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S-RNase in transgenic plants did not confer a gain of S -function (unlike the wild-
type protein); hence, intrinsic RNase activity is indeed an integral part of the function
of S-RNases.
S-RNases are glycoproteins that contain one or more N-linked glycan chains,
raising the question as to the function of these groups. One possibility, drawn by
analogy with lectins, is that allelic specificity might be encoded by the sugar moieties
in the glycan chains. Again this question has been addressed by site-directed mu-
tagenesis and plant transformation. An S-RNase gene was engineered in which the
asparagine codon essential for glycosylation of the sole N-glycosylation site of an
S-RNase was replaced with a codon for aspartic acid. Analysis of transgenic plants
expressing this non-glycosylated S-RNase demonstrated that the resultant protein
was indistinguishable from wild-type S-RNase with regards to rejecting self-pollen
(Karunanandaa et al. , 1994). Thus, the encoding of S -specificity resides not in the
glycan side chains but in the protein backbone of S-RNases. The function of the
glycan side chains is unresolved but there has been speculation that they may be
involved in protein stability.
As S -specificity is encoded in the primary structure of these proteins, efforts have
been made to determine which amino acid residues are involved in this function. A
sequence comparison by Ioerger et al. (1991) identified two hypervariable regions,
termed HVa and HVb. In addition to being highly polymorphic, these regions are
also highly hydrophilic and likely to be exposed on the surface of the molecules,
making them prime candidates to be regions involved in the encoding of specificity
(Ioerger et al. , 1991; Tsai et al. , 1992). Recent determination of the crystal structure
of S F11 -RNase by X-ray diffraction (Ida et al. , 2001) has confirmed that HVa and
HVb regions are indeed juxtaposed on the surface of S-RNases.
Several groups have employed transgenic approaches to obtain direct evidence
concerning regions and amino acid residues involved in the encoding of allelic
specificity. In these experiments, chimeric S-RNase genes have been constructed
and introduced into transgenic plants. In all cases, one allele of the S-RNase gene
was used as a 'backbone' and sequences of a particular region were exchanged
with the sequence of a corresponding region from another allele. The S -specificity
displayed by hybrid S-RNases was then assayed by crossing to plants carrying
the allelic progenitors of the chimaeras. The results of these experiments are su-
perficially contradictory. In two separate experiments, exchanges between pairs of
S-RNases with a high degree of sequence diversity led to the loss of the backbone
S -allele specificity with all regions exchanged (which covered the whole gene) (Kao
& McCubbin, 1996; Zurek et al. , 1997). Further, no gain of the S -specificity of the
donor allele was found, despite the fact that all hybrid S-RNases exhibited 'normal'
levels of RNase activity (suggesting that overall protein structure was probably
unaffected by the exchanges) (Kao & McCubbin, 1996; Zurek et al. , 1997). An
apparently different result was obtained in a separate experiment employing two
very closely related S-RNases ( S 11 and S 13 )of Solanum chacoense (Matton et al. ,
1997). These S-RNases differ by only 10 amino acids, three of these being in HVa
and one in HVb. Exchange of both hypervariable regions of S 11 -RNase with those of
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