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.

Differential Screening of a cDNA library using cDNA probes Amplified from Individual Embryos at Various Stages

Yuji Kohara

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.