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.

The act-4 Feminizing Element or: Sex and the Single-Stranded DNA

Scott M. Robertson, Dean Fraga and Philip Meneely

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 
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.