Variations in endosperm polypeptides among 16 cultivars of rice were analyzed by two-dimensional gel electrophoresis. One glutelin α-subunit (α₈) with a molecular mass of 32.5 kDa was exclusively present in indica cultivars. By contrast, the glutelin α₃ subunit, with a molecular mass of 32 kDa, was present only in japonica and javanica cultivars. Glutelin α-subunits α_5a and α_5b, which have a molecular mass of 32 kDa and slightly different isoelectric points, differed among rice subspecies, i.e. the former was detected in japonica cultivars and the latter was detected in indica cultivars, Internal amino acid sequences of the indica-specific glutelin α-subunits α_5b and α₈, were analyzed with a gas-phase protein sequencer. Of 46 amino acid residues determined, only two residues differed, and they corresponded with the sequences deduced from type I and type II cDNAs of glutelin sub family A, respectively. Nine polypeptides of higher molecular mass (35-90 kDa) also differed. In particular, two of these polypeptides designated B and C which were identical to Wx proteins with molecular masses of 58 kDa were strongly expressed in indica endosperms, but only weakly expressed in japonica and javanica. F₁ seeds obtained from a cross between japonica and indica cultivars showed an intermediate intensity of Wx proteins and contained all nine glutelin α-subunits including both the japonica and indica types. In this experiment, Wx proteins designated B and C and glutelin α-subunits could be used to identify differences between japonica and indica cultivars.

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We have accomplished the nucleotide sequence of the 1537 bp mitochondrial gene coding for small subunit (SSU) rRNA of yeast Hansenula wingei, and also determined the 5'-and 3'-termini by S1 nuclease mapping. Eight universally conserved (U) elements of the SSU rRNA were identified. Comparison of U regions among five fungal mitochondrial SSU rRNAs shows the striking similarity between H. wingei and Saccharomyces cerevisiae. The construction of the secondary structure revealed a core structure similar to the counterpart of Escherichia coli 16S rRNA. The secondary structure also enabled us to specify seven variable (V) regions differing from those of other mitochondrial SSU rRNAs in size, sequence and possible secondary structure. Molecular phylogenetic evaluation based on U regions of five fungi indicates that mitochondria of H. wingei and S. cerevisiae diverged from the same lineage. This suggests that the evolution of mitochondria-encoded genes does not directly correlate with the alteration of mitochondrial genetic system: genome size, gene organization and codon usage.

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To isolate mutations related to the ubiquitin system, I constructed a plasmid carrying the YUH1 and UBP1 genes (genes of ubiquitin-speeifie processing proteases) whose expressions were under the control of the galactose-inducible GAL1-GAL10 promoter. Cells of a strain carrying the plasmid were mutagenized with ethyl methanesulfonate. One mutant, which showed galactose-dependent growth at a high temperature (37°C), was isolated from about 380,000 mutagenized colonies. The mutation responsible for galactose-dependent growth at 37°C was a single nuclear recessive mutation designated as uby1-1. UBP1 and YUH1 as well as the GAL1-GAL10 promoter are required to suppress uby1-1. At the restrictive temperature, a uby1-1 mutant did not arrest at a specific phase of the cell cycle, but still lost viability. Even at the permissive temperature (30°C), the uby1-1 mutant grew somewhat slowly and showed pleiotropic phenotypes including hypersensitivity to stresses such as cadmium and canavanine, and sporulation defects. The genomic DNA fragments in a single-copy plasmid which complemented uby1-1 were isolated. Chromosomal mapping, sequencing and subcloning analyses indicated that the gene complementing uby1-1 is RSP5, which encodes a ubiquitin-protein ligase (E3) homologous to E6-AP (E6 associated protein). Deletion, complementation and linkage analyses revealed that UBY1 and RSP5 are the same gene. Therefore, the E3 protein encoded by RSP5 (UBY1) is required for vegetative growth, sporulation and stress response. The present procedure using suppression by co-overexpression of two cloned genes will be useful to isolate mutations of related genes and to analyze biochemical pathways and gene-interactions.

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RNA editing was examined in rice mitochondrial apocytochrome b (cob) transcripts. Nineteen C-U conversions were found, and most of them changed the polypeptide sequence encoded by genomic DNA sequence. Evidence for partial and excess editing was also found.

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