Worm Breeder's Gazette 9(1): 25
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.
A consensus sequence for Tc1 insertions: Using an oligonucleotide complementary to part of the Tc1 inverted repeat as a primer, we have sequenced the insertion sites of unc-22(st136::Tc1), 137::Tc1), and stP1: stP1 is a Tc1 dimorphism located about 0.05 mu left of unc-22 and is due to a recent transposition event. A comparison of these three sites and two sites described by Hirsh's laboratory (NAR 11:4201 and 7137) and a repeatedly targeted site in unc-54 described by Eide and Anderson ( PNAS 82:1756; pers. comm.) reveals the following common sequence motif: T(5/6) N N N N A(6/6) T(5/6) N T(6/6) [Tc1] A(6/6) N N T(5/6) The frequency of tagging a gene of interest probably reflects both the number and the location of this motif within a gene. For example, unc-22 is a much 'hotter' target than unc-54 for Tc1 insertion, but the difference in gene size, about three-fold, cannot really account for the difference in insertion frequency. A search for this motif in unc-54 reveals that it occurs ten times but only one of these is in an exon and this is the insertional 'hot spot' recovered by Eide and Anderson. For st136 and st137 of unc-22, 12 of 15 nucleotides around the insertion sites are identical. These two sites are out of reading frame, so this homology does not merely reflect a constraint imposed at the protein level. This observation suggests that the six bases identified above are perhaps only part of a preferred target site. We are sequencing more Tc1-induced unc-22 mutants to further examine this idea of sequence specificity. Heterogeneity in the Tc1 family: The Tc1 element at stP1 lacks the 3' Tha [site present in the Tc1 sequenced by Rosenzweig, et. al., (NAR 11:4201)]. We surveyed Tc1 elements isolated from a lambda 1059-BO library and found that about 10% of the Tc1 elements lacked this site. The restriction site heterogeneity reported by others (Harris, et. al. , WBG 8, 1 : 48; Eide and Anderson, pers. comm.) for HindIII and EcoRI is not correlated with this ThaI difference, which suggests there may be a great deal of microheterogeneity within the Tc1 family in the BO strain. Mapping a mutator: We have continued to genetically localize a factor which is necessary for germ-line excision and transposition of Tc1. From three-factor crosses we have positioned the factor to a region right of dpy-5, left of unc-29 and possibly near unc-37 on chromosome I. At present, we do not know whether we are mapping a single unit or a cluster of active factors but we have evidence the factor on chromosome I can itself transpose. RW7037, which has unc-22( st136::Tc1) as a marker and has been used for the mutator localization on chromosome I, was further outcrossed to N2 and RW7080 was isolated after recovering st136 animals in the F2. This strain has fewer copies of Tc1 (probably about 70), but greater activity for unc-22 reversion. Mapping experiments show that RW7080 has the activity on LGI as well as on at least one other chromosome. Using Ac/Ds and P factor/elements as our paradigm, we are searching through the LGI recombinants for a Tc1 that may be acting as an activator or a factor. A region of Tc1 homologous to the SV40 enhancer core: Recent results in yeast demonstrate that at least part of the transcriptional control exerted by Ty elements on nearby genes is due to an enhancer sequence (PNAS 82:5423 and 5428). For a lark, we examined Tc1 and found that there is a sequence that differs by 1 bp from the SV40 enhancer 'core' at position-107 relative to the putative Met start site at bp 523. The sequence in Tc1 is TTTGGAAAGT, and in SV40 is TGTGGAAAGT. Does it mean anything? Obviously, we do not know, but the fact that Tc1 can be incorporated into the mRNA of unc-22, leads one to wonder.