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I'm having a great deal of trouble understanding chromosomal walking, especially in regards to this article:

Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.

I can get the fact that you can use recombination frequency analysis with chromosomal markers to assess the relative position of a gene, but I'm just stumped as to what it exactly it means when they say that they cloned the gene using DNA segments flanking the two insertions (fru3 and fru4) as a basis for the chromosomal walk.

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How to perform a chromosomal walk

I believe that the first publication to describe this approach was Bender et al., Science, 1983. In that paper they explain how they "walked" along the chromosome to reach the Bithorax Complex (BX-C).

This approach requires two reagents:

  1. a cloned DNA fragment that you have mapped to a defined genetic interval, and possibly to a cytogenetic interval as well (this is only possible if the organism in question has cytogenetic tools).

  2. a genomic library of larger cloned DNA fragments.

Step 1. Use the DNA fragment to screen the library and isolate genomic clones.

Step 2. Purify each genomic clone and prepare enough DNA to construct a restriction map of the foreign insert (N.B., Bender et al., actually used heteroduplex mapping--an EM-based technique--as well) in each clone.

Step 3. Isolate a suitable DNA fragment, devoid of repetitive DNA, from one end of the clone that extends the farthest along the genome in the direction in which you wish to "walk".

Step 4. Congratulations, you have taken one "step." Now use the new DNA fragment as a probe and screen the genomic library again. Each time you screen the library you will recover all of the genomic clones that hybridize, or overlap, with the fragment you are using as a probe.

Step 5. --> go back to Step 2. above.

Each time you take another "step" you are isolating cloned DNA that is taking you closer to your target (the gene of interest). For the very first step you will not know which clone is extending in the desired direction (typically) so you need some type of complementary assay to decide which end of the overlapping clones' restriction map is the direction you want to pursue. In other words, you need to align the physical map to the genetic map. In Drosophila one can use in situ hybridization to polytene chromosome squashes to visualize where the genomic clones are derived from in the genome, and this lets you pick the correct clone at that first step.

Periodically during the walk you will need to test the new genomic clones by in situ to find out when you are getting close to your target (or if you have even walked past your destination).

Ideally you will have some chromosomal rearrangements that alter the physical and genetic maps, and let you unambiguously determine when the chromosomal walk has reached, or entered, the locus. Alternatively, a strong loss-of-function mutation in your target gene might be a deletion or deficiency that causes a RFLP, and isolating genomic DNA from such a mutant will let you use a Southern Blot to confirm that you have reached the gene.

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  • $\begingroup$ Thanks for your answer, but can you also explain what it means when they write that they used DNA flanking the fru4 and fru3 insertion alleles as starting points for the walk? Does it mean that these DNA segments had already been isolated and sequenced by others beforehand? $\endgroup$ – Inhibitor Jan 7 '16 at 7:15
  • $\begingroup$ I have not read the entire methods section of the paper you cited, but presumably further specific details are in there. The authors state that fru4 and fru3 are independent insertions of the P-element transposon. As described by Gerry Rubin (I think it is Bingham et al., Cell, 1982), if you have a cloned DNA fragment that hybridizes to a transposable element, then all you need to do is construct a genomic library from a strain carrying the insert and probe the library to isolate clones containing P-elements. One of those clones contains the fragment(s) used to start the walk. $\endgroup$ – mdperry Jan 8 '16 at 2:52
  • $\begingroup$ Okay, a more detailed scan reveals several more germane facts. Figure 1 is your friend. When coupled with the methods section "Molecular Cloning" you have all of the facts at hand. Their task was even simpler than I described because all four of the P-element insertions they describe, fru1, fru2, fru3, and fru4 are loss-of-function alleles of the fruitless gene, so they knew they had knock-outs. They were not walking to they gene, they were walking to recover all of the gene--it is huge (compared to an average fly gene). $\endgroup$ – mdperry Jan 8 '16 at 3:03
  • $\begingroup$ Thanks! Now it all starts to make sense. I have one more question though, if you don't mind. In the "Molecular Cloning" section they say that "The positions of the fru mutants were determined by Southern analysis and in situ hybridization to salivary gland chromosomes in the case of the fru1 inversion and the Df(3R)P14 and Df(3R)ChaM5 deficiency breakpoints." - I don't quite understand how they did this and how they mapped it. Did they make a genomic library of the particular mutant and probe it or something? $\endgroup$ – Inhibitor Jan 8 '16 at 6:12

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