BERGAMO, ITALY
Istituto Sperimentale per la Cerealicoltura

Molecular analysis of opaque2 alleles
--Hans Hartings, Nadia Lazzaroni, Vincenzo Rossi, and Mario Motto

Studies on genetic mutations that influence the accumulation of zein proteins have indicated the presence of several regulatory mechanisms controlling the expression of specific members of the zein multigene family. The opaque2 (o2) gene is one of the most widely studied loci involved in these regulatory mechanisms. The o2 mutation causes, in addition to a modification of endosperm appearance, a severe reduction (50-70%) in zein synthesis, leading to a concomitant enhancement of lysine content in the seed. A number of spontaneous o2 mutations have been described in past years. We describe the molecular analysis of ten recessive (o2) alleles of independent origin [o2-R, o2-m(r), o2-Columbian, o2-Agroceres, o2-261, o2-mh, o2-33, o2-Go2-Charentes, o2-Italian, and o2-Crow].

Southern analysis performed on the o2 alleles, with HindIII and EcoR1 endonucleases in combination with two molecular probes corresponding to the 5' and the 3' end of the O2 cDNA respectively, allowed division of alleles into 6 polymorphic groups. Results are summarized in Table 1.

Table 1. Summary of Southern analyses (fragment size in kb).
 
o2mutant
HindIII-probe1
EcoR1-probe1
EcoR1-probe2
wt normal
16.0
9.5
5.0
o2-R
12.0
7.5
5.0
o2-Italian
12.0
7.5
5.0
o2-m(r)
7.0
7.0
4.0
o2-Columbian
7.0
7.0
4.0
o2-Agroceres
7.0
7.0
4.0
o2-261
7.0
7.0
4.0
o2-mh
7.0
7.0
4.0
o2-33
16.0
7.0
12.0
o2-G
16.0
9.5
10.5
o2-Crow
16.0
9.5
5.0
o2-Charentes
17.0
5.5
10.5

Northern analysis revealed the presence of an O2-homologous transcript identical in size to the wild-type transcript for the o2-R and o2-Crow alleles. The cDNA of these two o2 alleles was isolated and studied at the sequence level. Comparison of the cDNA sequence obtained for the o2-R allele with the published O2 sequence revealed 17 base substitutions. Moreover, the o2-R sequence contained two insertions of, respectively, 2 and 7 nucleotides, 41 bp apart. Since no stop codons are present in this sequence stretch, these insertions merely give rise to a short frame-shifted stretch and consequent alteration of the deduced protein sequence in this region. Two deletions of 2 and 23 bases respectively in the central region of the o2-R sequence generate a second frame shift. This change in reading frame causes the premature termination of the coding sequence. As a consequence, the open reading frame of the o2-R gene is only 255 codons long,with a deduced polypeptide devoid of one acidic region and the basic and zipper domains. Comparison of the o2-Crow and wild-type O2 cDNA sequences disclosed 27 base substitutions. In addition, the o2-Crow sequence contains five deletions which measure 3, 6, 120, 1, and 4 nucleotides with respect to wild-type. Immediately following the 120 bp deletion, a 24 bp stretch devoid of homology with the O2 sequence is present in o2-Crow. Whilst the first three deletions merely cause the omission of, respectively, 1, 2, and 40 residues from the deduced protein sequence, the fourth deletion generates a frame shift, which causes the premature termination of the o2-Crow polypeptide. As a consequence, the deduced o2-Crow protein is terminated after the basic domain.

The nucleotide changes observed between the O2 and o2-R sequences and between the O2 and o2-Crow sequences were used to estimate the average number of nucleotide substitutions per site according to Kimura's three substitution model. The average distance calculated for the O2 and o2-R sequences is K = 0.0106, while O2 and o2-Crow yield an average distance of K = 0.0186. Taking these data, and considering a neutral nucleotide substitution rate of 5 x 10-9 substitutions/site/year, the o2-R and O2 alleles should have diverged from a common sequence approximately 1 million years ago. In a similar manner, O2 and o2-Crow diverged approximately 1.86 million years ago. 


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