Worm Breeder's Gazette 12(3): 86 (June 15, 1992)
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 reported previously that several X-linked elements that feminize triploid males all contain an octmer sequence which serves, in only one orientation, as a single-stranded DNA-binding site for a protein present in worm nuclear extracts (see Abstracts for 1991 Meeting, pg. 354). We have now purified this binding activity (termed FEBP) and are cloning the gene for this protein.
Our purification scheme was simplified by the observation that the sequence-specific ssDNA-binding activity survived boiling. Following removal of denatured proteins by centrifugation, the boiled extract was directly loaded onto an affinity column. The column consisted of a single-stranded oligonucleotide (containing the octamer in correct orientation and derived from the first intron of the act-4 gene) attached to an agarose matrix. Following elution at high salt the predominant protein visible on Coomassie-stained SDS gels was approx. 30-32 kd, with several other bands both above and below 30 kd also visible. These protein bands were individually cut from preparative SDS gels, eluted, denatured and renatured. Band shift activity was not associated with the predominant 30 kd protein, however, but instead with a single band at approx. 24 kd. This purified and renatured protein demonstrated single-strand specificity, octamer specificity (as assayed by competitions), and, when UV crosslinked to the single-stranded probe, gave the same band on a SDS gel as previously reported (30-32 kd). It was, nonetheless, still of some interest to us to know what the predominant 30 kd protein was, since until the denaturation/renaturation experiments it seemed a likely candidate itself for FEBP. Both proteins were therefore gel-purified following the affinity column and microsequenced (William Lane, Harvard Microchem).
30-32 kd protein: Based on the sequence of one trypsin peptide an unambiguous identification could be made. It is a member of a small family of DNA-binding proteins called Y-box binding proteins and appears to be identical to a Y-box binding protein identified and cloned by Fire and co-workers (WBG 12 (2), pg. 72). This protein was initially identified by virtue of its binding to an upstream regulatory sequence of the unc-54 gene. Exactly why this protein is binding to our ss-DNA affinity column is unclear, although reports on Y-box binding proteins in several other systems have detected interactions with single-stranded nucleic acids.
FEBP: Three different trypsin peptides of 14, 21 and 25 residues respectively have been sequenced. Protein database searches reveal no homologies to known proteins for two of the three peptides; the third peptide shows a small block of homology (7 of 8 or 9 of 14 identities) to the N-terminus region of E. coli SSB, a sequence non-specific single-stranded DNA binding protein. The significance of this result is unclear as this region appears to lie outside of the SSB DNA-binding domain. Genomic PCR as well as RT-PCR cloning using degenerate primers based upon these peptide sequences is underway. In addition, cDNA libraries are being screened with longer degenerate oligonucleotides. Mice have been injected with purified FEBP (as well as, separately, with the 30 kd protein) and preliminary ELISA results with purified antigens indicate immunoreactivity in the tested sera; fusions should follow the next boost or two. Rabbit antisera await either injection with coupled peptides or with cDNA expression products.
Fire and co-workers. WBG 12 (2), pg. 72