This proposal is concerned with nuclear genes from pea and tomato which encode the small subunit of ribulose-1,5-bisphosphate carboxylase. We will examine the hypothesis that gene families encoding the small subunit are evolving in concert by comparing the sequences of allelic and non-allelic small subunit genes in two different pea varieties. We will extend our previous sequence studies of promoter fragments for weakly-expressed pea genes to include promoter fragments from strongly-expressed genes. By Agrobacterium-mediated transformation we have previously shown that a 1 kb pea small subunit promoter fragment confers light-inducibility to a hybrid gene and similarly we have determined the effect of promoter-deletions on light inducibility. We will carry out further transformation studies and examine the hypothesis that the high-levels of expression of the pea small subunit genes are mediated by enhancer sequences. Furthermore, we will check if these putative enhancer sequences can act as """"""""inducers"""""""". For this we will construct hybrid promoters and ask if small subunit regulatory sequences (lacking a TATA box) can confer light-induciblity to a promoter which itself is not normally light-inducible. We will extend our studies of the small subunit genes to tomato with which, in contrast to pea, it is possible to regenerate plants from single somatic cells. We will isolate small subunit genes from tomato and characterize them in a manner similar to our studies with pea. In addition we will carry out homologous transformation studies with regenerated plants and determine whether sequences in addition to the 5' noncoding sequences are required for tissue specific expression that mimics the endogenous genes. We will develop an in vitro transcription system from an extract of pea leaf nuclei. We will examine the transcription response to promoter-deletions and we will compare the levels of transcription from weakly- and strongly-expressed pea small subunit genes. If the in vitro transcription system is successfully developed then we will use it to characterize cellular transcription factors responsible for mediating light-induction of the small subunit genes. The proposed comparative sequence studies combined with the transformation studies using in vitro modified genes will eventually provide us with a detailed understanding of the DNA sequences responsible for mediating light-induction of the small subunit genes. A more difficult task will be to characterize the predicted cellular factors which through interaction with these regulatory sequences also serve to mediate light-induction. The in vitro transcription studies outlined in this proposal will serve to initiate research aimed at characterizing these factors.
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