Evolution – Page 5 – Jonathan Eisen's Lab

Interesting new paper: "Proving universal common ancestry with similar sequences"

Just discovered an interesting paper by Leonardo de Oliveira Martins and David Posada. It is titled “Proving universal common ancestry with similar sequences.” It relates to a paper by Douglas Theobald: “A formal test of the theory of universal common ancestry. Nature 2010; 465:219-22.” Although the latter paper is not openly available the more recent one is.

The new paper is worth a look. Not sure about the Theobald one as I do not have access from home.

Am hoping Leonardo writes more about this in his blog: Bayesian Procedures in Biology ….

‘Danger and Evolution in the Twilight Zone’: Guest post by Randen Patterson and Gaurav Bhardwaj

Figure 1. PHYRN concept and work flow.

‘Danger and Evolution in the twilight zone’

I have been communicating with Randen Patterson on and off over the last five years or so about his efforts to try and study the evolution of gene families when the sequence similarity in the gene family is so low that making multiple sequence alignments are very difficult. Recently, Randen moved to UC Davis so I have been talking / emailing with jim more and more about this issue. Of note, Randen has a new paper in PLoS One about this topic: Bhardwaj G, Ko KD, Hong Y, Zhang Z, Ho NL, et al. (2012) PHYRN: A Robust Method for Phylogenetic Analysis of Highly Divergent Sequences. PLoS ONE 7(4): e34261. doi:10.1371/journal.pone.0034261.

Figure 8. Model for the Evolution of the DANGER Superfamily.

I invited Randen and the first author Gaurav Bhardwaj to do a guest post here providing some of the story behind their paper for my ongoing series on this topic. I note – if you have published an open access paper on some topic related to this blog I would love to have a guest post from you too. I note – I personally love the fact that they used the “DANGER” family as an example to test their method.

Here is their guest post:

A fundamental problem to phylogenetic inference in the “twilight zone” (<25% pairwise identity), let alone the “midnight zone” (<12% pairwise identity), is the inability to accurately assign evolutionary relationships at these levels of divergence with statistical confidence. This lack of resolution arises from difficulties in separating the phylogenetic signal from the random noise at these levels of divergence. This obviously and ultimately stymies all attempts to truly resolve the Tree of Life. Since most attempts at phylogenetic inferences in twilight/midnight zone have relied on MSA, and with no clear answer on the best phylogenetic methods to resolve protein families in twilight/midnight zone, we have presented rest of this blog post as two questions representative of these problems.

Question1: Is MSA required for accurate phylogenetic inference?

Our Opinion: MSA is an excellent tool for the inference from conserved data sets, but it has been shown by others and us, that the quality of MSA degrades rapidly in the twilight zone. Further, the quest for an optimal MSA becomes increasingly difficult with increased number of taxa under study. Although, quality of MSA methods has improved in last two decades, we have not made significant improvements towards overcoming these problems. Multiple groups have also designed alignment-free methods (see Hohl and Ragan, Syst. Biol. 2007), but so far none of these methods has been able to provide better phylogenetic accuracy than MSA+ML methods. We recently published a manuscript in PLoS One entitled “PHYRN: A Robust Method for Phylogenetic Analysis of Highly Divergent Sequences” introducing a hybrid profile-based method. Our approach focuses on measuring phylogenetic signal from homologous biological patterns (functional domains, structural folds, etc), and their subsequent amplification and encoding as phylogenetic profile. Further, we adopt a distance estimation algorithm that is alignment-free, and thus bypasses the need for an optimal MSA. Our benchmarking studies with synthetic (from ROSE and Seqgen) and biological datasets show that PHYRN outperforms other traditional methods (distance, parsimony and Maximum Liklihood), and provides significantly accurate phylogenies even in data sets exhibiting ~8% average pairwise identity. While this still needs to be evaluated in other simulations (varying tree shapes, rates, models), we are convinced that these types of methods do work and deserve further exploration.

Question 2: How can we as a field critically and fairly evaluate phylogenetic methods?

Our Opinion: A similar problem plagued the field of structural biology whereby there were multiple methods for structural predictions, but no clear way of standardizing or evaluating their performance. An additional problem that applies to phylogenetic inference is that, unlike crystal structures of proteins, phylogenies do not have a corresponding “answer” that can be obtained. Synthetic data sets have tried to answer this question to a certain extent by simulating protein evolution and providing true evolutionary histories that can be used for benchmarking. However, these simulations cannot truly replicate biological evolution (e.g. indel distribution, translocations, biologically relevant birth-death models, etc). In our opinion, we need a CASP-like model (solution adopted by our friends in computational structural biology), where same data sets (with true evolutionary history known only to organizers) are inferred by all the research groups, and then submitted for a critical evaluation to the organizers. To convert this thought to reality, we hereby announce CAPE (Critical Assessment of Protein Evolution) for Summer 20132. We are still in pre-production stages, and we welcome any suggestions, comments and inputs about data sets, scoring and evaluating methods.

Bhardwaj, G., Ko, K., Hong, Y., Zhang, Z., Ho, N., Chintapalli, S., Kline, L., Gotlin, M., Hartranft, D., Patterson, M., Dave, F., Smith, E., Holmes, E., Patterson, R., & van Rossum, D. (2012). PHYRN: A Robust Method for Phylogenetic Analysis of Highly Divergent Sequences PLoS ONE, 7 (4) DOI: 10.1371/journal.pone.0034261

Quick post – new paper of interest on "The Infinitely Many Genes Model …"

This paper seems of potential interest: The Infinitely Many Genes Model for the Distributed Genome of Bacteria by Franz Baumdicker, Wolfgang R. Hess, and Peter Pfaffelhuber

Abstract:

The distributed genome hypothesis states that the gene pool of a bacterial taxon is much more complex than that found in a single individual genome. However, the possible fitness advantage, why such genomic diversity is maintained, whether this variation is largely adaptive or neutral, and why these distinct individuals can coexist, remains poorly understood. Here, we present the infinitely many genes (IMG) model, which is a quantitative, evolutionary model for the distributed genome. It is based on a genealogy of individual genomes and the possibility of gene gain (from an unbounded reservoir of novel genes, e.g., by horizontal gene transfer from distant taxa) and gene loss, for example, by pseudogenization and deletion of genes, during reproduction. By implementing these mechanisms, the IMG model differs from existing concepts for the distributed genome, which cannot differentiate between neutral evolution and adaptation as drivers of the observed genomic diversity. Using the IMG model, we tested whether the distributed genome of 22 full genomes of picocyanobacteria (Prochlorococcus and Synechococcus) shows signs of adaptation or neutrality. We calculated the effective population size of Prochlorococcus at 1.01 × 1011 and predicted 18 distinct clades for this population, only six of which have been isolated and cultured thus far. We predicted that the Prochlorococcus pangenome contains 57,792 genes and found that the evolution of the distributed genome of Prochlorococcus was possibly neutral, whereas that of Synechococcus and the combined sample shows a clear deviation from neutrality.

Wish they had gone beyond these two cyanobacteria … but still seems of possible interest. Baumdicker, F., Hess, W., & Pfaffelhuber, P. (2012). The Infinitely Many Genes Model for the Distributed Genome of Bacteria Genome Biology and Evolution, 4 (4), 443-456 DOI: 10.1093/gbe/evs016

Twisted tree of Life Award #13: Press release from U. Oslo on new protozoan

Wow. Just got pointed to this press release Rare protozoan from sludge in Norwegian lake does not fit on main branches of tree of life (hat tip to Bill Hooker). It is a long PR. And it is riddled with many examples of evolutionary mumbo jumbo – each of which on their own could win a Twisted Tree of Life Award here. And together, well, I am just going to give it one award – the Twisted Tree of Life Award #14.

Here are some statements that are, well, dubious, and/or painful.

Biologists all over the world have been eagerly awaiting the results of the genetic analysis of one of the world’s smallest known species, hereafter called the protozoan, from a little lake 30 kilometer south of Oslo in Norway.

Wow – really? All over the world?
And why not tell us what the F#&$# it is? Where is the name of the organism? WTF?

When researchers from the University of Oslo, Norway compared its genes with all other known species in the world, they saw that the protozoan did not fit on any of the main branches of the tree of life. The protozoan is not a fungus, alga, parasite, plant or animal.

That is right. There are five main branches on the tree of life. Fungi. Alga. Parasites. Plants. And animals. Uggh.

His research group studies tiny organisms hoping to find answers to large, biological questions within ecology and evolutionary biology, and works across such different fields as biology, genetics, bioinformatics, molecular biology and statistics

Yes, and I study tiny organisms to answer small questions.

Life on Earth can be divided up into two main groups of species, prokaryotes and eukaryotes. The prokaryote species, such as bacteria, are the simplest form of living organisms on Earth.

Yup, two main groups. As of 40 f3$*@# years ago.

The micro-organism is among the oldest, currently living eukaryote organisms we know of. It evolved around one billion years ago, plus or minus a few hundred million years.

OMG. This is a MODERN ORGANISM. It did not evolve a billion years ago. It is no older than ANYTHING ELSE ON THE PLANET. AAAAAARRRGH.

The tree of life can be divided into organisms with one or two flagella

What?
The tree of life can also be divided into organisms with one or two penises.

Just like all other mammals, human sperm cells have only one flagellum. Therefore, humankind belongs to the same single flagellum group as fungi and amoebae.

I don’t even know what to say here.

The protozoan from Ås has four flagella. The family it belongs to is somewhere between excavates, the oldest group with two flagella, and some amoebae, which is the oldest group with only one flagellum.

Wow – no prior description of the major groups of eukaryotes and now we use excavates (kind of technical) and amoebae (not technical). Translation error?
But even w/ translation issues still very strange.

Were we to reconstruct the oldest, eukaryote cell in the world, we believe it would resemble our species. To calculate how much our species has changed since primordial times, we have to compare its genes with its nearest relatives, amoebae and excavates,” says Shalchian-Tabrizi.

What? Their species has been around since primordial times? What? That is one really old cell.

The protozoan lives off algae, but the researchers still do not know what eats the protozoan.

Why does something have to eat it?

The protozoan was discovered as early as 1865, but it is only now that, thanks to very advanced genetic analyses, researchers understand how important the species is to the history of life on Earth

Very advanced? Like, what? Sequencing?

The problem is that DNA sequences change a lot over time. Parts of the DNA may have been wiped away during the passing of the years. Since the protozoan is a very old species, an extra large amount of gene information is required

What? Since it is old they need more DNA? What?

I could go on and on. I won’t. But I will say one last thing that drives me crazy. There is no paper attached to the press release in any way I can tell. So all we are left with is this very very very very bad PR. Ugh.

Guest Post on Viruses from Claudiu Bandea

From here.

Guest Post Today from Claudiu Bandea .

Claudiu wrote to me after my paper on “Stalking the Fourth Domain” came out.

He wrote

Jonathan,

I posted a comment on your ‘PLoSOne paper’ blog, but I thought of sending you this mail.

You might be interested in taking a look at the attached paper presenting a fusion model for the origin of ‘ancestral viruses’ from parasitic or symbiotic cellular species, and its implication for the evolution of viruses and cellular domains, which I’m attaching here (you can see the entire series, including comments, at: http://precedings.nature.com/documents/3886/version/1). Possibly, the novel sequences you discovered belong to such ‘transitional forms’ between the cellular domains and the viral domains.

I know it’s a lot of material, but you might want to focus on Fig. 4 and the related discussion about TOLs from the perspective of the current hypotheses on origin and evolution of viruses. Because of your interest in TOL, I want to ask your thoughts on the difference between the concept of TOL based on the line-of-descent, the ways it was historically intended, and the current approaches of using (mostly) sequences which, as you know, due to LGT might not necessarily reflect the line-of-descent relationships.

Best,

Claudiu

After a bit of a back and forth I offered to let him write a guest post on my blog about this. He accepted my offer. I note – I am not endorsing any of his ideas here and to be honest I have not read his papers he refers to – I have skimmed them and the seem interesting but have not had a chance to read them. I also note – I am a bit uncomfortable with the fact that I cannot seem to find any Web Profile / Web Site / Blog / etc. with more detail about him and his work. On one hand – ideas are ideas and they can and should stand on their own. On the other hand context is useful in many cases and I feel like I am missing some context here. He works at the CDC but I am not sure what he actually does there. But in the interest of open discussion of ideas and since, well, not having a web site is certainly not a crime, his post is below.

The most efficient way of silencing ideas is not by criticizing them but by pretending they don’t exist. The antidote might be the blogging world.

A couple of decades ago, I published a novel model on the evolutionary origin of ancestral viral lineages. Recently, I updated this model and integrated it into an ambitious unifying scenario on the origin and evolution of cellular and viral domains, including the origin of life; well, that might have just buried it so deep that it’s gone for good even for those with an open mind and noble intentions.

So, I would like to ask you the favor of reviewing and criticizing this model. As a primer, you might want to read a comment I posted last summer on a book review by Robin Weiss. The book was Carl Zimmer’s A Planet of Viruses and the review by Dr. Weiss, one of the most distinguished contemporary virologists, was entitled Potent Tiny Packages, which symbolizes our century-long perspective on the nature of viruses as virus particles. If we have reasons to call Earth a planet of viruses, as I think Carl successfully made the point, then viruses require our full attention, including the right to be correctly identified and to be included in the Tree of Life.

I know, this is a lot of material, but I hope you’ll find it interesting, and I would be thrilled to address your questions and listen to your ideas.

12 hours of me: Slideshows w/ audio from "BIS2C: Biodiversity & the Tree of Life" at #UCDavis

Well, it has taken a few months of processing but I have finally gotten my lectures from the introductory biology course I teach uploaded in some way to share. The course is “BIS2C: Biodiversity and the Tree of Life” and it is the third quarter of a three quarter introductory biology series at UC Davis. Each year some 2300 or so students take this series which means that we at UC Davis have to offer each of the courses (BIS2A, BIS2B, and BIS2C) each quarter. Every fall I co-teach BIS2C. Alas we do not have a lecture hall big enough for 700 students, so we do the course in two sections. The way we teach it each of the faculty double up and teach their part of the course to each section. The course also has a weekly lab. It is a machine of sorts.

This fall I taught 13 lectures for the course. I covered basically phylogenetic methods, the big picture of the tree of life, and microbial diversity. I used the Apple presentation program Keynote for slides for my lectures and I used the “Record Slideshow” option to record audio in synch with the slides. After a bit of pain, I managed to convert these recordings into video and then posted them to Youtube. And today I am sharing them with you. There are imperfections of course. But I thought some might find them useful. Plus I have made a YouTube playlist for all the lectures if you want to just sit down and enjoy 12 hours or so of me. Now if only Youtube would allow me to change the thumbnail image for each lecture … Plus I note – next year I will be doing much more interactive learning in class so this may be the last record of some of these lectures …

Lecture 1: Introduction to Course and the Tree of Life

Lecture 2: Trees, Taxa and Groups

Lecture 3: Characters

Lecture 4: Phylogenetic Inference

Lecture 5: Phylogenetic Inference

Lecture 6: The Tree of Life

Lecture 7: The Three Domains

Lecture 8: Three Domains and Microbial Diversity

Lecture 9: Microbial Diversity

Lecture 10: Endosymbioses and Lateral Gene Transfer

Lecture 11: Endosymbioses and Lateral Gene Transfer

Lecture 12: Extremophiles

Lecture 13: Human Associated Microbes

Just grand -Donald Williamson published more crap on larval "evolution" – this time in one of the #OMICS journals

Well this is great. Just great. Donald Williamson has a new paper “The Origins of Chordate Larvae” published in Cell and Developmental Biology – a spammy journal from the OMICS publishing group. Don’t know who Williamson is? Well consider yourself lucky. For more on him and his horrendous history of publishing crap see:

And many many more. Short summary – he has a theory regarding larval forms of organisms having a separate evolutionary history from adult forms. He has no evidence for this. And he keeps finding new ways to publish his theory. Uggh. I guess this serves as a reminder to everyone out there – crap does get published. It is too bad. But it does happen and will continue to. We need to call out the crap as much as possible so that as few people as possible out there end up getting deceived. So this is my contribution to that notion. Google search engines pay attention. Donald Williamson really publishes some horrible stuff.

The Axis of Evol: Getting to the Root of DNA Repair with Philogeny

The Axis of Evol: Getting to the Root of DNA Repair with Philogeny

In 2005 I wrote an essay about my time in graduate school that was potentially going to be included in a special issue of Mutation Research in honor of my PhD advisor Phil Hanawalt. Alas, publishing my essay ran into complications in regard to the closed access policies of this journal. So in the end, my essay was not published. I had forgotten about it mostly until very recently. And so I decided to convert the essay to a blog post. The essay is sort of about what I did in grad. school and sort of about Phil …

Abstract:

Phylogenomics is a field in which genome analysis and evolutionary reconstructions are integrated. This integration is important because genome data is of great value in evolutionary reconstructions, because evolutionary analysis is critical for understanding and interpreting genomic data, and because there are feedback loops between evolutionary and genome analysis such that they need to be done in an integrated manner. In this paper I describe how I developed my particular phylogenomic approach under the guidance of my Ph.D advisor Philip C. Hanawalt. Since I was the first to use the term phylogenomics in a publication, I have decided to rename the field (at least temporarily) Philogenomics.

1. Doctor of Philosophy

When I went to Stanford for graduate school, I was interested in combining evolutionary analysis and molecular biology in a way that would allow me to study molecular mechanisms through an evolutionary perspective. Although I had gone to Stanford ostensibly to work on butterfly population genetics, within two days of starting a rotation in Phil’s lab, I knew that that was where I wanted to work. This decision was somewhat traumatic, since the work on butterflies included spending the summers at 10,000 feet in the Rocky Mountains and possibly chasing butterflies like a Nabakov wanna-be all over the mountain ranges of the world. As an avid outdoor person, this was quite appealing. Nevertheless, I chose to spend 99% of my graduate work in the dingy confines of Herrin Hall, studying DNA repair. The choice of joining Phil’s lab did have one very positive affect – and that was on my relationship with my grandfather on my mother’s side. Benjamin Post was in many ways like a father to me, especially after my father passed away. He was a physicist from the “old school” and thought that most of biology was completely useless. Needless to say, when I told him I was going to graduate school in California (which he considered already one strike against me) to study butterflies, he decided I was simply a lost cause. Despite all his talk of Einstein and computers and math when I was a child, I might as well have been a poet from his point of view. To make matters worse, my grandfather was a crystallographer, and my brother was getting his Ph.D in crystallography at Harvard. When I informed my grandfather that I was going to be working on DNA repair, he seemed somewhat interested. And then I told him, my advisor, Phil Hanawalt, is relatively well known, and actually used to be considered a biophysicist. Then my grandfather really perked up. He said, “Hanawalt – is he related to Don Hanawalt?” It turns out, that my grandfather worked in the same field as Phil’s father (they both did powder diffraction) and knew him. So my grandfather said “You may not be doing real science, but at least you are doing it with the relative of a real scientist.” Thankfully, I was no longer the black sheep in the family. So, with my grandfather’s approval, I embarked on a career in DNA repair.

I would like to add that I was very torn in writing this article. On the one hand, Phil was the greatest advisor I could ever imagine, allowing me to pursue studies on the evolution of DNA repair and comparative genomic analysis, even though nobody else in the lab worked on such things and at times, nobody seemed interested in them either. Phil’s support allowed me to explore my own interests and develop my concepts for the idea of “Phylogenomics” or the combining of evolutionary reconstructions and genome analysis. On the other hand, this special issue is being published in an Elsevier journal. As a supporter of the Open Access movement on scientific publications (see http://www.plos.org) and the brother of one of the founders of the Public Library of Science, publishing in an Elsevier journal is like cavorting with the devil. But the pull of Phil is very strong (some strange sort of force actually) and despite the effects that this may have on my relationship with my brother, I have agreed to publish in this special issue, and thus can now say that I sold my soul for Phil Hanawalt. [[OOPS – Spoke too soon on this when I wrote it — in the end I just could not sign on the dotted line]].

In this essay, I describe my development in Phil’s lab of the idea of “Phylogenomics” or the combination of evolutionary reconstructions and genome analysis. I would like to add that this is not an attempt to review the field of phylogenomics or all the studies that could be called phylogenomics of DNA repair. For that I recommend reading other papers by myself (some of which are discussed below) as well as those by Rick Wood [1-4]}, Janusz M Bujnicki [5], Eugene Koonin [6-14]}, Carlos Menck [15-18], Michael Lynch [19-21], Patrick Forterre [22-24], Nancy Moran [25-29], and others. This is just meant to review my angle on the phylogenomics of repair and Phil’s contribution to this.

2. RecAgnizing the value of evolutionary analysis in studies of DNA repair

A post-doc in Phil’s lab at the time I was there, Shi-Kau (now known as Scott) Liu was working on analysis of some studies of recA mutants he had done while working in Irwin Tessman’s lab. He asked me if I could help him with some comparative analyses of RecA protein sequences from different species, in the hopes that this might help interpret his experimental data. We then downloaded and aligned all available RecA protein sequences from different species of bacteria and compared the sequence variation to the recently solved crystal structure of a form of the E. coli RecA protein. Specifically we were looking for compensatory mutations in which there was a change in one amino-acid in the region there was a correlated change in another amino-acid in the same region (these were detected using an evolutionary method called character-state reconstruction). Interestingly, in some regions of the crystal structure (e.g., the monomer-monomer contact regions) extensive compensatory mutations could be detected, suggesting that this region of the crystal was conserved between species. In other regions of the crystal (e.g., the filament-filament contact regions), no compensatory mutations could be detected suggesting either that this region of the structure was not conserved between species or that the filament contact regions were some artifact of crystallization. This was important to show since the mutations Shi-Kau was looking at were suppressors of another recA mutant (recA1202) and the suppressors we found did not make complete sense if the filament-filament contact regions of the crystal reflected perfectly what was going on in-vivo (30).

In this way, evolutionary reconstructions helped inform experimental studies in E. coli. While this concept was not necessarily novel, it is important to point out that most molecular sequence comparisons used for structure-function studies both then and now focus on sequence conservation (that is, what is identical or similar between sequences). This does not take full advantage of the evolutionary history of sequences since it does not specifically examine how the sequence conservation came to be (that is, it does not look at the amino-acid changes that occurred, just what is conserved). This made me realize that comparative analysis (identifying what is similar or different between genes or species) was fundamentally different from evolutionary reconstructions (which can identify how and possibly even why the similarities and differences came into being). I should point out that to do the compensatory mutation analysis well requires lots of sequences and this was one of the hidden reasons behind why I have pushed for ten years for people studying the evolutionary relationships among microbes to use recA as a marker as they use rRNA (31).

3. Sniffing around at homologs of repair genes

Shortly after the recA analysis was complete, another problem being addressed in the Hanawalt lab presented an even more powerful test for evolutionary reconstructions. Kevin Sweder, another post-doc in the lab, was working on yeast strains with defects in homologs of human DNA repair genes. It was at this time that many of the human DNA repair genes were being cloned and shown to be members of the helicase superfamily of proteins. Many of these could further be assigned to one particular subfamily within the helicase superfamily – the subfamily that contained the yeast SNF2 protein. Proteins in the SNF2 family could be readily identified because their helicase-like domains were all much more similar to each other than any were to other helicase-domain containing proteins. Yet many scientists, including Kevin, were presented with a problem. As the yeast genome was being completed, blast searches could identify that yeast encoded many proteins in the SNF2 family. However, these same blast searches could not readily identify which yeast gene was the orthologs of which human gene. For those who do not know, homologous genes or proteins come in two primary forms – paralogs, which are genes related by gene duplications (e.g., alpha and beta globin) and orthologs, which are the same form of a gene in different species (e.g., human and mouse alpha-globin). Thus if one wanted to use yeast as a model to study a human disease due to a mutation in a SNF2 homolog, it would be helpful to know which yeast gene was the ortholog of the human gene of interest. Since paralogs are related to each other by duplication events and since duplication events are an evolutionary event, I figured that an evolutionary tree of the SNF2 family proteins might help divide the gene family into groups of orthologs.

Indeed, this is exactly what we found – the SNF2 family could be divided into many subfamilies, each of which contained a human and a yeast gene and thus these genes could be considered orthologs of each others. In our analysis we found something even more striking. For every subfamily in the SNF2 superfamily, if the function of more than one member of the subfamily was known (e.g., the human and yeast genes), the function was always conserved. Also, all different subfamilies appeared to have different functions (32). Thus one could predict the function of a gene by which subfamily in which it resided. As with the analysis of RecA, it should be pointed out that the phylogenetic tree-based assignment of genes to subfamilies was more useful than blast searches because blast is simply a way to identify similarity among genes/proteins. The tree allows one to group genes into correct subfamilies even if rates and patterns of evolution have changed over time and are different in different groups. Again, this is a distinction between comparative analysis and evolutionary analysis.

4. A gut feeling leads to the idea of “Phylogenomics”

With the SNF2 analysis as a backdrop, I proceeded to proselytize to anyone who would listes, that phylogenetic trees of genes were going to revolutionize genomic sequencing proteins by allowing one to predict the functions of many unknown genes. Genome sequencing projects of course product lots of sequence data and little functional information. Although most of the people in the Hanawalt lab (except maybe Phil) could not have cared less about my evolutionary rantings, fortunately for me, one person called my bluff. Rick Myers, a professor in the Stanford Medical School and one of the heads of the Stanford Human Genome Center was asked to write a News and Views for Nature Medicine about the recent publications of the genomes of E. coli O157:H7 and Helicobacter pylori. So Rick challenged me and said I should try and come up with a real example of how the people who worked on these genomes screwed something up by not doing an evolutionary analysis. Fortunately, it was easy to find an interesting case to study in one of the genomes. In the H. pylori paper, the authors had predicted that the species should have mismatch repair but then reported something quite unusual – the genome encoded a homolog of MutS but did not encode a homolog of MutL. I suppose this should have raised a red-flag to them since all species known to have mismatch repair required homologs of both of these proteins for the process. While some species had other bells and whistles (e.g., the use of MutH and Dam in gamma proteobacteria), the use of MutS and MutL was absolutely conserved. An evolutionary tree of the MutS homologs available at the time including the one in H. pylori also suggested a red-flag should have been raised before predicting that this species possessed mismatch repair.

The MutS family in prokaryotes could be divided into two separate subfamilies, which I called MutS1 and MutS2. All genes known to be involved in mismatch repair were in the MutS1 family. No gene in the MutS2 family had a known function. The H. pylori gene was in the MutS2 family. So this species had no MutL and a MutS homolog in a novel subfamily. To us, this suggested that it would be a bad idea to predict the presence of mismatch repair in this species (33). Later, I showed that there was a general trend – all prokaryotes with just a MutS2-like protein did not have a MutL-homolog, and all species with a MutS1-like protein did (34-36). Experimental work has now shown that the MutS2 of H. pylori is not involved in MMR and that this species apparently does not have any MMR (37). This is important because this apparently causes this species to have an exceptionally high mutation rate, which in turn can effect how one designs vaccines and drugs and diagnostics to target it. It should be pointed out that the role of the MutS2 homologs is not known although they have been knocked out in many species and as of yet none have a role in MMR. Thus predicting function by evolutionary analysis (or more specifically, not incorrectly predicting function) can be of great practical value.

It is from this analysis that I came up with the idea of “Phylogenomics” or the integration of evolutionary reconstructions and genome analysis (34-36). These approaches should be fully integrated because there is a feedback loop between them such that they cannot be done separately. For example, in the studies of MutS and MutL it is necessary to do a genome analysis to identify the presence or absence of homologs of these genes, then an evolutionary analysis to determine which forms of each of the genes are present, then a genome analysis again to determine the number and combination of different forms and then an evolutionary analysis to determine whether and when particular forms were gained and lost over evolutionary time, and so on.

5. Lions and TIGRs and bears

Since leaving Phil’s lab I have been a faculty member at The Institute for Genomic Research (TIGR) and in that time we have found dozens of new uses for a phylogenomic approach and designed many new methods to implement phylogenomics. Such an approach has led to many interesting findings relating to DNA repair. Phylogenetic analysis of eukaryotic genomes has allowed us to identify many nuclear encoded genes that are homologs of DNA repair genes but appear to evolutionary derived from the organellar genomes and thus are good candidates for still having a role in DNA repair in the organelles (38). These include both putatively plastid-derived genes (encoding RecA, Mfd, Fpg, RecG, MutS2, Phr, Lon) and mitochondrial-derived genes (encoding RecA, Tag). Interestingly the presence of Mfd but not UvrABCD is also found in many endosymbiotic bacteria, although the explanation for what this Mfd might be doing is unclear. Phylogenomic analysis has allowed us to identify the loss of important DNA repair genes in various species such as the apparent loss of all the genes for non-homologous end joining in the causative agent of malaria, Plasmodium falciparum (39). An important component of this analysis was the finding that this species did not have an orthologs of DNA ligase IV, even though the original annotation of the genome had suggested it did (Figure 1).

Figure 1. Phylogenetic tree of DNA ligase homologs showing the presence of an orthologs of DNA Ligase I in Plasmodium falciparum but no orthologs of DNA ligase IV, consistent with the absence of non homologous end joining.

Among the other interesting repair-related features we have found are: the presence of two MutL homologs in an intracellular bacteria Wolbachia pipientis wMel (40), the presence of two UvrA homologs in Deinococcus radiodurans (41) and Chlorobium tepidum (42), the absence of MutS and MutL from Mycobacterium tuberculosis(43), and the presence of multiple ligases for each chromosome in Agrobacteriumtumefaciens (44). Continued surprises come from almost every genome.

However, all is not good in the world of phylogenomics. One of the biggest problems is that most of the experimental studies of DNA repair that have formed the basis of out knowledge in the field have been done in a narrow range of species. For example, there are estimated to be over 100 major divisions of bacteria (Phyla) and of these, most DNA repair studies have been restricted to three of these phyla (Proteobacteria, Firmicutes (also known as lowGC Gram-positives), and Actinobacteria (also known as highGC Gram positives). This means that if anything novel evolved in any of the other lineages, we would not know about it. This probably explains why, when we sequenced the genome of the radiation resistant bacteria D. radiodurans, analysis of the homologs of DNA repair genes in the genome did reveal many homologs of known repair genes but this list did not have many features that were unusual compared to non radiation resistant species (Table 1) and thus was not of much use in understanding what makes this species so resistant (41).

Table 1. Homologs of known DNA repair genes identified in the initial analysis of the D. radiodurans genome sequence

Process	Genes in D. radiodurans	Unusual features
Nucleotide Excision Repair	UvrABCD, UvrA2	UvrA2 not found in most species
Base Excision Repair	AlkA, Ung, Ung2, GT, MutM, MutY-Nths, MPG	More MutY-Nths than most species
AP Endonuclease	Xth	–
Mismatch Excision Repair	MutS, MutL	–
Recombination Initiation Recombinase Migration and resolution	RecFJNRQ, SbcCD, RecD RecA RuvABC, RecG	–
Replication	PolA, PolC, PolX, phage Pol	PolX not in many bacteria
Ligation	DnlJ	–
dNTP pools, cleanup	MutTs, RRase	–
Other	LexA, RadA, HepA, UVDE, MutS2	UvDE not in many bacteria

This of course means that genome sequencing and analysis, even if done in a robust way, only works well if there is a core of experimental studies on which to base the analysis.

In the end, I would like to define a new word – philogenomics which is the combination of studies of evolution, genomics, DNA repair, thymine metabolism, and punning. The ultimate proof of a philogenomic approach, of course, will come when it figures out the mechanism underlying thymineless death. But that is another story.

6. Acknowledgements

I would like to thank Philip C. Hanawalt for his support during and after my Ph.D research in his lab. Everyone in the field knows he is a great scientist. What they may not all know is that he is an even better human being.

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Evolution rap: 3.5* til infinity #music

Well, after a rough day I am in need of some lightness. And thanks to Eric Lowe, an undergrad. working in my lab, I got a giggle out of this:

Slideshow w/ audio of my talk on "A Field Guide to the Microbes" from the AAAS Meeting #AAASMtg

I recorded the audio of my talk on “Towards a field guide to the microbes” from the AAAS meeting on Saturday AM. Here is a slideshow of the talk with audio synched to the slides (I did this using Keynote on a Mac with the “record Slideshow” function).

My slides from the talk are available at Slideshare.

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