II. 18. Biochemical evidence for two sub-families of genes at the Hor 2 locus.
P. R. Shewry, M. Kreis, B. G. Forde, S. Rahman and B. J. Miflin, Biochemistry Department, Rothamsted Experimental Station, Harpenden, Hertz, AL5 2JQ, U.K. "R"
The hordein storage proteins of barley endosperms appear to be coded for by three loci termed Hor 1, Hor 2 and Hor 3 which are located on chromosome 5 (Shewry and Miflin, 1982; Shewry et al., 1983). The Hor 2 locus is thought to be a cluster of structural genes which code for the 'B' hordein group. Analysis of the genomic DNA by hybridization to cloned cDNA sequences derived from mRNA for B hordein indicates the presence of at least 13 genes present on fragments with a total length of over 100 kilobases (Miflin et al., 1983). B hordein is a highly polymorphic mixture of polypeptides; these vary in their number and properties between cultivars, and can be classified into three groups on the basis of the patterns of peptides given by cleavage at methionine residues with cyanogen bromide (Faulks et al., 1980). One of these groups (called class III) was present in all eight cultivars studied while seven cultivars also had polypeptides of one of the other two groups (classes I and II). Several lines of evidence indicate that these groups are coded for by separate sub-families of structural genes. These can be summarized as follows:
(1) We have recently described the preparation and characterization of a library of about 50 cDNA clones which are related to mRNAs for B hordein (Forde et al., 1981). These clones can be broadly divided into two groups on the basis of their cross-hybridization behaviour. The polypeptides encoded by a number of the cDNA clones were identified by in vitro translation of mRNAs selected by hybridization of the clones to mRNA fractions from a range of cultivars. This showed that the two groups of cDNA clones coded for class I and II or class III polypeptides, respectively (Kreis et al., 1983). This suggests that class III polypeptides represent one structural sub-family and class I and II polypeptides a second. The second sub-family presumably has diverged to give two types of cyanogen bromide cleavage pattern.
(2) The relative amounts of polypeptides of the two sub-families are affected by mutant high lysine genes. This effect is most clearly seen in the cultivar Bomi where the sub-families of polypeptides are clearly separated by SDS-PAGE. Thus the Risø mutants 7 and 13 have reduced proportions of polypeptides of class III and class I, respectively (Køie and Doll, 1978). The mutant gene of Risø 1508 also appears to have a differential effect on the two sub-families and this difference is also present when the gene is transferred from Bomi to Sultan which has a different Hor 2 allele (unpublished results).
(3) The class I and class III polypeptides accumulate at different rates in developing endosperms of Sundance, those of class I accounting for an increasing proportion of the total (Rahman et al., 1982). This is associated with differences in the populations of mRNAs encoding them (unpublished results).
If it is accepted from the chemical evidence that each B hordein polypeptide is the product of a separate mRNA and gene, then these data clearly indicate that the Hor 2 locus consists of two sub-families of genes which are, to a limited extent, under separate regulatory control. This raises the question of whether these sub-families represent spatially distinct regions of the locus. Netsvetaev and Sozinov (1982) have reported that some 'B' hordein polypeptides are coded for not by Hor 2 (which they call Hrd B) but a closely linked locus called Hrd F. Although we have not observed recombination between our two sub-families of B hordein polypeptides in crosses (Shewry et al., 1980), it is possible that they correspond to the products of Hrd B and Hrd F. We are currently testing this hypothesis with further crosses.
References:
Faulks, A. J., P. R. Shewry and B. J. Miflin. 1981. The polymorphism and structural homology of storage polypeptides (hordein) coded by the Hor 2 locus in barley (Hordeum vulgare L.). Biochem. Gen. 19:841-858.
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Køie, B. and H. Doll. 1979. Protein and carbohydrate components in Risø high-lysine barley mutants. In: Seed Protein Improvement in Cereals and Grain Legumes, Vol. I. IAEA: Vienna, p. 205.
Kreis, M., S. Rahman, B. G. Forde, P. R. Shewry, J. Pywell and B. J. Miflin. 1983. Sub-families of hordein mRNA encoded at the Hor 2 locus of barley. Mol. Gen. Genet., Submitted.
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