CPSC 568: Recombinant DNA Technology Laboratory (Schedule)

Our efforts in this course will be mainly devoted to the molecular characterization of the maize Glossy15 gene. Glossy15 (gl15) encodes a DNA binding protein that regulates the juvenile versus adult differentiation of leaf epidermal cells during seedling development (see Moose and Sisco, 1996, Genes and Development, 10: 3018). In normal maize plants, epidermal cells within the first 5 - 6 leaves are juvenile: they synthesize leaf waxes and lack leaf hairs. Beginning with leaf 6, the leaf epidermis expresses adult traits such as trichomes. Gl15 activity is required for juvenile leaf epidermal cell differentiation, because loss-of-function mutations in the gl15 gene result in the formation of adult leaf epidermal cell characters such as trichomes in seedling leaves.

Nine different gl15 mutant alleles have been identified on the basis of their phenotype, but the molecular basis for these mutations is known for only one of these (gl15-m1). In addition, the gl15 gene itself is regulated differently among maize genotypes, because the timing of expression of juvenile and adult leaf epidermal traits varies among inbred lines. Your collective task in this course will be to characterize both mutant and wild-type alleles of the gl15 gene at the DNA sequence and mRNA expression levels. You will also use molecular markers to determine the genetic map location for the gl15 gene and another newly identified mutation that blocks the formation of trichomes, which is known to at least be on the same chromosome and regulated by the gl15 gene.

The wild-type gl15 gene has previously been cloned and characterized by DNA sequencing and mRNA expression analysis. In this course, we will amplify DNA sequences from genomic DNA that span the gl15 gene using PCR, clone the PCR products into plasmid vectors, and obtain their DNA sequences. We will also obtain a restriction enzyme map of the cloned PCR products using DNA blotting and hybridization techniques. The DNA sequences of the gl15 genomic DNA fragments will be compared by computer analysis and restriction mapping to that of the previously cloned gl15 gene. These comparisons will determine the nature and location of the DNA sequence changes among the different gl15 alleles. The ability of the mutant gl15 alleles to produce mRNA will be assayed by a reverse-transcriptase PCR technique. Finally, we will attempt to produce recombinant Gl15 protein using a bacterial protein expression system.

Planned Course Schedule