Date of Award
Doctor of Philosophy
M. Duane Enger
Hsp60 is a mitochondrial protein first identified in Tetrahymena thermophila and Saccharomyces cerevisiae. It belongs to a class of proteins called "molecular chaperones" which prevent aggregation and precipitation of partially or completely unfolded polypeptides;Two cDNAs for maize cpn60 were identified and sequenced. Southern blots of maize DNA showed two bands hybridizing with mt-cpn60 in maize inbreds. One copy was present in Arabidopsis. Both maize genes are contained within the nuclear genome. The chromosomal location of the cpn60 genes have been mapped by RFLPs. A maize B73 genomic library has been constructed and screened for cpn60 clones and two major families of clones that correspond to the two gene copies identified on genomic Southern blots have been identified and sequenced. The site of transcription initiation for each gene has been determined;Four minor families of cpn60-homologous clones were also identified. An Arabidopsis mt-cpn60 genomic clone has been isolated and partially sequenced. Sixteen introns have been identified in each maize gene, most of which correspond in position to introns in the partially sequenced Arabidopsis cpn60 gene;When maize cpn60II cDNA was expressed in yeast, maize cpn60 gene product was found to be properly imported into mitochondria and processed to the correct size mature protein. Hsp60 is an essential protein in yeast. The maize cpn60II gene was also capable of sustaining yeast cells in the absence of yeast hsp60 protein;Expression of maize cpn60 mRNA is developmentally regulated in young seedlings. Levels are high in imbibed embryonic axis of etiolated seedlings and decrease to a lower constitutive level by 8 days after imbibition. Expression increases several fold in response to cold and heat shock treatments. General patterns of cpn60I and II specific expression during development and high temperature stress are similar, but some differences are evident. Transient expression assays of cpn60I and cpn60II promoter sequences show similar patterns. Transgenic maize cells containing cpn60I or II gene promoters and putative mitochondrial targeting presequences fused to the GUS reporter gene exhibited cytological staining patterns which indicated that the GUS protein was targeted to the mitochondria.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Pamela Sue Close
Close, Pamela Sue, "Cloning and molecular characterization of two nuclear genes for Zea mays mitochondrial chaperonin 60 " (1993). Retrospective Theses and Dissertations. 10804.