Worm Breeder's Gazette 15(1): 72 (October 1, 1997)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Are hyp6 and hyp7 interconnected? Some observations from mosaic analyses using SUR-5GFP(NLS)

John Yochem, Trent Gu, Min Han

MCD Biology, University of Colorado, Boulder, CO 80309 U.S.A.

Worms transgenic for high copies of pTG96, a plasmid constructed to
have a translational fusion of all of the open reading frame of sur-5
to the GFP(NLS) gene present in pPD95.70 from Andy Fire!s lab, have
many brightly fluorescing nuclei. Although not all cells express pTG96,
the striking degree of localization of SUR-5GFP(NLS) to nuclei
suggested that it may serve as a useful marker for genetic mosaicism.
Analyses of three strains!MH1066 [lrp-1(ku156); him-8; kuEx76(pTG96;
lrp-1+)]; MH1067 [unc-36(e251); kuEx77 (pTG96; unc-36+)]; and MH1068
[ncl-1(e1865) unc-119(ed3); kuEx78(pTG96; ncl-1+; unc-119+)]! have
yielded promising results.
In each analysis, it was possible to identify certain mosaic L3s, L4s
and adults on normal NGM plates with a dissecting microscope. It was
particularly easy to pick animals with losses in P1 by examining plates
for green animals lacking fluorescence of their intestinal nuclei. One
third of these on average will have losses in E, one third in EMS, and
one third in P1.  (It is possible to refine one!s picking further by
assessing the fluorescence of body muscles and the terminal bulb of the
pharynx!a microscope capable of magnifying 100 fold is helpful). In
this way, a large part of the cell lineage can be examined for a gene!s
requirement in a short period of time. (Alas, identifying losses in AB
by means of a dissecting microscope is not as straight forward but can
be done.) The key feature of SUR-5GFP(NLS) is its affinity for and
retention in nuclei. It appears that SUR-5 also has an NLS that
enhances localization to nuclei. Two limitations of pTG96 are the lack
of expression by some cells and the possible presence of SUR-5
activity. A better marker might be a transcriptional fusion of GFP
containing many NLSs with the promoter from a more widely-expressed
gene, several of which were reported at the last meeting.
A strange observation was made during the analysis of the lrp-1 strain.
A total of five and only five animals were isolated as having losses in
AB based on a large set of cells from ABa and ABp. Each of them
displayed an expected but nevertheless striking pattern: all of the
nuclei of hyp7 fluoresced, including the nonvulval descendants of the P
cells. This was an expected pattern because, although most of the
nuclei of hyp7 descend from AB, the blastomere C also contributes to
hyp7, and the array had been inherited by C in each case. All of the
nuclei of hyp7 should therefore have access to SUR-5GFP(NLS) from the
common cytoplasm of the syncytium.  However, an unexpected pattern was
evident in each of the five cases: the six nuclei of hyp6, a syncytium
of nuclei from AB, also fluoresced, albeit somewhat more weakly than
the nuclei of hyp7. Six and only six presumed AB- mosaic animals have
now been isolated from the unc-36 strain, and in each case the nuclei
of hyp6 and hyp7 also fluoresced (as for the lrp-1 strain, C had
inherited the array in each case such that hyp7 should fluoresce).
There are several explanations for why hyp6 might fluoresce in these
eleven animals. In each case, there may have been a high degree of
perdurance in hyp6 of SUR-5GFP mRNA and/or protein present in the
zygote (although the animals were scored as L4s or adults, and hyp6 did
not fluoresce in animals in which only E or EMS inherited the arrays).
Also, the possiblity still exists that one or more nuclei of hyp6 may
have actually inherited pTG96 (but it seems improbable that the
inheritence would be so precisely confined to a only a few nuclei from
AB!no close relatives other than hyp7 nuclei fluoresced in each of the
eleven cases). hyp6 and hyp7 may therefore have connections between
them that permit proteins to be shared; thus, they may not be separate