GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP

Abstract

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.

Figure 1

Overview of the bidirectional link between ESTs and cDNA-AFLP expression profiles established by GenEST. (A) From EST to cDNA-AFLP expression profiles. The predicted TDF of EST GE54 with primer extensions E+TC/T+AG and a size of 138 nt was readily identified on a cDNA-AFLP gel. (B) From cDNA-AFLP to EST. A band on the gel was amplified with primer extensions E+AA/T+TA with a size of 251 nt. These identifiers were used to search the virtual TDF list generated by GenEST. The corresponding EST was identified. M, molecular ladder. D, S, H, U and P represent the five different developmental stages of the potato cyst nematode: D, unhatched J2 in diapause; S, unhatched J2 after diapause, rehydrated for 2 days in water; H, freshly hatched J2 in PRD; U, developing nematodes (J1) in gravid females 2 months post-inoculation; P, developing nematodes (J2) in gravid females 3 months post-inoculation.