Normal embryogenesis involves selective activation and deactivation of genes in a stage-specific manner through transcription regulatory genes. Studying the function of stage-specific genes and the regulation of their expression should help us understand the molecular basis of embryogenesis. We have isolated a gene whose transcription is activated during the differentiation of embryonic and embryonal carcinoma (EC) cells into parietal endoderm (PE). This gene encodes a secreted protein acidic and rich In cysteine (SPARC) that is believed to participate in cell migration and proliferation. Transcriptional enhancement or repression results from the synergistic action of the multiple cis-control elements interacting with trans-factors. We will investigate the transcriptional regulation of the SPARC gene by identifying cis-control elements through transient expression assays, detecting trans-factors and determining recognition sequences of the factors by gel retardation analysis and methylation interference, respectively. The recognition sequences are to be used to identify factors by South-Western method and to clone corresponding genes from expression libraries. We will analyze the developmental role of the SPARC gene by generating germ-line chimeras for breeding homozygous mutant mice with mutation at the SPARC locus by homologous recombination in ES cells and injection of the mutant ES cells into blastocysts. Phenotypic disorders will be examined to assess the effect of the mutation. Lastly, we will isolate the promoter from our cloned essential housekeeping gene encoding elongation factor-2 (Ef-2) to produce a vector for the ubiquitous expression of target genes which should enable the functional analysis of the target genes. The promoter will be used to study regulation of constitutive transcription and cell cycles. Since normal embryo development depends on concerted effects of housekeeping genes and stage-specific genes the results from this study should facilitate our understanding of how development is controlled by transcription regulators relevant to ubiquitous and differential expression and how a stagespecific gene participates in development.
