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Lecture 5 Next time, spend less time on the lecture below.

Benzer did a number of neat things. He asked what is the size of a mutant

M₁ muton 1

M₂ muton 2

M₃ muton 3

<=====> A (Rate of recombination between mutons 1 & 2)

<=====> B (Rate of recombination between mutons 2 & 3)

<==============> C (Rate of recombination between mutons 1 & 3)

Size of Muton 2 in recombinational units is C - (A + B).

This would be the max size of a muton. It could be smaller and one would really never know the true size of the smallest muton. However, the best estimate of the smallest size would require measurement of many muton sizes. It turns out that there is an experimental complication that precluded pushing this type of analysis: the rate of recombination is not to be exactly proportioned to nucleotide distance. Thus numbers are not accurate enough to make definitive arguments.

Another way to estimate the smallest size of a muton is to ask how many mutants one could put in distance C. Again this is an upper distance.

How small can a recon be? What is the smallest non zero recomb number can one observe. Need non-reverting mutants for this since this is measured on a background. In all of these analyses, needs lots mutants. Need them close together.

Benzer used deletions (These do not revert and do not recombine with three mutants, each of which will recombine with each other.)

Suppose you want a group of mutants that are close together. What Benzer did was take those mutants that would not recombine with a particular mutant but would recombine with themselves:

A X B -- can get w.t. but A X G -- no wild type

A X C -- can get w.t. B X G -- no wild type

A X D -- can get w.t. C X G -- no wild type

A X E -- can get w.t. D X G -- no wild type

A X F -- can get w.t. etc. E X G -- no wild type

B X C -- can get w.t. F X G -- no wild type

B X D -- can get w.t.

etc.

This would place A through F in an area that is included in the deletion covered by G.

How does one define a deletion.

Will not recombine with mutants which themselves will recombine. (worry about double mutants and inversions)
Will not revert or back mutate to wildtype.

Advantages of deletions:

1.) Do not revert. Hence, the presence of wildtypes arising from a cross of two deletions proves recombination. With point mutants, there is a background of gene reversion which can complicate interpretation of recombination especially when it occurs at very low frequency. This is a particular problem when one is determining the minimum size of a recon and one is studying recombination between two very close mutants.

2.) Have a finite size. This is particularly important when one wants a number of mutants in a very small region. Without deletions, one must cross all mutants in all combinations (n {n-1}/2) and sort out those that are close. With deletions, one can pick one deletion and determine how many of the mutants fall within the region covered by the deletion with just n crosses (n = the number of mutants).

3. Can be used to determine whether the gene is linear, as described below.

Benzer argued that if the gene is linear and if one lined up deletions in proper or dictionary order, as drawn below, once one found a combination that gave rise to recombinants, then all other crosses involving deletions further down stream must give rise to recombinants.

Once one found that mutant 1 and 3 recombined, then 1 and 4 had to. True also with 1 and 5. This would work only if the gene is linear. Benzer did this experiment with 147 deletions and the data fit with the idea that the gene is linear.

	1	2	3	4	5	going to the left or down from the diagonal once one finds a + one could never find a 0.
1	0	0	+	+	+
2	0	0	0	+	+
3	+	0	0	0	+
4	+	+	0	0	0
5	+	+	+	0	0

In the assigned reading, Benzer summarizes similar results but with hundreds of mutants. Be familiar with these. Understand this because it makes great exam questions!!!