New DNA Sequencing Technologies
Topic: Biology
All I have to say is - brilliant!

Two new awesome DNA sequencing technologies were reported this week in Nature and Science (as mentioned in a previous post).

So how do these work?

Well both techniques start by linking the genomic DNA fragments to a short DNA sequence (aka a primer or oligonucleotide) that has a biotin on the end. Biotin sticks very strongly to avidin and beads coated with avidin are used to trap the genomic DNA fragments - the ratio of DNA fragments to beads are fixed so that there is one fragment per bead. Now excess DNA primers (linked to biotin) are then added in excess to the bead. These primers are used to amplify the genomic fragment by PCR.

So now we have a population of beads each with many copies of a single stretch of DNA sequence. The beads can be immobilized to a sticky surface or inside microwells. These two techniques allow the monitoring of many beads in a conventional microscope.

OK for non-biologists - the following will be very technical ...

In method number one, "anchor primers" that are complementary to the linkage primer are annealed to the DNA-bead preparations. Then degenerate 9mers are mixed in - if the 9mer can bind to the genomic fragment that lies adjacent to the anchor primer the ligase will link the two ligated to the anchor primers. Depending on the identity of the query base (lets say base #4 of the degenerate sequence), the primer is conjugated to one of four fluorophores, each correlating with the identity of the query base (so blue for primers with base #4 being an A, Green for T, Red for C, Infrared for G). Conditions are tweaked until only perfect matches can base pair. After ligating the sequences with DNA ligase, the color of each bead is monitored by microscope. Thus identity of base #4 is revealed. Depending on how many beads you can visualize (potentially up to 500 000 per image) you will know the identity of base #4 for all those DNA fragments. Now you can wash off the anchor primer-query primer ligation product and use new anchor primer to ligate a different 9mer probe (but to querry a different base). Using some tricks the researchers inserted multiple primers into each sequence so that with this "ligation based sequencing" you can figure out the identity of potentially 20 to 100 nucleotides on every visualized bead. (500 000 beads X 100 nucleotides = half a billion nucleotides sequenced!) The authors claim that this sequencing method utilizes reagents off the shelf.

In the second paper, the authors annealed their anchor, then added DNA polymerase, pyrophosphatase and one type of nucleotide to their beads. If the primer can be extended with the given nuclerotide, DNA polymerase will utilize the nucleotide and release pyrophosphate that can be further processed into free phosphate by pyrophosphatase. This last reaction can be monitored on a microscope. So if the researchers add "T", every bead (this time in a microwell) whose first base has an A (thus needing a T to form the base pair) will catalyze the reaction ... and light up. Everything is then washed off the beads and the next nucleotide (say "C") is added. By monitoring which bead lights up during each round the authors can figure out each individual sequence simultaneously. Incredible!

Shendure et al., Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome. Science. (2005) Published online 4 August

Margulies et al., Genome sequencing in microfabricated high-density picolitre reactors. Nature (2005) Published online 31st July

Upsidedown Catfish
Topic: Biology
Got into an interesting conversation with "P" about upside-down catfish and the how evolution may (or may not) have selected this trait.

Here's an email that P sent to me ... enjoy:

"This Catfish IS Upside Down!"

"...why they swim upside down. The reason is quite simple: it's easier to eat that way!" "Food on top of the water or under logs is much easier acquired by a fish floating upside down with it's mouth fully directed toward the food!" "...it is speculated that when food became scarce on the bottom (where most catfish eat), some species inverted (swam upside down) to take advantage of a food supply that was available at the surface. As the catfish acquired neutral buoyancy, it became more difficult to resist that upside down force. In order to save energy, the catfish gave in to the upside down swimming!"

"Some scientists believe the upside down catfish took up inverted swimming as a means of protection. The theory is that because mid-water predators usually attack from below, the upside down catfish is better able to see the imminent attack, enhancing their chance of survival."

"Another oddity, which is attributed to the inverted swimming, is the fact that the upside down catfish's belly is darker than the rest of the fish. Most fish have a lighter underside, a feature developed by them in order to escape detection from predators lurking beneath them. The lighter underside against the light water makes for a less obvious target. However, the upside down catfish has a reversal of the normal shading."

"In the wild, upside down catfish are found in huge shoals of several thousand fish."

"... "If they die, do they float right side up?" The answer: surprisingly not! Belly up like other fish, like they have spent their entire lives!"

Upside-down Catfish Site
http://www.aquafriend.com/modules.php?op=modload&name=News&file=article&sid=41&mode=thread&order=0&thold=0

4th Nuclear RNA Polymerase Identified
Topic: Biology
Well remember central dogma of Biology (DNA => RNA => protein)?

There were 3 known polymerases in mammalian nuclei that copied DNA into RNA (this copying process is known as transcription).

Pol I: responsible for transcribing rRNAs (the RNAs that form the catalytic portion of the ribosome, the machine responsible for translating mRNA into protein - see this entry on why RNA can support catalytic reactions)

Pol II: responsible for transcribing mRNA (the common messenger RNA that is translated into proteins) and snRNA (small nuclear RNAs that are part of the RNA splicing machinery, see entry on mRNA splicing).

Pol III: responsible for transcribing one rRNA (the 5s rRNA) one snRNA (called U6) and the tRNAs (tiny t-shaped RNAs that plug into the ribosome and bind both to the mRNA sequence and to the appropriate amino acid - remember amino acids are the lego blocks of proteins).

But wait there is more! Eukaryotic cells are the result of a symbiotic relation ship between a large primordial cell and mitochondria (see organelle C). These "mitos" (as some researchers affectionately call them) are thought to be descendants of oxidative alpha-proteobacteria bacteria that could oxidize sugars to water and CO2, as proposed by Lynn Margulis (click here for a brief summary of her ideas). Mitochondria have their own DNA that encodes for their own mRNA - and thus they have their own RNA polymerases. The translation of mRNA into protein is slightly different in mitos than in the nucleus, so mitos also have their own tRNA.

OK now you are ready ...

In the latest issue of Nature, a fourth RNA polymerase has been discovered - named spRNAP-IV, for single-polypeptide RNA Polymerase IV ... or Pol IV. This protein is actually encoded by a mitochondrial gene! The gene is alternatively spliced into spRNAP-IV, that is transported to the nucleus and mtRNAP, an RNA polymerase responsible for translating mitochondrial genes. Like Pol II, spRNAP-IV seems to translate genes that encode mRNAs. An interesting side note is that many putative proteins required for mito function are encoded by nuclear genes ... it is thought that these genes once resided in the mitos but then were transfered to the nucleus. Perhaps when the mito genes were "transfered", the nucleus also had to import mito specific RNA polymerases to copy these mito derived genes into mRNA ... and Pol IV is a remnant of that event?

In anycase this study demonstrates that many basic cellular functions are yet to be discovered and it leaves open the question of what other basic machinery is out there to be found?

Ref: Kravchenko et al., Transcription of mammalian messenger RNAs by a nuclear RNA polymerase of mitochondrial origin. Nature 436:735-739

Autism and Hormones
Topic: Biology
Fascinating piece in today's NY Times about Autism and Testosterone by the researcher Simon Baron-Cohen.

From the article:

On average, males finish faster and score higher than females on a test that requires the taker to visualize an object's appearance after it is rotated in three dimensions. The same is true for map-reading tests, and for embedded-figures tests, which ask subjects to find a component shape hidden within a larger design. Males are over-represented in the top percentiles on college-level math tests and tend to score higher on mechanics tests than females do. Females, on the other hand, average higher scores than males on tests of emotion recognition, social sensitivity and language ability.

Many of these sex differences are seen in adults, which might lead to the conclusion that all they reflect are differences in socialization and experience. But some differences are also seen extremely early in development, which may suggest that biology also plays a role. For example, girls tend to talk earlier than boys, and in the second year of life their vocabularies grow at a faster rate. One-year-old girls also make more eye contact than boys of their age.

In my work I have summarized these differences by saying that males on average have a stronger drive to systemize, and females to empathize. Systemizing involves identifying the laws that govern how a system works. Once you know the laws, you can control the system or predict its behavior. Empathizing, on the other hand, involves recognizing what another person may be feeling or thinking, and responding to those feelings with an appropriate emotion of one's own.


His research has found that men tend to be systhemizers (type S) and women empoathizers (type E). He points out some studies that suggest that exposure of prenatal infants to testosterone in amnionic fluid influences their ability to display S-type and E-type behaviors. Testosterone exposure levels correlates with S-type behavior and inversely correlates with E-type behavior. Hmm, is Larry reading this? Although I warn you all not to make simplistic judgements based on these studies.


What does all this have to do with autism? According to what I have called the "extreme male brain" theory of autism, people with autism simply match an extreme of the male profile, with a particularly intense drive to systemize and an unusually low drive to empathize. When adults with Asperger's syndrome (a subgroup on the autistic spectrum) took the same questionnaires we gave to non-autistic adults, they exhibited extreme Type S brains. Psychological tests reveal a similar pattern.


Very interesting! Instead of a little Einsteins, testosterone exposure can produce a Rain Man.


FIRST, both mothers and fathers of children with autism complete the embedded figures test faster than men and women in the general population.

Second, both mothers and fathers of children with autism are more likely to have fathers who are talented systemizers (engineers, for example).

Third, when we look at brain activity with magnetic resonance imaging, males and females on average show different patterns while performing empathizing or systemizing tasks. But both mothers and fathers of children with autism show strong male patterns of brain activity.

Fourth, both mothers and fathers of children with autism score above average on a questionnaire that measures how many autistic traits an individual has. These results suggest a genetic cause of autism, with both parents contributing genes that ultimately relate to a similar kind of mind: one with an affinity for thinking systematically.

Eye Candy & Quiz
Topic: Biology
Well I was taking some phase pictures of hepatocytes (liver cells) that we plated on a collagen coated coverslip - nice cells, if I do say so myself. To the left is an image of a hepathocyte (or part of a hepathocyte), all those lines and squiggles being organelles ... compartments where specialized cellular functions take place.

So here is your quiz ...

What is organelle A (the big round slightly out-of-focus thing)?
What is organelle B (the dark dots)?
What is organelle C (the long squiggles)?

And finally, which of these organelles contains DNA?

Funny thing is that I just noticed the question mark in side of organelle A ...

Good luck!

Flying Snakes!
Topic: Biology
(Well maybe they just glide.)

I saw an interesting article in the current issue of Science on these curious serpents.

Apparently there are five related species (of the genus Chrysopelea) that jump out of trees and glide. To find out more visit http://www.flyingsnake.org, a website maintained by Jake Soka who studies these snakes at the University of Chicago.

Bug Tampers with Fly Sex
Topic: Biology
I was just alerted about an interesting "News item" in the July 7th edition of Nature that describes how a small bacterial parasite, Wolbachia, drastically alters how flies respond to genetic changes. This bacterium seems to be able switch the sex of infected embryos. In certain cases, the parasite actually rescued the viability of flies harboring lethal genetic mutations.

When looking at a large collections of mutant fly colonies housed at the Bloomington Stock Center at Indiana University, researchers identified that about 30% of the strains were infected with the bug!

This bug could cause problems for fly geneticists ... and others who read their papers!

So how many published results are just wrong? Well labs that study sex determination must be extra vigilant, and this field may be the most affected. I also wonder how many other unknown parasites exist out there that can drastically affect how our biological subjects respond to our experimental protocols?

Using the Nuclear Bomb to Test the Age of Neurons
Topic: Biology
Well some people are smart, and others are really clever.

In today's issue of Cell, researchers use the C14, released in the environment during the atom bomb testing (1943-1963), to determine the age of neurons.

This type of experiment is known as a pulse-chase experiment where a pulse of labeled chemical is added to a biological sample. To figure out how fast the sample is being replaced, researchers measure the amount of labeled sample remaining after different intervals. Thus in the 1943-1963 period, every human born incorporated trace amounts of C14 into their newly formed bodies. Then after 1963, the levels of C14 drooped ... thus anytime after 1963 the newly made proteins, lipids, sugars or DNA had less C14 in them. So to find out how fast a cell was replaced, all you need to do is to measure the amount of cells with labeled DNA (since DNA is only made at the "birth" of a cell).

Now in normal experiments researchers add radiolabeled chemicals to laboratory samples ... but in this case these clever researchers used a natural pulse of C14 (caused by atom bomb testing) to date long lived cells such as neurons ... pretty ingenious.

Ref: Kirsty L. Spalding, Ratan D. Bhardwaj, Bruce A. Buchholz, Henrik Druid, and Jonas Frisen. Retrospective Birth Dating of Cells in Humans. Cell (2005) 122:133-143

Lipid Rafts Seen (Again) at Last
Topic: Biology
One controversial field in cell biology, is the study of how different types of lipids segregate from the bulk lipids in biological membranes.

OK ... lets step back a bit. In 1972, a model of how lipids form a membrane was proposed by Singer and Nicolson ... the fluid mosaic model. The famous cartoon from their paper is seen here (right). In this model, biological membranes were composed of two layers (or a bilayer) of lipids. The membrane is stable as the lipid's hydrophilic (water loving) heads (circles in the pic on the right) are exposed to the solution and their hydrophobic (water fearing) acyl side chain (squiggles in the pic on the right) are buried in the membrane core. Membrane bound proteins (big blobs in the pic) would float in the plane of the membrane.

So membranes are simple ... right?

Well some weird results started to appear. The very next year John Yu et al. publish a paper describing how a fraction of membranes were resistant to being solubilized with cold detergent. This was eventually seen in cells by Deborah Brown who also copurified proteins that seem to like these "membrane microdomains". These enigmatic membranes acquired other names such as "lipid rafts" and DRMs for Detergent Resistant Membranes ... however it would seem like the more names they gave it the less they knew about it. Many thought that these microdomains were artifacts of cooling trhe samples. Others refered to them (with contempt) as oil slicks.

But this is all very controversial - most researchers are weary of these microdomains as they can be only isolated at low temperatures. However, researchers (such as John Silvius) have shown that you can create this partitioning of lipid domains in artificial vesicles (or liposomes). In these rafts, the lipids tend to be ordered and kick out any lipids that do not compress well. The ability of these ordered membrane patches to form is dependent on cholesterol. So do these rafts exist in cells (or as us biologists say, in vivo)? Well rafts turn out are too small to see with the light microscope ... but despite this fact, Schutz et al. saw them by labeling single lipids and adding just the right amount of these labeled molecules to muscle cells. Others have observed that in polarized cells raft components localize to the site of polarity (for more on polarity, click here).

Now in a recent issue of the Journal Cell (see cover left), Adam Douglass, from the Vale Lab, observed single protein molecules going in and out of microdomains on the surface of white blood cells. In fact they trick the white blood cell into polarize towards the coverslip and use TIRF Microscopy to visualize the polarized membrane with all it's lipid rafts. (I'll post something on TIRF some other day). Very cool. And check out the movies!

Ref:

Singer SJ, and Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science (1972) 175:720-731

Yu J, Fischman DA, Steck TL. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. Journal Of Supramolecular Structure (1973) 1:233-248

Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell (1992) 68:533-44

Schutz GJ, Kada G, Pastushenko V, and Schindler H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO (2000) 19:892-901

Douglass AD, and Vale RD. Single-Molecule Microscopy Reveals Plasma Membrane Microdomains Created by Protein-Protein Networks that Exclude or Trap Signaling Molecules in T Cells. Cell (2005) 121:937-950

Richard Axel
Topic: Biology
Two days ago I was talking to a rotation student in the lab about the Nobel laureate and Columbia Professor Richard Axel, then last night at another BBQ (this time at Ben's place), the biology of olfactory was brought up, and finally this morning flipping through the Columbia University Magazine, there was an article on ... Richard Axel.

My first recollection of Dr. Axel was in a graduate student class I attended at Columbia. A bald, tall, and lanky individual in a suit walked into the room, sat down on the front desk, crossed his legs and said "So what do you want me to talk about?" He gives the paradoxical appearance of aloofness mixed with intense concentration.

Apparently in high school (Stuyvesant High) he played center for the school basketball team ... and once faced-off against against Lew Alcindor (who later renamed himself Kareem Abdul-Jabar). Apparently Alcindor taunted him by calling him Einstein. Once in college (at Columbia University) he was hired to wash dishes for Bernie Weinstein's lab. Since he asked many questions but broke much or the glassware they fired him after the first day, and rehired him as a research assistant.

He originally wanted to study English or Biology, then the Vietnam War arrived. To avoid the draft he applied to med school - but then spent his entire subsequent career as a researcher as he disliked dealing with patients (and as a pathologist disliked dealing with corpses). Although he is now famous for his discovery (with his then postdoctoral fellow, Linda Buck) that the human genome contains about a thousand or so odorant receptors (all belonging to the G-couple receptor protein family), he was involved in many serendipitous but key scientific findings.

He cloned one of the key proteins involved in Cell Polarity, Rho (for Ras Homologue). What is strange is that he cloned it from Aplasia, the giant slug with giant neurons, first popularized by another Columbia Nobel Laureate. Dr. Axel was also an author on the paper that first described how to introduce foreign DNA into cells using lipid. From this paper, Columbia patented DNA transfection, the single most profitable Biotechnology patent to date (for you lab rats, think about that every time you use lipofectamine and other related products). His lab is currently investigating olfactory perception as a model of brain function.

There are many other Axel stories, but you can read up about some of them at the Nobel Site.

Ref:

Buck, L., and Axel, R., A Novel Multi gene Family May Encode Odorant Receptors: A Molecular Basis for Odor Recognition, Cell (1991) 65:175-187

Wigler, M., Pellicer, A., Silverstein, S. and Axel, R. Biochemical transfer of single-copy eukaryotic genes using total cellular DNA as donor. Cell (1978)14:725?731

Madaule, P. and Axel, R. A novel Ras-related gene family. Cell (1985) 41:31?40

RNAi is Hot!
Topic: Biology
I was flipping through today's NY Times' Science Section, and what do I see? An article devoted to RNA (well really the history of RNAi). Click here to view the original article.

It would seem like RNAi is a hot field or as we in science say ... and I kid you not we really do use this expression ... "it's sexy".

The History of Tubulin Detyrosination
Topic: Biology
Late last week I posted an entry on tubulin modification ... an area of research that one well respected cytoskeletal researcher described as "a cottage industry based on antibodies" ... Due to increased interest in the field, I'll recount here the tale of how tubulin modifications were first discovered.

In 1973 group of researchers (known to some affectionately as "the crazy Argentines") decided to test whether proteins were repaired in neurons, since in these cells, the nerve terminal is located far away from the cell nuclei (where the instruction material to make new proteins is found). To test this hypothesis, they gave mice drugs to stop protein synthesis from the canonical protein synthesis pathway (e.i. by the ribosome - see the entry on the Central Dogma of Biology) and then feed these poor mice radio-labeled amino acids. If the small criters "repaired" proteins using the fed amino acids, then the radio-label should incorporate into "fixed" proteins - since protein synthesis was inhibited, there should be no other radioacive incorporation. Sure enough one, and only one amino acid was being incorporated into neuronal protein ... tyrosine. Eventually the Argentines looked to see what was being modified, and it was a single protein, alpha-tubulin. One amino-acid, one protein.

Turns out that tyrosine is reversibly ligated to the end (or Carboxyl-terminal) of tubulin and this occurs in almost every cell in the body. The two modifying enzymes (the one that adds tyrosine to tubulin - the tubulin tyrosine ligase, and the one that takes tyrosine off - the tubulin carboxypeptidase) had been elusive for decades, until the tubulin tyrosine ligase was identified. As I mentioned in a previous blog entry, this enzyme is special in that it is one of a few enzymes (outside of the ribosome) that can catalyze the formation of a peptide bond.

Antibodies raised against tyrosinated and detyrosinated tubulin are very specific(they don't cross react) and can bind to their respective targets at incredible dilutions. These antibody studies suggested that the tubulin tail (where the reversible tyrosine is situated) is highly antigenic (i.e. is reactive against antibodies and thus very "bindable") and a change of a single amino acid can drastically change it's "affinity profile" (i.e. it's ability to bind stuff). The first surprise was when antibodies against the tyrosinated and detyrosinated forms were used to stain fixed cells. All the modifications (in this case detyrosinated tubulin) were segregated to a subset of microtubules that were oriented along the cell's "axis of polarity", such as towards the front in a migrating cell. Thus microtubule differentiation was discovered. Then came the famous discovery that microtubules, which are distributed in an astral patern in cells, grow and shrink from their plus ends (which are located on the periphery) and are inert at their minus ends (which are located in the cell center at the MTOC). Tim Mitchison and Marc Kirschner speculated about what this all means and came up with their famous review Beyond self-assembly: from microtubules to morphogenesis. The idea is simple - microtubule plus ends grow and shrink, probing the cellular space to find specialized targets (such as chromosomes, waiting to be pulled apart by microtubule plus ends). When they do find these targets, the microtubule plus ends are "captured" and now serve as a track conecting the cell center (where the microtubule minus ends are) and the specialized site (where the captured microtubule plus ends are). Elements like chromosomes can then be pulled to the new cell center. In the case of migrating cells, the specialized site (the front of the cell) captures and stabilize microtubules. It was then shown that the stable microtubules accumulate modifications (like detyrosination) and can be used by the cell to allow communication between the front of the cell and the cell center (see image right, modified microtubules in green/yellow are all poited towards the wound while non-modified microtubules in red are everywhere).

Well is tubulin detyrosination important after all? I won't go into details about how detyrosinated microtubules bind to stuff differently from tyrosinated "tubes" ... all I'll mention is that Jurgen Wehland and Didier Job's groups finally knocked out the tubulin tyrosine ligase (TTL) gene ... and the knock-out mice died due to a lack of neuronal organization in the cerebral cortex ... basically no modification = the brain is a mess. Why neurons - well as the crazy Argentines will tell you, those are long cells - and they have a lot of modified tubulin!

Ref:

1- The crazy observation that started it all off -
Barra HS, Rodriguez JA, Arce CA, Caputto R.J, A soluble preparation from rat brain that incorporates into its own proteins ( 14 C)arginine by a ribonuclease-sensitive system and ( 14 C)tyrosine by a ribonuclease-insensitive system. Neurochem. (1973) 20:97-108.

2- The first look at modified microtubules in cells -
Gundersen GG, Kalnoski MH, Bulinski JC. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell. (1984) 38:779-89.

3- The great hypothesis -
Kirschner M, Mitchison T., Beyond self-assembly: from microtubules to morphogenesis. Cell. (1986) 45:329-42.

4- The TTL knockout -
Erck et al. A vital role of tubulin-tyrosine-ligase for neuronal organization. PNAS (2005) 102:7853-7858.

Big Day for Tubulin Biochemistry
Topic: Biology
So today some mysteries surrounding tubulin biochemistry have been solved. Tubulin, the "lego block" of the microtubule polymer (see image right), can be chemically modified in many unusual ways.

In most cells, the majority of microtubules grow and shrink, while a subset of microtubules that are oriented towards a site of polarity (e.g. the front of migrating cells) are stable (they do not grow or shrink). These stable microtubules also accumulate tubulin modifications. Although these modifications have been known for quite a while (some for as many as 30 years!) the modifying/demodifying enzymes have been elusive ... to date only 2 are known (the tubulin tyrosine ligase - TTL, and the tubulin deacetylase - HDAC6). Well in the latest issue of Science Janke et al. describe the isolation of the tubulin polyglutamylase. Like the tubulin tyrosine ligase, the glutamylase is a rare enzyme in that it can form a peptide bond (besides those two the only other enzyme that has that function is the ribosome - the protein synthesizer). Well it turns out that the Polyglutamylase is a homologue (i.e. a relative) of the tubulin tyrosine ligase (and thus was originally called the tubulin tyrosine ligase like protein, or TTLL1). So now there are 3 tubulin modifying/demodifying enzymes known (just another 7-9 to go including the notorious tubulin carboxypeptidase!)

But that's not all!

Not to be outdone, the latest issue of Nature has a paper on why the tips of microtubules are frayed when they fall apart (or depolymerize). It turns out that it has to do with how the tubulin subunits bind a nucleotide cofactor. Thus if tubulin binds to GTP (and is thus growing) the tubulin monomers are less curved and can form proper barrels ... but when tubulin binds to GDP, it stacks differently, causing the microtubule lattice to curve, fray and thus come apart. So now we can explain, on a molecular level, what has been observed on electron microscope pictures of microtubules.

Ref:

Janke et al., Tubulin Polyglutamylase Enzymes Are Members of the TTL Domain Protein Family, Science 308:1758-1762

Wang et al., Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly, Nature 435:911-915

The Proteasome Falls Apart
Topic: Biology
The most important part of Biology (in my opinion) is to understand how molecular machines do their job. One of the biggest machines in the cell is the Proteasome - the protein shredder (see image left). Early studies indicated that protein breakdown requires energy (in the form of ATP) - this was surprising as protein hydrolysis should release energy and thus be a favorable reaction. In the end, the energy is used to attach a small molecule, named ubiquitin for it's "ubiquitousness". Ubiquitin then targets the protein in question to the Proteasome, a machine that brakes up the protein polymer into it's primary monomer components, amino acids. Protein degradation is such an important part of biology that the 2004 Nobel Prize in Chemistry was awarded for the elucidating this process. A minor point of controversy was that many people thought that Alexander Varshavsky was snubbed by the Nobel Prize Comitee...

Any way in the May 20th edition of Cell, Babbitt et al. describe how ATP induces a major conformational changes in the Proteasome - in fact the peripheral regulatory subunits dissociate leaving behind the core "barrel" like component. The fact that no one has noticed this until now is quite surprising.

Ref: Babbitt et al. ATP Hydrolysis-Dependent Disassembly of the 26S Proteasome Is Part of the Catalytic Cycle Cell 121:553-565

Systems Biology - Biology of the Future or Newest Fad?
Topic: Biology
Biology is unquestionably the most rapid moving scientific discipline in recent times. People will look back at this point in history (the past 20 years and perhaps the next 50 years) and declare it the golden age of biological discovery.

Having said this, biologists, like Canadians, have an inferiority complex ... that may not be warranted. Of who? Well just as Canadians look to their southern neighbours with anxiety, biology look down the halls of their local institutions to the physicist and mathematicians (chemists on the other hand are seen as either quaint or if they dabble in biopolymers, recreational-biologists).

The field of physics has solved most everyday mysteries ... a physicist working 50 years ago could recognize the majority of the current models today, concerning everything from matter to the structure of the universe. Many questions in contemporary physics involve the finding of new particles and recapitulating the first nanosecond of the Big Bang etc., and require enormous pools of researchers, time, money and other resources.

Although these large endeavors of modern experimental physics rarely produce true novel insight, these projects are covered in the news and popular science magazines. In contrast, big developments in Biology (such as how cells crawl or how cells divide) are rarely discussed in the media. So what is the puny self-doubting biologist to do?

Well the first fad in biology to address this issue was called "Interdisciplinary Research", which means "to look cool like physicist, we should start collaborating with them" . Unfortunately this revolution (or the "hardening") of biology already took place 60 year as ago after quantum mechanics was worked out and out-of-work physicists moved into biology. (See Shroedinger's book What is Life? - left) These physicists transformed a field that consisted mostly of observational scientists, into a quantitative endeavor. Scientists such as Linus Pauling used the tools of physics and chemistry to create molecular biology and analytic biochemistry. Flash forward 50 years - the idea of "Interdisciplinary research" was thrown around ... physicists, mathematicians and other individuals from the "serious sciences" were welcomed into genetics, biochemistry and cell biology labs ... biologists were told "become computer programmers" ... and well ... nothing revolutionary did happen this time.

So what could the biologists do to re-revolutionize their underappreciated discipline? Well they took a look at the atom smashers, the Hubble telescopes, the Interplanetary probes and the vast underwater reservoirs used to detect elusive particles produced by stars exploding eons away, and these biologists said ... we need to create Big Biology. So instead of inactivating one gene, now geneticists inactivated every gene. Instead of looking at how one gene was turned on, biochemists measured every gene's level on a "chip". Everything was now done on a grand scale, and if possible was automated using robots and other gizmos. This culminated with the sequencing of the human genome. Now people could study the whole genetic output of an organism - hence genomics. Biology finally had it's day in the spotlight! At this point every biologist wanted to be have the 'omics cache. Instead of studying one RNA transcript, you did experiments on all the transcripts ... and called it the transcriptome. Other big sounding -omic names came. The proteome. The kinome. The ubiquinome. The metabolome.

The problem with big biology is that often quality was sacrificed for quantity. I remember reading some paper where the authors were going to map every protein-protein interaction (by yeast two-hybrid). Looking through the results, every fourth molecule interacted with hexokinase (some boring metabolic enzyme) ... and it just didn't smell right. So in the end all this data was produced and ... then largely ignored. People were not happy.

Hmmm.

Now the latest buzz word in Biology is ... Systems Biology. Go to the May 20th edition of Cell to find out exactly what Systems Biology is. (OK, so maybe Marc Kirschner and company don't really know what they've started ...) In any case Systems Biology is so hot that WIRED magazine has a write up on the new subject.

The essence of Systems Biology is to regenerate life in silico ... in other words ... to create virtual models of biological processes on a computer screen. You see biology, unlike physics and chemistry (and most other sciences), has a diversity creating machine at it's core - evolution. Through this process, life creates intricate machines with very plastic adaptable parts, but at the same time these molecular devices retain the ability to self assemble (such as the cytoskeleton) .. for a good discussion on related ideas, see Marc Kirschner and John Gerhart's essay on Evolvability or their essay (with Tim Mitchison) on Molecular Vitalism. In a sense, Systems Biology is an attempt to understand these processes, as Marc Kirschner wrote, "in toto".

So will the Systems Biologists succeed? We'll see.

mRNA Splicing and Sexuality
Topic: Biology
A week or so ago, a lot of fuss was made over a gene that activated male sexual behavior in fruit flies. Of course this made the news because of the whole nature vs. nurture debate with regards to sexuality.

Well having looked over the original paper in the latest issue of Cell I realized that the gene in question (the fruitless gene) was alternatively spliced in males and females. This is worth writing about - not so much for a discussion on sexuality but to explain alternative splicing.

Going back to the Central Dogma of Biology (DNA => RNA => protein), genes encoded in DNA contain small coding parts called exons (which specify a code to make protein), interspaced with large junk parts called introns (see image right).

Due to the Nobel Prize winning work of Richard J. Roberts and Phil Sharp, who both worked on the RNA of Adenovirus, we learned that when DNA is first copied into RNA, all the junk is also copied to form pre-mRNA. Very rapidly the introns (junk) are spliced out to form mature mRNA (for messenger RNA). Sometimes certain exons (i.e. coding regions) are left out of the mRNA - this variance in the splice patterns, called alternative splicing, is needed to form particular versions of proteins needed in certain circumstances.

Recently everyone was shocked to learn that the human genome only has 30,000 genes (and even more so as the number has been recently downgraded to 23,000). Well this number doesn't take into account how this genetic information is processed into RNA. The pre-mRNA from a single gene can be spliced into many different forms (the most talked about being Dscam a gene that produce as many as 38,016 different neuronal cell receptors - due to the presence of 95 exons that can be alternatively spliced).

If we add all the small genes that encode micro-RNAs (miRNAs ... see my RNAi entry ) the actual number of RNA products from the genome goes up substantially.

So getting back to the fly study by Ebru Demir and Barry J. Dickson we can see that the processing of genes (in this case alternative splicing of a single gene) can result in modulating complicated phenotypic processes (such as mating behavior) and may underlie the diversity in nature that may not be directly apparent if one simply looks at the genes alone.

Ref: Demir, E, and Dickson, BJ, fruitless Splicing Specifies Male Courtship Behavior in Drosophila. Cell, 121:785-794

More Evidence that Birds and Dinosaurs are Related
Topic: Biology
Well the idea that Birds evolved from a lineage that branched off from Dinosaurs has been in the public eye ever since the remains of Archaeopteryx was discovered in the in Solnhofen limestone formation in Germany, in 1861.

Now more evidence is presented in the latest issue of Science, where Schweitzer et al. document how a bone from a female Tyrannosaurus-Rex resembles the bone of ovulating birds.

Basically they observe a build up of medullary bone tissue that usually occurs in birds during the egg-laying process. This extra store of calcium is used to form eggshells.

This finding can not only help scientists determine the sex of a subset of dinosaur specimens but is further proof of the common heritage of dinos and birds.

Ref: Schweitzer et al., Gender-Specific Reproductive Tissue in Ratites and Tyrannosaurus Rex, Science 2005 308:1456-1460

The RNA World
Topic: Biology
Well tomorrow I'm off to Banff where I will be attending the 2005 RNA Meeting followed by some trekking round the rockies with my parents.

RNA is the mid point of the "Central Dogma of Biology". It is a very interesting polymer in that it can both store information and catalyze reactions. It's interesting in that when scientists (like Linus Pauling) were first thinking about heredity and genes in the 1940s and 50s, they envisioned that enzymes must be able to copy themselves. Of course they were wrong - there is a clear division of labor in cells: DNA stores most of the information, and protein does most of the enzymatic work.

This separation of information and work is problematic when one imagine the origin of life.

Let's perform some thought experiments. How does the first DNA molecule copy itself? Well it obviously needs an enzyme (a protein) to do the work. Where does the information for making the protein come from? It comes from the DNA.

Before you give up - it's important to note that the circular paradox is not yet complete. How is the DNA's information converted to an amino acid polymer (aka protein)? The DNA is copied into mRNA (for message RNA) and translated into an amino acid sequence by a large enzyme complex made up of protein and RNA. It turns out however that most of the important components of this large complex (called the ribosome) is contained in the rRNA (or ribosomal RNA).

So there it is - RNA can both be the message and the enzyme - just as Pauling and company thought would apply to life today. Scientist envision this world, where life's information is encoded by RNA, and life's reactions are also carried out by RNA, as the "RNA World".

What makes RNA catalytic (i.e. able to act as an enzyme) is this extra hydroxy group (see image) that is not found on DNA (hence RNA = ribonucleic acid and DNA = deoxyribonucleic acid). Unfortunately this hydroxy group also makes RNA unstable, and in a sense suicidal (this group is somewhat reactive and will hydrolyze the RNA backbone, thus chopping up itself). Presumably life soon adapted to RNA's properties by storing information in the more stable DNA. Life also started using polymers of amino acids rather than RNA for enzymes probably because there are more amino acids (cells use 20 kinds) than ribonucleic acids (4 major kinds and a couple of minor ones). Other advantages are that amino acids are smaller and require less energy to synthesize than nucleic acids.

So having describe one possible scenario for the primordial soup, I think I'll pack up and leave.

Ciao.

The Nature of Cells
Topic: Biology
Well instead of reporting on advances in therapeutic cloning, which is just a technical advance for some and dizzy spells for others, here is an interesting article in the latest issue of Nature on how pressure is distributed throughout the cellular space.

Cells exhibit fluctuations in their outer membrane ... in other words the membrane on different parts of the cell periphery can expand and contract, whereas other portions of the cell membrane are relatively inert.

So what is the underlying principle that dictates this differentiation in the different regions of the cell? Well in one model, inner-cell pressure (or turgor) is uniform while the contact between the cytoskeleton and the outer membrane is locally disturbed. This results in local membrane expansion (or blebbing - see image left from Charras et al.). In a second model, inner-cell pressure is simply non-uniform. To address this question, Guillaume Charras visualized a melanoma cell line that undergoes a lot of "blebbing", and the cell's actin cytoskeleton (as seen in the image here).

He found that blebbing did not alter cell volume, suggesting that the increase in local volume in a bleb was due to the movement of liquid inside a cell. He also saw that the initiation of blebbing was due to a "rupture" between the membrane and the underlying actin cytoskeleton. Moreover the halting and retraction of blebbing correlated with the assembly of a new actin network directly under the blebbing membrane. Next he found that drugs that inhibit actin contraction (caused by the actin motor myosin) led to an increase in blebbing and an inhibition in bleb retraction. Local disparities in inner cell pressure over 15-30um (about 1/3 of a cell length) took about 10 secs to equilibrate.

With all this information (and for you Systems Biologists - some models), Charras et al. concluded that the inner pressure of cells was (most-probably) non uniform and can be modeled by a "contractile, elastic network infiltrated by cytosol".

From Charras et al.:

Our poroelastic model could have important implications for other types of cell motility, because it implies that hydrostatic pressure can be generated and used locally to power shape change in animal cells.

Indeed, this idea that hydrostatic pressure may influence cell motility is supported by studies on the importance of water channels on cell migration.

So in the end it is likely that disparities in membrane-cytoskeletal contact and in inner cell pressure both act to change the morphology of cells.

Click here to see the cool blebbing movies.

Ref: Charras et al. Nature 435:365-369

Echinoderms
Topic: Biology

This weekend we visited Plum Island on the Massachusetts coast just south of New Hampshire. About half the Island is a wildlife refuge where you can find many endangered species of birds like the Piping Plover.

On the beaches we collected many giant snail shells (as large as 4 inches in diameter) and the remains of other invertebrates such as sea urchin (Arbacia Punctulata) and starfish (Asterias Forbesi) both echinoderms (see photo). To read more on the echinodermata phylum follow this link to the Tree of Life Homepage.

Besides the missing leg, a mysterious feature of the starfish was a small round patch of minute bristles (the patch is about 3mm in diameter ? see arrow in the enlarged inset). After reading a bit, my understanding is that this patch is called the madreporite and is the only visible anatomical feature on starfish that does not follow the organism?s five fold radial symmetry (note its acentric position). It turns out that this structure is the entrance to a tubular network filled with water (or water vasculature) that is involved in providing turgor pressure. As the starfish moves it's muscles squeeze these tubes thus sending water to distal tubes found in it's tentacles. As the turger pressure increases, the tentacles expand and allow the starfish to extend these tentacles out so that it can move about. The madreporite's role is to filter out particles but allow water into the vasculature to maintain this turgor presure.

For more on how the water vasculature acts as the starfish's skeleton visit this interesting site maintained by Johnathan Dale who wrote a PhD thesis on how starfish can move (and chemotax).