Communicated by Motoo Kimura, National Institute of Genetics, Shizuoka-ken, Japan, September 9, 1994 ABSTRACT We analyzed the complete mitochondrial DNA (mtDNA) sequences of three humans (African, European, and Japanese), three African apes (common and pygmy chimpanzees, and gorilla), and one orangutan in an attempt to estimate most accurately the substitution rates and divergence times of hominoid mtDNAs. Nonsynonymous substitutions and substitutions in RNA genes have accumulated with an approximately clock-like regularity. From these substitutions and under the assumption that the orangutan and African apes diverged 13 million years ago, we obtained a divergence time for humans and chimpanzees of 4.9 million years. This divergence time permitted calibration of the synonymous substitution rate (3.89 x 10-8/site per year). To obtain the substitution rate in the displacement (D)-loop region, we compared the three human mtDNAs and measured the relative abundance of substitutions in the D-loop region and at synonymous sites. The estimated substitution rate in the D-loop region was 7.00 x 10-8/site per year. Using both synonymous and D-loop substitutions, we inferred the age of the last common ancestor of the human mtDNAs as 143,000 ± 18,000 years. The shallow ancestry of human mtDNAs, together with the observation that the African sequence is the most diverged among humans, strongly supports the recent African origin of modern humans, Homo sapiens sapiens.Light will be thrown on the origin of man and his history (1).

Modification of genes through homologous recombination, termed gene targeting, is the most direct method to characterize gene function. In higher plants, however, the method is far from a common practice. Here we describe an efficient and reproducible procedure with a strong positive/negative selection for gene targeting in rice, which feeds more than half of the world's population and is an important model plant. About 1% of selected calli and their regenerated fertile plants were heterozygous at the targeted locus, and only one copy of the selective marker used was found at the targeted site in their genomes. The procedure's applicability to other genes will make it feasible to obtain various gene-targeted lines of rice.

Mutagenized Arabidopsis seedlings (ecotype Columbia) were screened for the ability to grow photoautotrophically on solid medium containing 200 mM NaCl. A novel mutant line, designated pst1 (for photoautotrophic salt tolerance1 ), was obtained. There were no significant differences between pst1 and wild-type plants with regard to their ability to induce proline as an osmoregulatory solute. In addition, the content of monovalent cations in pst1 plants grown with or without salt stress was equal to that in the wild type. We observed that light, even at moderate intensities, increased the effects of salt stress on wild-type plants. The pst1 seedlings were nearly 10 times more tolerant to methyl viologen than were wild-type seedlings. We also found that the activities of the active oxygen scavengers superoxide dismutase and ascorbate peroxidase were enhanced significantly in pst1 plants. The pst1 plants also were tolerant to other stresses, such as high light intensity and toxic monovalent cations. The recessive nature of the pst1 mutation indicates that the potential for salt-stress tolerance is blocked in wild-type Arabidopsis. INTRODUCTIONArid areas are expanding gradually but steadily on earth. It is known that salt accumulation caused by unsuitable irrigation practices decreases crop productivity (Epstein et al., 1980). Under these conditions, high concentrations of NaCl arrest plant development and lead to plant death. Several interacting events are triggered in plants by salt stress, including the inhibition of enzyme activities in metabolic pathways, decreased uptake of nutrients in roots, decreased carbonuse efficiency, and the denaturation of protein and membrane structures. However, the complete response of plants to salt stress has not been explained systematically (Benzel and Reuveni, 1994).Several mechanisms of defense and adaptation to water deficiency resulting from salt stress have been well studied. Adaptation to water deficiency can involve the accumulation of osmolytes, such as proline (Igarashi et al., 1997), glycinebetaine, and sugar alcohols (Bohnert et al., 1995), or lateembryo-abundant (Lea) proteins (Dure, 1993). With this knowledge, researchers have attempted to improve salt tolerance in plants by increasing levels of osmolytes. For example, osmolyte concentrations have been manipulated by constitutively expressing genes for a protective protein (Xu et al., 1996) or genes for key enzymes involved in the production of osmosolytes occasionally associated with protective activities (Tarczynski et al., 1993;Thomas et al., 1995;Hayashi et al., 1997;Karakas et al., 1997).Photosynthesis, one of the most important metabolic pathways in plants, is a target of salt stress. Abscisic acid produced in response to salt stress (Leung et al., 1994) decreases turgor in guard cells and thus limits the CO 2 available for photosynthesis. During water stress, reduction of chloroplast stromal volume (Gupta and Berkowitz, 1988) and generation of active oxygen species (Price and Hendry, 1991) also are thought ...

SummaryWhile characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, nonautonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.

DNA methylation is a type of epigenetic marking that strongly influences chromatin structure and gene expression in plants and mammals. Over the past decade, DNA methylation has been intensively investigated in order to elucidate its control mechanisms. These studies have shown that small RNAs are involved in the induction of DNA methylation, that there is a relationship between DNA methylation and histone methylation, and that the base excision repair pathway has an important role in DNA demethylation. Some aspects of DNA methylation have also been shown to be shared with mammals, suggesting that the regulatory pathways are, in part at least, evolutionarily conserved. Considerable progress has been made in elucidating the mechanisms that control DNA methylation; however, many aspects of the mechanisms that read the information encoded by DNA methylation and mediate this into downstream regulation remain uncertain, although some candidate proteins have been identified. DNA methylation has a vital role in the inactivation of transposons, suggesting that DNA methylation is a key factor in the evolution and adaptation of plants.

Transposon display (TD) is a powerful technique to identify the integration site of transposons in gene tagging as a functional genomic tool for elucidating gene function. Although active endogenous DNA transposons have been used extensively for gene tagging in maize, only two active endogenous DNA transposons in rice have been identified, the 0.43-kb element mPing of the MITE family and the 0.6-kb nDart element of the hAT family. The nDart transposition was shown to be induced by crossing with a line containing its autonomous element aDart and stabilized by segregating aDart under natural growth conditions, while mPingrelated elements were shown to transpose in cultured cells, plants regenerated from an anther culture, and γ-ray-irradiated plants. No somaclonal variation should occur in nDart-promoted gene tagging because no tissue culture was involved in nDart activation. As an initial step to develop an effective tagging system using nDart in rice, we tried to visualize GC-rich nDart-related elements comprising 18 nDart-related sequences of 0.6-kb and 63 nDart-related elements longer than 2 kb in Nipponbare by TD. Comparing the observed bands in TD with the anticipated virtual bands of the nDart-related elements based upon the available rice genome sequence, we have improved our TD protocol by optimizing the PCR amplification conditions and are able to visualize approximately 87% of the anticipated bands produced from the nDart-related elements. To compare the visualization efficiency of these nDart-related elements with that of 50 mPing elements and a unique Ping sequence in Nipponbare, we also tried to visualize the mPing-related elements; all mPing-related elements are easily visualized. Based on these results, we discuss the parameters affecting the visualization efficiencies of these rice DNA transposons. We also discuss the utilization of nDart elements in gene tagging for functional genomics in rice.

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An endogenous 0.6-kb rice DNA transposon, nDart1, has been identified as a causative element of a spontaneous mutable virescent allele pyl-v conferring pale-yellow leaves with dark-green sectors in the seedlings, due to somatic excision of nDart1 integrated into the OsClpP5 gene encoding the nuclear-coded chloroplast protease. As the transposition of nDart1 depends on the presence of an active autonomous aDart element in the genome, the plants exhibiting the leaf variegation carry the active aDart element. As several mutable alleles caused by nDart1 insertions have subsequently been identified, nDart1-promoted gene tagging has been proven to be an effective system. At present, the nDart/aDart system appears to be the only endogenous rice DNA transposon system whose transposition activity can be controlled under natural growth conditions without any artificial treatments, including tissue cultures. To apply the nDart/aDart tagging system in various cultivated rice varieties, we explored the presence and distribution of an active autonomous aDart element in 19 temperate japonica, 30 tropical japonica, and 51 indica varieties. Only eight temperate japonica varieties were found to bear a single copy of an active aDart element, and no aDart activity could be detected in the indica varieties examined. Six of seven japonica varieties appear to carry the active aDart element at the identical site on chromosome 6, whereas the remaining one contains aDart on chromosome 5. Leaf variegations in the plants with the mutable pyl-v allele and the excision frequencies of endogenous nDart1 elements indicated that the aDart element on chromosome 6 is more active than that on chromosome 5. The findings described here are an important step in the development of a new and efficient nDart1-promoted gene-tagging system in various rice cultivars.