Worm Breeder's Gazette 12(2): 70 (January 1, 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.

Sex Determination and DNA-binding: An Update

Scott M. Robertson, Enrique Patino, Philip M. Meneely

FHCRC, Seattle, WA 98104

The actin-4 feminizing element, located in the first intron of the act-4 gene, is one of a number of cloned C. elegans genomic sequences which derive almost exclusively from the X chromosome and which are capable of feminizing triploid males. A conserved octamer is common to all of these feminizing elements and, when subcloned into a plasmid vector and microinjected, is capable of feminization. This octamer serves as a protein binding site only when single-stranded and in an antisense orientation relative to the act-4 transcription unit (i.e. TTTCAATA).

The binding activity is resistant to DNase and RNase digestion, but is very sensitive to Proteinase K digestion. A major aid in the purification of this binding activity from either whole cell or nuclear extracts came with the observation that the binding activity was resistant to boiling. Therefore in one fast and simple step the vast bulk of proteins are removed while most of the binding activity (as assayed by gel shift using a 44 base single-stranded oligo derived from the act-4 intron sequence and containing the octamer) is retained. However, when the boiled extract is passed over an affinity column consisting of this 44 base oligonucleotide all activity is lost. From this came the realization that we were dealing with at least two components - a component (termed 'add-back') in the flow-through of the affinity column that is required in conjunction with a component that binds and elutes at high salt from the affinity column ('binder') for a gel shift. Either factor alone is incapable of effecting a gel shift. The 'binder' fraction shows two bands in a Coomassie-stained gel: one at approx. 31 kd and the other at approx. 21 kd. UV-crosslinking experiments had detected a single polypeptide of approx. 30-32 kd covalently attached to the labelled 44 base oligonucleotide. Scaled-up purifications are being carried out for the purposes of both protein micro-sequencing and antibody production. As stated above, the binding activity present in the crude protein extract is resistant to boiling; however, the isolated 'binder' is sensitive to boiling and the isolated 'add-back' is resistant. The simplest explanation for these observations involves an interaction between these two components in the crude extract, rendering the complex temperature-stabile and that during and/or following interaction of the 'binder' with its appropriate DNA target the 'add-back' component dissociates. Very similar results are seen in the yeast gal-4 system (M. Parthun, U. of Indiana; pers. comm.) where an affinity column consisting of the GAL-4 target sequence (dsDNA!) separates two activities, both of which are needed for a gel-shift with the same target sequence. The yeast/ GAL-4 affinity column flow-through can replace our 'add-back' fraction and vice versa, and the yeast flow through fraction is also resistant to boiling. This suggests that our 'add-back' component is highly conserved. We are characterizing it further, including its evolutionary conservation.

A light micrococcal nuclease digestion of purified nuclei (for the purpose of preparing chromatin) led to the unexpected observation that the majority of the gel-shift/binding activity was found in the enzyme digestion supernatant, and not in the later extraction supernatants with the bulk of the DNA and protein. We feel it likely that the binding activity was solubilized due to the overriding preference of micrococcal nuclease to cut at single-stranded DNA, particularly A/T-rich regions, suggesting an in vivo interaction of this protein with single-stranded DNA. In vivo footprinting experiments will confirm whether a single-strand specific interaction occurs and whether this entails the conserved octamer in the act-4 intron or in any of the other identified feminizing elements.

Titration of the act-4 feminizing element DNA in the injection assay told us three things: 1) there is a sharp transition between DNA concentrations at which no feminization was observed and concentrations at which we began to observe intersexes, consistent with expectations based on previously published analyses of the X/A ratio; 2) the smallest number of sequences injected still capable of feminizing (approx. 2,000-10,000 copies) was physiologically very relevant; and 3) the amount of DNA being injected was so small that transmission was unlikely, implying that the effect was probably a maternal one. Both the X chromosome duplications and the dosage compensation dumpys (dpy's -21, -26, -27 & -28 ), which feminize 2X:3A males, also exhibit maternal effects. These results have led to a model whereby titration of a maternally supplied protein in the oocyte, associated preferentially with the X chromosome, occurs immediately following fertilization. This titration, and therefore the relative distribution of this protein on the X chromosome(s) will differ according to whether the sperm contains or does not contain an X chromosome. The distribution of this protein between two X chromosomes leads to the hermaphrodite mode of development whereas the accumulation of this protein on one X chromosome leads to the male mode of development. Still open questions within this model are whether these effects are direct or indirect in relationship to genes like xol-1 and the sdc's (X-linked genes!) and whether the protein is functioning as a specific/general transcription factor or conversely as a structural component of chromatin involved in.transcriptional activation.