Happy New year!

Happy New Year to all molecular biologists out there! We hope 2010 is filled with peace, good health, love, happiness and also publications, grant money and fascinating results!

[If you then want to discuss those provocative results here at MolBio Research Highlights, all the better :) ]

Happy 2010!!

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I need a vacation

Yesterday I realized I need a vacation, when instead of asking if a party was open bar, I said
"Open Access"....



On the other hand, will I be able to relax on my vacations?

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Quotes from the science blogosphere

(...) I need to start acting like a politician when I particpate in the question and answer session of a talk- i.e. if I don't like the question, I should talk about a question that I want to answer instead of the one that was asked.

[...]

I think many of us, even though we are no longer students, are used to being tested and grilled and answering questions like students. But in fact, we are under NO obligation to entertain retarded questions during talks. Fuck that shit. We are in charge of our own show, and we call the shots..

Candid Engineer in Academia, on a post on her blog entitled "Conference Questions and Getting Angry"

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“Filling the gaps in the tree of life” and “Stochasticity in gene expression” in my Picks of the Week from RB.

Another week has gone by and some very interesting molbio blog posts have been aggregated to Researchblogging.org. Every week [see my opening post on the matter], I'll select some blog posts I consider particularly interesting in the field of molecular biology [see here to get a sense of the criteria that will be used], briefly describe them and list them here for you to check out.

Note that I'm only taking into consideration the molbio-related blog posts aggregated under "Biology".

Congratulations to everyone who got their post selected.

This will be the last "Picks of the Week" of 2009 and I want to take the opportunity to thank everyone for their support. This has been a great experience and "Picks of the Week" has been a tremendous success.

Now, the Picks....

1) The GEBA (Genomic Encyclopedia for Bacteria and Archaea) project is “aimed at systematically filling in the gaps in sequencing along the bacterial and archaeal branches of the tree of life”.

The prokaryotic genome sequences that have been obtained (currently, there are more than 1,000 complete or nearly complete genome sequences of microorganisms available) reflect a selection based on “clinical or functional importance (to us), ease, biotechnological or pharmaceutical potential, etc”, and have covered a wide range of biological and functional diversity, but not a phylogenetic one. It represents a biased view of the tree of life and limits our understanding of the evolution, physiology, and metabolic capacity of these fascinating groups.

So, what is GEBA?

In a nutshell, it’s a large-scale systematic (and phylogeny-driven) effort to sequence bacterial and archeal genomes from across the tree.

This project represents the first systematic attempt to use the tree of life itself as a guide to sequencing target selection.

Iddo Friedberg at Byte Size Bio discusses the first paper derived from this project, reporting the genome sequences of 56 culturable species of Bacteria and Archaea, which were selected to maximize phylogenetic coverage.

Also, you will find a video from Jonathan Eisen, one of the project leaders, talking about GEBA.

2) The different steps in gene expression are stochastic biochemical events and this randomness can lead to substantial cell-to-cell variability in RNA and protein levels and have phenotypic consequences even within a clonal population of cells. Notably, just last week a friend of mine (who is a grad student at Ben Lehner’s Lab at CRG and was visiting for a few days) and I, were discussing the impact single-cell studies can have on our understanding of the mechanisms of gene expression, so the selected post comes at a perfect time!

Tim Sampson at "The Times Microbial" @ Phagehunter.Org (Formerly known at "Blogging for Bacteriophages") discusses an article which was published in Science a few years ago addressing the impact these variations can have on a process as important as cell fate in bacteria.
This will be the first of a two-part series of posts on this fascinating subject.


That's it for this week. Stay tuned for more MolBio Research Highlights!

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Some of the articles discussed in this week's selected posts:

Wu, D., Hugenholtz, P., Mavromatis, K., Pukall, R., Dalin, E., Ivanova, N., Kunin, V., Goodwin, L., Wu, M., Tindall, B., Hooper, S., Pati, A., Lykidis, A., Spring, S., Anderson, I., D’haeseleer, P., Zemla, A., Singer, M., Lapidus, A., Nolan, M., Copeland, A., Han, C., Chen, F., Cheng, J., Lucas, S., Kerfeld, C., Lang, E., Gronow, S., Chain, P., Bruce, D., Rubin, E., Kyrpides, N., Klenk, H., & Eisen, J. (2009). A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea Nature, 462 (7276), 1056-1060 DOI: 10.1038/nature08656

Maamar H, Raj A, & Dubnau D (2007). Noise in gene expression determines cell fate in Bacillus subtilis. Science (New York, N.Y.), 317 (5837), 526-9 PMID: 17569828

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Method of the year 2009

Nature Methods has selected induced pluripotent stem (iPS) cells as its Method of the Year 2009, a contest we discussed a few months ago [See The Method of the Year].

The fantastic discovery that somatic cells can be reprogrammed to a pluripotent state by the expression of a transcription factor cocktail has had a great impact for basic research, diagnosis and cell therapy. Indeed, these cells have potential uses in in vitro disease modeling, toxicology and pharmacology, and regenerative medicine.

Be sure to check Nature's Special Feature, in which "a series of articles—and the related video —showcase how induced pluripotency is coming into its own in 2009 as a tool for discovery in both basic and disease biology and explore the incredible impact this area promises to have in biological research".

Here's the aforementioned video:

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Season's Greetings from MolBio Research Highlights!

Merry Xmas to all readers and followers.... I hope you got lots of acceptance letters and research funding :-)

More seriously now, we wish you lots of love, joy and happiness in this Holiday Season!


--
[Image credit: I only put the hat on this fantastic picture of F. Crick. Original image from Siegel RM, Callaway EM (2004) PLoS Biol. 2004 Dec;2(12):e419]

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MolBio Research Highlights turns 1

It was on Tuesday 23rd, 2008, on a hot summer afternoon, that MolBio Research Highlights was launched.

As I've mentioned before (See So what exactly is this blog about? and The 100th post at MolBio Research Highlights), this blog was created out of necessity and we are very excited about the way it has turned out.

MolBio Research Highlights, was initially born to compile articles, tools, websites and news in molecular biology I considered of interest [See So exactly, what is this blog about?]. Its first posts simply listed Pubmed entries of stand-out papers in the field in a way to compile them all in one place, instead of emailing them to the people I thought could be interested in them. With time, however, I decided I wanted to comment a little more extensively on the items I was posting and eventually these comments got lengthier and richer, as I sometimes discussed not only the item being highlighted but also related stuff.

Having been around for a year now, I wanted to round up the major events of the past 12 months. MolBio Research Highlights turned into a blog with a philosophy that makes it stand aside among science-related blogs (it is written by scientists for scientists) and we are extremely pleased with the way this project has unfolded.

So, what has happened in this first year?

As I mentioned, we started discussing articles, news, tools and websites in molecular biology, more extensively which made the blog a lot more useful for fellow scientists.

We joined ResearchBlogging.org which gave the blog a lot more exposure and helped us learn about many other interesting science-related blogs.

We were featured a few times in the Cancer Research Blog Carnival and even hosted its 27th Edition.

I was interviewed by a Chilean nation-wide newspaper, about scientists that blog.

Some of our posts have had wide exposure in the blogosphere, with many "views":

What is Epigenetics? An operational definition
Are we training pit bulls to review our manuscripts?
Cancer Stem Cells: the root of all evil?
A few ideas for grad students in the life sciences to keep in mind ,

just to name a few.

We started "Picks of the Week", where we highlight the best molbio-related blog posts aggregated on ResearchBlogging.org. This has been around for roughly 20 weeks now and has been a great success.

We have several followers in Twitter and Friendfeed, accounts that were created mainly to promote the blog.

We would like to thank everyone who has supported us during this first year and we hope to have been an interesting addition to the science-related blogosphere.

Also, we would like to encourage fellow grad students to write for this blog and discuss new research and advances in molecular biology. The doors are open, just send me an email. You don’t have to be a regular writer to be featured here; if you occasionally find an article you’d like to write about, you are most certainly welcome to contact us with the idea.

Thanks again to everyone who has made this possible.

[Image credit: Chez Peg]

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“Chameleon sequences’’, “surveying the human microbiota” and more, in my picks of the week from RB

Another week has gone by and some very interesting molbio blog posts have been aggregated to Researchblogging.org. Every week [see my opening post on the matter], I'll select some blog posts I consider particularly interesting in the field of molecular biology [see here to get a sense of the criteria that will be used], briefly describe them and list them here for you to check out.

Note that I'm only taking into consideration the molbio-related blog posts aggregated under "Biology".

Congratulations to everyone who got their post selected.

1) Cancer is a complex genetic disorder and emerging genomic technologies have enabled us to scan entire cancer genomes for mutations that may be associated with this disease.

Keith Robison at Omics Omics! discusses two recent papers on this matter. The first, using massively parallel sequencing technology , reports the sequencing of “a small-cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking” and the second one, reports the genome sequencing of a malignant melanoma and a lymphoblastoid cell line from the same person, “providing the first comprehensive catalogue of somatic mutations from an individual cancer”.

These papers are in line with emerging results suggesting that cancer genomes are composed of a few commonly mutated genes and many infrequently mutated ones.

2) Can a single amino acid substitution, in the right context, completely change the fold of a protein? What may be the implications of this for the evolution of protein structure over biological time?
Michael Clarkson at Discount Thoughts comments on a recent PNAS paper that suggests that we are still far from having “a complete quantitative understanding of protein structures and their transitions”.

Be sure to check this blog post and the cited article (it’s Open Access!).

3) The human body hosts complex and tremendously diverse microbial communities. Geek! discusses a new Science article reporting an intensive survey of human-associated bacterial communities using a multiplexed barcoded pyrosequencing approach, with the aim of addressing three general questions regarding the biogeography of the human microbiota in healthy adults:

How is bacterial diversity partitioned across body habitats, people, and time?
How does diversity at a variety of skin locales compare to that found in other body habitats?
Do skin communities assemble differently at different sites?

Apparently, each of us has our own “microbial signature”.

That's it for this week. Stay tuned for more MolBio Research Highlights!

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Some of the articles discussed in this week's selected posts:

Pleasance, E., Stephens, P., O’Meara, S., McBride, D., Meynert, A., Jones, D., Lin, M., Beare, D., Lau, K., Greenman, C., Varela, I., Nik-Zainal, S., Davies, H., Ordoñez, G., Mudie, L., Latimer, C., Edkins, S., Stebbings, L., Chen, L., Jia, M., Leroy, C., Marshall, J., Menzies, A., Butler, A., Teague, J., Mangion, J., Sun, Y., McLaughlin, S., Peckham, H., Tsung, E., Costa, G., Lee, C., Minna, J., Gazdar, A., Birney, E., Rhodes, M., McKernan, K., Stratton, M., Futreal, P., & Campbell, P. (2009). A small-cell lung cancer genome with complex signatures of tobacco exposure Nature DOI: 10.1038/nature08629

Alexander, P., He, Y., Chen, Y., Orban, J., & Bryan, P. (2009). From the Cover: A minimal sequence code for switching protein structure and function Proceedings of the National Academy of Sciences, 106 (50), 21149-21154 DOI: 10.1073/pnas.0906408106

Costello, E., Lauber, C., Hamady, M., Fierer, N., Gordon, J., & Knight, R. (2009). Bacterial Community Variation in Human Body Habitats Across Space and Time Science, 326 (5960), 1694-1697 DOI: 10.1126/science.1177486

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Quotes from the science blogosphere

"We've all been there- in the lab, working hard on an experiment, trying to achieve the goals of a larger project. That's what we do, right? Day in and day out. But there seems to be a small problem:

The shit is not working.

Now, I will be upfront about the fact that my shit almost never works at first. As such, I am generally nonplussed but such failure. Minor annoyances in the grand scheme of things- I often tell my coworkers that I will attempt a new assay or method 5-7 times before I give up. Inevitably, the problem will sort itself out when I change my seeding density or the timing alleged by the assay "instructions" or the color of the shirt I wear when I run the experiment".

(my emphasis)

-Candid Engineer in a post on her blog on how to proceed when you are against experiments that just won't work.

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What do you use for your DNA and protein sequence analysis ?

DNA and protein sequence analysis software are routinely used in molecular biology labs.

Sequence assembly, alignments, primer design, in silico cloning and many other tools are now included as part of most software packages for sequence analysis and data management.

I've prepared a little poll to check the software preferences of the readers of MolBio Research Highlights.

[Image credit: Biology Reference]

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The most cited biology articles of 2009

These are the 5 papers in biology, published in the last two years, which received the most citations during 2009, according to Thomson Reuters (thanks to TheScientist.com for the info!)

5. A M. Wernig, et al., "In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state," Nature 448: 318-24, 2007.

4. E. Birney, et al., "Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project, "Nature, 447: 799-816, 2007.

3. A. Barski, et al., "High-resolution profiling of histone methylations in the human genome," Cell, 129: 823-37, 2007.

2. K.A. Frazer, et al., "A second generation human haplotype map of over 3.1 million SNPs," Nature, 449: 854-61, 2007.

1. K. Takahashi, et al., "Induction of pluripotent stem cells from adult human fibroblasts by defined factors," Cell, 131: 861-72, 2007.

Three Nature articles and two Cell ones... well, I guess there's no surprise there.

Here's a cartoon from Jorge Cham's Piled Higher and Deeper series.

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"A tale of two membranes", "follow the (Spliced) Leader" and more in my picks of the week from RB

Another week has gone by and some very interesting molbio blog posts have been aggregated to Researchblogging.org. Every week [see my opening post on the matter], I'll select some blog posts I consider particularly interesting in the field of molecular biology [see here to get a sense of the criteria that will be used], briefly describe them and list them here for you to check out.

Note that I'm only taking into consideration the molbio-related blog posts aggregated under "Biology".

Congratulations to everyone who got their post selected.

1) Nitric oxide synthases are highly regulated heme-based enzymes that oxidize L-arginine to nitric oxide (NO) and L-citrulline. NO has been described as an important signaling molecule and cytotoxin in eukaryotes. Interestingly, bacteria can also produce NO from arginine thanks to a structurally and mechanistically related enzyme termed bacterial nitric oxide synthase (bNOS), which is present in many Gram-positive species. J. Kandler at "Blogging for Bacteriophages" discusses a recent article reporting a physiological role for bNOS. It appears that NO generated by bNOS "increases the resistance of bacteria to a broad spectrum of antibiotics through both the chemical modification of toxic compounds and the alleviation of oxidative stress".

2) Bacteria are commonly classified as either Gram-positive or Gram-negative according to their ability to retain a particular stain (crystal violet) after being washed with an organic solvent, which reflects differences in the composition of their cell walls. Gram-negative bacteria have two lipid membranes surrounding a thin peptidoglycan wall (and don’t retain the stain), while Gram-positive bacteria have a much larger peptidoglycan cell wall and lack an outer lipid membrane (and do retain the stain).

LabRat discusses a thought-provoking article supporting the position of the root “of the tree of all life” within the eubacteria, specifically with the Chlorobacteria (green non-sulfur) representing the earliest diverging eubacterial lineage. An interesting idea put forward in the article is that eubacteria with two membranes arose first, with those having only one membrane (like gram positive bacteria) evolving from them.

3) Spliced-leader (SL) trans-splicing is a nuclear RNA processing reaction where a small leader sequence, derived from an SL RNA, is attached to the most 5′ exon of an independently transcribed pre-mRNA. It was first discovered in trypanosomes, but was later shown to be present in dinoflagellates, cnidarians, rotifers, nematodes, flatworms and urochordates. SL trans-splicing has a patchy phylogenetic distribution and enigmatic evolutionary origins.

This patchy distribution poses an interesting question: is SL trans-splicing an ancestral eukaryotic trait that has been lost in multiple lineages or did it arise independently in the various groups where it occurs?
Lucas Brouwers at Thoughtomics comments on a recent article providing evidence for multiple independent origins of trans-splicing in Metazoa.


That's it for this week. Stay tuned for more MolBio Research Highlights!

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Some of the articles discussed in this week's selected posts:

Gusarov I, Shatalin K, Starodubtseva M, & Nudler E (2009). Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics. Science (New York, N.Y.), 325 (5946), 1380-4 PMID: 19745150

Cavalier-Smith T (2006). Rooting the tree of life by transition analyses. Biology direct, 1 PMID: 16834776

Douris V, Telford MJ, & Averof M (2009). Evidence for multiple independent origins of trans-splicing in Metazoa. Molecular biology and evolution PMID: 19942614

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Quotes from the science blogosphere

"I have, on at least one occasion been unfortunate enough to receive several barely readable manuscripts (in which I strongly suggested someone look over the manuscript for proper English, in addition to offering my own grammatical and typographical corrections) from a particular journal. When they came calling again, I told them to forget it, they had filled their quota of reviews with me for the year and they needed to find some other poor sucker willing to read some horribly written manuscripts".

(my emphasis)

This was written by Thomas Joseph, as a comment on a fascinating blog entry at DrugMonkey's blog about rejecting manuscripts solely based on it being poorly written (bad use of English). Check it out. C'mon, go there now... I give you permission to leave my blog (for a minute or two) :-)

An example of horrid English use....

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"Moving through mucus", "quick death-tagging" and more, in my picks of the week from RB

Another week has gone by and some very interesting molbio blog posts have been aggregated to Researchblogging.org. Every week [see my opening post on the matter], I'll select some blog posts I consider particularly interesting in the field of molecular biology [see here to get a sense of the criteria that will be used], briefly describe them and list them here for you to check out.

Note that I'm only taking into consideration the molbio-related blog posts aggregated under "Biology".

Congratulations to everyone who got their post selected.

1) Helicobacter pylori is a gram-negative bacterium that colonizes the epithelium of the human stomach. Although most of the infected individuals are asymptomatic (~80%), it can cause peptide ulcers and gastric cancer.

H. pylori can survive in the highly acidic environment of the human stomach through a urease-dependent mechanism. On his first appearance at Picks of the Week, Tim Sampson at Blogging for Bacteriophages discusses a recent article shedding light on the mechanism that allows this bacterial pathogen to move through the viscoelastic mucus gel that coats the stomach wall, an important aspect of its pathogenesis.
Apparently, its motility is directly related to the mechanism with which it copes with low pH.

2) The ubiquitin-proteasome system is the major cytosolic proteolytic system in eukaryotes and plays important functions in different aspects of cell biology, like cell cycle control, apoptosis, inflammation, signal transduction, protein quality control, and many others. Basically, it degrades proteins that have been modified by the attachment of a chain of ubiquitin molecules, and this latter process occurs through a series of steps.

An unanswered question has been how this works. Is the ubiquitin chain formed first, and then transferred to the target en bloc? Or are single ubiquitin transferred one at a time, sequentially, first to the target protein and then to the previously-attached ubiquitins?

Ian York at Mystery Rays from Outer Space, briefly comments on an impressive new article reporting the “detection of sequential polyubiquitylation on a millisecond timescale”. The authors show, by measurements with millisecond time resolution, that substrate polyubiquitylation proceeds sequentially, that is, one at a time.

3. It is now generally accepted that mitochondria are dynamic organelles, constantly undergoing both divisions and fusions. Interestingly, mutations in genes involved in such processes can lead to neurodegenerative diseases. LabRat (on her 4th Pick in a row!) gives a brief overview of the mechanisms underlying its dynamic nature.

That's it for this week. Stay tuned for more MolBio Research Highlights!

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Some of the articles discussed in this week's selected posts:

Celli, J., Turner, B., Afdhal, N., Keates, S., Ghiran, I., Kelly, C., Ewoldt, R., McKinley, G., So, P., Erramilli, S., & Bansil, R. (2009). Helicobacter pylori moves through mucus by reducing mucin viscoelasticity Proceedings of the National Academy of Sciences, 106 (34), 14321-14326 DOI: 10.1073/pnas.0903438106

Pierce, N., Kleiger, G., Shan, S., & Deshaies, R. (2009). Detection of sequential polyubiquitylation on a millisecond timescale Nature, 462 (7273), 615-619 DOI: 10.1038/nature08595

Benard G, & Karbowski M (2009). Mitochondrial fusion and division: Regulation and role in cell viability. Seminars in cell & developmental biology, 20 (3), 365-74 PMID: 19530306

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Meeting report: "The Architecture of Life"

A few weeks ago, the Institute for Research in Biomedicine (IRB) Barcelona held its first ever PhD student symposium and I had the pleasure of being part of its organizing committee, along with a few other graduate students. In this post I want to present part of the exciting science discussed there, but first I’d like to briefly highlight the importance of science communication in the life of a scientist

Science, for me, is not just sitting at the bench performing experiments and generating data. An important part of the life of a scientist is the actual communication of his research, not only to the rest of the scientific community, but also, and perhaps just as important, to the non-scientific population.

Regarding the former, I had the chance of getting firsthand experience in organizing a scientific meeting, something for which scientists rarely get any instruction during their careers. This was an exciting experience and I highly recommend it, despite all the hard work involved: in this case, the pros definitely outweighed the costs.

The concept behind this symposium was straightforward: let PhD students organize a meeting for other PhD students. We had to handle every detail, like coming up with the meeting’s theme, contacting scientists of different fields and putting together its website.

Since this was our first time being a part of such a project, we decided to give it a broad orientation, such that would encompass most of the fields studied in our institute. In the end, we booked 8 speakers with very different scientific backgrounds divided in 4 sessions: DNA and RNA, Proteins, Cells and Tissues/Organisms (as you can see, the categories cannot be broader).

We decided on “The Architecture of Life” for the meeting’s theme and so, we intended to start of from the basic building blocks and, throughout the meeting, work our way to whole organisms. I won´t go into much detail regarding the talks, but I do want to give you a brief idea of the wide array of interesting topics addressed.

Starting everything off was Gene Myers and Eric Miska.

As some of you may know, Gene Myers pioneered the BLAST algorithm (which most of us use on a daily basis), and during his talk he gave an overview of its inner workings. He then went on to discuss how he developed the shotgun sequencing strategy (which nowadays is pretty much standard) and he concluded his talk by presenting some of his current work regarding high-throughput image analysis software.

Eric Miska talked about non coding RNAs, with a particular focus in miRNA studies. He works on C elegans, and he showed some very interesting projects aimed at studying the complex biology of miRNAs in this organism. Notably, he showed that the repressive function of certain miRNAs can be stably inherited. If you are interested in this area I recommend you keep an eye on Eric’s work.

The second session focused on protein biology, and we had the opportunity to listen to this year´s Noble laureate Ada Yonath, as well as to the cutting edge science of Tanja Kortemme.

Ada´s talk focused on the ribosome, and she gave an extensive structural overview on how this amazing macromolecular machine works. Furthermore, she talked about how structural insights have led her group to hypothesize about the evolutionary history of the ribosome.

Tanja Kortemme´s talk focused on “protein engineering”. By protein engineering I mean that her group is actually designing and testing new interactions between proteins through a combination of computational structure predictions and old-school classic chemistry, which is mainly used for validation purposes. I think she gave a wonderful talk I consider her work to be very impressive.

The third session combined talks given by two scientists working in very different areas: Wolfgang Baumeister and Magdalena Zernicka-Goetz.

In his talk, Wolfgang discussed how the application of EM tomography can help elucidate very complex biological processes. For example, by applying the methodology developed in his lab, they can literally count the number of ribosomes present in a particular subcellular compartment (he showed some beautiful results of studies done in neuron synapses in culture). This can also be used to count other macromolecular complexes present in a particular subcellular location.

Next up was Magdalena Zernicka-Goetz, who, in my opinion, gave the best talk in the symposium. Her lab is studying how the first cell-fate decisions are made in the mouse embryo and their question is quite straightforward: how do identical cells end up with such different phenotypes? In her talk, Magdalena summarized the major breakthroughs her lab has made in the last few years. For those who are more interested in the subject I recommend that you read her recent review in Nature Reviews Genetics (doi:10.1038/nrg2564).

The final session, centered on developmental biology, also had two great speakers: Darren Gilmour and Steve Cohen.

Darren´s work focuses on cell migration using zebrafish as a model. His group aims to understand the molecular and biophysical properties underlying the development of the zebrafish lateral line system, an attractive model system for studying cell migration during organogenesis. Interestingly (and I’m leaving out a lot of details), it appears that the leading cells have distinct signaling pathways activated (compared to the lagging ones), that when disturbed inhibit the movement of the entire group of cells.

The final talk was given by Steve Cohen, who is currently studying miRNAs in Drosophila. One of his most attractive projects involves knocking out every single miRNA from the Drosophila genome. Shall this area of research pique your interest, keep your eyes open for his upcoming work and also check out some of his already published research (for example, see PLoS One 2007;2(11):e1265)

That’s it for this brief overview of the symposium we organized, “The Architecture of Life”. This was a great experience and I sincerely hope these sorts of initiatives are imitated in other institutions worldwide.

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A few great biologists...

Here's the latest cover from Genetics (Nov 2009 issue):

From the journal:

About the Cover
Some members of the band of geneticists who established and extended the guiding principle of biology.

Here's the key in case there are some you don't recognize:

1 James F. Crow
2 Theodosius Dobzhansky
3 Ronald Aylmer Fisher
4 Motoo Kimura
5 John Burdon Sanderson Haldane
6 Hermann Joseph Muller
7 Sewall Green Wright
8 Carl Erich Correns
9 William Bateson
10 Carl Linnaeus
11 Frederick Sanger
12 Gregor Johann Mendel
13 Alfred Russel Wallace
14 middle-aged Charles Robert Darwin
15 Erasmus Darwin
16 young Charles Robert Darwin
17 old Charles Robert Darwin
18 Alfred Day Hershey
19 Francis Harry Compton Crick
20 James Dewey Watson
21 Oswald Theodore Avery
22 Rosalind Elsie Franklin
23 Max Delbrück
24 Salvador Edward Luria
25 Joshua Lederberg
26 Margaret Oakley Dayhoff
27 Linus Carl Pauling
28 Emile Zuckerkandl
29 Calvin Blackman Bridges
30 Alfred Henry Sturtevant
31 Thomas Hunt Morgan
32 Sydney Brenner
33 Seymour Benzer
34 Barbara McClintock
35 Thomas Henry Huxley
36 George Wells Beadle
37 Edward Lawrie Tatum
38 Edward B. Lewis
39 A bulldog

[H/T: Sandwalk]

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An alternative cloning strategy: yeast recombinational cloning

As part of my Ph.D thesis, I have to generate a lot of transcriptional fusions (constructs in which a promoter of choice is cloned in front of a reporter gene in order to evaluate transcriptional regulation. Such plasmids can then be transformed into your model organism to study this regulation in vivo).

Traditionally, this involves amplifying the region of interest (in my case a promoter region) by PCR using primers that harbor the recognition site of particular restriction enzymes. Sometimes you can use the PCR product directly in a digestion reaction with the proper enzymes (although it’s recommended that you column purify your PCR product first) in order to clone it into the vector of interest, but many prefer to clone it first into pGEMT. In order to do the latter, you’ll have to do a TA tailing, as high fidelity enzymes (used for cloning) typically produce blunt ends. Once again, it’s recommended that you column purify your PCR product before tailing.

Once tailed, you can clone the PCR product into pGEMT and transform E. coli in order to propagate the plasmid, which can now be sent out for sequencing. After everything checks out, you would have to set up a digestion reaction in order to get the segment out of pGEMT and clone it into the final plasmid. After that digestion, and in order to get this segment into the final plasmid, you would digest the target plasmid with the same enzymes, set up a ligation reaction and then use an aliquot of the reaction to transform E. coli.

Ideally you’ll use two different enzymes for target plasmid linearization, which would avoid plasmid re-circularization, but sometimes things are not that easy. If you can only use one enzyme for linearization (due to plasmid design, enzyme availability in your lab, etc.) then you are going to have to dephosphorylate the linearized plasmid (typically using Calf intestinal phosphatase) after digestion to avoid re-circularization. Some phosphatases can be heat inactivated, but others can’t. In any case, after digesting and dephosphorylating your plasmid, you’ll have to do a phenol:chlorophorm extraction followed by ethanol precipitation to get your dephosphorylated plasmid, which can now be used for ligation with the segment of interest. After setting up this ligation, an aliquot is used for transformation into E. coli.
You can then check your plasmid by colony PCR of the antibiotic-resistant colonies resulting from the transformation.

At this point you’ll realize that generating several constructs following this protocol can take some time. This strategy, then, does not fit my cloning needs.

We’ve taken a different approach to satisfy most of our cloning requirements, which makes use of my favorite organism: Saccharomyces cerevisiae.

The cloning strategy we use routinely in our lab is called Yeast Recombinational Cloning (YRC), a strategy which has been around for some time (Ma et al., 1987) and has been refined over the last ~13 years (Oldenburg et al., 1997; Gibson, 2009).

The concept behind this strategy is simple: if the segment you’d like to clone into a particular plasmid bears homology to defined plasmid sequences, you can directly “ligate” it into the linearized vector by in vivo recombination: yeast machinery will take care of it. This alleviates the need for an in vitro ligation reaction.

Initially, it was shown that a DNA restriction fragment containing appropriate sequence homology could serve as a substrate for such “recombinational repair” (it’s called repair, as you’ll repair the gap in the linearized plasmid). However, an important advance in the use of such recombination-based methods for gene cloning in yeast, involved the use of PCR (rather than restriction assays) to generate the DNA fragment to be used.

As it was shown that the length of sequence homology needed to promote efficient recombination between the segment of interest and the plasmid was small (~20-40 bp), it was quickly realized that these sequences could be included as part of PCR primers used to amplify a segment of interest. This “recombination-mediated PCR targeting” is very efficient and I consider it now one of my favorite techniques.

So, how do you generate your plasmid through YRC? The linearized target plasmid containing a selectable marker (e.g. URA3 ) is co-transformed into yeast with the PCR fragment of interest: this fragment has 20-40 nt of homology at each end to the region of the plasmid at which recombination is to occur. These nucleotides were added to the fragment as part of the primers. You don’t even have to purify the PCR product before transformation; an aliquot taken directly from the PCR reaction tube works fine. By homologous recombination, some of the cut plasmids are recircularized (due to the integration of the segment of interest into the plasmid) and the plasmid can now be propagated in yeast. Recombinants are then selected as Ura+ transformants (See figure 1).

FIGURE 1. Basic outline of yeast recombinational cloning. Figure based on the one at the “Yeast Model Systems Genomics Group” website.

You can then do yeast colony PCR on the Ura+ transformants to check for your plasmid. Note that the linearized plasmid will not lead to Ura+ transformants, as such a plasmid cannot be propagated.

This protocol may take longer (in days) than the traditional approach outlined at the beginning of this post (as you'll have to wait for the transformed yeast to grow in selective media), but it has significantly less steps, uses less restriction enzymes (as you'll only use them to linearize the target plasmid), uses no ligase, does not require any sort of purification and the only methods involved are digestion, PCR and yeast transformation (the latter is very simple, efficient and requires reagents typically found in a molbio lab. We use the Liac/SS Carrier DNA/PEG method with excellent results).

Further, this approach is more amenable to high-throughput cloning than the traditional strategy (See Colot et al., 2006).

In addition, homologous recombination in yeast can be used to build complex constructs from multiple overlapping constituent parts (see figure 2). Generating such constructs through traditional approaches would take considerably longer. Note that the target vector doesn’t necessarily have to be the final vector. You can use YRC to assembly this complex construct and then the DNA insert can be cut out of the yeast vector and placed into any other vector.

FIGURE 2. Multiple overlapping segments can be cloned into a target plasmid (in this case a shuttle plasmid) through homologous recombination in yeast.

OK, enough about the methodology. How is this strategy used for the generation of my transcriptional fusion constructs? The target plasmid we use is linearized just upstream of the coding region of a reporter gene. Due to proper primer design, the PCR amplicon of the promoter of interest bears homology to particular plasmid sequences that allow its integration upstream of this reporter gene through homologous recombination. The particular target plasmid that we use can be propagated both in yeast and bacteria (i.e it’s a shuttle plasmid) and can also be transformed into our model organism, where by homologous recombination, will integrate in a particular locus in the genome so that we can study the promoter’s biology in vivo.

So, in summary, we have generated a yeast strain which contains a plasmid with our promoter of interest controlling the reporter gene. Now, it’s time to obtain that plasmid in working concentrations, sequence it and then transform it into our model organism.

How do we do this? How do we get the resulting plasmid out of yeast? That will be the matter of a follow-up post, so stay tuned!

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Some of the articles discussed in this post:

MA, H., KUNES, S., SCHATZ, P., & BOTSTEIN, D. (1987). Plasmid construction by homologous recombination in yeast Gene, 58 (2-3), 201-216 DOI: 10.1016/0378-1119(87)90376-3

Oldenburg KR, Vo KT, Michaelis S, & Paddon C (1997). Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic acids research, 25 (2), 451-2 PMID: 9016579

Gibson, D. (2009). Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides Nucleic Acids Research, 37 (20), 6984-6990 DOI: 10.1093/nar/gkp687

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