The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [¹⁴C]-labeled G3P to synthesize [¹⁴C]-labeled LPA, whereas those from ΔplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a ΔygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

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Postzygotic reproductive isolation (RI) often arises in inter-subspecific crosses as well as inter-specific crosses of rice (Oryza sativa L.). To further understand the genetic architecture of the postzygotic RI, we analyzed genes causing hybrid sterility and hybrid breakdown in a rice inter-subspecific cross. Here we report hybrid male sterility caused by epistatic interaction between two novel genes, S24 and S35, which were identified on rice chromosomes 5 and 1, respectively. Genetic analysis using near-isogenic lines (NILs) carrying IR24 (ssp. indica) segments with Asominori (ssp. japonica) genetic background revealed a complicated aspect of the epistasis. Allelic interaction at the S24 locus in the heterozygous plants caused abortion of male gametes carrying the Asominori allele (S24-as) independent of the S35 genotype. On the other hand, male gametes carrying the Asominori allele at the S35 locus (S35-as) showed abortion only when the IR24 allele at the S24 locus (S24-ir) was concurrently introgressed into the S35 heterozygous plants, indicating that the sterility phenotype due to S35 was dependent on the S24 genotype through negative epistasis between S24-ir and S35-as alleles. Due to the interaction between S24 and S35, self-pollination of the double heterozygous plants produced pollen-sterile progeny carrying the S24-ir/S24-ir S35-as/S35-ir genotype in addition to the S24 heterozygous plants. This result suggests that the S35 gene might function as a modifier of S24. This study presents strong evidence for the importance of epistatic interaction as a part of the genetic architecture of hybrid sterility in rice. In addition, it suggests that diverse systems have been developed as postzygotic RI mechanisms within the rice.

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Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1,700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function.

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Differentiation into specific embryo cell types correlates with the processes that lead to the accumulation of seed storage proteins in plants. The α subunit of β-conglycinin, a major component of seed storage proteins in soybean, accumulates at a higher level in cotyledons than in the embryonic axis in developing embryos. To understand the mechanisms underlying this phenomenon, we characterized the upstream region of the α subunit gene in terms of transcriptional control using transgenic Arabidopsis thaliana plants carrying reporter gene constructs comprising the 1357-bp upstream sequence of the α subunit gene and the β-glucuronidase (GUS) gene. Analysis of the time-course-dependent pattern of GUS expression revealed that the expression was first confined to the cotyledons and occurred later in the entire embryo during embryogenesis. The level of GUS expression was higher in cotyledons than in the embryonic axis throughout the period of its expression, coincident with the distribution of the α subunit protein in soybean embryos. By testing progressively shorter promoter fragments, the cis-acting elements responsible for transcriptional activation in the cotyledons and the embryonic axis were both localized to the region spanning –245 to –161 relative to the transcription start site. It is also concluded that the upstream region up to –245 is sufficient to control the spatial and temporal pattern of transcription, while further upstream regions influence transcription rate without affecting the transcriptional pattern. Overall, these results indicate that the unequal distribution of α subunit protein within the embryos is established primarily as a consequence of differential transcriptional activation controlled by a short proximal promoter region of the gene in different embryonic tissues.

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