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.
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.