Rapid analysis for the isolation of novel genes encoding putative effectors to the position-specific regulatory element of murine Hoxa-7

Abstract

Hox genes are known to play a critical role in pattern formation during vertebrate development by being expressed at the specific time and in the specific position along the antero-posterior body axis. In order to understand the regulatory mechanism for the position-specific expression of murine Hoxa-7, yeast one-hybrid system was applied. DNA fragment conferring a position specificity to the Hoxa-7 gene was placed just upstream from the yeast CYC1 promoter and lacZ gene in a reporter. Selection of LacZ positive clones after cotransformation of the reporter and mouse embryonic cDNA library as an effector, which was designed to be expressed as fusion proteins to the GAL4 activation domain, allowed us to isolate putative factors interacting with the position-specific regulatory element of murine Hoxa-7. A total of 28 positive clones were screened from 5 × 105 yeast transformants. About 70% of the clones turned out to be novel and most of the candidate clones selected in this study showed a temporally restricted expression pattern during embryonic development, suggesting that this method could provide an efficient way for isolating novel genes whose expressions are temporally regulated during embryogenesis.