Evaluation of an automated DNA sequencing system developed in RIKEN

For the advancement of Human Genome Project, we have developed an automated DNA sequencing system HUGA-I. It is composed of several automated instruments and transfer robots connecting them. In this paper we describe the results of the performance evaluation test of HUGA-I. Although some of the system units showed good performances, the total performance of the HUGA-I was about 1/6 of the designed value. By revealing principal reasons of this poor performance, we would like to contribute to the automation in genome analysis, particularly in human genome analysis.Since the sequence technology advanced remarkably in these years, the system units of HUGA-I become older than those which are now commercially available and the throughput of it is out of our expectations. Nevertheless, we believe that it is meaningful to introduce the exact performance of HUGA-I and present the bottle neck points in the automating sequencing processes. Because, automation in the gene analysis is ultimately important, in particular for the analysis of large genomes such as the human genome. The aims of this paper are to introduce the results in performance evaluation of HUGA-I and to elucidate the bottle neck points in the automation of sequencing processes.The authors express their sincere thanks to Mr. Morisada Hayakawa and Mrs. Nobuko Kato for their technical asistance.     

An on-line advisory control system for the lactic acid fermentation process

A fuzzy expert system was developed for online diagnosing and controlling of bioprocesses. The system was constructed in object-oriented Smalltalk/V for diagnosing and controlling of bioprocesses. Lactic acid fermentation with an industrial strain ofLactobacillus casei was chosen as the model system. The performance of the fuzzy expert system and the knowledge base utilizing experts' knowledge and several facts obtained from the experiments were successfully validated with on-line fermentations. The fuzzy expert system could diagnose a fault on-line and give reasonable advice to the process operator. In order to achieve the diagnosing faculty, a database, a knowledge base, and both backward and forward chaining procedures were implemented employing the object-oriented programming environment. A defuzzifier was implemented in the system to achieve on-line control. In order to realize a decision-making system with a human operator and a fuzzy expert system, a new control strategy namedAdvice was also introduced. Several cultivations were carried out in order to collect knowledge on the effects concerned with inoculum properties to the process and to construct a database including standard time-course profiles. The performance of the fuzzy expert control system was successfully tested with on-line experiments.Visiting scientist from HUT at RIKEN     

Sialosylcholesterol Effects on Reconstitution of Microfilament and Glia Filament

Abstract: The effects of α-sialosylcholesterol (α-SC) on formation of either microfilament or glia filament of rat astrocytes were investigated using a reconstitution system. Polymerization of the depolymerized microfilament preparation that had been extracted from a crude cytoskeletal fraction of rat astrocytes, in the presence of 100 m M KCI and 10 m M MgCI₂, was suppressed in a dose-dependent manner by α-SC. α-SC inhibited polymerization of G-actin in a similar manner. The intensity of a-SC inhibition of G- actin polymerization was as great as that of microfilament polymerization, suggesting that the inhibition of microfilament polymerization by α-SC was due to the direct action of α-SC on actin, the main component of microfilament. α-SC depolymerized partly the polymerized microfilament preparation, which resembled F-actin (microfilament-like filaments). α-SC suppressed, in a dose-dependent manner, polymerization of a glia filament preparation that had been extracted from astrocyte cytoskeletons in the presence of phalloidin. An increase in the amount of added α-SC (up to 15 n M ) decreased the amount of the larger glia filament-like filaments, which were 10 nm thick and centrifuged down at 16,000 g for 30 min, and increased that of smaller ones precipitated only after centrifugation at 100,000 g for 1 h. The lower the concentration of the depolymerized glia filament extract, the greater was the inhibition by α-SC of the polymerization. α-SC repressed polymerization of vimentin, the dominant component of glia filament. Vimentin polymerization was more strongly inhibited by α-SC than polymerization of glia filament was. The findings suggested that α-SC suppressed polymerization of glia filament through a direct action on vimentin and that the glia filament-associated proteins increased its structural stability in the presence of α-SC.     

Presence of β-cyanoalanine synthase in unimbibed dry seeds and its activation by ethylene during pre-germination

Evolution of HCN from both rice ( Oryza sativa ) and cocklebur ( Xanthium pennsylvanicum ) seeds increased during a pre-germination period and preceded the evolution of (C₂H₄). These two species were adopted as the representatives of starchy and fatty seeds, respectively. Ethylene promotes seed germination of many species. However, HCN evolution declined abruptly when the radicles emerged and before the peak in C₂H₄ evolution. More-over, both rice and soybean ( Glycine max ) seeds showed some activity of β-cyanoalanine synthase (CAS, EC 4.4.1.9) even in the unimbibed dry state. The activities of CAS in the lower seed of cocklebur and in soybean seeds increased rapidly after emergence of the radicle. However, the CAS of rice seeds, with high activity in the dry state, exhibited a bimodal change, gradually decreasing until radicle emergence had occurred, but then increaing. It is thus likly that HCN evolution during initial imbibition may be derived from cyanogenic reserves and controlled by both pre-existing and subsequently-developing CAS. The exogenous application of C₂H₄ stimulated the activities of CAS in both rice and upper cocklebur seeds and reduced their cyanogen contents. Therefore, the decline of HCN evolution after germination seems to be due to the increased activities of CAS by endogenously produced C₂H₄.     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

The effect of temperature,light-spectrum,desiccation and salinity gradients on the photosynthetic performance of a subtidal brown alga,Sargassum macrocarpum,from Japan

The effect of temperature, light-spectrum, desiccation and salinity gradients on the photosynthesis of a Japanese subtidal brown alga, Sargassum macrocarpum (Fucales), was determined using a pulse amplitude modulation-chlorophyll fluorometer and dissolved oxygen sensors. Temperature responses of the maximum (F_v/F_m in darkness) and effective (ΔF/F_m′ at 50 μmol photons m⁻² s⁻¹; = Φ_PSII) quantum yields during 6-day culture (4–36°C) remained high at 12–28°C, but decreased at higher temperatures. Nevertheless, ΔF/F_m′ also dropped at temperatures below 8°C, suggesting light sensitivity under chilling temperatures because F_v/F_m remained high. Photosynthesis–irradiance responses at 24°C under red (660 nm), green (525 nm), blue (450 nm) and white light (metal halide lamp) showed that maximum net photosynthesis under blue and white light was greater than under red and green light, indicating the sensitivity and photosynthetic availability of blue light in the subtidal light environment. In the desiccation experiment, samples under aerial exposure of up to 8 h under dim-light at 24°C and 50% humidity showed that ΔF/F_m′ quickly declined after more than 45 min of emersion; furthermore, ΔF/F_m′ also failed to recover to initial levels even after 1 day of rehydration in seawater. Under the emersion state, the ΔF/F_m′ remained high when the relative water content (RWC) was greater than 50%; in contrast, it quickly dropped when the RWC was less than 50%. When the RWC was reduced below 50%, ΔF/F_m′ did not return to initial levels, regardless of subsequent re-hydration, suggesting a low capacity of photosynthesis to recover from desiccation. The stenohaline response of photosynthesis under 3-day culture is evident, given that ΔF/F_m′ declined when salinity was beyond 20–40 psu. Adaptation to subtidal environments in temperate waters of Japan can be linked to these traits.     

A new snailfish of the genus Careproctus (Cottoidei: Liparidae) from the Pacific coast of southern Japan

Ichthyological Research - A new snailfish, Careproctus tomiyamai, is described on the basis of four specimens collected from Suruga Bay, Tosa Bay, and the Hyuga-nada Sea, southern Japan...     

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.