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I am a bit confused about what targeted gene disruption means. I was reading this article in which they compare the Pyrococcus Furiosus genome with a genetically tractable strain P. Furiosus variant genome, called COM1. When they described how COM1 strain was obtained they say:

The COM1 strain was obtained by targeted gene disruption of the pyrF locus (PF1114) using a plasmid designed for double-crossover recombination (37).

Since the definition from Springer about targeted gene disruption is simply:

Experimental replacement of an endogenous gene by a mutated (non‐functional) version within the otherwise unchanged genome is called targeted gene disruption.

I concluded that it is the replacement of the gene, that is called pyrF in this case, by the experimenter with a non-functional version within the genome of the genetically tractable P. Furiosus variant. So I thought it was a sort of "inactivation" of a gene.

Then, in this discussion about What is the relationship between a “main” strain genome and its variant genome in archaea?, someone kindly suggested me to read the original paper about how the COM1 strain was discovered. Here I find some contradictions with the idea that I built about "targeted gene disruption of the pyrF locus".

In this paper, they say that

In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA.

Then also that:

there have been no reports of genetic manipulation of the P. furiosus chromosome. Here we used simvastatin and 5-FOA selections to generate a ΔpyrF strain, designated COM1

And, finally, an other important extract:

Here we report the construction of a deletion of the pyrF locus in the P. furiosus chromosome and the discovery that this strain, designated COM1, is remarkably efficient and naturally competent for uptake of both circular DNA and linear DNA.

I have to say that this article was too difficult for me and full of details that I am not interested in, so I did not read it in deep, but I selected these extracts that describe in simple words how COM1 was obtained. However, from these I get an idea of how COM1 is obtained that is different from the one I thought at the beginning by Springer.

Since the Wikipedia definition of gene deletion mutant is:

In genetics, a deletion (also called gene deletion, deficiency, or deletion mutation) (sign: Δ) is a mutation (a genetic aberration) in which a part of a chromosome or a sequence of DNA is left out during DNA replication. Any number of nucleotides can be deleted, from a single base to an entire piece of chromosome.

Considering this and the other two extracts that I reported, now my idea of how COM1 was obtained is that the pyrF was deleted (by the scientists) and that part was (naturally) substituted by a marker (that is "a DNA sequence with a known physical location on a chromosome") which is a linear DNA since this COM1 strain is remarkably efficient and naturally competent for uptake of both circular DNA and linear DNA.

Now my question is: is the Springer definition (linked above) "universal" or by targeted gene disruption one can intend not only "artificial" genetic manipulation but also deletion of a gene and natural replacement that could also lead to a new pyrF gene (in this case) that is functional (so not non-functional) ? What does it mean when I say "The COM1 strain was obtained by targeted gene disruption of the pyrF locus (PF1114)"?

I apologize for the long question and I thank you in advance.

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You have it mostly right, however, you are misunderstanding what has gone on here a little.

The definition from Springer is correct - a targeted gene disruption is a direct replacement of the gene with a non-functional one. However, to do so you also need some method of detecting that your gene disruption worked and will persist. This is most commonly done by introducing marker(s) along with the disrupted gene in a form known as a cassette.

In the case that you present it seems that they have used simvastatin as a selection agent and 5-fluroorotic acid (5-FOA) as a reporter to show that while the gene has been disrupted (i.e. non-functional), expression from that location is still happening as it should.

The method they used for introducing this cassette is a recombination between a plasmid and the Pyrococcus genome. This is (or was) a common method of altering the genome of many organisms to effect some functional change. The plasmid will have sites flanking the cassette that match the host (in this case Pyrococcus genome) and these are used to get the genome to recombine in the manner described in the pictures here.

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  • $\begingroup$ Thank you so much @bob1 ! $\endgroup$
    – Manuela
    Jun 14 at 10:12

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