The laminin B2 chain promoter contains unique repeat sequences and is active in transient transfection

We previously isolated cDNA clones coding for the entire murine laminin B2 chain. In this report, we have screened a mouse genomic library with a 5' portion of the laminin B2 chain cDNA and isolated two genomic clones which contain the first and second exons. The transcription initiation site was determined by primer extension and S1 nuclease mapping. Exon 1 contained 641 bp (base pairs) including 229 bp of 5'-untranslated segment, sequences coding for the signal peptide and the N-terminal portion of the protein, while exon 2 contained 305 bp. Nucleotide sequencing of 830 bp of the 5'-flanking region of the gene showed several interesting features including the presence of 9 "GC" boxes, a stretch of 9 nearly identical repeats of 11 nucleotides between -200 and -450, and a sequence which is similar to the cAMP consensus sequence. There was no TATA box or CAAT box. A recombinant plasmid containing the 830 bp promoter segment coupled to the chloramphenicol acetyltransferase gene was constructed and transfected into various cells. Differentiated F9 cells transfected with this construct showed twice as much chloramphenicol acetyltransferase activity as the undifferentiated cells. The 830-bp B2 laminin promoter was also active in NIH-3T3 cells which produce little laminin, but was not active in human HT-1080 cells. These results indicate that this structurally unique promoter contains DNA sequences that help regulate the gene during differentiation.