Expressed Sequence Tag Projects
BDGP EST Submitted Collections
Project to screen for novel secreted and transmembrane proteins during development. 1,600 ESTs Completed 1997.
7297 ESTs from the Brian Oliver laboratory at the National Institute of Diabetes and Digestive and Kidney Diseases.
2001 EST Project
We have finished another EST project at the Drosophila Sequencing Center at Lawrence Berkeley National Laboratory. Our primary goal was to expand the Drosophila Gene Collection by sequencing an additional 160,000 ESTs. The ESTs for this project were generated from existing as well as newly constructed cDNA libraries.
We sequenced cDNA clones from the old GM and SD libraries, as well a new AT Drosophila testes library, which is in the new plasmid vector pOTB7. Clones from new normalized embryonic (RE) and head (RH) Drosophila cDNA libraries made at Riken have also been sequenced.
1999 EST Project
Our first EST project was motivated by the short-term goal of producing at least 80,000 Drosophila ESTs from several different cDNA libraries. We completed our goal ahead of schedule in March, 1999
These ESTs were then clustered by sequence similarity in order to choose the best cDNA clones for the Drosophila Gene Collection.
All BDGP ESTs are available at dbEST
All of the cDNA clones that are submitted to dbEST are available from Research Genetics.
CK EST Project
The goal of the CK library EST project aimed to characterize secreted and transmembrane proteins involved in intercellular communication during development. The project was conducted both at the BDGP and in the laboratory of Corey Goodman at U.C. Berkeley.
The CK library - named for its primary author Casey Kopczynski - consists of clones made from rough endoplasmic reticulum-bound mRNA, and is therefore enriched in clones encoding membrane and secreted proteins. To increase the representation of rare cDNAs in the library, the library was normalized using a novel method based on cDNA hybridization to genomic DNA-coated beads.
cDNAs were chosen for sequencing using a specialized screen that involves whole embryo in situ hybridization. A total of 2518 individual cDNAs from the normalized library were screened by in situ hybridization, and 917 of these cDNAs represent genes differentially expressed during embryonic development.
Sequence analysis of 1001 cDNAs revealed that 811 represent genes not previously described in Drosophila. Expression pattern photographs and partial DNA sequences have been assembled in a database and are publicly available from this web site. A total of 1691 CK ESTs have been submitted to dbEST.
The work of Kopczynski et al. is reported in PNAS 95(17):9973-8. You can read it [HERE].
Notice: Chimeric cDNA Clones
Cautionary note on the presence of chimeric clones in some libraries. During synthesis of the cDNA libraries the linkers used to clone the double-stranded cDNA into the pOT2 vector are cleaved with restriction enzymes. It appears that this reaction did not go to completion during synthesis of the GH and LP libraries. The result is that blunt end ligations between 2 different cDNAs can occur. We estimate that about one-quarter of the clones in the LP and GH libraries contain cDNAs derived from two distinct transcripts ligated together into a single pOT2 plasmid. In contrast, such chimeric cDNA are much less frequent in other libraries.