Wow. I step away for a while, then return to a thread I thought wasn't getting enough attention only to find it has blown up to the point half the responses are from Ambassadors and Administrators!! More has happened here since I've been gone than I can process without some further rumination time, but I appreciate the discussions and dialogue, especially those who are saying that "viruses don't exist" need to step back from the edge for a while.
For the moment, I will simply lay out my own direct observations based on experiments that I personally performed that proved to me that viruses, or some other otherwise unknown phenomenon of DNA exchange of information involving some conglomeration of DNA and proteins, are real. For background, I worked on a doctoral-level project that involved the isolation and characterization of a virus which included isolation and sequencing of DNA and proving that transfer of DNA was occurring between bacterial species by means that could only be explained by current viral transduction theories as we currently understand them.
(Disclaimer: I did not receive a doctorate, only a Master's degree - because the British technician I was collaborating with at Los Alamos National Laboratories had to leave because her visa expired, but only after a fire that summer (1999 I believe) razed a large area there forcing her egress and because the DNA I provided her was not of sufficient purity to do her work properly due to problems I had with the ultracentrifuges, which I did not solve in time. I needed that sequence information in order to publish my characterization findings in a journal. Had I been successful, I would have had a virus known simply as "TP21" renamed after me as "KetoneCopperii". Sigh.)
The subject of my thesis was a bacterium known as
Bacillus thuringiensis, subspecies
kurstaki, which harbors a lysogenic bacteriophage named "TP21" (for "
Thuringensis Phage #21). This bacterium is one of three in the Bacillus anthracis family, which also includes
Bacillus cereus. A lysogenic bacteriophage is a virus that usually maintains its DNA within its host organism as a separate entity in a plasmidic state within the bacterium it inhabits; with a "plasmid" simply being a circular piece of extrachromosomal DNA. When the bacteria divides, it produces copies of the bacteriophage plasmid along with its other plasmids and chromosome.
For the argument I'm going to make here, this information is critical. Some of it is gong to be very specific and scientific. Pay attention! Because stupid comments/questions are now being taxed at $50 here apparently after reading the latest C's session. I personally don't think that fine is steep enough, but whatever.
The goal of my project was to isolate the DNA of the TP21 virus/plasmid, characterize it along with the means of viral transmission involved, and then alter or add DNA information that could potentially aid the virus to both infect the closely related bacterial species
Bacillus anthracis and deliver a genetic payload that would shut down the production of a certain anthrax toxin in vivo. ( Background information:
Sequence and Organization of pXO1, the Large Bacillus anthracis Plasmid Harboring the Anthrax Toxin Genes )
This is going to get technical. Just a warning!
So, what I did first was isolate the DNA plasmid in
Bacillus thuringiensis kurstaki using gels (the DNA band containing the plasmid was quite distinct from the other plasmids and chromosomal DNA present within
B. thuringiensis) and further characterized it by restriction enzyme analysis. Each enzyme - and I used at least 20 - cuts DNA at very specific short DNA base pair sequences which usually only occur at a few specific points within any given unique DNA strand, which is a way to map a piece of DNA to get its general layout, structure, and size and to generate markers that you can point to in a separation gel to say, "that is a viral DNA fragment".
(TECHNICAL - avoid unless you're a masochist: A separation gel is an agar gel that you can add DNA to and use electric charge to move DNA through, with large pieces moving much slower than small pieces due to resistance, and a standard using many different but exactly sized pieces of DNA is also run so you can determine the sizes of each band of DNA. When analyzing a specific piece of DNA, using one restriction enzyme, you should see several pieces repeatedly that when taken as a whole add up to the entire length of the piece of DNA you are analyzing, with each band representing a site where that restriction enzyme DNA marker exists. When done in concert with other enzymes, you can thus generate a restriction enzyme map of any piece of DNA, and from that information, define its structure and from that point forward use that information to determine that yes, this DNA piece is from this virus, because it's replicatable.)
After doing that, I then treated a growing culture of
Bacillus thuringiensis kurstaki with Mitomycin C (
https://www.sciencedirect.com/topics/chemistry/mitomycin-c ) , which elicited a response whereby the bacteria started producing some kind of DNA/protein product which I could isolate using ultracentrifugation at very high speeds. Taking that resulting pellet, I treated it with proteases to destroy protein structures that contained the viral DNA (if I didn't do that step first, I didn't get DNA - hard lesson learned), then I removed the proteins and crystallized the freed DNA using a cold alcohol process if I remember correctly. After isolating the DNA, I treated it with the same restriction enzymes that I treated the plasmid DNA with, and I saw the exact same size fragments in the gels as I saw in the treated plasmid DNA. (An aside: had I done this first, then sent it to the tech in Los Alamos, I may have a doctorate today because this DNA was CLEAN of any other cellular DNA fragments. Alas...)
Not only that...my restriction enzyme analysis proved that these DNA particles were circularly permuted and terminally redundant (
Chromosome Structure in Phage T4, III. Terminal Redundancy and Length Determination on JSTOR ) - which means, each DNA particle had some extra genes present on it, but not the SAME genes on each DNA fragment. Some process involved in producing these DNA particles was creating fragments that had at least 20% repetition, which matched the previously observed phenomenon that some kind of viral production mechanism was stuffing viral head proteins with a bit more DNA than they could hold, based on the fact that the DNA was being created using some kind of rolling toilet paper mechanism, to be...illustrative.
So, SOMETHING was creating viral DNA genomes and stuffing it into some kind of protein container that held more than a single genome of DNA. That was good news to me, because it meant that if I could indeed create a genetic construct that could be incorporated within this virus to shut down toxin production in
Bacillus anthracis, then I had room on the DNA strand being shoved into the capsids to hold my genetic construct! Get out the champagne!!
After that, my next step was to try to find a mutation of TP21 that could infect
Bacillus anthracis while the work on creating the gene product to stop toxin production continued. On that front, I did find that it was possible to infect some strains of anthrax with TP21 based on agar plate studies, but I could never grow one of those colonies enough to try to isolate TP21 DNA from any of them. That said, all of this happened using isolates I obtained from
Bacillus thuringiesis kurstaki, and only after processing ultracentrifugation products of
B. thuringiensis that had been subjected to Mytomycin C treatment; and the DNA I recovered from those pellets matched that from
B. thuringiensis plasmids that had not been treated with Mitomycin C directly. So, some process was causing that plasmid DNA to be replicated and placed into some kind of protein container that I had to personally liberate from before confirming by restriction analysis that it was, indeed, the same DNA sequence; and when I used some of that pellet to infect other strains of Bacillus, I saw evidence on the agar plates I was using that whatever was in those pellets did, indeed, infect them. My control plates did not produce the same results.
So, THERE.
) times more to understand the details…
