"Homozygosing" and chromosome mapping
Use of the cross with yw; Sp/SM6a; Gl/TM3.

First, the following definitions are needed to follow the presentation:

p(w+) = transposed insert, ? p(w+) means we do not know which chromosome the insert is located on.

¡ = single virgin female, ¢ = single male

¡¡ = multiple virgin females, ¢¢ = multiple males

Sp = "Sternaplural", multiple bristles on the sternum near the leg, 2nd chromosome.

Gl = "Glued" eyes, distorted and sparkly appearing eyes, 3rd chromosome.

SM6a = 2nd chromosome balancer, Cy marker (Curly wings)

TM3 = 3rd chromosome balancer, Sb marker (Stubble bristles)

w = the white gene which normally resides on the X chromosome

y = the yellow gene which gives the light body color resides on the X chromosome


Cross 1:
¢¢  yw; p(w+)/+ X ¡¡ w/w; Sp/SM6a; Gl/TM3

If there are no w+ males among the offspring, the insert is on the X chromosome. There is no need to go farther with these flies. You can establish homozygous lines using single pair matings of flies derived from the interse stock. Note that a fly line carrying the insert on the X chromosome can be extremely useful for generating additional transformed lines. The insert can be mobilized using a dysgenic cross, and new lines resulting from movement of the insert from the X to an autosome can be easily identified by screening for lines that have lost the sex linkage. In general, X-linked inserts should not be used for evaluating promoter activity since the level of expression can be influenced by dosage compensation in males.

Note that the balancer stock lacks the yellow mutation (y) so the flies will have a darker body color than the injection strain. There is no reason to suspect that this will affect the hsp70 promoter so we have not taken the time to introduce y into the balancer stock. This should be easy to do and would be a good side project so that this inconsistency in cross 1 can be eliminated.

If there are w+ males among the offspring, the insert is on an autosome. Then from the offspring of cross 1, three separate crosses are set up:


Cross 2A:
¢¢  w; ?p(w+)/SM6a; ?p(w+)/TM3 X ¡¡ w/yw; ?p(w+)/SM6a; ?p(w+)/TM3
(Curly, Stubble X Curly, Stubble)

Cross 2B:

¢¢  w; ?p(w+)/SM6a; ?p(w+)/Gl  X ¡¡ yw/yw
(Curly X yellow, white)

Cross 2C:

¢¢  w; ?p(w+)/Sp; ?p(w+)/TM3 X ¡¡ yw/yw
(Stubble X yellow, white)

Obviously, the males in crosses 2A, 2B, and 2C should be w+ (colored eyes)


From the offspring of Cross 2A, establish a homozygous line by crossing straight-winged, long-bristled, colored eyed flies as follows:

Cross 3A:

¢¢  w; ?p(w+)/?p(w+); ?p(w+)/?p(w+) X ¡¡ yw/w; ?p(w+)/?p(w+); ?p(w+)/?p(w+)

As a back-up, I recommend setting up a vial of ¢¢  CySb X ¡¡ CySb (Cross 3B: Curly, Stubble X Curly Stubble). This vial should produce Cy+Sb+ flies that could be used to repeat Cross 3A should the first attempt fail. If you still fail to get Cy+Sb+ flies, there is a good chance that the p(w+) is homozygous lethal.

The sequence of Cross 1 and 2A render the 2nd and 3rd chromosomes homozygous. It remains to be determined from Crosses 2B and 2C which chromosome the insert is on. Consequently, ?p(w+) appears for both the 2nd and 3rd chromosomes in the above description.

If Cross 2B fails to produce any colored-eyed, curly-winged flies, then the insert is on the 2nd chromosome. If Cross 2C fails to produce any colored-eyed, stubble-bristled flies, then the insert is on the 3rd chromosome. The results of Crosses 2B and 2C should be consistent with each other. If not, there might be multiple inserts or the insert might be on the 4th chromosome. The existence of multiple inserts should become apparent from the Southern blot.