Zucker-Aprison E et al. (1986) Worm Breeder's Gazette "the heptamers in the vitellogenin promoters: an update."

Blumenthal TE, Zucker-Aprison E, Cane J

[Worm Breeder's Gazette, 1986]

There are two heptamers repeated multiple times upstream of the C. enin genes. In order to obtain evidence for or against the functional importance of these heptamers we have investigated their evolutionary stability. We sequenced the homologous promoters from C. briggsae and the only C. enin promoter remaining unsequenced vit-3. This brings the total number of nematode vitellogenin promoters sequenced to eleven. The data are shown schematically in the Figure. Box 1 TGTCAAT, is shown as rectangles; Box 2 CTGATAA is shown as ovals. In general the heptamers have beer very highly conserved while the sequences between them have diverged considerably. In particular there has been strong conservation of the Box 1 heptamers centered at -45 ( just upstream of the TArA box)those at -180 (which we have suggested may be at the 5' border of the promoter regions) and at least one Box 1 between -45 and -180. In each case the locations and sequences of at least these Box 1 heptamers and at least one Box 2 heptamer per promoter have been conserved. This data strongly supports the hypothesis that Box 1 and Box 2 are involved in vit promoter function. We are initiating experiments to determine the relationship between the two heptamers and the three modes of regulation: sex stage and tissue. {Figure 1} We have noted previously the presence of Box 2 in the vertebrate vitellogenin promoters. Klein-Hitpass et al. (Cell 46 1053) have tested the idea that this heptamer plays a role in Xenopus vitellogenin gene induction by estrogen in a transient expression assay using a human mammary cell line. They present evidence that it is a binding site for an activator protein required for estrogen induction but not the site for estrogen receptor itself. They name it the 'elegans box'. Does this mean that the regulatory protein that binds to this sequence in the C. e is also present in human mammary cells?

McLafferty SA et al. (1984) Worm Breeder's Gazette "30,000 fold purification of the levamisole receptor over crude wild type specific activity."

McLafferty SA, Skimming J, Lewis JA

[Worm Breeder's Gazette, 1984]

We have purified the levamisole receptor to a specific activity more than 30,000 times that present in total homogenates of the wild type worm. We calculate the purified material is about 5% pure, assuming one [3H] MAL binding site per 100,000 daltons protein (not a bad assumption considering the number of genetic loci affecting receptor binding). In our best efforts, we've ended up with about a 15% final yield of the receptor activity in our starting material. From 270 grams of worms, we get 110 g of total protein of which we figure about 5 g is receptor. We're ready to try making monoclonal antibodies against the levamisole receptor. The mouse probably deserves about twice as much purified material per injection. That's four large carboys of worms per shot the way we grow worms (see our article on growing worms in quantity in this issue). Our numbers work out to the rather frightening fact that the levamisole receptor is about 1 part in a million of total worm protein in the wild type, so if we somehow manage to get monoclonals, we're going out for a long beer to celebrate. Our purification method is pretty simple. We start with the minor advantage of working with lev-1(x61), a levamisole-resistant strain that contains about 1.6 times the amount of total activity as wild type and we grow our worms until they are dauers, which increases the specific activity about 2 fold over what's present in extracts made from the average well-fed culture. We obtain a washed pellet of insoluble material from our total extract and extract that with 1% Triton X-100, providing another 5 fold or so purification. We chromatograph the detergent extract on our very own Made-in-Missouri affinity column containing a trimethylammonium compound attached to a long spacer arm. After washing away unbound protein, the levamisole receptor is eluted from the column with a choline wash. In our best purifications, we've obtained a 100 fold purification and a 30% yield at this step. The keys to success here are an affinity column that contains only an alkyl chain for some distance from the trimethylammonium group and the use of Pharmacia N-hydroxysuccinimide- activated CH-Sepharose 4B to make the column. The roughly equivalent material from BioRad doesn't work. The only obvious explanation is that the BioRad material is cross-linked and of somewhat smaller pore size (a nominal exclusion limit of 5x10+E6 versus 20x10+E6 for the Pharmacia gel). The last step is a Concanavalin A affinity column, which provides us with a further 26 fold purification and a 65% yield in this step. The purification is summarized in the following table showing a 60,000 fold enrichment over the crude wild type specific activity, a somewhat better than average effort. [see Figure 1]