In article <Dovn60.I26 at udcf.gla.ac.uk>, gbga14 at ucdf.gla.ac.uk wrote:
> Can anyone suggest an unc that has been cloned that would be
> good to use as a marker (by rescue) for constructing free arrays.
> Ideally I want an unc that can be scored during most stages
> and in a double background with a relatively strong dpy.
> Obviously it would also be good if the wild type cloned
> gene was relatively small and available on a plasmid
> rather than a cosmid. One final requirement -
> temperature sensitivity would prove problematic. (One that sings,
> dances and pours petri dishes would also be good!)
The unc-119 gene (on LGIIIR) is very suitable. I can score for rescue as
early as a day after injection, since mutants are Unc as L1, and wild types
can easily be obtained as L1s by following their characteristic wild type
'trail' in the bacteria. The gene is small (less than 5 kbp rescuing
genomic, or 2.5 kbp 'mini-gene'). The phenotype of all extant alleles is
an unconditionally null strong Unc with high viability, and the animals are
very easy to inject: Dried agarose pads can even be made to a slightly
lower concentration, which seems to reduce dessication and hence increase
survival. The other nice thing is that the wild type transgenics quickly
outgrow the Unc non-transgenics, and since unc-119 mutants are unable to
form dauers, if you forget to transfer a strain for a month, the only
dauers on a starved plate must be transgenic. (This is an immense
improvement over rol-6D and, probably, unc-22 antisense.) And as if these
aren't already good reasons to use this gene as a marker, rescue of unc-119
appears insensitive to dose - even very high amounts of rescuing DNA do not
appear to induce a phenotype of their own.
There may a problem with your application, however. Double mutants of
unc-119 may not be viable in strong Dpy backgrounds because unc-119 is
slightly dumpy itself. For example, dpy-18 unc-119 is okay, but dpy-20
unc-119 double mutants are about as long as they are wide, and so are quite
sterile (not to mention impossible to inject)! Of course a ts allele of
the Dpy would alleviate this problem.
(Unfortunately, despite repeated attempts, these rescued Unc's neither
dance nor pour Petri dishes. However, I once thought I saw one singing
"Hello My Baby" but I have been unable to repeat this result with anyone
else watching.)
I can distribute the canonical Tc1 allele unc-119(e2498) and a rescuing
clone (pDP#MM016) for use as a transformation marker to anyone who'd like
to use it.
Morris Maduro
morris_maduro at biology.ualberta.ca
