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Martha Chase, Max Delbruck, and the American Phage Group

Martha Chase is a bit of an enigma. Her career started promisingly enough working in a productive field amongst productive scientists; but following her PhD, her health precipitated setbacks in both her career and her home life from which she did not recover.

She received her bachelor’s degree in 1950 from the College of Wooster. Later that same year, she joined the laboratory of Alfred Hershey in Cold Spring Harbor to work as his lone laboratory assistant asking questions about the mechanisms of life using a viral model system. The virus they used was the bacteriophage T2, a fascinating conglomeration of proteins and DNA that specifically infects bacteria. Through her work with Hershey, she became linked to the remarkable influence of a network of biologists nucleated around the German-American biologist, Max Delbrück, called the ‘American Phage Group.’

Delbrück, himself a Nobel Prizewinner, led a rich intellectual life amongst an elite group of academic luminaries. The Chemist, Karl Friedrich Bonhoeffer, was a close friend and mentor to him during his younger years steering him into the study of physics where he became associated with Wolfgang Pauli and Niels Bohr. It was Bohr’s influence that put him on the path to Biology through its relationship with Physics. Again, not to be on the outside looking in, he became assistant to Lise Meitner who had worked with Nobel Laureate Otto Hahn to discover fission of Uranium (Meitner is often regarded as missing out in the Nobel for anti-Semitic reasons), and with Otto Frisch, who recognized that fission must be accompanied by a massive energy release tying it to both the potential for energy production and a potential massive destructive power. Delbrück initially came to the States to study genetics in drosophila, but made a deeper mark studying viruses, eventually earning a Nobel Prize in 1969 for his work with Salvador Luria and Alfred Hershey, largely thanks to the diligent work of Martha Chase.

Many of the Phage Group’s members are credited with landmark advances in our understanding of molecular biology. Luria, working with Delbrück, demonstrated that mutation of bacteria occurred in a strictly Darwinian sense, i.e. that bacteria could mutate to resist viruses even without the virus being present. This is a fundamental distinction from Lamarck’s notion that evolution was driven by need, rather than by selection of completely random events. It was at this time that he took on and trained his first PhD student, James Watson (who also did something important – I forget what).

In 1949 Renato Dulbecco came to Caltech to join Delbrück’s group with the focus of understanding how some viruses would lead to tumors. Along with David Baltimore and Howard Temin, Dulbecco shared the 1975 Nobel Prize for discovering how these viruses would reverse transcribe their RNA genome into DNA and integrate it into the host’s chromosome.

Matthew Meselson and Franklin Stahl, also working with the phage group, demonstrated that DNA replication is a semi-conservative process retaining one strand from the ‘parent’ DNA and one ‘new’ strand synthesized as a complement to the ‘parent.’ This work did not earn them a Nobel Prize, although it provided early support for Watson and Crick’s DNA structure and remains a landmark experiment in biology that every student is taught.

As evidenced by the sheer number of Nobel Prizes shared by members of this group, the Phage Group and its associates dominated the fields of bacterial genetics and molecular biology. But before those experiments were performed and Prizes collected, the physical molecule carrying genetic material was yet to be discovered.

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Frederick Griffith (see reference 1)

Frederick Griffith was the first to point the way to this molecule by showing bacteria’s mysterious ability to transfer new characteristics (Darwin’s Traits) between organisms. But, tragically, left his work incomplete due to his death in London during WWII. A number of stories exist regarding his whereabouts when he died. Regardless of its veracity, I personally like the one that suggests that he was working late in the lab when it was bombed by the Nazis.

 

Before his death, in the 1920s, Frederick Griffith demonstrated that some element of a bacterium, that is released upon its death, was sufficient to carry genetic information from one strain of bacteria to another. Specifically, he demonstrated that ‘smooth’ pneumococcus, which secreted a glycocalyx, could transfer this trait to ‘rough’ bacteria that lacked the glycocalyx. Clinically, this was very important because the rough type pneumococcus was easily handled by the immune system, while the smooth type colonized the heart and killed the host. He called this element the ‘Transforming Principle,’ but died before he could identify it specifically.2

 

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see reference-2

Experiments showing that the transforming principle was probably DNA were performed by Avery, MacLeod, and McCarty at the Rockefeller Institute in 1944. Despite being both elegant and thorough, many thought these experiments lacked the appeal needed to be convincing.

 

Knowledge of Avery’s work supported the case for DNA as the genetic material, but Protein remained a persistent contender because, with its 20 physiological amino acids, its capacity to carry the information associated with genes seemed more reasonable. DNA, on the other hand, was an arrangement of only four bases, a simplicity that obfuscated its coding potential. One compromise hypothesis suggested that perhaps DNA served as a scaffold for the information-carrying proteins, although Avery’s experiments showing that protein-free DNA preps could transform bacteria strongly argued strongly against this model.

So, the issue remained to be effectively demonstrated denying Avery and his co-workers Nobel. A more satisfying answer, reaffirming Avery’s discovery, was to come from the ever-productive phage group in the hands of Martha Chase.

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Martha Hershey and Alfred Chase (see reference 3)

Working together, as laboratory technician for Alfred Hershey, the two performed their eponymous experiment in 1952 with the purpose of identifying what served as the genetic material in phages.

 

Hershey intended to use the T2 bacteriophage to assess this question, in part because it contained no other molecules such as fats or sugars, making it an exceedingly simple model (See illustration of method, panel A) but also because electron micrographs already hinted of protein ‘ghost’ particles left outside of the cell while new phages were being assembled within. Indeed, by involving only DNA and unglycosylated proteins, it was possible to label the DNA and Protein elements individually using radioactive Phosphorous-32 to mark the DNA and radioactive Sulfur-35 to mark proteins. These isotopes worked well because they were trackable by following the radioactivity, while each was specific to its target due to the natural, exclusive distribution of Sulfur and Phosphorous in Protein and DNA, respectively.

 

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A bacterium with phages attached to its surface and phage capsids assembling within the cell. (attribution unknown)

The basic experiment was simple in theory. T2 bacteriophage was grown in media containing either nucleotides with Phosphorous-32 or amino acids with Sulfur-35. In the first condition, only the phage DNA was radiolabeled. In the second condition, only the protein was labelled (see illustration of method, panel B). Once the phage was prepared, it was allowed to attach to fresh bacteria for a time period known to allow for the passage of genetic material. At this time, the bacterial cultures were moved into a kitchen blender and pulsed to remove the material that did not enter the host cells (the ghosts). Centrifugation permits the separation of the ghosts and any other viruses in the supernatant from the (infected) bacterial cells. The only thing remaining was to check to see where the radioactive elements were: the supernatant fraction that did not enter the cell, or within the cell, where the genetic material was (see illustration of method, panel C). Like most experiments, much of the work invested in the project occurred prior to the actual experimentation in order to optimize each condition.4

 

 

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Illustration of Method for Hershey-Chase Experiment

The answer was clear, Radioactive Sulfur was never found inside of infected cells, only in the supernatant. Radioactive Phosphorus was overwhelmingly found within the cell. With this elegant experiment, the question was answered, and DNA was widely recognized as the genetic material setting up the next, obvious Nobel Prize: what is the structure of DNA? And does this structure reveal any of its properties?

 

At the University of Southern California, Martha continued to study phages under Giuseppe Bertani (Joe to his friends), ultimately following him to the Karolinska Institute in Stockholm, Sweden where she completed her PhD thesis on “Reactivation Of Phage-P2 Damaged By Ultraviolet Light” in 1964.5,6,7 Her obituary, which is one of only a few primary sources of information on Chase, describes life after earning her PhD as plagued with personal troubles arising from short-term memory loss that likely contributed to the end of her scientific career and possibly her marriage.

Despite the fact that this work represented the accomplishment of Hershey in the 1969 Nobel Prize along with Delbruck and Luria, Chase did not share in this honor. As a technician in the lab, it may be that her hands performed many (if not all) of the Hershey laboratory’s experiments, but technicians are rarely (if ever?) included in the Prize on the assumption that it is unlikely that they are major theoretical contributors to the work. Her name, however, will forever be associated with this experiment, serving as a lasting reminder of her contribution to molecular biology.

References

  1. Photograph of Frederick Griffith, photographer unknown
  2. “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Deoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III.” January 1944. Exp. Med., 79: 137-158.
  3. photo: Martha Chase and Alfred Hershey, 1953. Attribution unknown. I found both of these images at https://varietyofrna.wikispaces.com/Hershey+and+Chase
  4. Link to Hershey and Chase’s J. Exp Med paper: http://jgp.rupress.org/content/jgp/36/1/39.full.pdf
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827068/#
  6. http://www.the-scientist.com/?articles.view/articleNo/22403/title/Martha-Chase-dies/
  7. http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll18/id/368326/rec/7
  8. See http://www.nobelprize.org/ for a listing of Nobel Prizes, Biographies, Acceptance Speeches, and even games.
 
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Posted by on October 3, 2016 in Uncategorized

 

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One Quarter in

US08395894-20130312-D00000.pngI’ve been working for an intellectual property firm now for three months and I thought it might be time to update my impressions of the field.

When I entered this position (as a technical patent analyst)at the start of the year, I had little background in the area. I have a few patents on work that I’ve done in my various biotech positions which I did work on assembling the data for the attorneys working in our company / the university. However, that didn’t really provide much insight into what a patent is, or what really went into making it work. In fact, one of the only things I learned is that lawyers (or at least some of them) won’t shy away from exploiting your ignorance of their system in order to get what they want. In my case, this was signing over my rights to the company I had worked for after they had laid me off.-That’s a whole other discussion though!

Screen Shot 2016-03-25 at 10.23.32 PM.pngIn the past, my writing had always been of the sort that presented data and built a story around that data in a way that was essentially persuasive in nature. Patent Applications do present data, and they can tell something of a story, but they are not meant to be persuasive documents. No one reads them for the purpose of evaluating the data to see if you’ve missed something, forgotten some important principle, or are making an invalid argument. You simply show what you have, make claims based on both the data and your ideas about it and determine if it’s:

  1. Patentable subject matter
  2. Novel work
  3. Not Obvious

Have you presented enough information such that a representative person skilled in the art can now replicate the invention(i.e. are they enabled)?

Is there an industrial application for this? Not because everything in the world needs to be commercialized, but because it is not worth the time and expense of protecting something that can’t be stolen from you in a way that you have suffered financial harm.

The last question is important because a patent is a deal between the inventor and the society. In general, the US government, at least, does not look kindly on monopolies (e.g. ma Bell). However, what a patent does is give the inventor a period of time when they can legally monopolize their invention. In exchange for this, the inventor supplies all the information one would need to recreate the invention. The public gets something and the inventor gets something. Ideas are shared, but there is still incentive to invent without sinking all your money into research and then having someone copy your work and sell it

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huh?

cheap.

What a patent doesn’t do is try to get you to believe that what someone is claiming as part of their invention is actually a real thing. Of course, it’s easier to get a patent on something that you have in hand, but this is not necessarily required.

Getting back to the point of having patents, this is why your brand name prescription drugs cost so much. For every life-saving medicine, there are hundreds, thousands, millions of other ineffective drugs that had to be tested along the way.That testing costs a lot of money. Moreover, it takes years of research to develop a drug to the point where it is reliably safe and effective to use. Why invest in that, if you can’t pay for the failures with your successes? The monopoly on the invention makes this worth it; it gives you time to recoup your investments and even make a profit.burger-labeled-2.jpg

One thing that has been interesting is learning more about what is, and what is not parentable in the US (point 1 from above). This remains an active question. Can a gene be patented? Can something like a gene be patented?

What if that gene is a naturally occurring thing? What if it is synthetic? What does it mean to be a synthetic gene? You can’t patent something that you didn’t invent. So, the general principle is that a simple DNA sequence, as it occurs in nature, is as unpatentable as is an abstract idea like algebra.

Some insight into trials that have been getting to this question…

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“Nor do we consider the patentability of DNA in which
the order of the naturally occurring nucleotides has been
altered. Scientific alteration of the genetic code presents a
different inquiry, and we express no opinion about the
application of §101 to such endeavors. We merely hold
that genes and the information they encode are not patent
eligible under §101 simply because they have been isolated
from the surrounding genetic material.
* * *
For the foregoing reasons, the judgment of the Federal
Circuit is affirmed in part and reversed in part.
It is so ordered.”
And, of course there will be dissents…
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Nor are patents the only kind of intellectual property. Trademarks and Copyrights are also protected by the same office. I don’t have to work with those, but there are those around me who do. I just have to admit that I’ve learned nothing about them yet- and may never.
And, as always, I keep asking myself… “is this system really serving the public good?” I definitely think that patent protection is important for there ever to be innovation that requires significant effort and expense. But I am still struggling with the fact that much of what we do is cut the world’s ideas into smaller and smaller pieces assigning each piece’s ownership to one group or another. As such, it is a lawyer’s game, where the rules have been made so byzantine that following them is nearly impossible without great expense.
 
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Posted by on March 25, 2016 in Uncategorized

 

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Back from the Dead

Halloween seems like a good time to resurrect old blog posts that haven’t seen the sunlight for several years. Creeping out of the tomb is my first blog post about Genes, DNA, Memes, and GMO foods. Rather than post it here, I decided to post it over on my Medium site to see if it can catch some new eyes.

Take a look: Linked Memes

 
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Posted by on October 26, 2015 in Uncategorized

 

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Fine – Octopi are awesome, I get it.

Screen Shot 2015-08-17 at 11.49.37 PMBut aliens?

Doubtful.

The Irish Examinier posts, ‘Don’t freak out, but scientists think octopuses ‘might be aliens’ after DNA study.‘ I guess this is just an eye-catching title to bring in readers for a pretty straight-forward article about how octopi are different from other animals. This article is referring to new data published in Nature following  DNA analysis of the octopus, Octopus bimaculoides.

Octopi can escape confinements, like this one that was sealed inside a jar with a screwcap:

They can move over land as well as in the water (especially when motivated by food):

They can mimic other animals:

And use camouflage to hide:

Briefly, though, I would like to take a second to look critically at the basic claim – not because it’s realistic, but because it’s useful to think about what we would or wouldn’t expect to find in a real alien.

claim:      “octopus DNA is highly rearranged – like cards shuffled and reshuffled in a pack – containing numerous so-called “jumping genes” that can leap around the genome. ”

answer: it’s  interesting that their DNA is rearranged in ways that we don’t see in other species, but it’s still DNA, right? And it still follows the same ‘universal codon’ rules dictating what codons (3 letter nucleotide sequences) call for what amino acids. That all life uses the same DNA and rules for its use is one of the most convincing pieces of evidence that all life on earth is related to one another.

claim:    Octopi have “eight prehensile arms, [a] large brain and … clever problem-solving abilities”

answer: This all just makes them interesting specimens, not alien.  Albert Einstein was extraordinarily smart and was not caught up in group-think (at least early in his career). This made his a great scientist, not an extraterrestrial.

claim:     “Analysis of 12 different tissues revealed hundreds of octopus-specific genes found in no other animal, many of them highly active in structures such as the brain, skin and suckers. ”

answer: This is actually great evidence of a large gap between octopi and other organisms, perhaps even stumbling upon new genes or gene combinations that allowed them to rapidly evolve away from homologies with their closest phylogenetic neighbors. Perhaps this phylogenetic tree might be hinting at such a separation for mollusks?

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claim:  “Hox genes – which control body plan development – cluster together in almost all animals but are scattered throughout the octopus genome. ”

answer: Pretty cool. But I’ve always wondered what kept these genes together in other animals rather than why are they scattered in the octopus. Difference is always intriguing though, so I get why this is notable. from the paper that elicited the Irish Examiner article, Albertin, et al, comes this cartoon of the arrangement of hox genes in other species compared to the scattering across several chromosomes in octopi:

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Rather than call them aliens, which I agree might grab the interest of Discovery Channel viewers, I prefer Albertin, et al’s description, “Our analysis suggests that substantial expansion of a handful of gene families, along with extensive remodelling of genome linkage and repetitive content, played a critical role in the evolution of cephalopod morphological innovations, including their large and complex nervous systems.”

 
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Posted by on August 17, 2015 in Uncategorized

 

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CRISPR: Accelerating the pace of molecular biology

CRISPR stands for Clusters of Regularly Interspaced Short Palindromic Repeats. Dr. Jennifer Doudna was one of the first researchers to see these short palindromic repeats in bacteria and archaea where she speculated that they were being used as a form of molecular immune system to protect these organisms from viruses.

Even bacteria get sick, so having a protection against invading viruses is a matter of life and death to a cell. Recall that viruses are essentially genetic material that will reproduce itself again and again after it hijacks a cell. Viruses may have protein coats or membranes to protect them outside of the cell, but inside, they are little more than DNA. If this DNA can be damaged or destroyed, then the virus is rendered harmless.

Screen Shot 2015-07-27 at 9.54.10 PMTo the right is a clip from Dr. Doudna’s video illustrating the repeated elements (in black) flanking a variety of ‘other DNA’. This ‘other DNA’ is what the cell will use to identify  foreign DNA – presumably from retaining the genomic material from an earlier exposure either in the lifetime of the single cell or its parents.

So, how does it actually work?
Two videos do an excellent job of explaining how CRISPR works. A short, simple video from MIT gives a non-technical explanation (a good place to start).

MIT’s McGovern Institute

Jennifer Doudna explains the system in greater detail…

Basically, the natural system uses two RNA molecules to target specific DNA sequences in the genome and recruit a protein that acts as an endonuclease to cleave this target:

crRNA – a ‘targeting’ molecule
tracrRNA – an adaptor RNA that recruits CAS9 to the bound crRNA
CAS9 – an endonuclease enzyme that will bind and cleave DNA once recruited by the RNAs

Doudna’s lab improved the system by combining the two RNA molecules into a single RNA that still effectively recruits CAS9 but is easier for researchers to manipulate in the lab. This last element is essential because manipulating this RNA sequence gives researchers the power to target any DNA sequence in the cell.

As stated above, the system was originally identified in prokaryotic organisms where it appears to allow targeting of the viruses that attack them. CRISPR uses ‘stored’ DNA as the targeting RNA and then brings in CAS9. CAS9 binds to the targeted DNA and cleaves it resulting in one of two possibly outcomes. 1) the virus is destroyed and is no longer a problem, 2) the virus is cut, but then repairs itself – hopefully in a way that introduced fatal mutations.

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How might this translate into clinical medicine?
The possibilities are endless, however a few low-hanging fruit present themselves immediately. Among these are therapies for sickle cell anemia (and a host of other blood disorders). Because sickle cell anemia is caused by a single base pair mutation, it is conceivable that hematopoietic (i.e. blood) stem cells can be isolated, the faulty gene repaired, and then re-introduce the corrected stem cell back into the body (possibly after the faulty stem cells have been ablated).

The newly altered and re-introduced stem cells now do the rest of the work for you by finding their place in the body where they reside while continually producing cells with the desired genetic changes.

The key is that these RNA molecules are quite simple to make exactly and in pure form (i.e. they can be manufactured chemically rather than needing cells to do the job for us and then we have to clean up all the extraneous contaminants). Most labs will design the molecules in-house and then order the constructed molecules from a ‘core lab’ that specializes in doing just that.

Jacob Corn, of UC Berkeley has compiled a simple protocol that anyone with a modicum of molecular biology training could follow. Find that protocol here.

 
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Posted by on July 27, 2015 in Uncategorized

 

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An Epidemiological Method: Using RFLP to Identify Strains of Pathogens

An excellent classroom resource for a case study in epidemiology is presented by the CDC. This study walks students through an outbreak of E. coli O157:H7 in Michigan.

The purpose of this study is to provide student investigators with the opportunity to walk through the procedures and rationale behind investigating the etiology and to develop experiments testing hypotheses generated by the students.

I am using this exercise as an end-of-semester project for my microbiology students to work through collaboratively now that we have completed our discussion of Paul Offit’s Vaccinated.

The study begins:

PART I – OUTBREAK DETECTION

 

Escherichia coli O157:H7 was first identified as a human pathogen in 1982 in the United States of America, following an outbreak of bloody diarrhea associated with contaminated hamburger meat. Sporadic infections and outbreaks have since been reported from many parts of the world, including North America, Western Europe, Australia, Asia, and Africa. Although other animals are capable of carrying and transmitting the infection, cattle are the primary reservoir for E. coli O157:H7. Implicated foods are typically those derived from cattle (e.g., beef, hamburger, raw milk); however, the infection has also been transmitted through contact with infected persons, contaminated water, and other contaminated food products.

Infection with E. coli O157:H7 is diagnosed by detecting the bacterium in the stool. Most laboratories that culture stool do not routinely test for E. coli O157:H7, but require a special request from the health care provider. Only recently has E. coli O157:H7 infection become nationally notifiable in the U.S. Outside the U.S., reporting is limited to a few but increasing number of countries.

In the last week of June 1997, the Michigan Department of Community Health (MDCH) noticed an increase in laboratory reports of E. coli O157:H7 infection. Fifty-two infections had been reported that month, compared with 18 in June of 1996. In preliminary investigations, no obvious epidemiologic linkages between the patients were found.   The increase in cases continued into July.

Students are then asked a number of introductory questions and then presented with the following problem:

Compare the DNA fingerprints in Figure 2 from seven of the Michigan E. coli O157:H7 cases. Each isolate has its own vertical lane (i.e., column). Controls appear in lanes #1, 5, and 10. Which Michigan isolates appear similar?

This question requires some background in DNA Fingerprinting (aka Restriction Fragment Length Polymorphisms, or RFLPs), which I want to take some time to explain.

As the source material states, The purpose of this test is to identify common strains of organisms through their DNA banding pattern. “Different DNA composition will result in different PFGE banding patterns. Bacteria descended from the same original parent will have virtually identical DNA and their DNA fingerprints will be indistinguishable. Identification of a cluster of isolates with the same PFGE pattern suggests that they arose from the same parent and could be from the same source. “ (emphasis mine).

The method involves two core techniques. First, DNA from the target organism must be isolated and cut with one or more restriction enzyme(s). This will create a number of DNA fragments, where the precise number and size of fragments is determined by the sequence of that organism’s DNA.

As an example, let’s imagine a 10,000 base pair (bp) chromosome that we intend to cut with the restriction enzyme, EcoRI. EcoRI recognizes and cuts double stranded DNA at a specific sequence of 6 bases.

Image

Figure: DNA cut by the Restriction Enzyme, EcoRI. A. DNA sequence with EcoRI recognition site highlighted and cut pattern illustrated. B. Enzyme binds to DNA at the recognition site. C. DNA has been cleaved.

On average, this enzyme will cut a random sequence of DNA every 4096 bases (this can be estimated by 4 raised to the power of n, where n = the number of bases in the enzyme’s recognition sequence , or 46 = 4096 in this case.) In our example, this suggests that a 10,000 bp chromosome will have two EcoRI sites by random chance.

The circular chromosome should be cut twice by this enzyme, resulting in two fragments of DNA (see note #2, below). Let’s say the two bands are 4000 bp and 6000 bp.

We can see these two fragments by running them through agarose, which works as a molecular sieve, to separate the two fragments by size

How does this work?

DNA is a negatively charged molecule with that charge spread uniformly across the length of the fragment. Therefore, there is no difference in charge between our two fragments, except in proportion to their length. This means that as they run through the sieve, the only difference between the molecules comes from their lengths. As any sieve, smaller objects go through easier, while larger ones are held up.

ImageThe result is that the two fragments will appear as distinct bands on a gel, with the smaller fragment running farther through the agarose that the larger. (here, the smaller band at the bottom of the gel has migrated farther toward the positive electrode)

If someone new were to become infected with this bacteria, we could isolate it from them, digest the DNA and get the same banding pattern. A closely related bacteria may have one additional EcoRI site. This would result in one of the two bands being cut into two smaller fragments, meaning that the two strains could be easily distinguished.

Back to the question posed above…

Given this, examine the following compilation of samples. Controls appear in lanes #1, 5, and 10. Which of the remaining isolates appear similar?

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Definitions:

  1. Restriction Enzyme or Restriction Endonuclease– an enzyme that can recognize and cut DNA.
  2. Recognition Sequence – the sequence of bases that a restriction enzyme recognizes and binds to.

 

Notes:

  1. In my example, we are using the restriction enzyme, EcoRI, to cut DNA from E. coli. As the name suggests, EcoRI actually derives from E.coli, where it functions as a defence against invading DNA, i.e. a virus. In order to do this successfully, E. coli will either not have any EcoRI restriction sites in its own DNA, or it will protect them by methylation so that the enzyme does not destroy the host’s own DNA. I am ignoring the possibility that the DNA we are dealing with in our experiment may not be cleavable with this enzyme.
  2. Also note, that bacterial chromosomes are circular, rather than linear – interestingly, this means that they are not actually ‘chromosomes’ at all. Again, let’s ignore this.
 
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Posted by on April 18, 2014 in Uncategorized

 

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Your Inner Fish Crawls off the Page

ImageI’ve been assigning Neil Shubin’s Your Inner Fish as a reading and discussion assignment in my General Biology classes for several years now. I believe that it’s a good introduction to understanding how the process of science works in the real world, it does a good job communicating the methods and findings of a number of complex experiments, and it also walks through the history of ideas and how new information changed these ideas over time.

If I can get students to think about all these things and perhaps do a little extra digging (into the research), then I’ve down my job.

Episode I of the adaptation of this book  just aired this week and I was very impressed by the way the material was put together- refining the story from the book a little- and coming up with a standard documentary supported by computer graphics that really add to the story rather than looking tacky of fake. In fact, I think the graphics really transform the material into a living experience.

The story is told in two converging arcs. In one, we follow Shubin’s field work, where he decided that he was interested in finding the remains of one of the earliest organisms to crawl out of the water and establish terrestrial life. Prior work suggested that the earliest tetrapod ancestor on land emerged from the Devonian Seas about 370 Million Years Ago. Shubin and colleagues identified an ancient river delta of about this age in the Canadian Arctic and set out to locate some fossils.

ImageThe other story walks us through the idea of relationship with other life on Earth. What suggests this relationship? What evidence is there for it? How long does it go back?

As I said above, I have liked this adaptation very much so far and I am already planning to bring at least parts of this video into my classroom to supplement our discussions.

More on this later…

 
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Posted by on April 10, 2014 in Uncategorized

 

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A quick look at mRNA Splice Variants

-Beadle and Tatum Redux

-Beadle and Tatum Redux

In my microbiology class this past week, we were discussing how prokaryotes and eukaryotes differ in their handling of DNA, RNA and gene regulation. Mostly, we focused on how the presence of the nuclear membrane in eukaryotes separates the processes of transcription and translation and what this results in. Briefly, bacteria are prokaryotic life forms that lack a defined nucleus (among other differences). Because of this, when bacteria transcribe mRNA, it is immediately available for translation – the DNA, RNA polymerase and Ribosome all exist in shared space. Below is a classic image of a strand of DNA(stretching left to right) in E. coli being transcribed into RNA. The RNA molecules extend away from the DNA and appear to travel up or down away from the DNA in this micrograph. Along the length of the RNA, we see dense ribosomes which are busy synthesizing proteins.

Image

In Eukaryotes, the nucleus encases the DNA, the RNA polymerases and mRNA. mRNA can be completely synthesized and modified in a number of ways before they are exported from the nucleus to the cytoplasm, where ribosomes will translate the message into protein.

One of the modifications of Eukaryotic mRNA we spoke about was splicing. Splicing is a means of snipping segments of non-coding introns out of the mRNA leaving a mature mRNA with a continuous strand of exons. One interesting possibility this enables is the production of alternative sequences made from differential splicing of the immature mRNA. These alternative mRNAs are known as splice variants. At this point, I was asked for an example of a gene that is handled in this way and was caught flat-footed.

Hmm. Perhaps this is something that I’d heard so much about in classes but never in the ‘real world’. I’ll have to look.

One of the first things I found was this discussion of splice variants suggesting that this was not a biologically significant event. i.e., the RNA may be alternatively spliced, but do these splice variants actually result in functional proteins with different properties. The author poses a challenge to find examples of splice variants that are ‘real’. The ensuing discussion is a good one.

What would this looks like?

Image

Regardless, I found a paper with some good figures that may help students understand how this phenomenon (at least putatively) occurs.  Here’s the best figure presenting a diagram of the different mRNAs created and gels and sequence data indicating that these exist.

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The above Figure shows the presence of distinct RNA species, although that, alone, does not mean that these RNA are ever made into protein. To do that, western blots of protein extracted from various tissues is shown below.

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What is left to find is whether each of these two proteins actually does something. Are both forms required? Are their functions distinguishable?
My quick look through the literature did not uncover any evidence for this last question. If anyone out there knows the literature on this, I would love a push in the right direction. It doesn’t matter what gene we’re looking at, just that it is an example of alternative splicing and that each of the splice variants is actually made and has some identifiable and distinct function.
 
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Posted by on April 4, 2014 in Uncategorized

 

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More on the Lac Operon

A while ago I wrote two posts about the Lac Operon here. The first pointed to an animation by McGraw Hill Publishers that did a pretty good job illustrating how the operon works. In the second post, I highlighted the notion of polycistronic messages (more than one gene per mRNA molecule) and how this allows for control of a number of related genes at once – a trait not shared by eukaryotic cells. In that second post, I also finished with a graph of how cells grow in the presence of glucose and lactose.

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Cell Growth in the presence of glucose + lactose – As glucose is depleted, cells adjust to lactose digestion

One feature of that graph (reproduced here) that is notable is a little bump in the growth rate as glucose runs out and the cell converts to lactose digestion. A second important feature is that the rate of growth slows when the cell is burning lactose as its primary fuel.

 

Together, these features suggest that the cell is regulating lactose digestion very closely. In fact, there are two primary mechanisms of this regulation to appreciate. The first is that the lactose-digesting enzymes are controlled together on an operon that is regulated by lactose itself (or at least we can assume so for simplicity’s sake). In the absence of lactose, no lactase enzymes are made and no lactose is used as fuel. The reason for this is obvious when you look at the slope of cell growth under glucose metabolism (left) and lactose metabolism (right). Clearly, growth is SLOWER when lactose is used as fuel.

Therefore, so long as there is glucose, it is pointless to digest lactose at the same time. So it is best to only turn on the lac operon in the ABSENCE of glucose – regardless of whether lactose is present of not.

If glucose is absent and lactose is absent, turning on lactase enzymes is still useless. However, slow growth is better than no growth. So we should have a mechanism to turn on the operon when there is lactose in the environment.

Here’s a matrix of ideal regulation:

Screen Shot 2014-03-31 at 2.18.05 PM

How can a little, mindless bacteria achieve this exquisite control?

Simple: By using two regulators. One for glucose and one for lactose. Only when both conditions (glucose-, lactose+) are met do we make lactase.

Structure of the Lac Operon

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First, lactose itself serves as an inducer. In the absence of lactose, a regulator protein binds to a DNA site between the polymerase binding site (the promoter) and the structural genes (the enzymes). When the regulator binds, its presence physically prevents the progress of RNA Polymerase.

When lactose is present, it binds the repressor protein in a way that causes its shape to change in a way that can no longer bind the DNA. The repressor then drifts away from its binding site allowing RNA Polymerase a clear shot to the structural genes.

operon2

However, RNA Polymerase is not always parked on the promoter waiting for the repressor to be removed. Its binding requires another protein to help stabilize its interaction with the DNA. This second protein is the CAP protein. The Catabolite Activated Protein. However, CAP alone will not bind either. It requires a signaling molecule called cyclic AMP (cAMP). cAMP is readily broken down when glucose is in the cell, so it only accumulates when glucose is absent. In that case, cAMP accumulates and binds to the CAP protein, which then binds to the CAP site. This site is located adjacent to the promoter, but on the side away from the structural genes. When CAP binds, it assists in recruiting the RNA polymerase to the promoter.

operon3

Therefore, if only one condition is met, it is insufficient to promote gene transcription. Only when the CAP+ cAMP protein is bound will the Polymerase be recruited. And only when lactose is present, will the repressor protein let the Polymerase pass.

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In terms of the matrix we set forth above, we can see that these molecular interactions result in exactly the regulation that is optimal:

post-operon

 

 
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Posted by on March 31, 2014 in Uncategorized

 

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A quick followup on that lac operon post

Last week I posted a quick link about operons for my micro class to check out before taking their quiz on bacterial gene regulation This post is intended to complement that one. To go back to that post, click here. If there’s one thing to remember about operons it is that bacteria, lacking a nuclear membrane, regulate their genes differently than Eukaryotes. Having a nuclear membrane separates transcription and translation into two distinct compartments allowing for more subtle tweaking of Eukaryotic mRNAs before they are exported for translation.

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Click on this figure to go to a good description of how polycistronic genes work

One thing this does is it makes it very beneficial to package genes with related function closely on the genome and use a single regulatory region to control them all together. They wind up getting packed so closely together that they are actually expressed as a single messenger RNA – known as a polycistronic (meaning ‘many gene’) message.

Upstream of this polycistronic cassette are regulatory elements. One element common to all regulatory elements is the promoter. The promoter consists of several elements which ‘promote’ the binding of an RNA polymerase to the DNA. Additional regulatory elements exist to ensure that this polymerase only transcribes the genes if they are needed. In doing so, the cell conserves energy and components (e.g. Amino Acids) for only necessary processes.

In the case of the paradigm lac operon, lactose is a fuel source, but not as good as glucose. Therefore, enzymes to digest lactose are only needed when lactose is present, but glucose is not. In order to interrogate both conditions, two additional regulatory elements are present.

First, the operator sequence. This sequence binds a repressor protein that physically blocks the polymerase’s path in the absence of lactose. However, if lactose is present, the sugar binds to the repressor, causing a conformational (shape) change that causes the protein to release its grip on the operator sequence.

Second, a catabolite activator protein (CAP) will only bind to the DNA behind the RNA polymerase if cAMP is present. Let’s not get too distracted, other than to say that cAMP levels are high in the ABSENCE of glucose, and low when that sugar is present. When cAMP binds to the CAP protein it can now bind the DNA and do it’s other job: making a nice binding site for the RNA polymerase. Without CAP, the polymerase binds very inefficiently.

Together, the production of lactase enzymes (those that digest lactose) is exquisitely controlled in a way that conserves the most energy.

ImagePs – take a look at this graph and tell me why (not mechanistically, but rationally) the cell does not make lactase enzymes when both glucose and lactose are present.

 
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Posted by on March 15, 2014 in Uncategorized

 

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