Cloning, immunoselection and characterization of cDNA clones of the two non-allelic sucrose synthase genes

cDNA clones of the two non-allelic sucrose synthase (SS) genes, Ss2 and Sh, have been isolated from lgt11 expression libraries derived from immature kernel poly(A)' RNA of the sh bz-m4 (sh-deletion) and ShlSh genotypes respectively. Recombinant clones containing the longest Ss2 (lshD13) and Sh (lSh9) cDNAs, each of approximately 2.5 kb size, were characterized and comparatively analysed. Proteins were analysed on polyacrylamide gels to identify the chimeric expression of the SS cDNAs fused to b-galactosidase gene in E. coli klysogens. The SS and b-galactosidase epitopes were recognized on Western blots using antisera against both proteins. Although the lSh9 expresses as a sucrose synthase-1 (SS1)-b-galactosidase fusion protein (~200 kD: 115 kD b-galactosidase and 92 kD SS) in llysogens, the lshD13 failed to form such a chimeric protein and instead showed a ~70 kD SS2 polypeptide. Another Ss2 cDNA clone, lshD12, which is ~50 bp shorter at both ends than lshD13, also did not form any fusion protein and only ~70 kD SS2 polypeptide could be seen. The reason for the lack of fusion protein in lshD13 and lshD12 lysogens is not known.

The following evidence indicates that the clone pshD13 (lshD13 cDNA insert subcloned in pUC19) contains the Ss2 cDNA sequence: 1. The pshD13 was immunoselected from the cDNA expression library of the sh bz-m4 stock which is known to have a deletion at the Sh locus (Burr and Burr, Genetics 98:143, 1981; Dooner, CSHS Quant. Biol. 45:45 7, 1981). The residual activity in the endosperm of this genotype is due to the Ss2 locus (Chourey, MGG 184:372, 1981). 2. The pshD13 hybridized to endosperm poly(A)+ RNA of the sh-deletion genotype as a single sharp band (~2900 b) which is also present in the Sh genotype. A second weak hybridizing band in the Sh genotype (2750 b) is considered due to cross-homology with Sh transcript as it comigrates with the band seen in the Sh poly(A)+ RNA using Sh cDNA as a probe. 3. pshD13 showed homology to the restriction fragments of Sh cDNA and Sh genomic clone (Werr et al., EMBO J. 4:1373, 1985). 4. An identical pattern of hybridization was observed when pshD13 was used as a probe on different restriction enzyme digests of sh-deletion and Sh genomic DNA. One such 6.3 kb BamHI hybridizing fragment cloned in EMBL4 from the sh-deletion stock, pGshD6, also showed different enzyme cleavage sites (Fig. 1) as compared to 16.3 kb BamHI Sh genomic clone (Werr et al., EMBO J. 4:1373, 1985).

The pSh9 (lSh9 cDNA insert subcloned in pUC19) can be characterized as Sh cDNA clone by the following criteria: 1. It did not hybridize to poly(A)+ RNA of the sh-deletion genotype at the Sh transcript position. 2. pSh9 did not hybridize to genomic DNA from the sh-deletion strain at the Sh position. 3. pSh9 contains the expected restriction sites of the Sh cDNA (Werr et al., EMBO J. 4:1373, 1985). 4. It shows homology to Ss2 cDNA.

The restriction enzyme cleavage site maps of pSh9 and pshD13 are very different (Fig. 1). There are unique restriction sites in both clones, i.e., NciI in pshD13 and PvuII, ClaI, and BglI in pSh9. Among the common restriction sites, the SstI and BglII are also located --550 bp apart in SstI-BglII fragments of the two cDNAs. Southern cross-hybridization studies also revealed more homology around this region based on the intensity of hybridizing fragments. The sequences 5' to SstI restriction site on two cDNA clones are diverged.

Genetic mapping analysis using the first Ss2-null mutant isolated among the Sh-revertants upon Ds excision from sh-m5933 allele indicates a tight linkage between Sh and Ss2 on chromosome nine (Chourey et al., CSH meeting p. 65, 1986; and Chourey et al., manuscript in preparation). The recent molecular mapping data, obtained in collaboration with Ben Burr (Brookhaven Lab) using B-A translocation stocks and the Ss2 cDNA clone (pshD13) as a probe, suggest that the Ss2 locus is close to the bronze (bz) locus on chromosome nine (Gupta et al., manuscript in preparation).

Figure 1.

M. Gupta, P.S. Chourey and P.E. Still

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