Worm Breeder's Gazette 11(4): 12
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.
Using the method reported previously (WBG 11, #1, p.67) with several modification, I have prepared amplified cDNA from single embryos at various stages (every 2 hr after 1st cleavage). The attempt for subtractive hybridization (ibid.) was not productive due to its trickiness, so I performed differential screening of a cDNA library. The set of amplified cDNA were labeled and probed Julie Ahlinger's embryonic cDNA library. I expected a big difference in the pattern of positive clones between far different stages, but there was not a big difference. This is probably because many (most?) of the cDNA clones have artificial rearrangement due to insufficient methylation of EcoRI sites during library construction: many of such rearranged clones carry parts of abundant cDNA. However, by comparing the signals in autoradiograms very carefully, several clones seemed to be differentially expressed. To examine these, aliquots of the amplified cDNA were dot-blotted onto strips of nylon membrane and then hybridized with [32P]-labeled cDNA insert. The results were quite encouraging. For example, clone 4-3 gave a very strong signal at 1. 5hr embryo (1.5hr after 1st cleavage), a weak signal at 3.5hr embryo and virtually no signal at 0hr and other stages. Clone 4-1 also gave a strong signal at 7.5hr embryo. A control clone 1-1 which did not show differential expression gave strong signals at similar level at all stages. These hybridization patterns were essentially reproducible in another experiment using amplified cDNA from another series of embryos. Clones 4-1 and 4-3 were mapped near zyg-11(LGII) and ceh-16 (LGIII) by probing the YAC filters. Now I am trying to identify the cell lineage in which these genes are expressed using the protocol for in situ hybridization by David Greenstein (personal communication and WBG 11 #3 p.78). Homologous clones to clones 4-1 and 1-1 appeared in the library at the ratio of 8 (0.016%) and 35 (0.07%) out of 50,000 pfu, respectively. Much less abundant clones could be analyzed in this assay. So, I picked up 16 low abundant cDNA clones at random and probed the test strips with them. Out of them one clone gave a signal only at 0hr embryo, one clone at 5.5hr embryo, three clones at early stages (0 and 1.5hr embryo) and other clones showed a similar result to that of clone 1-1 more or less. Being encouraged by these results, attempts to characterize all clones of a cDNA library which is normalized to some extent are in progress toward construction of a 'true' gene library of C. elegans. For this purpose, I wish to know more about the validity of the test strips, so, please send me cDNA clones which contain their 3' ends (N.B. By my method up to 1 Kb from 3'-end is amplified). I should probe the test strips with them and give the results back to the senders.