Most, if not all, multimeric enzymes in the chloroplast(ct) are made up of subunits whose structural genes are dispersed in two different cellular compartments (nucleus and ct). Our long term objective is to understand how the expression of these genes are coordinated and regulated. As a first step toward this goal, we wish to gather more data on the fine structures of ct genes themselves. Although ct DNA has the potential to code for more than 100 polypeptides, only two of them have been identified so far. This proposal deals with the identification and characterization of cucumber ct genes encoding polypeptide subunits of the photosystem (PS) II complex. Polypeptide subunits that are made inside the ct will be determined by pulse-labeling in the absence or presence of specific inhibitors. Antibodies will be raised against the ct-synthesized subunits which are likely to be ct gene products. Restriction fragments of cucumber ct DNA will be used to select for specific mRNA from a total ct RNA mixture. The selected mRNA will be translated in a reticulocyte lysate system and the products identified by immunoprecipitation with antibodies to PS II polypeptides. These experiments will identify ct genes encoding PS II polypeptides and localize them to specific restriction fragments of known map positions. The 5' initiation site of each mRNA encoding PS II polypeptide will be mapped by a modification of the S1 technique. Each gene will be characterized with respect to its direction of transcription and whether it contains any introns. The 3' and 5' regions of the gene will be sequenced by the Maxam-Gilbert technique to see if there are consensus sequences that may be important in gene regulation. To study ct gene expression and regulation in vitro we will establish a DNA-dependent soluble transcription system using plastid lysates. Truncated fragments of the carboxylase large subunit (LS) gene and the 32 kd polypeptide gene will be used as templates. We predict transcription of both genes in the ct lysate but only the LS gene in the etioplast lysate. We hope to exploit this differential transcriptional response of lysates from the two plastid types of isolate a factor that is specifically required for the transcription of the 32 kd polypeptide gene in ct.
