Mol Gen Genet (1983) 190:421-426 © Springer-Verlag 1983 Isolation and Analysis of a Genomic Clone Encoding Sucrose Synthetase in Maize: Evidence for Two Introns in Sh Edward Sheldon ~, Robert Ferl 2, N. Fedoroff 3, and L. Curtis Hannah 1 1 Vegetable Crops Department and 2 Department of Botany, University of Florida, Gainesville, FL 32611, 3 Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA USA Summary. The shrunken locus of maize which codes for the major endosperm sucrose synthetase has been an object of extensive genetic investigations. To extend these studies, a genomic maize library was probed with a cDNA clone of endosperm sucrose synthetase. The first clone character- ized contains DNA sequences found also in wild type maize but missing in a shrunken genetic deletion. This observation and the similarity in the restriction map of this clone to that observed when total maize DNA is probed with sucrose synthetase cDNA showed that this clone contains the shrun- ken locus. Comparison of the restriction map of this clone to that of the cDNA suggested the presence of two introns. DNA sequencing verified their presence and showed that their two junction sequences partially resemble the consen- sus junction sequences. Observations of others have shown that this region of the gene, here shown to be one of the two introns, is altered by association with the transposable element Dissociation. Introduction The sucrose synthetase genes of maize have several attrac- tive attributes for studies of gene regulation. The endosperm specific sucrose synthetase gene (Shrunken), located on chromosome 9, (Chourey and Schwartz 1971) codes for an abundant protein (Schwartz 1960) composed of four identical subunits (Su and Preiss 1978). The physiological role of this enzyme is apparently the catabolism of sucrose (Chourey and Nelson 1976; Chourey and Nelson 1979; Cobb 1982). Another sucrose synthetase has been identified in the endosperm and other tissues of maize. Although the two enzymes are similar in size, number of subunits, Kms for all reactants, pH optima for the forward and back reac- tions, and cross react immunologically, they are encoded by two separate loci (Chourey and Nelson 1976; Chourey 1981). The two loci, however, are regulated differently in that only the endosperm specific enzyme undergoes dramat- ic increases in activity during endosperm development (Chourey 1981). Thus, from the viewpoint of genetic regula- tion, an attractive feature of these genes is that two regions similar in structure are regulated differentially. In addition, the Shrunken locus has been of interest in the study of transposable elements. In her early studies, McClintock (1956) showed that the (Activator-Dissociation) Offprint requests to. L.C. Hannah Ac-Ds transposable element system caused mutations at the Sh locus. Recently this has been studied at the nucleic acid level. Several groups (Burr and Burr 1981; D6ring et al. 1981; Chaleff et al. 1981) have taken advantage of the abundant sucrose synthetase mRNA in the endosperm to construct cDNA probes. Using these cDNA probes, re- striction maps have been generated from digests of total DNA of Sh stocks with and without controlling elements. The positions of alterations induced by the Ds controlling element within the gene have been determined (Burr and Burr 1981 ; D6ring et al. 1981; Chaleff et al. 1981; Burr and Burr 1982; Fedoroffet al. 1983a; 1983b). As part of our efforts to characterize the two sucrose synthetase genes and the mechanism of their expression, we have isolated and characterized a Sh genomic clone con- taining the sequence encoding the sucrose synthetase mRNA at the Sh locus. Restriction mapping and DNA sequence analysis provide evidence for the existence of two introns near the 3' end of the mRNA coding region. The intron/exon junction sequences are in general agreement with recently proposed (Mount 1982) consensus sequences. Of particular interest to the study of controlling element mutations is the observation that the rearrangement break- points in two Ds-associated mutations at the Sh locus (Cha- left et al. 1981; Burr and Burr 1982) map within one of the two introns identified in the present study. Materials and Methods Enzymes. DNA polymerase I and DNase I were from Boehringer Mannheim. Restriction endonucleases, ligase and DNA polymerase I (large fragment) were obtained from Bethesda Research Laboratories. Lysozyme was from Sigma. Enzymatic reactions were performed essentially as recommended by the manufacturers. Screening for Sucrose Synthetase Coding Genomic Clones. Approximately 106 clones with Black Mexican maize DNA inserts averaging about 1.5 x 104 bp were screened using a sucrose synthetase-coding cDNA clone made from size- enriched poly A RNA of immature endosperms from W23XK55 as described in detail elsewhere (Fedoroff 1982a; Chaleffet al. 1981). The Black Mexican maize DNA library was prepared using the EcoRI site of the Charon 4A bacteriophage vector as described by Sheldon (1982). The screening method was the plaque hybridization proce- dure of Blattner et al. (1977). Hybridization was overnight