Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Action of red light on indole-3-acetic-acid status and growth in coleoptiles of etiolated maize seedlings

Brief irradiation of intact etiolated seedlings of maize (Zea mays L.) with red light (R; 30 W cm^-2, 10 min) reduces the amounts of diffusible and free (solvent-extractable) indole-3-acetic acid (IAA) obtainable from excised coleoptile tips. The effect is transient, the lowest level (30% of the dark control) occurring at about 3 h after irradiation. The free-IAA content of the whole coleoptile and the diffusible-IAA yield from the base of the same organ are similarly reduced, whereas the conjugated-IAA content of the coleoptile is not affected. These results support the view that R inhibits the production of IAA at the coleoptile tip. It is further shown that R inhibits biosynthesis of [³H]IAA from [³H]tryptophan supplied to the coleoptile tip. The shapes of the fluence-response curves obtained for the reduction of the diffusible-IAA yield by R and far-red light (FR) indicate the participation of two photoreactive systems. One has thresholds at 10^-3 W s cm² of R, five orders of magnitude less than the minimum required for the appearance of spectrophotometrically measurable far-red-absorbing form of phytochrome (Pfr) in vivo, and 10^-1 W s cm^-2 of FR; its response is linear to the logarithm of fluence exceeding five orders of magnitude. The other system is seen above 10² W s cm^-2 as an increase in the slope of the fluenceresponse curve; its response is FR reversible and related to the Pfr level of total photoreversible phytochrome. Both systems inhibit biosynthesis of IAA from tryptophan. Elongation of the coleoptile is stimulated by R; the stimulation is most apparent in the apical region, and is saturated with a fluence at which bo detectable pfr is formed. Farred light can also saturate this response. Since the endogenous IAA concentration in the coleoptile appears not to be in the inhibitory range, it is concluded that the stimulation of coleoptile elongation is not the result of changes in free-IAA levels.Abbreviations FR far-red light - IAA indole-3-acetic acid - Pfr phytochrome in the far-red-absorbing form - Pr phytochrome in the red-absorbing form - R red light     

Torque Generation of Enterococcus hirae V-ATPase

V-ATPase (V_oV₁) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V_oV₁ for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V_oV₁ (EhV_oV₁) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV_oV₁ exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV_oV₁ only showed the “clear” state without apparent backward steps, whereas EhV₁ showed two states, “clear” and “unclear.” Furthermore, EhV_oV₁ showed slower rotation than EhV₁ without the three distinct pauses separated by 120° that were observed in EhV₁. When using a large probe, EhV_oV₁ showed faster rotation than EhV₁, and the torque of EhV_oV₁ estimated from the continuous rotation was nearly double that of EhV₁. On the other hand, stepping torque of EhV₁ in the clear state was comparable with that of EhV_oV₁. These results indicate that rotor-stator interactions of the V_o moiety and/or sodium ion transport limit the rotation driven by the V₁ moiety, and the rotor-stator interactions in EhV_oV₁ are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV₁. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV_oV₁.     

Population structure and transmission dynamics of Plasmodium vivax in the Republic of Korea based on microsatellite DNA analysis

Background

In order to control malaria, it is important to understand the genetic structure of the parasites in each endemic area. Plasmodium vivax is widely distributed in the tropical to temperate regions of Asia and South America, but effective strategies for its elimination have yet to be designed. In South Korea, for example, indigenous vivax malaria was eliminated by the late 1970s, but re-emerged from 1993. We estimated the population structure and temporal dynamics of transmission of P. vivax in South Korea using microsatellite DNA markers.

Methodology/Principal Findings

We analyzed 255 South Korean P. vivax isolates collected from 1994 to 2008, based on 10 highly polymorphic microsatellite DNA loci of the P. vivax genome. Allelic data were obtained for the 87 isolates and their microsatellite haplotypes were determined based on a combination of allelic data of the loci. In total, 40 haplotypes were observed. There were two predominant haplotypes: H16 and H25. H16 was observed in 9 isolates (10%) from 1996 to 2005, and H25 in 27 (31%) from 1995 to 2003. These results suggested that the recombination rate of P. vivax in South Korea, a temperate country, was lower than in tropical areas where identical haplotypes were rarely seen in the following year. Next, we estimated the relationships among the 40 haplotypes by eBURST analysis. Two major groups were found: one composed of 36 isolates (41%) including H25; the other of 20 isolates (23%) including H16. Despite the low recombination rate, other new haplotypes that are genetically distinct from the 2 groups have also been observed since 1997 (H27).

Conclusions/Significance

These results suggested a continual introduction of P. vivax from other population sources, probably North Korea. Molecular epidemiology using microsatellite DNA of the P. vivax population is effective for assessing the population structure and transmission dynamics of the parasites - information that can assist in the elimination of vivax malaria in endemic areas.     

Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

In vitro synthesis of phase-specific flagellin of Salmonella

Chromatography of Salmonella flagellin at pH 8 on DEAE-cellulose separated at least four serologically distinct kinds of flagellin, a, enx, i and 1,2, eluting in that order with increasing concentration of sodium chloride. By this chromatographic technique, the preincubated cell-free extract of Escherichia coli given saltprecipitable RNA of Salmonella was shown to synthesize flagellin characteristic of the flagellar antigen type of the cells from which the RNA was derived. Two of the in vitro synthesized flagellins specifically reacted with their corresponding antiserum.When RNA was extracted from the cells of the diphasic strain propagated from a single colony, expressing either phase 1 or phase 2, the in vitro synthesized flagellin was predominantly the same as that produced by the original colony. Translation of messenger RNA specific for phase 1 flagellin was not inhibited by the presence of messenger RNA specific for phase 2. RNA extracted from the cells of a diphasic strain without any selection directed synthesis of both phase 1 and phase 2 flagellins in the ratio expected if the culture was at equilibrium with respect to phase variation. Experimental evidence is presented to support the hypothesis that phase variation is due to the alternative synthesis of phase-specific messenger RNA.     

Blue-light-dependent osmoregulation in protoplasts of Phaseolus vulgaris Pulvini.

Blue light was found to induce shrinkage of the protoplasts isolated from first-leaf lamina pulvini of 18-day-old Phaseolus vulgaris. The response was transient following pulse stimulation, while it was sustainable during continuous stimulation. No apparent difference was found between flexor and extensor protoplasts. Protoplasts of the petiolar segment located close to the pulvinus showed no detectable response. In the plants used, the pulvinus was fully matured and the petiole was ceasing its elongation growth. When younger, 12-day-old, plants were used, however, the petiolar protoplasts did respond to blue light. The pulse-induced response was similar to that in pulvinar protoplasts, although the response to continuous stimulation was transient and differed from that in pulvinar protoplasts. No shrinkage was induced in pulvinar protoplasts when the far-red-light-absorbing form of phytochrome was absent for a period before blue-light stimulation, indicating that the blue-light responsiveness is strictly controlled by phytochrome. Inhibitors of anion channels and H(+)-ATPase abolished the shrinking response, supporting the view that protoplasts shrink by extruding ions. The response of pulvinar protoplasts is probably involved in the blue-light-induced, turgor-based movement of pulvini. The blue-light responding system in pulvini is suggested to have evolved from that functioning in other growing organs.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究(简报)

正常情况下,染色质和染色体在细胞内呈高度致密状态,在光镜和透射电镜下常呈浓染的斑块状。由于方法学上的困难,至今对染色质乃至染色体的微细结构,仍缺乏清楚的了解。特别是关于染色质如何凝缩形成染色体方面,现仍存在有争论。扫描电镜的冷冻割断技术,曾被用于对游离细胞间期核染色质的观察,并取得了较好的     

Structure of a central stalk subunit F of prokaryotic V-type ATPase/synthase from Thermus thermophilus

The crystal structure of subunit F of vacuole-type ATPase/synthase (prokaryotic V-ATPase) was determined to of 2.2 A resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY. The structure was successfully placed into the low-resolution EM structure of the prokaryotic holo-V-ATPase at a location indicated by the results of crosslinking experiments. The crystal structure, together with the single-molecule analysis using fluorescence resonance energy transfer, showed that the subunit F exhibits two conformations, a 'retracted' form in the absence and an 'extended' form in the presence of ATP. Our results postulated that the subunit F is a regulatory subunit in the V-ATPase.