Positional cloning of dzr1: Physical analysis of the 22-kDa c-zein cluster region
--Llaca, V and Messing, J

Zeins, the storage proteins in maize, constitute 50-60% of the total protein in mature seeds. They are expressed in the endosperm, under strict developmental control. They are classified into four major groups: 1) c-zeins are 19-kDa and 22-kDa proteins encoded by many genes grouped in separate clusters, where pseudogenes are also present. Conversely, the other three groups, 2) i-zeins (15 kDa), 3) h-zeins (16 kDa and 27 kDa), and 4) t-zeins (10 kDa and 18 kDa) are encoded by unique or a few genes (Heidecker et al., Genomics 10:719, 1991). The suboptimal nutritional value of maize for both humans and livestock is due to a large extent to the abundant expression of t-zeins, which are deficient in lysine, tryptophan and methionine. The maize inbred line BSSS53 has a 30% higher level of methionine than standard lines (Phillips et al., Crop Sci 21:601, 1981). This increase is due to overexpression and accumulation of the 10-kDa -zein, which has an unusual high (23%) content of methionine. The overexpression of the high-methionine zein is postranscriptionally regulated in trans by the product of the dzr1 (delta-zein regulator 1) gene. This gene shows allele-specific parental imprinting (Chaudhuri and Messing, Proc. Natl. Acad. Sci. U.S.A. 91:4867, 1994). dzr1 is tightly linked to a cluster embodying most of the genes and pseudogenes for the 22-kDa -zeins. This cluster spans 3.4 cM on chromosome 4 (Chaudhuri and Messing, Mol. Gen. Genet. 246:707, 1995).

Figure 1. Maximum size of the 22-kDa c-zein cluster. A) Genetic map of the cluster (chromosome 4S) as previously described (Chaudhuri and Messing, Mol. Gen. Genet. 246:707, 1995). B) Southern blot of gel shown in A), and hybridized to a 500 bp 22-kDa c-zein specific probe.

Figure 2. A) Long-range restriction map of the 22-kDa c-zein cluster for 4 restriction endonucleases. B) Southern blot of gel shown in A), and hybridized to a 500 bp 22-kDa c-zein probe. Single- and double-digestions are indicated on top of each lane.

Figure 3. Partial cosmid overlaps for the two zein subclusters and the php20725 intermediate region in the 22-kDa c-zein cluster region.

As part of our initial approach to isolate and characterize dzr1, we are constructing the complete physical map of the region where the 22-kDa -zein cluster and the dzr1 gene are located. This map should facilitate the cloning of the dzr1 gene, which would provide novel approaches to increase the nutritional value of maize. This study is also expected to contribute to the understanding of imprinting in maize endosperm and the evolution of clustered gene families in cereals.

Long-range restriction analysis of the cluster. We have optimized high-molecular-weight DNA isolation techniques and used pulsed field gel electrophoresis to make a long-range restriction map of the chromosomal region where the 22-kDa c-zein cluster is located. We wanted to determine the maximum size of the locus and the relationship between genetic and physical distance in the region. As Figure 1 shows, 22-kDa c-zein-specific probes hybridize to a single MluI fragment of 350kb, and to two SalI fragments, of 200kb and 100kb. Further restriction mapping (Figure 2) indicates that the cluster has a maximum size of 225-250kb and is divided into two subclusters of genes. The two clusters are 3.4 cM apart. One restriction fragment length polymorphism (RFLP) marker, php20725, maps between the subclusters, at 1.1 from one subcluster and dzr1, and 2.3 cM from the other. By Southern hybridization analysis we have estimated that there are 15-17 22-kDa c-zein related sequences (i.e., genes and pseudogenes) for the inbred line BSSS53.

Cosmid analysis. To provide a more detailed restriction map, we have constructed an overlapping, representative cosmid library (>8 genome equivalents) for BSSS53. The library has been amplified in 1700 independent sublibraries. We are isolating cosmids harboring 22-kDa zein-related sequences and the RFLP single copy marker php20725 in order to create an overlap of the whole region. Thus far, we have isolated 15 independent recombinant cosmids for the region. Thirteen cosmids have been ordered into three partial overlaps which cover a total of 200kb and include at least 14 different 22-kDa zein-related sequences (Figure 3). We have subcloned and sequenced 22 zein sequences to identify overlaps and identify in particular the gene 22/6 through its specific amino acid sequence. The 22/6 c-zein gene is located at less than 0.1 cM from dzr1

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