Characterization and Reverse Genetic Establishment of Cattle Derived Akabane Virus in China

Background: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed in the tropical and temperate zones of Asia and Africa and is considered as a great threat in herbivores.Results: An AKAV defined as TJ2016 was firstly isolated from the serum of cattle in China in 2016. Sequence analysis of the S and M segments suggested that the isolated TJ2016 was closely related to the strains JaGAr39 and JaLAB39, which belonged to genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ-2016 S, M, and L segment was separately constructed into the TVT7R plasmid under the control of T7 promoter which named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. Then, the three constructed plasmids were transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to rescue AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to have similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells.Conclusion: We successfully isolated a AKAV strain TJ2016 from the serum of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool which can be used for further AKAV pathogenesis and even vaccine studies.

miR-7 AND miR-34a sequence cloning and expression in a1235 glioblastoma cell line

miRNAs are small non-coding RNAs which have an important role in signalling circuits regulating different cell processes. miR-7 and miR-34a are known as tumour suppressors, and both of them can interfere with cell proliferation, differentiation, apoptosis and migration. We constructed plasmids containing pri-miRNA sequences for these two miRNAs and introduced them into the A1235 glioblastoma cell line. Clones containing increased expression of processed miR-7 and miR-34a were obtained. The proliferation and sensitivity to alkylation agent of transfected cells were similar to those of control cells. Our results indicate that an increase in miR-7 and miR34 expression alone in A1235 glioblastoma cells is not sufficient to change their proliferation or sensitivity to the influence of alkylating agents.

The variation of promoter strength in different gene contexts

BackgroundPromoter engineering has been employed as a strategy to enhance and optimize the production of bio-products. There have been many effortless studies searching the best promoter for biological application. However, whether promoter strengths stay unchanged in different gene contexts remains unknown.ResultsSix consecutive promoters at different strength levels were used to construct six different versions of plasmid backbone pTH1227, followed by inserted genes encoding two polymer-producing enzymes. Some of promoter strengths in the presence of inserted sequences did not correspond to the reported strengths in a previous study. When removing the inserted sequences, the strengths of these promoters returned to their reported strengths. These changes were further confirmed to occur at transcriptional levels. Polymer production using our newly constructed plasmids showed polymer accumulation levels relatively corresponding to the promoter strengths reported in our study.ConclusionOur study revealed the essence of re-assessing promoter strength in a specific gene context. Different gene contexts could result in the variation of promoter strengths, hence this might lead to different outcomes in downstream applications.

Human Gyrovirus Apoptin as a Potential Selective Anticancer Agent: An In Vitro Study

Background: Selective therapy has always been the main challenge in cancer treatments. Recently, it has been shown that Human Gyrovirus-derived protein apoptin (HGV-Apoptin) has selective cytotoxic effects on cancer cells similar to its homologue, Chicken Anemia Virus-derived Apoptin (CAV-Apoptin). However, apoptotic effects of Human Gyrovirus apoptin have been only evaluated on a few cancerous cell lines and need to be further investigated. In this study, we have evaluated the apoptotic effects of HGV-Apoptin and CAV-Apoptin expression on lung cancer (A549) and normal (HEK-293) cell lines, in order to provide more information about the specificity of these proteins on cancerous cells. Methods: Target cells were transfected by the calcium-phosphate precipitation method with constructed plasmids expressing HGV-Apoptin and CAV-Apoptin proteins as well as the control plasmid. Transfection efficiency was followed and imaged by fluorescence microscopy. Quantification of apoptosis was performed by flow cytometry. Measurements were compared by paired Student t-test. Results: Cells were successfully transfected with control and constructed plasmids. Flowcytometry analysis showed that A549 cells transfected with HGV-Apoptin and CAV-Apoptin expressing plasmids, undergone the apoptosis compared to A549 cells transfected with control plasmid (P<0.001). None of the plasmids could induce apoptosis in HEK-293 cells. Conclusion: Human Gyrovirus-derived apoptin (HGV-Apoptin) similar to its homologue, chicken anemia virus derived Apoptin (CAV-Apoptin) can induce apoptosis in Non-small-cell lung carcinoma cell line A549, but not in normal human embryonic kidney cell line HEK-293, which can be introduced as a promising novel specific antitumor agent.

Clone, Expression, and Purification of a Hyperthermophilic Endoglucanase Gene Cel12B from Thermotoga maritima

The thermostability and catalytic activity of endoglucanase from thermophilic Thermotoga maritima were improved by evolutionary molecular engineering. The cel12B gene from Thermotoga maritima was cloned and expressed, and the expressed Cel12B protein was purified by His-tag affinity chromatography. The constructed plasmids were screened on LB plate with ampicillin using Escherichia coli BL21 (DE3) as a host. The results showed that both the enzyme activity and yield of the Cell12B protein expressed in pET15b-cel12B were 2 times as that of the protein expressed in pET20b-cel12B.

Identification of three crucial histidine residues (His115, His132 and His297) in porcine deoxyribonuclease II

DNase II is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different histidine (H)-to-leucine (L) mutants for porcine DNase II and examined the enzyme properties of the expressed mutant proteins. Of the mutants, all but H132L were secreted into the medium of expressing cells. Six of the mutated DNase II proteins (H41L, H109L, H206L, H207L, H274L and H322L) showed enzyme activity, whereas the H115L, H132L and H297L mutants exhibited very little activity. The H115L and H297L mutants were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II mutants; these mutants were inactive, but their DNase activities could be rescued with imidazole, indicating that His115 and His297 are likely to function as a general acid and a general base respectively in the catalytic centre of the enzyme. In contrast with the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for His132 with lysine resulted in the misfolded form being retained in endoplasmic reticulum.

Signature-Tagged Transposon Mutagenesis Studies Demonstrate the Dynamic Nature of Cecal Colonization of 2-Week-Old Chickens by Campylobacter jejuni

ABSTRACT We have constructed plasmids to be used for in vitro signature-tagged mutagenesis (STM) of Campylobacter jejuni and used these to generate STM libraries in three different strains. Statistical analysis of the transposon insertion sites in the C. jejuni NCTC 11168 chromosome and the plasmids of strain 81-176 indicated that their distribution was not uniform. Visual inspection of the distribution suggested that deviation from uniformity was not due to preferential integration of the transposon into a limited number of hot spots but rather that there was a bias towards insertions around the origin. We screened pools of mutants from the STM libraries for their ability to colonize the ceca of 2-week-old chickens harboring a standardized gut flora. We observed high-frequency random loss of colonization proficient mutants. When cohoused birds were individually inoculated with different tagged mutants, random loss of colonization-proficient mutants was similarly observed, as was extensive bird-to-bird transmission of mutants. This indicates that the nature of campylobacter colonization in chickens is complex and dynamic, and we hypothesize that bottlenecks in the colonization process and between-bird transmission account for these observations.

Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

ABSTRACT To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production inE. coli.

Hybrid Motor with H+- and Na+-Driven Components Can Rotate Vibrio Polar Flagella by Using Sodium Ions

ABSTRACT The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacteriumRhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether theR. sphaeroides motor genes can function in place of thepomA and pomB genes of V. alginolyticus, we constructed plasmids including bothmotA and motB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/ormotB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroidescan generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.

Differential organization of desmin and vimentin in muscle is due to differences in their head domains.

In most myogenic systems, synthesis of the intermediate filament (IF) protein vimentin precedes the synthesis of the muscle-specific IF protein desmin. In the dorsal myotome of the Xenopus embryo, however, there is no preexisting vimentin filament system and desmin's initial organization is quite different from that seen in vimentin-containing myocytes (Cary and Klymkowsky, 1994. Differentiation. In press.). To determine whether the organization of IFs in the Xenopus myotome reflects features unique to Xenopus or is due to specific properties of desmin, we used the injection of plasmid DNA to drive the synthesis of vimentin or desmin in myotomal cells. At low levels of accumulation, exogenous vimentin and desmin both enter into the endogenous desmin system of the myotomal cell. At higher levels exogenous vimentin forms longitudinal IF systems similar to those seen in vimentin-expressing myogenic systems and massive IF bundles. Exogenous desmin, on the other hand, formed a reticular IF meshwork and non-filamentous aggregates. In embryonic epithelial cells, both vimentin and desmin formed extended IF networks. Vimentin and desmin differ most dramatically in their NH2-terminal "head" regions. To determine whether the head region was responsible for the differences in the behavior of these two proteins, we constructed plasmids encoding chimeric proteins in which the head of one was attached to the body of the other. In muscle, the vimentin head-desmin body (VDD) polypeptide formed longitudinal IFs and massive IF bundles like vimentin. The desmin head-vimentin body (DVV) polypeptide, on the other hand, formed IF meshworks and non-filamentous structures like desmin. In embryonic epithelial cells DVV formed a discrete filament network while VDD did not. Based on the behavior of these chimeric proteins, we conclude that the head domains of vimentin and desmin are structurally distinct and not interchangeable, and that the head domain of desmin is largely responsible for desmin's muscle-specific behaviors.