A thermostable α-amylase (EC 126.96.36.199, α-1, 4-glucan-glucanhydrolase) from a new Bacillus sp-JF2 strain was purified and characterized. The molecular weight of the α-amylase was about 110, 000 by the method of polyacrylamide gel gradient electrophoresis and that of the subunit was about 50, 000 by the method of SDS polyacrylamid gel gradient electrophoresis. The α-helix content of the enzyme protein was 41% by the method of the circular dichroism. The enzyme protein molecule contained calcium ion, but the calcium ion did not closely relate with the activity site of α-amylase. The temperature for the high enzyme activity was 85 to 90°C. Calcium and magnesium ions did not highly affect enzyme activity, but Fe2+, Cu2+, Zn2+ and Ag+ ions inhibited the activity.
We have previously shown that an alkaline extract from cones of pine trees could significantly inhibit HIV-1 replication in vitro. Ultraviolet, infrared, thin-layered chromatography and nuclear magnetic resonance analyses showed that polymeric phenylpropenoids might be the active ingredient in the alkaline extract that inhibited HIV-1 replication. We synthesized polymeric phenylpropenoids by dehydrogenation of caffeic acid, p-coumaric acid, ferulic acid and coniferyl alcohol. All 4 synthetic isopolymers had anti-HIV activity comparable to that found in the alkaline pine cone extract. Additionally, 3 of the 4 polymers blocked infection of cell by herpes simplex type-1 virus. Among the monomers, only caffeic acid had anti-HIV activity, but was also toxic to cells. None of the monomers had impact on herpes simplex virus infection.
The technique of Contour-clamped Homogenous Electric Field (CHEF) gel electrophoresis has been used to separate intact chromosomal DNA of different species of the yeast genera Saccharomyces and Zygosaccharomyces. Various strains of the same species had similar chromosomal band patterns. However, differences between individual bands indicated that strain-specific chromosome length polymorphism is common in these organisms. To test conspecificity, a new, DNA-DNA hybridization technique was developed using individual whole chromosomes as templates to prepare randomly primed, radioactive probes. Under our conditions of stringency, species-specific hybridization reactions were achieved with these probes. The method is an efficient tool to study the genetic diversity of a given yeast species. Also, it can assist yeast species identification.
Plasmid pRt032 which has a 14-kb HindIII fragment containing nodulation genes from Rhizobium leguminosarum bv. trifolii ANU843 was transferred into R. fredii USDA 193. A transconjugant CT24 was isolated whose host range was extended to white clover. However, nodulation on the original host soybean was strongly inhibited in CT24 as compared with that of wild-type R. fredii USDA 193. The inhibition of nodulation on soybean was investigated with R. fredii USDA 193 carrying a derivate of pRt032 (nod218) in which the lac operon and Tn5 (MudI1734) was inserted downstream of the nodA promoter. This strain, AF218, restored efficient nodulation on the soybean. The NodD-mediated induction of the R. leguminosarum bv. trifolii nodA-lacz fusion in R. fredii USDA 193 was also monitored during the growth cell cycle following exposure to 7, 4′-dihydroxyflavone (DHF), clover extracts, and soybean extracts for 3h. The nodA induction was observed in ratios of 11:7:3, respectively. However, nodA induction was inhibited using 7, 4′-dihydroxyisoflavone (daidzein), an inducer of R. fredii nod genes. The factor that induces the R. leguminosarum bv. trifolii nod gene expression in soybean extract is not the daidzein, but other unidentified plant factors. In strain CT24, heterologous common nod genes are expressed. These genes interfere with R. fredii nod gene products, causing poor nodulation on its original host soybean.
Two marine bacteria capable of producing extracellular β-1, 4-mannanases (strain nos. MA-129 and MA-138) were isolated from sea environment. A large amount of β-mannanases were induced with konjac powder from both strains and also with mannose from strain no. MA-138. Both strains showed high enzyme production when incubated with shaking at 25°C for 24h. The culture fluid of strain no. MA-129 was fractionated into one active peak and that of strain no. MA-138 two active peaks by ion-exchange chromatography. Every enzyme fraction had pH optimum of 7.0-7.5 and hydrolyzed β-mannan and glucomannan to form several oligosaccharides. Both strains belonged to genus Vibrio.
DNA of plasmid pKYM replicates in the extract prepared following the method of Fuller et al. (Fuller, R. S., Kaguni, J. M., and Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA, 78, 7370-7374), when it contains the Rep protein of pKYM. Although the synthesis of DNA was completely inhibited by the DNA gyrase inhibitor, a substantial quantity of DNA was synthesized even in the absence of RNA synthesis and the accumulated DNA contained a strand-specific single-stranded circular molecule. The kinetic analysis of the DNA synthesis in the absence of RNA synthesis suggests that the single-stranded DNA appeared after the synthesis of double-stranded molecule. Since the DNA synthesized under the condition which allowed the synthesis of RNA is a fully twisted double-stranded molecule, the synthesis of RNA will initiate the DNA synthesis on the single-stranded molecule. These results support the idea that pKYM replicates via a rolling-circle mechanism.