The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks

Background  

Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure.     

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.     

Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis,saturation mutagenesis,and in vitro recombination of the type II polyhydroxyalkanoate synthase gene

Type II synthase (PhaC1(Ps)) for polyhydroxyalkanoate (PHA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1(Ps) in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Escherichia coli. Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.     

Turnover of myelin lipids in aging brain

Turnover rates of myelin membrane components in mouse brains were determined by a method using stable isotope-labeling and mass spectrometry. The half-replacement times based on incorporation rates of newly synthesized molecules for young adult mice were 359 days for cholesterol, 20 days for phosphatidylcholine, 25 days for phosphatidylethanolamine, 94 days for cerebroside and 102 days for ganglioside GM1. The turnover rates of half-lives of myelin components were calculated from the decay curves of initially labeled molecules, and they were about the same as the half-replacement times. Individual components were thus revealed to be metabolized at different rates, and their turnover rates were differently affected by aging. As was observed with phospholipids, myelin pools appeared to be compartmentalized into rapidly and slowly exchanging pools. The turnover rates of cerebroside and GM1 decreased between the young and adult periods and slightly increased in senescence. The latter phenomenon may indicate an enhanced myelin turnover in senescence. The present study reveals the dynamic aspects of myelin membrane turnover during the life span of mouse.     

DFL2, a new member of the Arabidopsis GH3 gene family, is involved in red light-specific hypocotyl elongation

A new GH3-related gene, designated DFL2, causes a short hypocotyl phenotype when overexpressed under red and blue light and a long hypocotyl when antisensed under red light conditions. Higher expression of this gene was observed in continuous white, blue and far-red light but the expression level was low in red light and darkness. DFL2 gene expression was induced transiently with red light pulse treatment. DFL2 transgenic plants exhibited a normal root phenotype including primary root elongation and lateral root formation, although primary root elongation was inhibited in antisense transgenic plants only under red light. The adult phenotypes of sense and antisense transgenic plants were not different from that of wild type. DFL2 promoter activity was observed in the hypocotyl. Our results suggest that DFL2 is located downstream of red light signal transduction and determines the degree of hypocotyl elongation.     

A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso)flavonoids in Lotus japonicus

Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6'-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6'-deoxychalcone and 6'-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1-cCHI3) from a model legume, Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that four CHI genes (CHI1-3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed in Escherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae.     

Production of functional lectin in Pichia pastoris directed by cloned cDNA from Aleuria aurantia

A plasmid bearing a nucleotide sequence of fucose-specific lectin of Aleuria aurantia was constructed and expressed in a methylotrophic yeast, Pichia pastoris. The product showed almost the same hemagglutinating activity as the lectin produced in Escherichia coli, the properties of which were quite similar to the native one. Because of glycosylation of the product, the molecular mass was larger than that of the native one, and it acquired higher thermostability.     

An extra large insertion in the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion effects and relation to cellular proteolysis

The polyhydroxyalkanoate (PHA) synthase (PhaC(Da)) from Delftia acidovorans DS-17 (formerly Comamonas acidovorans) has a unique large insertion consisting of 40 amino acid residues in the alpha/beta hydrolase fold region. In order to examine whether this insertion is necessary for enzyme function, we generated a mutant gene where the nucleotides encoding the insertion sequence were deleted [phaC(Da)del(342-381)]. The ability of the mutant PhaC(Da) lacking the insertion sequence to produce PHA in recombinant Escherichia coli JM109 was compared with that of wild-type PhaC(Da). The results revealed that the mutant enzyme had approximately one fourth the activity of the wild-type enzyme. However, there was no significant difference in PHA content accumulated in cells harboring either the mutant PhaC(Da) or wild-type PhaC(Da) nor were there any differences in the molecular masses of the produced polymers. Therefore, we have concluded that the characteristic insertion is not indispensable for PHA synthesis. Also, slight cellular proteolysis in E. coli was found specifically for wild-type PhaC(Da) by Western blot analysis. This result prompted us to further examine the proteolytic stability of PhaC(Da) in D. acidovorans. Consequently, it has been suggested that the insertion region of PhaC(Da) is susceptible to cellular proteolysis during accumulation of PHA.