Studies with a cDNA clone for endosperm sucrose synthase

We are studying the gene for endosperm sucrose synthase with the aim to isolate and characterize McClintock's "controlling element" Ds which is found in the vicinity of gene Sh and has given rise to mutable sh alleles, probably by the formation of adjacent deletions which bring gene Sh into closer contact with Ds.

mRNA of 23-day-old maize endosperm was translated both in wheat germ extracts and rabbit reticulocyte lysates. A protein with an apparent molecular weight of 88,000 comigrates in SDS polyacrylamide electrophoresis with sucrose synthase. This protein is precipitated with an antiserum against sucrose synthase, and shows the same protease digestion pattern as the enzyme. It is not synthesized with mRNA extracted from sh/sh mutant kernels lacking sucrose synthase. By these criteria, the protein is the in vitro translation product of sucrose synthase mRNA.

The separation of mRNA in methylmercury hydroxide agarose gels and subsequent translation indicates a length of sucrose synthase mRNA of 2,800 nucleotides, which is compatible with the coding length necessary for a protein of a molecular weight of 88,000 and untranslated sequences.

A cDNA clone for maize endosperm sucrose synthase of 620 nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A)RNA in pBR322, and identifying the appropriate clone by hybrid-promoted translation.

Plasmid DNA containing the cloned cDNA was nick-translated and used as a probe for Southern blots against DNA obtained from wildtype maize and mutants. Using restriction endonucleases EcoRI and BamHI, we obtained a single band hybridizing to a probe from the wildtype and each of three mutant DNAs. In the case of EcoRI, the size of the band was different in each case. No hybridization was obtained with the DNA of a mutable bz, which according to the hypothesis advanced above should be a deletion extending from Ds beyond Sh to the beginning of Bz. These findings support an earlier report by B. Burr and F. Burr.

M. Geiser, J. Wostemeyer, H.-P. Doring, U. Behrens, E. Tillmann, A. Merckelbach, M. Muller and P. Starlinger

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