The PaJaMo Experiment and the Lac I Repressor

I have come to the end of a very interesting course based on the history of molecular biology.  Since molecular biology has been my course of study for the past few years, I really enjoyed the readings from the primary articles written mostly in the 1950's and 60's.  This paper was focused on the famous experiments performed by Jacques Monod, Francois Jacob, and Arthur Pardee which showed the first significant observation of the intermediate molecule between DNA and protein.  See the bibliography for links to the papers that I found so interesting.  Now my bit.

 
In 1959, molecular biology was firing on all cylinders.  There was a great interest in the Western world to describe the phenomena of the molecules of life.  One of the epicenters of this fascination was in Paris at the Pasteur Institute.  Jacques Monod and Franscois Jacob working in related departments brought their related expertise together, Monod bringing his understanding of the Lac region of the E. coli K12 strain and Jacob bringing his understanding of microbial genetics.

Leading up to 1959, Watson and Crick had proposed in 1953 the structure of DNA.  Following logically from their double helical structure, the “Central Dogma” was considered and officially enunciated by Crick in 1958.  While the logic of the age called for DNA to act as a template of RNA synthesis which would in turn act as template for protein synthesis, the observation of the intermediate RNA and the nature of this RNA were unknown.

            The Pasteur Institute was well-positioned for making great scientific discoveries.  Before Arthur Pardee took his sabbatical leave from UC Berkeley to visit the Pasteur Institute, he was preceded by a number of American post-doctoral fellows who converged on the Pasteur Institute to hone their skills and their logic in preparation for illustrious scientific careers.  American visitors to the Pasteur Institute included A.M. Pappenheimer Jr., Martin Polock, Melvin Cohn, Irving Zabin, Gunther Stent, and others.  And Americans weren’t the only ones visiting the Pasteur Institute.  Collaboration was an ideal of the science in those days allowing Crick to visit from England, and even Eastern Europeans came to Paris, such as Boris Magasanik.

At the Pasteur Institute an environment of learning by logic and experimentation had instituted the atmosphere whereby the theories of genetic regulation could be tried, tested, and improved from their basic heritage up to nearly what we now know of the phenomena.

      There were a variety of other theories upon which insights were gained as a result of the PaJaMo experiment.  Beadle and Tatum’s “one gene-one protein” theory had a narrow definition in the years before the Operon theory.  Monod hypothesized that the induction of proteins was from manipulating preexisting protein structures rather than nascent protein synthesis that each protein arose from a different mixture of pre-protein products.  The inducer-repressor theories of regulation were in their infancy.  The RNA intermediate of the central dogma, was at this time no more than a good guess following the logic of other findings.

      In order to test the resounding theories of the day, Pardee, Jacob, and Monod used the bacterial conjugation experiments of Jacob, the Beta-galactosidase system characterized by Monod, and the innovative experimental tweaking of Pardee. 

Jacob had discovered with Elie Wollman in 1955 that Hfr strains of bacteria are able to attach themselves to F^- strains and inject genetic information.  Using this technique, the PaJaMo experiment crosses Hfr strains and F^- strains of a variety of genotypes for the z and i genes.

Monod had previously described the nature of the products of the z and i genes.  The z gene represents the commonly mutated region of E. coli DNA that produces mutants that do not create Beta-galactosidase under any circumstance.  The i gene represents the commonly mutated region in E. coli that causes constant expression of Beta-galactosidase even in the absence of galactoside inducers.

Pardee had been studying similar repressible systems at UC Berkeley and brought a twist on data acquisition in the Beta-galactosidase system which raised the measurement output from twelve experimental values a day to something nearer one hundred in the same time.  While the he claims the measurements are cruder, the extra data points allowed for faster experimentation and rapid progress (Tribute p135).

The most interesting cross that they performed was an Hfr z⁺i⁺ x F^- z^-i^- of which the zygotes were ultimately z⁺ z^- i⁺ i^-.  However, what they found was that for the first two hours these zygotes acted as z⁺i^- mutants which constantly express Beta-galactosidase.  Then at two hours, they observed that without the addition of inducer, the z⁺ z^- i⁺ i^- zygotes would phenotypically show their genotype and Beta-galactosidase would stop being synthesized.  While inconclusive on the question of nature of RNA, it is among the first observations of a cytoplasmic intermediate that arises quickly, allows consistent expression, and degrades quickly.  Repression of the DNA by the newly synthesized repressor stops the ultimate synthesis of Beta-galactosidase by means that were not then understood, but were supportive of the logical assumption of the RNA intermediate.

By the end of the PaJaMo experiment, the nature of the repressor was of greatest interest.  In fact, the article finishes with a direct question: “What is the chemical nature of the repressor?  Should it be considered a primary or a secondary product of the gene?”  Not the most literary way of finishing a paper, but certainly it emphasized the intense interest that these scientists had in finding the true nature of the repressor (Pardee et al. 1959). 

By 1961 when Jacob and Monod published their review of genetic regulatory systems (1961) they were certain that this repressor must be a primary product of the gene, or in other words they believed it to be RNA.   Then, by 1965 in Monod’s Nobel lecture, he stated emphatically that the Lac i repressor is a protein.  We know today from molecular biology textbooks that the Lac i repressor is a protein.  So what happened to make Jacob and Monod so sure of their RNA repressor.

Later in the same year as the PaJaMo experiment, Pardee performed some errant experiments with Louise Prestidge that should have stopped protein synthesis altogether.  Unfortunately, this article was unavailable on the internet.  Since it was published in 1959, databases do not cover it, and there was no electronic version that I could find.  The main finding however, was mentioned in Jacob and Monod’s review that we read for class in which they cited Pardee as eliminating protein as the repressor product of the Lac i gene.

The struggle of finding when the repressor was definitively characterized as a protein is difficult as well.  Since the characterization took place before the internet, the laborious task of searching actual paper copies of the articles is necessary.  Only the most famous of these early articles are captured in digital copies today.  For this reason, and the time constraints of finals, I have not as yet found the article that characterizes the repressor as a protein.

Realistically, the mistake of an RNA repressor was small in comparison to the great strides that this experiment took in describing the regulatory nature of Lac i on the Lac z gene.  Students of any introductory molecular biology course become well versed in the Lac operon theory, the inducer-repressor theory, and their applicability to the regulation systems in other organisms.  Monod is quoted as saying “Whatever is true for E. coli is true for an elephant.”(p 181 “Tribute”)  While we now know of many instances where Monod is wrong, we also see the many more ways in which he was right.

Bibliography

            Jacob, F., Monod, J. Genetic Regulatory Mechanisms in the Synthesis of Proteins. (1960)

Jacob, F. Genetics of the bacterial cell. Nobel Lecture ,December ll, 1965

Monod, J. From enzymatic adaptation to allosteric transitions. Nobel Lecture, December 11, 1965.

Pardee, A. B., Jacob, F., Monod, J. The Genetic Control and Cytoplasmic Expression of “Inducibility” in the Synthesis of b-Galactosidase by E. coli (1959)

Ullman, A., Origins of Molecular Biology: A Tribute to Jacques Monod. (2003)

FASTA DNA Codes

I've been reading Fasta files a lot recently and I've noticed that these files of DNA sequence are made up of more than just A's, C's, G's, and T's. Because sequencing errors or frequent SNP variants can make it hard to give a clear consensus sequence, each of these other letters represent the ambiguities in the given DNA sequence.

Here's a nice website with a table that shows what all these other letters represent.

And for you frequent FASTA readers that want to memorize the ambiguous DNA code, I've created a Quizlet for just that purpose.

Choose a Study ModeScatterLearnFlashcards

Study these flash cards

Follow this link to enjoy all the tools on quizlet.com

The Cost of Being Sick

What did you do for your Christmas Vacation? I ate lots of food, play board games with family, watch movies and television, and took naps. Nothing unexpected. It was nice to take a break after the stress of Finals, and just "let go" of myself. But how are the cultural expectations of overeating and under-exerting affecting our national structure?

Christmas in Blackfoot was nice. I had no school work, and I got to follow a few doctors around in preparation for medical school (still a ways down the road).

Dr. Anthony K. Davis is a specialist in minimally-invasive surgery (he makes smaller scars), and many of his surgeries fall into bariatric surgery (weight loss surgery). Many of the patients that see Dr. Davis hope to lose weight in order to improve their quality of life and lower their health risks. Dr. Davis is perfect for these patients, because he empowers his patients with tools, some surgical and others habitual, and teaches them how to use their tools to fight their weight problems. He said to one patient, "You've been gaining this weight for 20 years, so you can't expect it to be gone over night." There is no twice-daily pill that will solve our health problems.

In conjunction with my experience with Dr. Davis, my wife and I measured our health by the BMI index and the waist-to-hip ratio. These online calculators told me that at 6'0 and 185 pounds, I was overweight and my waist-to-hip ratio gave me a moderate risk for heart disease (it's not as surprising after reviewing photographic evidence).

Then I found a book from 2003 called "The Cost of Being Sick" by Nicholas J. Webb that predicted today's healthcare calamities. Webb also discusses the cure: healthy diet and regular exercise.

From my 3 days following doctors, this cure comes as no surprise. While following two orthopedic surgeons I observed that 90% of their patients are obese. The studies that Webb references back up the claims that obese people need more healthcare thereby creating a strain on the healthcare system, not to mention the physical and emotional strain on the individuals from being overweight.

Since Christmas, I've turned back the alarm clock to give me time to exercise in the morning. Last year, I tried to go it alone with push-ups at home and infrequent jogging. This year, some friends of mine have been playing basketball on weekday morning for a half hour. Suzie and I have been buying more fresh foods and eating canned beans and fruits while monitoring the sweets. These are far from perfect practices, but we have to start somewhere. We all have to start somewhere. Amazingly, we're saving money already, because we're more conscientious of our food and our time. Healthstyle changes have created an upward spiral for us. And when the world around us seems in an ever downward spiral, we each need that daily boost from regular exercise, healthy diet, and financial relief.

Reflections of December

My first semester of blogging is over. Reflecting on the past months I can't remember what learning was like before this semester. I remember spending money on books just to sell'em and forgot'em after a couple months. I remember spending lots of time in Microsoft word trying to perfect my assignments for lots of classes. Everything I did was based on fitting professors requirements and getting the grade.

Now learning is very different to me. I like doing homework because there is something I can do to better the world around me. My education is fading from the me-centered, impress-the-teacher-to-get-the-grade sentiment to an attitude of improvement that lasts longer than the deadline. Case in point, to study for a final exam, I translated content from a PowerPoint lecture to a Prezi that I can invite my professor to edit. The Prezi will make his lectures a little more visually appealing, and it helped me study for the test. (I got a 95% on the final; I haven't completely got past caring about grades.)

Digiciv was a remarkable experience because it taught me to learn in a whole new way. The focus of a civilizations course is the history of civilization and at times I feel like a normal civ course would have been easier. In my other civ course, the grade was decided by three written exams that hurt my hand and the lectures beat my brain with boredom. However, I didn't have to do hardly anything, and I got the grade. Digiciv has taught me to keep on learning and to spread my knowledge. Education is not about stuffing my head with soon-to-be irrelevant facts just to dump them on a paper and forget them; education is about learning to make a difference in the world through whatever path I choose. I can't say that I have mastered all the historical content discussed in the course, but I've learned how to skim works and decide if it's worthwhile to share.

There is so much that I've been thinking about in context to this class so I made a Prezi to map my thoughts.

So what does the future hold for me? I plan to use this blog as a launch pad to further collaboration with Backpack 2.0 and other educational ventures. I've just got into a lab for the winter semester which will afford many blog posts as I learn what novel research in Bone Morphogenic Proteins is like. I feel like the blog format is nice because I can publish my ideas from the start and ask for reviews from my research advisor and other professors with whom I have a relationship. Digiciv opened my eyes to what the real world holds and I know that the principles that I've learned will govern my profession as I collaborate with peers and strengthen professional relationships through openness.

And to finish, I want to link to my favorite posts and tell a little bit about why I felt they were good posts.

1. MCAT Reformation - I really liked this post because it shows my efforts towards social discovery. I haven't published the group enough, but I figure that can come later when I'm not bogged down in finals. Just making the group was the first step and I really like the idea of saving a couple thousand dollars and helping other people do the same.
2. Web 2.0 Project continues - The post wasn't absolutely amazing, but it had an applicable screenshot to make it a little less boring and it examines my opinions between two web 2.0 formats (blogs and wikis).
3. Stupidity is Science + Sweet Stupidity - I liked these posts because first I like science; it's where I feel most comfortable. Secondly, I revisit an old idea on my blog in "sweet stupidity" and show an application of that idea (rereading research materials). It was nice to see my application of my own ideas.

I think the greatest thing about this course (and especially the blog that it requires) is that I can see myself growing from post to post and blogging for this class has helped me make connections in other classes. I know that I have grown from this class because I am accountable forever (or at least until the internet crumbles) for the things that I wrote. Accountability has made my education a sharing experience instead of just a learning experience. Thank you for reading this post and for growing and sharing with me this semester.

Open Healthcare

So I've just finished a pre-med class that constituted doctors coming once a week to tell us about the health profession. One professional after another would say in reference to the healthcare bill "No one knows what's in the bill!"

I think that someone must know what is in that bill. (As a side note, I don't believe that anything good can come from nearly 2000 pages of compromises of the simple notion "Love thy neighbor".) So how can we publicize it. I figure we need to read it. (We also need to set a word limit on crazy politicians who think anyone is going to read a bill with a quarter million words in it.)

Bills of this size make politics closed by the huge barrier-of-entry that requires one to read it for days of ones life. Bills like this one try to make democracy a monarchy where power lies in the hands of rich politicians (who typically spend millions of dollars to get elected).

So here it is, a crazy bill (seen above in one hand) and a crazy idea. Check out the open congress website for the bill and then read a page yourself from the full text. I'll read page one. This bill affects us. Take this chance to read a little for yourself.

Education from the Bottom Up

I just watched this awesome video that Kristen Cardon found and published through Diigo. Sugata Mitra spends 17 minutes showing how kids in developing countries are teaching themselves via the internet. If you're the type that wants to bridge the gap and make things more equal, then watch this movie.



This video shows the internet to be the most powerful means of teaching children what they want to know. I think it validates Marshall McLuhan's idea that "the media is the message". Students around the globe are hearing the messages that interest them and if Sugata Mitra is right, then we really can change the whole world in a matter of years, in a single generation.

MCAT Reformation

I attended a seminar on MCAT preparation. I'm considering going to medical school, but I'm not made of money. Now, I do recognize that expenses for a good education are an investment. I can understand the $230 price tag on the MCAT, but another $1800 for prep courses is too much for my budget.

I feel like Martin Luther in a way. I'm pounding my 95 theses on the internet door (I do know how to hack the MCAT Prep class sites; and I'm not that hard core). Getting into medical school should be hard, but don't believe that it should cost a lot of money. So I'm eliminating the monetary barriers to success. I first sent an e-mail to the MCAT officer in BYU's premed club to solicit ideas for forming study groups. I've also begun collecting free online resources in a Diigo group appropriately named "MCAT Prep Resources".

Next semester I'll become a member of the premed club and seek out premed students who want to save money on MCAT preparations. By finding free resources in groups and studying in groups the knowledge of the group will spread to the group members.

My hope is to reform the way students look at test preparations, turning it into a weekly study habit from the start of freshmen year until the week of their MCAT. The power to form groups and benefit from the social knowledge is far greater than any attribute tested in any assessment test. The reform isn't to save money. The reform is to change minds.