the time looking up all the details? Whenever I get a question like that I assign the task to the student. It's a good exercise for them to search through the scientific literature to find the answer to their own question. It also helps them appreciate why their Professor may not have had the answer at her fingertips.

2. For the sake of simplicity, I'm ignoring regulation. Some enzymes in the pathway might be regulated in which case the steady-state concentrations might not correspond to the equilibrium concentrations. This doesn't make much difference when it comes to addressing Bora's questions.

Wordle

 
Eva Amsen is writing her thesis. It is very easy to get distracted when you are writing your thesis—everyone needs a break from time to time. Eva found a fabulous website while she was surfing the net looking for references to put in her thesis and she blogged about it on [Expression Patterns].

The website is called Wordle. Here's what it does ...

Wordle is a toy for generating “word clouds” from text that you provide. The clouds give greater prominence to words that appear more frequently in the source text. You can tweak your clouds with different fonts, layouts, and color schemes. The images you create with Wordle are yours to use however you like. You can print them out, or save them to the Wordle gallery to share with your friends.

I fed it my essay on What Is a Gene? and here's what it gave me ...

Each time you try you get a different configuration of words so it's worthwhile to experiment a bit in order to get a pleasing layout. You can change fonts, colors, background and other things afterwards. Isn't this great?

Here's how Wordle handles another essay Evolution by Accident.



Here's another example with Theistic Evolution: The Fallacy of the Middle Ground.


This is so much fun. It must be bad for you. Send me your favorite Wordles, they must be created from something you wrote.

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With or Without God

 
Come to the Centre for Inquiry's lecture by Gretta Vosper.

With or Without God: Why the Way We Live is More Important than What We Believe

Starts: Friday, June 20th at 7:30 pm
Ends: Friday, June 20th at 9:30 pm
Location: Centre for Inquiry Ontario, 216 Beverley St, Toronto ON (1 minute south of College St at St. George St)

Lecture and Book Launch:
Gretta Vosper, United Church Minister at West Hill United Church, Toronto, and founder and Chair of the Canadian Centre for Progressive Christianity

In Gretta Vosper's church there are no prayers, no miracles-performing magic Jesus and no omnipotent God at all. Vosper's book argues that the Christian church, in the form in which it exists today, has outlived its viability and either it sheds its no-longer credible myths, doctrines and dogmas, or it's toast. With a humanist worldview, Vosper proposes a radical change at the heart of faith. The new church she envisions will play a viable and transformative role in the shaping of a future society. What will save the church from certain demise, Vosper argues, is a new emphasis on just and compassionate living.

A catered receptions shall precede the talk at 6pm exclusively for Friends of the Centre.

Canadian Centre for Progressive Christianity:

MacLeans Magazine coverage "The Jesus Problem":

Globe and Mail coverage "Taking Christ Out of Christianity"

Cost: $6 general, $4 students, FREE for Friends of the Centre

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Monday's Molecule #76

 
Name this molecule, being as specific as you can.

There's a direct connection between today's molecule and a Nobel Prize. The prize was awarded for discovering the basic structure of the molecule, although not at the level of detail depicted here. That came later.

The first person to correctly identify the molecule and name the Nobel Laureate(s), wins a free lunch at the Faculty Club. Previous winners are ineligible for one month from the time they first collected the prize. There are four ineligible candidates for this week's reward. You know who you are.

THEME:

Nobel Laureates
Send your guess to Sandwalk (sandwalk (at) bioinfo.med.utoronto.ca) and I'll pick the first email message that correctly identifies the molecule and names the Nobel Laureate(s). Note that I'm not going to repeat Nobel Laureate(s) so you might want to check the list of previous Sandwalk postings by clicking on the link in the theme box.

Correct responses will be posted tomorrow. I may select multiple winners if several people get it right.

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

UPDATE: The molecule is immunoglobulin G (IgG) and the Nobel Laureates are Gerald Edelman and Rodney Porter (1972). The first correct answer was from Jon Turnbull who beat everyone else by more than one hour! Honorable mention (and a free lunch) goes to Haruhiko Ishii of UCSD. Not only did he identify the molecule as IgG, he also showed that it was very likely to be Mab231, a mouse monoclonal anti-canine lymphoma antibody composed of IGg2a heavy chains and κ light chains [PDB 1IGT].

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Café Scientifique and Nature Network Pub Night

 
CAFÉ SCIENTIFIQUE PRESENTS
The future of medicine: help, hope or hype? (download the poster)

What lies in the future for medicine and health care? Over the next 50-100 years, how will we conquer illnesses and stay healthy? Join the discussion and debate at the next Café Scientifique, The future of medicine: help, hope or hype?, where experts will peek at the potential for robotics, genomics, alternative therapies and personalized medicine to cure our ills.

Experts:

• Dr. Tony Pawson – Distinguished Investigator, Samuel Lunenfeld Research Institute of Mount Sinai Hospital
• Karl Schroeder – Science fiction author and futurist
• Dr. Calvin Gutkin – Executive Director and CEO, The College of Family Physicians of Canada

Wednesday, June 18, 2008, from 6 to 8pm
Duke of York Pub – ground floor
39 Prince Arthur Avenue -Close to the St. George subway (Bedford exit)

FREE

Presented by the Samuel Lunenfeld Research Institute of Mount Sinai Hospital and Ontario Science Centre, with generous support from Canadian Institutes of Health Research.

Café Scientifique is a place where, for the price of a cup of coffee or a pint of beer, anyone can join discussions that explore the latest ideas in science and technology.

The members of the Toronto hub of Nature Network will meet afterwards in the Duke of York (same place as Café Scientifique) [see Eva Amsen's posting on easternblot]. You get two stimulating meetings for the price of one (i.e. free!).

If you haven't yet joined the Toronto hub of Nature Network you should sign up here. Current members of the Toronto hub are here.

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Kansas vs Darwin

 
Jeff Tamblyn, the director of Kansas vs Darwin will be in town this week for the ReelHeART International Film Festival. The film will be shown on Thursday evening. Here's the trailer, details below ...

Kansas vs. Darwin screening Thursday, June 19, 7:00 PM
ReelHeART International Film Festival
RHIFF MAIN PROGRAM B Tickets $8
INNIS THEATER 222
Innis College, University of Toronto
2 Sussex Avenue [1 block south of Bloor Street, on St. George Street]
Toronto, ON M5S 1J5
Advance Sales on line April 21, 2008 at www.reelheart.com

Kansas vs. Darwin
Director, Jeff Tamblyn, USA

Kansas vs. Darwin is a smart, funny, feature-length documentary about the Kansas state school board hearings on evolution. Features intimate revealing interviews with all major players on both sides, and exclusive, multi-camera footage of the hearings. Far more than a political film, Kansas vs. Darwin skillfully weaves multiple themes into a gripping dialectic, putting you face to face with, and inside the heads of, those who oppose your most closely held beliefs. Challenging and entertaining, it’s packed with fascinating characters who will leave you in admiration and astonishment, embarrassment and exasperation, as they feverishly pursue their goals, sometimes stumbling over their own eagerness in the attempt to win the most important battle of their lives.

I'm going. Contact me if you plan to attend and you want to meet up for dinner before the show.

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Fernando

 
Fernando was one of ABBA's biggest hits. There's a lot of debate about which war it refers to. The song mentions crossing the Rio Grande and that prompts many people in America to think of the Mexican revolution of 1910-1920. However, there aren't many examples of fighting that took place near the Rio Grande and there aren't too many examples of revolutionaries who crossed into Mexico from the USA.

Most people assume the song is about the Spanish civil war and the reference to the Rio Grande is just a generic reference to a river. Keep in mind that ABBA is a European group and the Spanish Civil War is still fresh in the memories of many europeans. For many it was glorious, but losing, fight against fascism.

The song refers to Fernando, a man who fought on the losing side against tyranny and fascism. Fernando was a revolutionary and a guerrilla fighter. He is now old and gray like many of the freedom fighters from all over Europe who went to Spain in the 1930's.

John McCain likes ABBA. I hope he appreciates that this song is about people who fought to defend their country from foreign domination. (Franco was supported by Hitler and Mussolini.)

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