Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.     

Study on fibroin heavy chain of the silkwormBombyx mori by fluorescencein situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized theFib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified thatFib-H has only one locus, thus providing a temporary solution to the problem about its localization.     

Factors contributing to cerebral hypomyelination in the growth hormone-deficientlittle mouse

We attempted to delineate the events leading to hypomyelination in the brain of thelittle mouse, a promising murine model of isolated growth hormone deficiency. At 20 days of age, the mutant mouse brain weighed less than its normal counterpart, and this difference in brain weight persisted. Increase in CNPase activity was found to be suppressed in the cerebrum throughout the developmental stage, but not in the other parts of the brain. Differences in cerebral DNA content between thelittle and normal mice first became apparent on the 10th day of age. Thereafter, the rate of increase in thelittle brain consistently lagged behind the normal. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the normal cerebrum is most active, was approximately half that of the controls in all parts of thelittle brain. These findings indicate that the hypomyelination of the mutant cerebrum might result from reduced oligodendroglial proliferation due to growth hormone deficiency.     

Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

Efficient Protein Expression in <Emphasis Type="Italic">Bombyx mori</Emphasis> Larvae of the Strain d17 Highly Sensitive to <Emphasis Type="Italic">B. mori</Emphasis> Nucleopolyhedrovirus

The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a “factory” for large-scale expression using the BmNPV bacmid system.     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.     

Activation process of pepsinogen.

The activation process of pepsinogen was analyzed by a combination of computer simulation and experiment. In order to investigate in detail the behavior of the basic schemes proposed in the previous study, further computer simulations were conducted. Some experiments were performed based on the information obtained. The changes in the UV difference spectrum in the early stage was measured by the stopped-flow technique and the conversion of pepsinogen to pepsin [EC 3.4.23.1] was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, on the basis of the experimental results, the most reasonable scheme was selected and modified. As a result, a scheme for the activation process of pepsinogen was obtained (Scheme 8). On the basis of the above analyses, it was assumed that the first step and the third step are pH-dependent based on the change in the UV spectrum, that the second step is a nonlinear reaction containing a looped reaction with a dimeric intermediate (in this step, peptide fragments are released and pepsinogen is converted to a pepsin-like molecule), and that the third step is an equilibrium reaction involving proton binding.     

Isolation and some properties of extracellular d-glucosyltransferases and d-fructosyltransferases from Streptococcus mutans serotypes c, e, and f

Extracellular d-glucosyltransferases (GTase) and d-fructosyltransferases (FTase) were isolated from Streptococcus mutans IB (serotype c), B14 (e), and OMZ175 (f) by chromatofocusing, followed by hydroxyapatite column chromatography. The GTases isolated from serotypes c, e, and f are basic proteins (pI 7.4). The serotype c and e enzymes have two protein components having M_r 173 000 and 158 000 and the enzyme of the serotype f one component having M_r 156 000. The GTases of all the serotypes showed a K_m value for sucrose of 10–14mm and an optimum pH 5.5–6.0 for enzyme activity, and their activities were enhanced by the presence of primer Dextran T10. The α-d-glucans synthesized by the purified GTases are water soluble and primarily consist of (1→6)-α-d-glucosidic linkage (41–66 mol/100 mol) and α-d-(1→3,6)-branch linkage (6–20 mol/100 mol), but significant proportions of α-d-(1→3), α-d-(1→4), and α-d-(1→3,4) linkages (11, 6, and 14 mol/100 mol, respectively) were detected in the serotype c α-d-glucan. The isolated FTases of the serotypes c, e, and f are acidic enzymes (pI 4.6) and consist of two components having M_r 84 000 and 76 000 for the serotype c enzyme, and 106 000 and 84 000 for the serotypes e and f enzymes, respectively. The K_m value for sucrose was 6, 10, and 17mm for the serotypes c, e, and f enzymes, respectively, and the optimum pH of enzymic activity 5.5–6.0. Reactivity with Concanavalin A, susceptibility to acid hydrolysis, and paper chromatography of the hydrolyzates suggested that the water-soluble β-d-fructans synthesized by the purified FTases were of the inulin-type and had chemical structures somewhat different among the serotypes.