Cloning and characterization of two nuclear genes for maize mitochondrial chaperonin cpn60

--P.S.Closea,b, S.P. Briggsb, B.A. Bowenaa,b, T.K. Prasada and R.G. Hallberga,c

aDept. of Genetics/Zoology, Iowa State Univ.

bPioneer Hi-Bred International Inc.

cDept. of Biological Sciences, Syracuse Univ.

Cpn60 (hsp60) is an abundant mitochondrial protein first described in Saccharomyces cerevisiae and Tetrahymena thermophila cells (McMullin and Hallberg, Mol. Cell. Biol. 8:371-380, 1988; Reading et al., Nature 337:655-659, 1989). It belongs to a family of proteins homologous to the E. coli protein groEL. This family also includes the chloroplast Rubisco-binding protein. These proteins are designated 'chaperonins' because they are implicated in the folding of peptides and the assembly of oligomeric protein complexes. A cpn60 protein has been identified in Zea mays and found to be structurally and metabolically indistinguishable from hsp60 in yeast and Tetrahymena (Prasad and Hallberg, Plant Mol. Biol. 12:609-618, 1989; Prasad, TK et al., Mol. Cell. Biol. 10:3979-3986, 1990). Protein expression is elevated after heat shock and in developing maize seedlings (Prasad and Hallberg, Plant Mol. Biol. 12:609-618, 1989). A partial cDNA clone was isolated from a lambda gt11 expression library using cpn60 antibody. This clone was used to isolate longer cDNA's.

Genomic Southerns reveal two copies of cpn60 in all maize inbreds screened. One copy is present in the Arabidopsis genome. Both maize genes are encoded in the nuclear genome. A maize B73 genomic library has been constructed and screened for cpn60 clones. Two major families (I and II) of overlapping clones have been identified and sequenced. These clones correspond to the two gene copies identified on genomic Southern blots. Both genes contain multiple (17) introns in identical positions. We used RFLP mapping to locate cpn60-1 to the short arm of chromosome 5 and cpn60-2 to the long arm of chromosome 1. One family of cpn60 genomic clones from Arabidopsis thaliana has been identified and partially sequenced. Introns are present in the Arabidopsis cpn60 genomic clone.

An RNA of approximately 2kb is extremely abundant and developmentally regulated in germinating seedlings. Transcript level is high in seedlings 24 hours post imbibition and decreases progressively to a lower constitutive level as seedlings age. Low levels of transcript were detected in seedling tissue up to two weeks old and mature leaf tissue. Steady state transcript levels increased significantly following heat shock treatment (3 hours, 39 C). We have isolated and sequenced two unique cDNA clones for cpn60 in maize. These correspond to Families I and II of overlapping genomic clones. The two cDNA's are 95% identical within coding regions but diverge significantly in the 3' untranslated regions. Gene specific probes are being utilized to study differential regulation of Families I and II.

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