Worm Breeder's Gazette 10(3): 82

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.

Embryonic cDNA Expression Libraries

Irene Schauer and W.B. Wood

We have constructed two C.  elegans embryonic cDNA libraries in 
lambda gt11, one from predominantly early embryonic RNA and one from 
total embryonic RNA of him-8 embryos.  Characterization of these cDNA 
libraries to date is as follows.  The early embryonic cDNA library was 
made with poly A+ RNA from an N2 embryo population that was about 85% 
<30 cells.  The library was amplified from 2x10+E6 independent 
recombinants in two batches.  The final library is 82-85% recombinants.
Before amplification, 16 random recombinants were picked and DNA was 
prepared.  All but 2 of these contained detectable inserts ranging 
from 0.4 -4 kb, with an average of 1.1 kb.  Eight of the inserts were 
used as probes on C.  elegans genomic Southern blots; 6 hybridized to 
single bands and 2 to pairs of bands.  The library was screened with 
four previously characterized clones of unidentified genes, three that 
are expressed in the early embryo and one expressed maternally, and 
positives were found for all of them.  As might be expected, the 
maternally expressed gene is especially abundant in this library.  One 
of these cDNAs has so far been characterized further and found not to 
be full length.  The him-8 cDNA library was made with poly A+ RNA from 
a him-8 embryo population that ranged from very early to well into 
morphogenesis.  This library was also amplified in two batches from a 
total of 1x10+E6 independent recombinants.  The final libraries are 
37% and 55% recombinants.  Random recombinants were not checked from 
this library, but positive recombinants have been identified with a 
her-1 probe and a generic actin probe.  For two her-1 positives, 
identified by Carol Trent, the inserts are about 65% and 75% full 
length.  The generic actin probe yielded positives at a frequency of .
05-.07%, as might be expected for an abundant mRNA.  Five positives 
were plaque-purified, and DNA was prepared.  Insert sizes ranged from 
0.8 to 1.7 kb (full-length actin mRNA's are 1.6 to 1.7 kb, depending 
on the gene).  These five inserts were also used as probes on genomic 
Southern blots.  Four yielded only the expected actin bands, while the 
fifth had a single additional band which may be unrelated to actin.  
Thus the majority of recombinants in both libraries appear to have 
inserts derived from single cDNA species.  There is, however, one 
known problem with the him-8 library and possibly, to a lesser degree, 
with the early embryonic library.  The two her-1 inserts and two or 
three of the five actin inserts cannot be cut out with EcoRI.  
Together with the low frequency of full length clones, this suggests 
that there may have been some EcoRI* activity during linker removal.  
In summary, both libraries contain a reasonable frequency of 
recombinants containing simple inserts of C.  elegans cDNA.  The 
frequency of full length clones is low, and analysis of inserts may be 
complicated by loss of EcoRI sites.  These libraries are available 
upon request.