Deletion analysis of regions at the C-terminal part of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040

Cycloisomaltooligosaccharide glucanotransferase (CITase) belongs to glycoside hydrolase family 66. According to the sequence alignment of enzymes in the same family, we divided the structure of CITase into five regions from the N terminus to the C terminus: an N-terminal conserved region (Ser1-Gly403), an insertion region (R1; Tyr404-Tyr492), two conserved regions (R2; Glu493-Ser596 and R3; Gly597-Met700), and a C-terminal variable region (R4; Lys701-Ser934). CITase catalyzes the synthesis of cycloisomaltooligosaccharides (CIs) with 7-17 glucose units (CI-7 to CI-17) from dextran. In order to clarify the functions of these C-terminal regions (R1-R4), we constructed 15 deletion mutant enzymes. M123Δ (R4-deleted), MΔ234 (R1-deleted), and MΔ23Δ (R1/R4-deleted) catalyzed CI synthesis, but other mutants were inactive. M123Δ, MΔ234, and MΔ23Δ increased their K(m) values against dextran 40. The wild-type enzyme and M123Δ produced CI-8 predominantly, but MΔ234 and MΔ23Δ lost CI-8 production specificity. The k(cat) values of MΔ234 and MΔ23Δ decreased, and these mutants showed narrowed temperature and pH stability ranges. Our deletion analysis suggests that (i) R2 and R3 are crucial for CITase to generate an active form; (ii) both R1 and R4 contribute to substrate binding; and (iii) R1 also contributes to preference of CI-8 production and enzyme stability.     

3′ cis-Acting Elements That Contribute to the Competence and Efficiency of Japanese Encephalitis Virus Genome Replication: Functional Importance of Sequence Duplications,Deletions, and Substitutions

The positive-strand RNA genome of Japanese encephalitis virus (JEV) terminates in a highly conserved 3′-noncoding region (3′NCR) of six domains (V, X, I, II-1, II-2, and III in the 5′-to-3′ direction). By manipulating the JEV genomic RNA, we have identified important roles for RNA elements present within the 574-nucleotide 3′NCR in viral replication. The two 3′-proximal domains (II-2 and III) were sufficient for RNA replication and virus production, whereas the remaining four (V, X, I, and II-1) were dispensable for RNA replication competence but required for maximal replication efficiency. Surprisingly, a lethal mutant lacking all of the 3′NCR except domain III regained viability through pseudoreversion by duplicating an 83-nucleotide sequence from the 3′-terminal region of the viral open reading frame. Also, two viable mutants displayed severe genetic instability; these two mutants rapidly developed 12 point mutations in domain II-2 in the mutant lacking domains V, X, I, and II-1 and showed the duplication of seven upstream sequences of various sizes at the junction between domains II-1 and II-2 in the mutant lacking domains V, X, and I. In all cases, the introduction of these spontaneous mutations led to an increase in RNA production that paralleled the level of protein accumulation and virus yield. Interestingly, the mutant lacking domains V, X, I, and II-1 was able to replicate in hamster BHK-21 and human neuroblastoma SH-SY5Y cells but not in mosquito C6/36 cells, indicating a cell type-specific restriction of its viral replication. Thus, our findings provide the basis for a detailed map of the 3′ cis-acting elements in JEV genomic RNA, which play an essential role in viral replication. They also provide experimental evidence for the function of 3′ direct repeat sequences and suggest possible mechanisms for the emergence of these sequences in the 3′NCR of JEV and perhaps in other flaviviruses.Japanese encephalitis virus (JEV), a mosquito-borne flavivirus of the family Flaviviridae, is serologically related to several significant human pathogens, including West Nile virus (WNV), Kunjin virus (KUNV), St. Louis encephalitis virus, and Murray Valley encephalitis virus. It is also phylogenetically close to other clinically important human pathogens, including yellow fever virus (YFV) and dengue virus (DENV) (11, 67). JEV is the leading cause of viral encephalitis in Southeast Asia, including China, Japan, Korea, the Philippines, Thailand, and India, and it has begun to expand throughout the Indonesian archipelago and as far as Australia (21, 43). Despite the fact that JEV is generally asymptomatic, ∼50,000 cases are reported annually, and the disease has a mortality rate of ∼25%, mainly in children and young adults (29, 63). Thus, the geographic expansion and clinical importance of JEV infection have drawn increasing attention from the international public health community (44, 71).Like other flaviviruses, JEV is a spherical enveloped virus (∼50 nm diameter) with a single-stranded positive-sense RNA genome that contains a 5′ cap structure but lacks a 3′ polyadenylated tail. Its genomic RNA of ∼11,000 nucleotides (nt) consists of a single long open reading frame (ORF) with two noncoding regions (NCRs) at the 5′ and 3′ ends (41, 84). The ORF is translated into an ∼3,400-amino acid polyprotein precursor, which is co- or posttranslationally cleaved by a cellular protease(s) or a viral protease complex into 10 mature proteins: (i) three structural proteins, the capsid (C), premembrane (prM; which is further processed into pr and M), and envelope (E) proteins; and (ii) seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, as arranged in the genome (13, 41, 84). The nonstructural proteins, together with cellular factors, form a viral replicase complex that directs the replication of the genomic RNA in the cytoplasm of the host cell in association with perinuclear membranes (40, 74). For the synthesis of the genomic RNA to take place, this replicase complex must specifically recognize viral cis-acting RNA elements, defined by primary sequences or secondary/tertiary structures. These RNA elements are found in various locations within the genome but most frequently are located in the 5′- and 3′NCRs (23, 47). The identification and characterization of these cis-acting RNA elements is critical for understanding the complete cycle of JEV genome replication.The availability of the complete nucleotide sequence of YFV genomic RNA (57) has led to the identification of three major conserved elements in the 5′- and 3′-terminal regions of the genomic RNA that contain the short primary sequences and secondary structures required for flavivirus RNA replication. (i) Both ends of the genomic RNA terminate with the conserved dinucleotides 5′-AG and CU-3′ (9, 10, 32, 45, 57, 72, 73) in all flaviviruses except an insect cell fusing agent virus (12). Mutations substituting another nucleotide for one of these four nucleotides in KUNV or WNV replicon RNA are known to abolish or compromise RNA replication (35, 69). (ii) A 3′ stem-loop structure (3′SL) has been recognized in all flaviviruses within the ∼90-nt 3′-terminal region of the genomic RNA (9, 45, 57). The structural and functional importance of this 3′SL in RNA replication has been demonstrated in several flaviviruses (9, 18, 49, 50, 61, 70, 82, 86). (iii) The presence of short 5′ and 3′ cyclization sequences (5′CYC and 3′CYC, respectively) in all mosquito-borne flaviviruses suggests that flavivirus genomes can cyclize via 5′-3′ long-range base-pairing interaction, since the 3′CYC upstream of the 3′SL is complementary to the 5′CYC in the 5′ coding region of the C protein (30). The role of these CYC motifs in RNA replication has been well characterized via cell-based assays in many mosquito-borne flaviviruses, including KUNV (34), WNV (42), YFV (8, 14), and DENV (2, 22, 49), and in cell-free systems in the case of WNV (51) and DENV (1, 3, 79, 80). Other RNA elements that have recently been shown to be important for RNA replication in DENV and WNV include an additional pair of complementary sequences (designated 5′- and 3′UARs) that participate in genome cyclization (3, 4, 17, 87) and a 5′ stem-loop structure (designated 5′SLA) present within the 5′NCR that promotes RNA synthesis in association with the 3′NCR (22).In all flaviviruses, the 3′NCR of the genomic RNA is relatively long (∼400 to ∼800 nt), with an array of conserved primary sequences and secondary structures. Although significant progress has been made in identifying cis-acting elements within the 3′NCRs that are essential for RNA replication, most of these elements (i.e., the 3′CYC, 3′SL, and CU-3′) are limited to the ∼100-nt 3′-terminal region that is highly conserved in these viruses (see recent reviews in references 23 and 47). However, the functional importance of the remaining 5′-proximal region of the 3′NCR, which differs in sequence between the various serological groups, is poorly understood. In particular, comparative sequence analyses and genetic algorithm-based computer modeling have suggested that in addition to the well-studied ∼100-nt 3′-proximal region, the remaining ∼474-nt 5′-proximal region of the 574-nt JEV 3′NCR also contains several RNA elements that may play critical roles in the viral life cycle (52, 55, 56, 68). To date, however, experimental evidence for the functional importance of these potential RNA elements in JEV genomic RNA replication is lacking.In the present study, we have identified and characterized the 3′ cis-acting RNA elements within the JEV 3′NCR and shown that they play an essential and/or regulatory role in genomic RNA replication. In particular, we have constructed and functionally characterized genome-length JEV mutant cDNAs with a series of 5′-to-3′ or 3′-to-5′ progressive deletions within the 3′NCR. In addition to identifying particular mutations within this region that affect either the competence or efficiency of genomic RNA replication, we found that the serial passaging of these mutants in susceptible BHK-21 cells produced a large number of pseudorevertants bearing a wide variety of spontaneous point mutations and sequence duplications, some of which were capable of restoring the replication competence of the defective mutants or enhancing replication efficiency. In addition, we assessed the replication of these mutants in three different cell types (BHK-21, SH-SY5Y, and C6/36 cells). Collectively, these data offer new insights into the functional importance of 3′ cis-acting RNA elements that regulate the cell type-dependent replication of JEV and perhaps other closely related mosquito-borne flaviviruses. Our findings also provide experimental evidence for the emergence of functional 3′ direct repeat sequences that are duplicated from the coding region and 3′NCR of JEV genomic RNA.     

A novel cyclic isomaltooligosaccharide (cycloisomaltodecaose, CI-10) produced by Bacillus circulans T-3040 displays remarkable inclusion ability compared with cyclodextrins

Cyclodextrans (CIs) are cyclic isomaltooligosaccharides and only CI-7, CI-8, and CI-9 were known. CI-7, CI-8, and CI-9, consisting of seven, eight, and nine glucoses, respectively, bound by alpha-(1-->6) linkages, are known to be produced by T-3040 strain of Bacillus circulans. However, we have found, using 13C NMR and mass spectrometry, that this strain also produces CI-10, CI-11 and CI-12. These large CIs are very soluble in water and inhibit the glucan synthesis of glucansucrases to the same degree as do the smaller CIs. The CIs were thought to be poor at forming inclusion complexes with chemical compounds, due to their flexible alpha-(1-->6)-glucosidic structure. Among these six CIs, CI-10 was much better at forming an inclusion complex, and it ability to do so was as good as cyclodextrins, as determined by its ability to stabilize the color of Victoria blue B. Therefore, CI-10 may be the most commercially useful CI.     

A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4⁺ T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4⁺ T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> sister chromatids often show complete alignment or separation along a 1.2-Mb euchromatic region but no cohesion “hot spots”

Sister chromatid cohesion is a prerequisite for correct segregation and possibly other functions of replicated chromosomes. Except for yeast, no details are known about arrangement of cohesion sites along interphase chromosomes. Within nuclei of several higher plants, sister chromatids are frequently not aligned at various positions along chromosome arms. Therefore, we tested whether preferential alignment positions (“cohesion hot spots”) and constant extension of and distances between aligned sites occur in plants. Along a ~1.2-Mb contig from the bottom arm of chromosome 1, the sister chromatid positions of 13 individual BAC inserts were found to be aligned for ~67–77% of homologues in 4C Arabidopsis thaliana nuclei. The differences between the 13 BAC positions were not significant at the P < 0.01 level. This suggests variability of alignment positions between cells and indicates the absence of cohesion “hot spots”. Similar as for single BACs, FISH with the entire contig indicated complete alignment for ~69% and complete separation of sister chromatids for ~31% of homologues in 4C nuclei. Partial alignment or separation was barely detectable. When three BAC inserts from a 760-kb region were tested simultaneously, alignment or separation of only the central BAC occurred in 3.3% and 3.5% of replicated chromosomes, respectively. Thus, we assume that sister chromatids can be separated or aligned within a Mb range in differentiated cells. However, the minimum extension of aligned sites or distances between them may (in rare cases) fall below ~500 kb.     

Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells

A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4 weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50 kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300 mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of ∼8 U μg⁻¹ for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ^®SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.     

Zika virus: An emerging flavivirus

Zika virus (ZIKV) is a previously little-known flavivirus closely related to Japanese encephalitis, West Nile, dengue, and yellow fever viruses, all of which are primarily transmitted by blood-sucking mosquitoes. Since its discovery in Uganda in 1947, ZIKV has continued to expand its geographic range, from equatorial Africa and Asia to the Pacific Islands, then further afield to South and Central America and the Caribbean. Currently, ZIKV is actively circulating not only in much of Latin America and its neighbors but also in parts of the Pacific Islands and Southeast Asia. Although ZIKV infection generally causes only mild symptoms in some infected individuals, it is associated with a range of neuroimmunological disorders, including Guillain-Barré syndrome, meningoencephalitis, and myelitis. Recently, maternal ZIKV infection during pregnancy has been linked to neonatal malformations, resulting in various degrees of congenital abnormalities, microcephaly, and even abortion. Despite its emergence as an important public health problem, however, little is known about ZIKV biology, and neither vaccine nor drug is available to control ZIKV infection. This article provides a brief introduction to ZIKV with a major emphasis on its molecular virology, in order to help facilitate the development of diagnostics, therapeutics, and vaccines.     

Production of ginkgolides and bilobalide from optimized the<Emphasis Type="Italic">Ginkgo biloba</Emphasis> cell culture

The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures ofGinkgo biloba were investigated. Callus induced from the leaf petioles exhibited distinct morphological and physiological responses. The cell biomass and ginkgolides content varied among the cell lines brownish callus lines produced high levels of ginkgolides and bilobalide in spite of poor cell growth. Among the culture media used, MS medium showed significant effect on cell growth and ginkgolides production. Low concentration of sucrose (3%) improved cell growth, while higher sucrose levels (5 and 7%) improved ginkgolides production. Cultivation of callus cultures above 28°C dramatically reduced their growth rate; however the cell lines grown at 36°C showed increased levels of bilobalide content. A 2.5-L balloon type bubble bioreactor (BTBB) was successfully developed for the cell growth and ginkgolides production.     

Production of adipic acid by short- and long-chain fatty acid acyl-CoA oxidase engineered in yeast Candida tropicalis

Bioprocess and Biosystems Engineering - In this study, to produce adipic acid, mutant strains of Candida tropicalis KCTC 7212 deficient of AOX genes encoding acyl-CoA oxidases which are important...