30
G. Jansen et al.
study. As all deletions were induced by random mutagenesis, all alleles
were backcrossed six times to a wild type Bristol N2 background, before
phenotypic analysis. The loss-of-function allele of gpa-16 has only
recently been isolated. As only a non-backcrossed allele was available,
this mutant has not been characterized phenotypically.
2.3.4 Overexpression Constructs
Mutants that putatively overexpress one of the Ga genes (gpa-l XS, 4XS,
5XS,6XS, 7XS, 8XS, lOXS, and llXSj were generated by introducing
extra copies of the wild type gene as transgenes (Jansen et al. 1999). The
Ga subunit genes were subcloned so that no other complete gene was
present. Marker dpy-20 DNA (Han and Sternberg 1991) was used at a
concentration of 100 f-lg/ml and test DNA at a concentration of
50 f-lg/ml. The transgenic arrays were integrated by irradiating transgenic animals with 40 Gy of y radiation from a 137Cs source. Before
phenotypic analysis all transgenic strains were outcrossed at least twice.
2.3.5 Behavioral Assays
Nematode chemotaxis to soluble compounds was assessed by a newly
developed assay (SRWicks and RHA Plasterk, in preparation). Briefly,
young adult nematodes were placed on a plate at the intersection of four
quadrants, filled with buffered agar either containing a dissolved attractant (75 mM NaAc, 75 mM NaCl, or 75 mM glucose) or no attractant.
The distribution of the worms over the four quadrants was determined at
10, 20, 30, 40, 50, 60, and 90 min. A chemotaxis index was calculated
(CI=(A-C)/A+C: A is the number of worms over attractant X, C is the
number of worms over attractant Y, or control). In this assay worms can
be presented with the choice between an attractant and only buffered
agar or with a choice between two salts (NaAc vs NaCl). In the latter
case wild-type animals show a preference for CI in the beginning, but in
time prefer Ac (Fig. 5A). Subtle changes in the animals' behavior can
now be detected more easily.
Soluble compound avoidance assays (C.l. de Vries and RHA PI asterk, unpublished results) were performed using 150 mM and 15 mM
G. Jansen et al.
study. As all deletions were induced by random mutagenesis, all alleles
were backcrossed six times to a wild type Bristol N2 background, before
phenotypic analysis. The loss-of-function allele of gpa-16 has only
recently been isolated. As only a non-backcrossed allele was available,
this mutant has not been characterized phenotypically.
2.3.4 Overexpression Constructs
Mutants that putatively overexpress one of the Ga genes (gpa-l XS, 4XS,
5XS,6XS, 7XS, 8XS, lOXS, and llXSj were generated by introducing
extra copies of the wild type gene as transgenes (Jansen et al. 1999). The
Ga subunit genes were subcloned so that no other complete gene was
present. Marker dpy-20 DNA (Han and Sternberg 1991) was used at a
concentration of 100 f-lg/ml and test DNA at a concentration of
50 f-lg/ml. The transgenic arrays were integrated by irradiating transgenic animals with 40 Gy of y radiation from a 137Cs source. Before
phenotypic analysis all transgenic strains were outcrossed at least twice.
2.3.5 Behavioral Assays
Nematode chemotaxis to soluble compounds was assessed by a newly
developed assay (SRWicks and RHA Plasterk, in preparation). Briefly,
young adult nematodes were placed on a plate at the intersection of four
quadrants, filled with buffered agar either containing a dissolved attractant (75 mM NaAc, 75 mM NaCl, or 75 mM glucose) or no attractant.
The distribution of the worms over the four quadrants was determined at
10, 20, 30, 40, 50, 60, and 90 min. A chemotaxis index was calculated
(CI=(A-C)/A+C: A is the number of worms over attractant X, C is the
number of worms over attractant Y, or control). In this assay worms can
be presented with the choice between an attractant and only buffered
agar or with a choice between two salts (NaAc vs NaCl). In the latter
case wild-type animals show a preference for CI in the beginning, but in
time prefer Ac (Fig. 5A). Subtle changes in the animals' behavior can
now be detected more easily.
Soluble compound avoidance assays (C.l. de Vries and RHA PI asterk, unpublished results) were performed using 150 mM and 15 mM