Examination of the lipid composition of spore membranes of Bacillus subtilis Marburg, extracted after treatment of spores with dithiothreitol/urea and NaOH followed by lysozyme digestion, revealed that the spore membranes had significantly higher cardiolipin (CL) content than the membranes of exponentially growing cells. Analysis of the membranes of coat-defective, cotE::cat and gerE::cat mutant spores, which are susceptible to lysozyme digestion without chemical treatment, confirmed that spore membranes contain a high level of CL. After addition of the germinants L-alanine or AGFK (a combination of asparagine, glucose, fructose, and KCl), the turbidity of wild type spore suspensions decreased to 50% within 30 min. Suspensions of spores with only trace amounts of CL, however, showed no decrease in turbidity when L-alanine was added and the initial decrease in turbidity with AGFK was slight (14% after 60 min). These results indicate that CL is involved in an early step of germination, related to the functioning of germinant receptors. This is the first conspicuous in vivo evidence that CL in bacterial membranes has a specific role, in which it cannot be replaced by other anionic phospholipids.

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A number of cold responsive (Cor)/late embryogenesis abundant (Lea) genes are induced by both low temperature (LT) and dehydration. To understand the molecular basis of cold acclimation and its relationship with drought stress response in wheat seedlings, we isolated a DREB2 homolog Wdreb2, which is the candidate gene for a transcription factor of the Cor/Lea genes. The Wdreb2 expression was activated by cold, drought, salt and exogenous ABA treatment. Detailed expression studies of Wdreb2 indicated the involvement of two distinct pathways of its activation, a drought and salt stress-responsive pathway and a cold-responsive pathway. The transient expression analysis showed that the Wrab19 expression was directly activated by the WDREB2 transcription factor in wheat cells. Three transcript forms of Wdreb2 (Wdreb2α, Wdreb2β and Wdreb2γ) were produced through alternative splicing. Under drought and salt stress conditions, the amount of the Wdreb2β form remained fairly constant during 24-hour treatment, while those of the Wdreb2α and Wdreb2γ forms showed transient increases. On the other hand, the LT treatment resulted in increased transcript levels of all three forms of Wdreb2. Thus, under the LT and drought/salt stress conditions the amount of the WDREB2 transcription factors in wheat is differentially controlled by the level of transcription and alternative splicing.

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CACTA is a class 2 transposon, that is very abundantly present in plant genomes. Using Rim2/Hipa CACTA transposon display (hereafter Rim2/Hipa-TD), we analyzed several A-genome diploid Oryza species that have a high distribution of the CACTA motifs. High levels of polymorphism were detected within and between the Oryza species. The African taxa, O. glaberrima and O. barthii, both showed lower levels of polymorphism than the Asian taxa, O. sativa, O. rufipogon, and O. nivara. However, O. longistaminata, another African taxon, showed levels of polymorphism that were similar to the Asian taxa. The Latin American taxon, O. glumaepatula, and the Australian taxon, O. meridionalis, exhibited intermediate levels of polymorphism between those of the Asian and African taxa. The lowest level of polymorphism was observed in O. glaberrima (32.1%) and the highest level of polymorphism was observed in O. rufipogon (95.7%). The phylogenetic tree revealed three major groups at the genetic similarity level of 0.409. The first group consisted of three Asian taxa, O. sativa, O. rufipogon and O. nivara. The second group consisted of three African taxa, O. glaberrima, O. barthii, O. longistaminata, and an American taxon, O. glumaepatula. The third group contained an Australian taxon, O. meridionalis. The clustering patterns of these species matched well with their geographical origins. Rim2/Hipa-TD appears to be a useful marker system for studying the genetic diversity and species relationships among the AA diploid Oryza species.

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We investigated DNA variation within and between two closely related conifers, Cryptomeria japonica and Taxodium distichum, at nuclear loci encoding ferredoxin, glutamyl-tRNA reductase, lycopene beta cyclase, and phosphoribosylanthranilate transferase. Average nucleotide diversity at silent sites was estimated to be 0.0035 (SE 0.0012) in C. japonica and 0.0058 (SE 0.0006) in T. distichum. One population in C. japonica was differentiated from the others but generally there was not much differentiation among populations or varieties within the two species. However, the two species seemed to differ in frequency spectra of DNA polymorphisms. Excesses of intermediate-frequency variants were found in C. japonica, whereas excesses of both rare and high-frequency variants were found in T. distichum, which suggested different histories of population structures in the two species. Deviations from the standard neutral expectations in DNA polymorphisms were found by applications of neutrality tests. The results show that actions of selection to respective loci seem to differ between the two species, indicating differences of interaction among evolutionary factors.

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The Colombian jaguar population is thought to contain two different subspecies, Panthera onca centralis and Panthera onca onca. The genetic structure of this population was evaluated using 12 microsatellite loci (n = 62 samples). In addition, 22 jaguar DNA samples from Guatemala, Paraguay, Perú, Bolivia, Venezuela and Brazil were analyzed for these microsatellite loci (n = 84 samples). The results of this study indicate six primary themes. First, the levels of gene diversity were very high. Second, the majority of the loci analyzed showed an absence of Hardy-Weinberg equilibrium, probably due to the Wahlund effect (= population subdivision). Third, several microsatellite loci showed significant heterogeneity between the two supposed subspecies in the country. Nevertheless, gene flow was present between them, and heterogeneity was relatively low, although the assignment analyses showed good classification of the jaguars studied into their respective subspecies. Fourth, the long-term historical effective population sizes were calculated through a maximum likelihood procedure for single and multi-step mutation models. Fifth, seven out of twelve DNA microsatellites studied significantly deviated from a single-step mutation model. However, the overall mean multi-step mutation percentage for these 12 DNA microsatellites was only 6%. Therefore, 94% of mutations were uni-step. Sixth, no bottleneck events were detected in the Colombian jaguar population overall.

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RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.

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β-conglycinin, a major component of seed-storage proteins in soybean, comprises three subunits: α, α', and β. Expression of these genes is spatially regulated in a stringent manner and occurs during seed development. To understand the mechanisms that control expression of the α subunit gene, we analyzed the nucleotide sequence of the 2.9-kb region upstream of the gene. The upstream sequence up to –1357 or a series of its 5'-deleted derivatives was fused to the β-glucuronidase (GUS) gene. These reporter gene constructs were introduced into Arabidopsis thaliana plants via Agrobacterium-mediated gene transfer. Prominent GUS activity was detected in developing seeds of the T3 generation when 245 bp or longer sequences of the upstream region were fused to the GUS gene. We found a clear association of decreased GUS activity with a stepwise deletion of a region containing the RY sequence from the original construct. These results are consistent with the notion that multiple sequence elements including the RY sequences are involved in the seed-specific transcriptional activation of the β-conglycinin α subunit gene in soybean.

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Gene targeting is a powerful method of specifically modifying genes of interest. It has been most consistently successful in the 129 mouse strain, because the embryonic stem (ES) cells of 129 mice are relatively easy to culture. In gene-targeting experiments, the use of ES cell-derived genomic clones as a source of homology arms is desirable, because the genetic variation among mouse strains results in a reduced frequency of homologous recombination. In this study, we generated an arrayed mouse 129/Ola BAC library derived from E14.1 ES cells, one of the frequently used ES cell lines. More than 135,000 BAC clones with a mean insert size of 110 kb were isolated. This library is estimated to represent a 5.5-fold mouse genome coverage. The BAC clones can be screened within 2 days by PCR. Considering that all 8 loci so far examined are contained in this BAC library, we believe it will be a useful resource for gene targeting studies using E14 ES cells as well as for genome analysis.

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