SummaryThe Spo11 protein is a eukaryotic homologue of the archaeal DNA topoisomerase VIA subunit (topo VIA). In archaea it is involved, together with its B subunit (topo VIB), in DNA replication. However, most eukaryotes, including yeasts, insects and vertebrates, instead have a single gene for Spo11/topo VIA and no homologues for topo VIB. In these organisms, Spo11 mediates DNA double-strand breaks that initiate meiotic recombination. Many plant species, in contrast to other eukaryotes, have three homologues for Spo11/topo VIA and one for topo VIB. The homologues in Arabidopsis, AtSPO11-1, AtSPO11-2 and AtSPO11-3, all share 20-30% sequence similarity with other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not well understood. Previous genetic evidence suggests that AtSPO11-1 is a true orthologue of Spo11 in other eukaryotes and is required for meiotic recombination, whereas AtSPO11-3 is involved in DNA endo-reduplication as a part of the topo VI complex. In this study, we show that plants homozygous for atspo11-2 exhibit a severe sterility phenotype. Both male and female meiosis are severely disrupted in the atspo11-2 mutant, and this is associated with severe defects in synapsis during the first meiotic division and reduced meiotic recombination. Further genetic analysis revealed that AtSPO11-1 and AtSPO11-2 genetically interact, i.e. plants heterozygous for both atspo11-1 and atspo11-2 are also sterile, suggesting that AtSPO11-1 and AtSPO11-2 have largely overlapping functions. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1 and AtSPO11-2 in meiotic recombination and AtSPO11-3 in DNA replication.

UGA is a nonsense or termination (opal) codon throughout prokaryotes and eukaryotes. However, mitochondria use not only UGG but also UGA as a tryptophan codon. Here, we show that UGA also codes for tryptophan in Mycoplasma capricolum, a wall-less bacterium having a genome only 20-25% the size of the Escherichia coil genome. This conclusion is based on the following evidence. First, the nucleotide sequence of the S3 and L16 ribosomal protein genes from M. capricolum includes UGA codons in the reading frames; they appear at positions corresponding to tryptophan in E. coli S3 and L16. Second, a tRNA"rP gene and its product tRNA found in M. capricolum have the anticodon sequence 5' U-C-A 3', which can form a complementary base-pairing interaction with UGA.We recently have sequenced a part of the Mycoplasma capricolum ribosomal-protein gene cluster that codes for polypeptides highly homologous to the Escherichia coli ribosomal proteins S3 and L16. The sequence contains four UGA codons in the reading frames; three appear at the sites corresponding to tryptophan, and one, at a site corresponding to arginine in the E. coli proteins. No "universal" UGG codon for tryptophan has so far been found. We have also isolated a clone containing a pair of M. capricolum tRNA genes, the sequence of both of which resembles that of tRNATrp of E. coli. The anticodon sequence of one of these tRNA genes is 5'-T-C-A-3', which can base-pair with both opal codon UGA and universal tryptophan codon UGG. That of the other is 5'-C-C-A-3', which may base-pair exclusively with UGG. These two tRNA genes are expressed in the cell. All these findings suggest strongly that, in M. capricolum, UGA codes for tryptophan using the opal tRNAUCA but not tRNACCA.RESULTS AND DISCUSSION UGA Codons in M. capricolum S3 and L16 Genes. As reported in a previous paper (1), we isolated the recombinant plasmid pMCB1088 containing a 9-kilobase-pair fragment of M. capricolum DNA. The fragment contains the genes for at least nine ribosomal proteins-S3, S5, S8, S14, S17, L5, L6, L16, and L18-as deduced from its encoded protein sequences being highly homologous with the corresponding E. coli ribosomal protein sequences (refs. 1 and 2; unpublished results). Fig. 1 shows the complete nucleotide sequence of a 629-base-pair (bp) HindIII fragment which is a part of the insert of pMCB1088 (see refs. 1 and 2). The DNA corresponds to the 3' half of the S3 gene and about 90% of the L16 gene from the 5' terminus. When the M. capricolum sequences are aligned with the E. coli protein sequences (3, 4) ( Fig. 1), four UGA (opal) codons are found within the reading frames. The possibility that these UGA codons are termination signals can be excluded by their occurrence in the regions having extensive sequence homologies with the E. coli proteins. More importantly, three out of the four UGA codons appear at the positions corresponding to tryptophan in the E. coli proteins. This suggests that UGA is a sense codon, probably for tryptophan, in M. capricolum. No UGG codon for tryptop...

Interactions between homologs in meiotic prophase I, such as recombination and synapsis, are critical for proper homolog segregation and involve the coordination of several parallel events. However, few regulatory genes have been identi®ed; in particular, it is not clear what roles the proteins similar to the mitotic cell cycle regulators might play during meiotic prophase I. We describe here the isolation and characterization of a new Arabidopsis mutant called solo dancers that exhibits a severe defect in homolog synapsis, recombination and bivalent formation in meiotic prophase I, subsequently resulting in seemingly random chromosome distribution and formation of abnormal meiotic products. We further demonstrate that the mutation affects a meiosis-speci®c gene encoding a novel protein of 578 amino acid residues with up to 31% amino acid sequence identity to known cyclins in the C-terminal portion. These results argue strongly that homolog interactions during meiotic prophase I require a novel meiosis-speci®c cyclin in Arabidopsis.

How plant organs grow to reach their final size is an important but largely unanswered question. Here, we describe an Arabidopsis thaliana mutant, brassinosteroid-insensitive4 (bin4), in which the growth of various organs is dramatically reduced. Small organ size in bin4 is primarily caused by reduced cell expansion associated with defects in increasing ploidy by endoreduplication. Raising nuclear DNA content in bin4 by colchicine-induced polyploidization partially rescues the cell and organ size phenotype, indicating that BIN4 is directly and specifically required for endoreduplication rather than for subsequent cell expansion. BIN4 encodes a plant-specific, DNA binding protein that acts as a component of the plant DNA topoisomerase VI complex. Loss of BIN4 triggers an ATM-and ATR-dependent DNA damage response in postmitotic cells, and this response coincides with the upregulation of the cyclin B1;1 gene in the same cell types, suggesting a functional link between DNA damage response and endocycle control.