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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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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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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.

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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?

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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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Codon Usage Bias – Part I

To the molecular biologists:

Optimize ye codons while ye may

For time is a-flying

And this clone you have in R & D today

Tomorrow will be … in manufacturing- and it’s just impossible to change anything at that point, so forget it.

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I”m no rocket surgeon

 Codon Usage Bias – Part I

I read an article yesterday about codon bias that has been stuck in my head ever since. The article, appeared as a ‘Perspectives’ piece in the 13 Dec 2014 issue of Science, with the title, ‘The Hidden Codes That Shape Protein Evolution.’

This article addresses some details not often considered in how DNA directs the synthesis of proteins.

 I spend a lot of time in my classes discussing the basic mechanism by which DNA –>RNA –> Protein, known as the Central Dogma. A lot gets left out of these lectures in order to keep it simple, which sometimes keeps the way I think about the flow of information pretty simplified as well.

Fortunately, this article rattled my cage enough to open my mind to the myriad influences that go into the stuff of life. Here, Weatheritt and Babu, look at how DNA sequences may be under selective pressures independent of just the proteins they encode.

I’ve done a fair amount of molecular biology in my life, including cloning genes and moving them into other organisms for expression as drugs or drug components. One example of this was in a lab where we used live-vectors as immunogens in order to take advantage of the uniquely broad immune response this single-cell pathogen elicits. The immune responses we wanted to trigger / amplify were typically against human tumor proteins or the products of human viruses (e.g. HIV, HPV), however the organism we were using as a vaccine was a bacteria.

As I said above, I usually teach the Central Dogma in a way that omits many of the complications seen in the real world. So, when we look at a codon chart, we see the redundancy (multiple DNA codons make the same amino acid) to illustrate how a change in the DNA sequence can often fail to change the protein sequence at all. These are called ‘silent mutations’.

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The way these codon charts work is by triangulating a position in the middle of the chart using the bases depicted along three of the four edges. For example, the codon AUG is read by locating the ‘A’ on the left margin, the ‘U’ on the top margin, and the ‘G’ on the right margin. The location this identifies is an amino acid called Methionine (abbreviated as met) on this chart.

Notice that if the first two bases in a codon are CU, then it does not matter what the third base is, no matter what, this codon will call for a Leucine (leu).. This means is the sequence of RNA is CUU originally, but mutates to CUC, there will be no change in the protein.

What codon optimization addresses is the fact that different organisms tend to prefer some codons over others, even if they encode the same amino acid. This has been appreciated for many years now so when a molecular biologist takes a protein (e.g. from a human tumor) that they want made by bacteria and they redesign the DNA sequence in a way that codon preferences are maximized in the organism that will express the protein.

This figure examines the percentage of times a gene uses a particular codon to make Leucine. In the bacteria, E. coli, CTG is used nearly 50% of the time. Meanwhile, in the yeast, S. cerevisea, TTA and TTG are preferred.

 (Note that T in DNA = U in RNA)

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————

So, what does this mean? Consider a simplified example…

I want to clone this protein from a yeast and grow it up in bacteria:

Met – Leu – Leu   [stop]

ATG- TTA – TTG – TAG

 We would take this DNA from the yeast and then modify the sequence by changing the two Leucine codons into the preferred sequence in bacteria (CTG):

Met – Leu – Leu   [stop]

ATG- CTG – CTG – TAG

The result should be a sequence of DNA that the bacteria will be able to optimally translate into protein. 

———

This has worked out to be much longer and more technical than I intended – and I haven’t even addressed the new ideas brought up in the Science article.

Therefore, I’m going to stop here and continue tomorrow with part II

 
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Posted by on February 9, 2014 in Uncategorized

 

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eteRNA RNA folding game

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Proper Folding Achieves Lowest Energy Conformation

My general biology students were asked yesterday to check out eteRNA, an interesting online game designed around problems of RNA folding. RNA is a fascinating molecule for a number of reasons.

  • What we might think of immediately, mRNA as an information carrying molecule, is just one of its many jobs.
  • In addition to this, RNA serves as a delivery molecule in the form of tRNA. These molecules are capable of both ‘reading’ the mRNA message, through codon:anti-codon interactions, and they also recruit and deliver Amino Acids that correspond to the codon in question. 
  • RNA also functions as ribozymes, enzymes comprised entirely, or in part, of RNA. An example of this is the ribosome that is mostly rRNA with only a small protein contribution.

EteRNA explores the plasticity of RNA function by demonstrating the capacity of this molecule to fold into a variety of shapes. As I frequently remind my class, FORM DICTATES FUNCTION. This is true of all things in biology (and perhaps beyond). When a molecule is formed correctly, it carries out its function appropriately. When that form is altered due to mutation, misfiling or other denaturing processes, the function is also altered. This may be for better or worse, but mostly for worse.  Depending on this function, the ‘fitness’ of the cell for survival / reproduction may be affected leading to selection for or against this cell.

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Eterna

I set up a group titled: FortScott_Treml that I invite my students (or anyone else) to join. For my students, anyone who completes the the tutorials will receive 5 extra credit points. Anyone who earns a puzzle master badge will earn another 5.

 
 
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Posted by on November 13, 2013 in Uncategorized

 

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