Worm Breeder's Gazette 11(5): 45
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.
Previously we reported (WBG 11(2): 70) an act-4 intron sequence capable of feminizing triploid males. This sequence contains an 8 base-pair sequence common to all such feminizing elements. This octamer alone, when subcloned into a plasmid vector and microinjected, is capable of feminization. The octamer serves as a protein binding site when single-stranded (but not when double-stranded) and in an antisense orientation relative to the act-4 transcription unit. Our ongoing studies are concerned with: a) defining the binding interaction more thoroughly and determining its significance to the feminization effect; and b) purifying the binding activity. a) To physically characterize the protein/single-stranded DNA interaction we are taking two approaches. First, single-stranded DNA- footprinting techniques are defining the location and the extent of the binding site within the band-shift probes. Methylation interference studies have confirmed an involvement of the octamer in the binding, as well as suggesting several protein contact points in the immediate vicinity of the octamer. KMnO4 footprints are in progress. Second, we are using the combination of asymmetric PCR and band-shifting to select out from a pool of oligonucleotides with the octamer randomized those oligos capable of binding to the protein. These oligos are then sequenced as a pool, enabling the derivation of a consensus binding site and a measure of the relative sequence requirements at each site in the octamer. A series of single, double and multiple base changes specifically within the octamer have been generated and are being assayed for their effects upon both the in vitro band shift and feminization following microinjection. In particular, one mutant with 6 of the 8 bases changed shows little or no binding activity in vitro and has no feminizing activity in vivo. Other mutants have been detected with intermediate binding activities and are presently being assayed for feminization. We have assayed whether this binding activity is directed against RNA by generating the appropriate sense and antisense RNA transcripts of the act-4 sequence containing the octamer. As yet we have detected no evidence for RNA-binding, consistent with binding observed to the antisense DNA strand. b) South-western blots of worm nuclear extract probed with an antisense act-4 sequence containing the octamer detect a single polypeptide in SDS gels of approximately 30-32,000 daltons. This means that the protein is apparently binding as a monomer, raising the possibility of probing expression libraries (in progress). UV- crosslinking studies also detect a protein in SDS gels of approx. 30- 32,000 daltons. The crude worm extract has been fractionated and the binding activity purified approximately 50-fold by passage over an anion exchange column followed by a single-stranded DNA cellulose column. An affinity column composed of a concatamer of an antisense act-4 sequence containing the octamer is being prepared for final purification. The partially purified binding activity shows a predominant band at 30-32,000 daltons. Renaturation/binding studies are underway to determine whether this protein band contains the binding activity. The final goal is to purify enough protein to enable micro-sequencing and the eventual cloning of the gene based upon these oligopeptide sequences, as well as possibly generating an antiserum against this protein.