Worm Breeder's Gazette 10(2): 144
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.
We have isolated polyadenylated RNA from a series of mutator and non- mutator strains with various rates of Tc1 germline transposition, and analyzed these in Northern hybridizations using a Tc1-specific RNA probe. RNA from the following strains has been analyzed: Bristol (30 copies of Tc1/inactive), RW7414 (80 copies/inactive), RW7406 (80 copies/active), Bergerac(BO) (300 copies/active), DH424 (300 copies/inactive), and TR679 (>300 copies?/very active). The two RW strains were constructed in the Waterston laboratory by a series of backcrosses of Bergerac to Bristol. One, RW7406, retains the mut-4 locus on LGI, whereas the other, RW7414, has no mutator. An actin III- specific probe was used to normalize the amounts of polyadenylated RNA present in each lane. We detect a Tc1 transcript of 1.3 kb in all of the above strains except Bristol. There is also much additional hybridization in most of the lanes, including a possible transcript of 3.8 kb in TR679. However, there is also significant cross hybridization of the Tc1 probe (but not the actin probe) to ribosomal RNA, even in poly A+ RNA preparations, and even after hybridization and washing under stringent conditions. This complicates the interpretation of the additional bands. The amount of the 1.3 kb Tc1 transcript appears to be greater in strains where Tc1 is active in the germline. The relative levels, normalized to the amount in Bergerac(BO), are as follows: Bristol, <0. 1 (undetectable); DH424, 0.25; RW7414, 0.5; RW7406, 1.5; Bergerac (BO), 1.0; TR679, 5. The level of hybridization in TR679 is high, about half the level of hybridization of our actin probe to the three major actin mRNA's. Dividing by the number of Tc1 elements in each strain, and once again normalizing to the level in Bergerac(BO), the levels of Tc1 transcript per Tc1 element are: Bristol (inactive), <1; DH424 ( inactive), .25; RW7414 (inactive), 1.89; RW7406 (active), 5.7; Bergerac(BO) (active), 1.0; TR679 (active), 5.0. These data are preliminary and subject to considerable error, including errors in the estimates of the number of Tc1 elements in the various strain backgrounds, but they suggest that activation of Tc1 transcription may accompany activation of Tc1 transposition in the germline. In the Tc1 sequence of Rosenzweig et al., the distance between the putative TATA and polyadenylation sequences of Tc1 is 1090 bp. We are presently mapping the 5' and 3' ends of the 1.3 kb transcript in ribonuclease protection studies, and will investigate further the nature of the 3.8 kb transcript. We are also attempting to raise antibodies to the putative Tc1 open reading frame protein using as immunogens both peptides and protein synthesized from an expression vector. We will use these to study expression of Tc1 at the protein level.