We have isolated 5 cDNA clones (din2, din6, din9, din10 and din11) corresponding to genes, the transcripts of which accumulated in leaves of Arabidopsis thaliana kept in the dark. These cDNA clones encode proteins similar to beta-glucosidase (EC 3.2.1.21, din2), asparagine synthetase (EC 6.3.5.4, din6), phosphomannose isomerase (EC 5.3.1.8, din9), seed imbibition protein (din10) and 2-oxoacid-dependent dioxygenases (din11). Accumulation of the transcripts from din6 and din10 occurred within 3 h after plants were transferred to darkness. The transcripts from din2, din9 and din11 were only detected after 24 h of dark treatment. We also observed the accumulation of the din transcripts in senescing leaves. Application of a photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1-1-dimethyl-urea, induced the expression of the din genes under illumination. Application of sucrose to detached leaves suppressed the accumulation of the din transcripts in the dark. These results indicate that expression of these genes partly depends on cellular sugar level. The sugar-modulated expression of the din genes suggests that dark-induced expression of these genes might be related to sugar starvation occurring in leaf cells in the dark, when the photosynthesis is hindered.

Nucleoside 3'-O-bicylic oxazaphospholidine derivatives were designed as monomer units for a solid-phase synthesis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-ODNs). The trans-isomers of appropriately designed nucleoside 3'-O-bicyclic oxazaphospholidine derivatives were generated exclusively by the reaction between the 3'-OH of the corresponding protected nucleosides and 2-chloro-1,3,2-oxazaphospholidine derivatives. The resultant trans-oxazaphospholidine isomers were configurationally stable, and their diastereopurity was not impaired by epimerization in the presence of an acidic activator during the condensation on a solid support. As a result, the formation of both (Rp)- and (Sp)-phosphorothioate internucleotide linkages by using the oxazaphospholidine monomers and the acidic activator proceeded without any loss of diastereopurity (diastereoselectivity > or = 99:1). In addition, ab initio molecular orbital calculations showed that the epimerization of oxazaphospholidine derivatives was most likely to proceed via an edge inversion process that was accelerated by N-protonation. The calculations rationalized not only the relations between the ring structure and the configurational stability of the oxazaphospholidines observed in this study but also the observations reported in the literature that the inversion of tricoordinated organophosphorus compounds were accelerated by acids or nucleophiles.

Branched-chain ␣-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1␤ and E2 subunits of BCKDH, respectively, from Arabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.The mammalian branched-chain ␣-ketoacid dehydrogenase (BCKDH) 1 is a mitochondrial multienzyme complex that is composed of three subunits carrying different enzymatic activities: branched-chain ␣-ketoacid decarboxylase (E1; EC 1.2.4.4), dihydrolipoyl transacylase (E2; no EC number), and dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4). The E1 subunit is further composed of two E1␣ and two E1␤ subunits. This enzyme complex also contains two specific regulatory enzymes, a kinase and a phosphatase (1). E1 catalyzes the oxidative decarboxylation of branched-chain ␣-keto acids, which are derived from branched-chain amino acids by transamination. E2 catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. E3 is a flavoprotein and reoxidizes the reduced lipoyl sulfur residues of the E2 subunit (2).BCKDH in mammals is thought to be a key enzyme in the catabolism of the branched-chain amino acids, i.e. valine, leucine, and isoleucine. As end products, leucine is converted to acetyl-CoA and acetoacetate, valine to succinyl-CoA, and isoleucine to succinyl-CoA and acetyl-CoA (3). Through this pathway, these amino acids serve as substrates for energy production via acetoacetate and succinyl-CoA. It has been firmly established that nutritional conditions play an important role in modulating the activity of BCKDH through a phosphorylation-dephosphorylation mechanism (4). From this point of view, BCKDH is critically important in the pathway for energy utilization, rather than being merely a system for the catabolism of a small group of amino acids.BCKDH has been extensively studied in mammals because defects in the genes for this enzyme cause maple syrup urine disease, an inborn disorder of metabolism in humans (3). In the plant kingdom, however, we have come across only one case reporting the detection...