The distribution of mitochondria during conjugation of the ciliated protozoan Tetrahymena thermophila was surveyed using a mitochondrial stain and fluorescence in situ hybridization (FISH). When the mitochondria-specific stain, Mito-Tracker, was used, the majority of mitochondria were detected in the cortex; their distribution was not changed during conjugation. On the other hand, FISH using mitochondrial large subunit (LSU) rRNA as a probe showed an unusual distribution of signals during conjugation. Unexpectedly, the signals were detected throughout the cytoplasm of conjugating cells. These signals were not observed in pre-mating cells and in exconjugants. The cytosolic localization of mitochondrial rRNA was supported by northern blot analysis using post-mitochondrial RNA fraction at the later stages of conjugation. These observations suggest selec-tive mitochondrial breakdown or transport of LSU rRNA into cytosol. The biological significance of the conjugation-specific appearance of the cytosolic mitochondrial rRNA is discussed.

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First and second backcrosses of amphidiploid hybrids (2n = 4x = 32, genomes AAFF) between shallot (Allium cepa Aggregatum group) and A. fistulosum were conducted to produce A. cepa – A. fistulosum alien addition lines. When shallot (A. cepa Aggregatum group) was used as a pollinator, the amphidiploids and allotriploids set germinable BC₁ and BC₂ seeds, respectively. The 237 BC₁ plants mainly consisted of 170 allotriploids (2n = 3x = 24, AAF) and 42 hypo-allotriploids possessing 23 chromosomes, i.e., single-alien deletions (2n = 3x–1 = 23, AAF-nF). The single-alien deletions in the BC₁ progeny showed dwarfing characteristics and were discriminated from the allotriploids (2n = 24) and hyper-allotriploids (2n = 25) by means of flow cytometric analysis. The chromosome numbers of 46 BC₂ seedlings varied from 16 to 24. Eight monosomic additions (2n = 2x+1 = 17, AA+nF) and 20 single-alien deletions were found in these BC₂ seedlings. Consequently, six kinds of A. cepa – A. fistulosum alien chromosome additions possessing different chromosome numbers (2n = 17, 18, 20, 21, 22, 23) were recognized in the BC₁ and BC₂ populations. A total of 79 aneuploids, including 62 single-alien deletions, were analyzed by a chromosome 6F-specific isozyme marker (Got-2) in order to recognize its existence in their chromosome complements. This analysis revealed that two out of 62 single-alien deletions did not possess 6F. One (AAF-6F) out of the possible eight single-alien deletions could be identified at first. The present study is a first step toward the development of a useful tool, such as a complete set of eight different single-alien deletions, for the rapid chromosomal assignment of genes and genetic markers in A. fistulosum.

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To understand the origin and evolution of common wheat, chloroplast (ct) and nuclear DNA variations were studied in five hexaploid and three tetraploid wheat subspecies. Based on chloroplast simple sequence repeats at 24 loci, they were classified into two major plastogroups. Plastogroup I consisted of 11 plastotypes, including the major plastotype H10 that occurred at the highest frequency (59%) in common wheat. Plastogroup II consisted of five plastotypes and occurred in eight out of 27 accessions of T. aestivum ssp. spelta and one accession of ssp. aestivum. As for nuclear DNA variations, AFLP data using 10 primer sets revealed two major clades of a phylogenetic tree constructed by UPGMA (unweighted pair-group method with arithmetic mean), one consisting of common wheat and the other of emmer wheat. The clade of common wheat was further divided into two major and six minor subclades. One of the major subclades consisted only of non-free-threshing ssp. spelta accessions, which were grouped into two clusters, one consisting only of accessions with plastogroup I ctDNA and the other with both plastogroups I and II. T. aestivum ssp. macha, another non-free-threshing common wheat, formed the other cluster. Taken together, our data indicate the existence of at least two maternal lineages in common wheat and support the hypothesis that European spela wheat originated in Europe separately from other groups of common wheat.

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According to the similarity of the amino acid sequences in their catalytic domains, eukaryotic protein kinases have been classified into the five main groups: ‘AGC’, ‘CaMK’, ‘CMGC’, ‘PTK’ and ‘other’. The AGC group, represented by the cyclic nucleotide-dependent kinases (PKA and PKG), the calcium-phospholipid-dependent kinases (PKC) and the ribosomal S6 protein kinases, are poorly characterized in plants except for a few cases. In this study, in order to gain a better understanding of plant protein kinases in the AGC group, three cDNAs encoding novel protein kinases, RsNdr1 and RsNdr2a/b, were cloned from radish and characterized by molecular and biochemical methods.
The deduced amino acid sequences of RsNdr1 and RsNdr2a/b contained all 12 conserved catalytic subdomains which are characteristic of the eukaryotic Ser/Thr protein kinases. A cell lysate from E. coli overexpressing RsNdr1 fusion protein had protein kinase activity toward a conventional protein substrate (myelin basic protein), whereas that from E. coli harboring a fusion plasmid encoding kinase-dead RsNdr1 or RsNdr2 did not show any protein kinase activity. A phylogenetic tree for 17 protein kinases from various organisms showed that the RsNdrs are more closely related to the protein kinases in a particular subgroup of the ‘AGC’ (fungal cot1-like and animal Ndr kinases) than to the authentic ‘AGC’ protein kinases, such as PKA, PKC or ribosomal S6 kinase.

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The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred ~2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.

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To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana. Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence. From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations. All mitochondria in these mutants were longer and/or larger than wild-type mitochondria. The approximate chromosomal loci of the mutations of seven mutants that grew well were determined. The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant. Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci. At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology.

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In order to understand the microspore and pollen development, recently, we have isolated a number of anther-specific genes in the model legume, Lotus japonicus. From these anther-specific genes, we identified one novel microspore-specific gene, LjImfb-c82. In order to determine the molecular characterization of LjImfb-c82, full-length cDNA clone was first isolated and sequenced. It encoded a protein of 286 amino acids (LjHIR1), which had sequence similarity to Hypersensitive-Induced Response like protein. LjHIR1 was specifically expressed in microspore on the in situ hybridization experiment. From the sequence similarity to prohibitin-domain protein, the LjHIR1 might be related to ion channel regulation in microspore development.

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