I t was intended to pick up small pieces from different parts of the cell contents and to compare the amounts of virus present. This involved having a ready test for the presence of minute quantities of virus: it was imagined th a t inoculation would provide such a test, but this soon proved totally inadequate. I t was then thought th a t the amounts injected were too small to contain the minimal dose necessary to cause infection. But when quantities of virus, certainly sufficient to produce the diseased con dition, were injected by micro-pipette directly into cells, in no case was more than one-tenth of the expected number of infections obtained (Sheffield 1936). The inoculation test was therefore temporarily abandoned, and a serological test similar to th a t designed by Matsumoto (1935) was tried. This too proved unsatisfactory. In order to carry out such a test with minute quantities of antigen it was necessary to watch the reaction under dark-ground illumination where it was found impossible to dis tinguish with certainty between an antiserum-antigen precipitate and the various granules and globules of the cytoplasm. A ring test was then tried in the micro-pipette, a drop of antiserum being picked up after the antigen. The end of the pipette containing the ring was broken off and mounted. No ring was visible by transm itted light, and it was very difficult to make pipettes of the exact size to give a mount of the thickness necessary for dark-ground examination. I t was then decided to abandon the finer experiments planned, to collect larger quantities of extracted material, and to inoculate these into leaves which would show local lesions. Purified virus was to be used as a standard with which to compare the extracted material. This method was applied to Solanum nodiforum in fected with aucuba mosaic disease.
ization, the membrane was washed with 6⫻ SSPE–1% SDS four times at room temperature for 10 min each time. The final wash was done with 1⫻ SSPE–1% SDS for 3 min at 50°C. The membrane was autoradiographed using Kodak XAR film. Construction of a plasmid DNA standard for Alu PCR. AMLV-infected PBMC DNA was amplified by Alu PCR using primers AMLV F1-A3, AMLV F2-Tag3, and AMLV F3-Tag3 as described above (see Fig. 4A, panel E). A 1.2-kb amplified fragment (see Fig. 4B, panel E, lane 2) was purified from the agarose gel using a Qiaquick column (Qiagen, Santa Clarita, Calif.). The frag- ment was subcloned in pCRII vector DNA (Invitrogen, Carlsbad, Calif.), and nucleotide sequences were determined. The AMLV long terminal repeat (LTR) was identified near Alu in the cellular DNA. See Fig. 4A, panel E, for the orientation of the AMLV LTR with respect to the Alu repeat. The AMLV LTR-Alu DNA was used to introduce the EAV LTR to create an AMLV-EAV two-LTR–Alu standard DNA. The EAV LTR fragment was PCR amplified from CEF total cellular DNA using primers EAV F17 and EAV R15, which contain AscI and Bsu36I restriction sites, respectively (EAV F17, 5⬘-AGCTCTAGAGG CGCGCCTGTTGTAATAGGCGTG-3⬘; EAV R15, 5⬘-TACCGGTACCTGAG GCTTGTTGCCTTTCGCAGC-3⬘). The amplified fragment was gel purified, and the EAV LTR (5⬘ 3 3⬘) was cloned into AscI and Bsu36I sites present in the cellular sequences located between the AMLV LTR and Alu in the AMLV LTR-Alu DNA. See Fig. 5, panel I, C, for the orientation of the EAV LTR with respect to the Alu repeat. The two-LTR–Alu DNA was used to determine the sensitivity of Alu PCR amplification with EAV primers.
Teratogenic effects of lithium on cultured rat embryos have also been reported (Klug et a l, 1992), including blebs in the cranial region, poor yolk sac circulation and abnormal development of the otic and optic vesicles and dorsal recess of the otocyst. These effects were not reversed by addition of inositol which is perhaps not surprising because lithium is an uncompetitive inhibitor, binding only to the enzyme-substrate complex, and therefore causing a greater degree of inhibition at higher substrate concentrations. In mouse embryos, similar malformations were not reported (Hansen et a l, 1990) but there was an increase of cranial NTD at the highest concentration used, 5 mM. This would be predicted if lithium depletes the level of inositol by inhibition o f inositol phosphate recycling, since inositol deficiency is known to cause cranial NTD (Cockroft et a l,