1. Intragenic mapping of tra-1In order to determine the relative location of recessive (masculinizing) and dominant (feminizing) mutations of the sex determining gene
tra-1 III, I have carried out a variety of intragenic recombination experiments. One protocol utilizes alleles that were generated by reverting the dominant allele
e1575. This was first partly reverted to a weaker dominant
e1575e1816, and recessive mutations were then introduced in cis by selecting for loss of the dominant feminization phenotype. Some of the resulting triple mutants contain an amber mutation, e.g. the leaky amber
e1825. Heterozygotes with flanking markers were constructed (genotype
vab-7 +
dpy-18/+ 75e1816e1825)+), and their progeny were screened for female recombinants. Screening was facilitated by introducing a homozygous
unc-32 marker, because females are conspicuous in starved Unc-32 populations. Out of a total of 18 intragenic recombinants, all had the genotype
vab-7 75). Thus, the order must be
e1825 -
e1575, with the modifying mutation
e1816 either left of
e1825 or very close to it. The distance between
e1825 and
e1575 is ca. 0.2 map units, which is very large. Either
tra-1 is a huge gene, or it experiences map expansion (since
tra-1 is not in the LGIII cluster). Another amber allele,
e1828, is much more tightly linked to
e1575, ca. 0.02 map units. Using other protocols,
e1099 (the reference non-amber null) W25 found to map ca. 0.03 map units to the left of
e1575; also, several different
tra-1(d) alleles were all found to map to the right of
e1099 (and in one case to the right of
e1575). Thus far, all dominants tested map to the right of all recessives. . The gene
tra-3 can be expressed in an XO germline The gene
tra-3 is required for hermaphrodite sex determination, but is not required in males, because null mutations of
tra-3 cause partial masculinization of XX animals and have no effect on XO animals. The gene also shows a strong maternal effect:
tra-3/tra-3 XX daughters of
tra-3/+ hermaphrodites are normal hermaphrodites, and the masculinization is only seen in the next generation of self-progeny. However, there is no paternal rescue: mating
tra-3 hermaphrodites with
tra-3/+ XO males gives rise to
tra-3/tra-3 XX cross progeny which are just as masculinized as their self-progeny sisters. The absence of paternal rescue could be explained by l) lack of
tra-3 expression in XO animals, or 2) the small size of the sperm, which probably contributes little more than a genome to the zygote. Tests for
tra-3 expression in XO animals were carried out using feminizing mutations of fem-l and
tra-1, which are believed to act downstream of
tra-3 in the sex determination pathway, and therefore should not affect
tra-3 expression. Females of genotype fem-l -1 + XO , and
tra-1(d)/+; constructed and crossed with tra- 3 XO males. Both crosses yielded several XX animals which were fully fertile hermaphrodites of normal appearance, but whose progeny were all Tra-3 abnormal males. Therefore, in the XO parents of these animals,
tra-3(+) must have been expressed, at least in the germline. This result lends support to the notion that the
tra-3 gene or gene product is not regulated, but is a necessary cofactor for the action of another gene,
tra-2 (which probably is regulated; see Hodgkin, Genetics 96; Doniach, Genetics 114).