Chimeric cDNA Research Articles

An Adapted GeneSwitch Toolkit for Comparable Cellular and Animal Models: A Proof of Concept in Modeling Charcot-Marie-Tooth Neuropathy.

Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch™ technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch™ cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco-Marie-Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical "teardrop" distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

DNAJB1–PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma

A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.

C-terminal region of human p53 attenuates buffalo p53N-terminal-specific transactivation of p21 promoter by modulating tetramerization of the protein.

Here, we have studied in p53 null H1299 lung carcinoma cells, the dominant-negative effect of human p53 (h-p53) on buffalo p53 (b-p53) induced nuclear transactivation-dependent function. Recently, we have isolated and cloned the full-length cDNA of buffalo p53. Buffalo and human p53 proteins exhibit a high degree of structural and functional similarities. In transiently transfected H1299 cell line b-p53 appeared to be more sensitive to Mdm2-mediated degradation as compared to h-p53, although its ability to transactivate p21 promoter was stronger than that ofthe human counterpart. This higher transactivation ability of b-p53 was lost in the presence of h-p53 suggesting, a dominant-negative effect of h-p53 on b-p53's transactivation of p21 promoter. Both human and buffalo p53 proteins could hetero-oligomerize but the b-p53 could tetramerize much faster than the h-p53. A chimeric cDNA construct of human p53 was made where the 1-260bpN-terminus was replaced with buffalo p53 counterpart and expressed in H1299 cell line. The tetramerization ability of the chimeric p53 protein was comparable to that of h-p53. Properties of b-p53 like stronger p21 transactivation and super sensitivity to Mdm2 mediated degradation were lacking in the chimeric protein. Thus, it is suggested that faster ability of tetramerization as well as higher transactivation property of buffalo p53 is determined by the interplay of N- and C-terminal domains through macromolecular interactions.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen.

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Barley Yellow Mosaic Virus VPg Is the Determinant Protein for Breaking eIF4E-Mediated Recessive Resistance in Barley Plants.

In this study, we investigated the barley yellow mosaic virus (BaYMV, genus Bymovirus) factor(s) responsible for breaking eIF4E-mediated recessive resistance genes (rym4/5/6) in barley. Genome mapping analysis using chimeric infectious cDNA clones between rym5-breaking (JT10) and rym5-non-breaking (JK05) isolates indicated that genome-linked viral protein (VPg) is the determinant protein for breaking the rym5 resistance. Likewise, VPg is also responsible for overcoming the resistances of rym4 and rym6 alleles. Mutational analysis identified that amino acids Ser-118, Thr-120, and His-142 in JT10 VPg are the most critical residues for overcoming rym5 resistance in protoplasts. Moreover, the rym5-non-breaking JK05 could accumulate in the rym5 protoplasts when eIF4E derived from a susceptible barley cultivar was expressed from the viral genome. Thus, the compatibility between VPg and host eIF4E determines the ability of BaYMV to infect barley plants.

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones.

The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS) during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution.

Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization.

(1,3;1,4)-β-Glucan Biosynthesis by the CSLF6 Enzyme: Position and Flexibility of Catalytic Residues Influence Product Fine Structure.

Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-β-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4)-linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-β-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-β-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.

Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNTs chromatography

Tissue-type plasminogen activator (tPA) is a serine protease that plays a crucial role in the fibrinolytic system. We increased the activity of tPA by splicing the active site of dodder-cuscutain gene to human tPA. The chimeric cDNA of tPA was constructed by Splicing by Overlap Extension Polymerase Chain Reaction (SOEing-PCR) method and transferred to the hairy roots of tobacco using different strains of Agrobacterium rhizogenes. Chimeric-tPA was purified by lysine-sepharose chromatography and specific aptamers were designed using SELEX method. Multi wall carbon nanotubes were functionalized with selected aptamers, packed in a column, and used for purification. The results demonstrated that selected aptamer having KD values of 0.320 nM and IC50 of 28.9 nM possessed good affinity to tPA, and the chimeric-tPA was properly purified by aptamer-chromatography. Hairy roots expressing chimeric-tPA and normal-tPA produced 900 and 450 ngmg−1 of total protein, respectively. The activities of chimeric–tPA and normal–tPA were 90 and 60 IUml−1, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.

Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the E(rns) genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation.

Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity.

Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (domains A, B, and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines and/or antivirals.

Identification of two amino acids within E2 important for the pathogenicity of chimeric classical swine fever virus

Our previous study demonstrated that a chimeric classical swine fever virus (CSFV) vSM/CE2 containing the E2 gene of the vaccine C-strain on the genetic background of the virulent CSFV strain Shimen (vSM) was attenuated in swine but reversed to virulence after serial passages in PK15 cells. To investigate the molecular basis of the pathogenicity, the genome of the 11th passage vSM/CE2 variant (vSM/CE2-p11) was sequenced, and two amino acid mutations, T745I and M979K, within E2 of vSM/CE2-p11 were observed. Based on reverse genetic manipulation of the chimeric cDNA clone pSM/CE2, the mutated viruses vSM/CE2/T745I, vSMCE2/M979K and vSM/CE2/T745I;M979K were rescued. The data from infection of pigs demonstrated that the M979K amino acid substitution was responsible for pathogenicity. Studies in vitro indicated that T745I and M979K increased infectious virus production and replication. Our results indicated that two residues located at sites 745 and 979 within E2 play a key role in determining the replication in vitro and pathogenicity in vivo of chimeric CSFV vSM/CE2.

Megaprimer-mediated capsid swapping for the construction of custom-engineered chimeric foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the selection of appropriate candidate vaccine strain and its adaptation in cell culture to yield high titer of virus is a cumbersome process. An attractive approach to circumvent this tedious process is to replace the capsid coding sequence of an infectious full-genome length cDNA clone of a good vaccine strain with those of appropriate field strain, to produce custom-made chimeric FMD virus (FMDV). Nevertheless, the construction of chimeric virus can be difficult if the necessary endonuclease restriction sites are unavailable or unsuitable for swapping of the capsid sequence. Here we described an efficient method based on megaprimer-mediated capsid swapping for the construction of chimeric FMDV cDNA clones. Using FMDV vaccine strain A IND 40/2000 infectious clone (pA(40/2000)) as a donor plasmid, we exchanged the capsid sequence of pA(40/2000) with that of the viruses belonging to serotypes O (n = 5), A (n = 2), and Asia 1 (n = 2), and subsequently generated infectious FMDV from their respective chimeric cDNA clones. The chimeric viruses exhibited comparable infection kinetics, plaque phenotypes, antigenic profiles, and virion stability to the parental viruses. The results from this study suggest that megaprimer-based reverse genetics technology is useful for engineering chimeric vaccine strains for use in the control and prevention of FMD in endemic countries.

Directed Expression of a Chimeric Type II Keratin Partially Rescues Keratin 5-null Mice

The crucial role of structural support fulfilled by keratin intermediate filaments (IFs) in surface epithelia likely requires that they be organized into cross-linked networks. For IFs comprised of keratins 5 and 14 (K5 and K14), which occur in basal keratinocytes of the epidermis, formation of cross-linked bundles is, in part, self-driven through cis-acting determinants. Here, we targeted the expression of a bundling-competent KRT5/KRT8 chimeric cDNA (KRT8bc) or bundling-deficient wild type KRT8 as a control to the epidermal basal layer of Krt5-null mice to assess the functional importance of keratin IF self-organization in vivo. Such targeted expression of K8bc rescued Krt5-null mice with a 47% frequency, whereas K8 completely failed to do so. This outcome correlated with lower than expected levels of K8bc and especially K8 mRNA and protein in the epidermis of E18.5 replacement embryos. Ex vivo culture of embryonic skin keratinocytes confirmed the ability of K8bc to form IFs in the absence of K5. Additionally, electron microscopy analysis of E18.5 embryonic skin revealed that the striking defects observed in keratin IF bundling, cytoarchitecture, and mitochondria are partially restored by K8bc expression. As young adults, viable KRT8bc replacement mice develop alopecia and chronic skin lesions, indicating that the skin epithelia are not completely normal. These findings are consistent with a contribution of self-mediated organization of keratin IFs to structural support and cytoarchitecture in basal layer keratinocytes of the epidermis and underscore the importance of context-dependent regulation for keratin genes and proteins in vivo.

Mapping the miRNA interactome by cross-linking ligation and sequencing of hybrids (CLASH).

RNA-RNA interactions have critical roles in many cellular processes, but studying them is difficult and laborious. Here we describe an experimental procedure, termed cross-linking ligation and sequencing of hybrids (CLASH), which allows high-throughput identification of sites of RNA-RNA interaction. During CLASH, a tagged bait protein is UV-cross-linked in cell cultures to stabilize RNA interactions, and it is purified under denaturing conditions. RNAs associated with the bait protein are partially truncated, and the ends of RNA duplexes are ligated together. After linker addition, cDNA library preparation and high-throughput sequencing, the ligated duplexes give rise to chimeric cDNAs, which unambiguously identify RNA-RNA interaction sites independent of bioinformatic predictions. This protocol is optimized for studying miRNA targets bound by Argonaute (AGO) proteins, but it should be easily adapted for other RNA-binding proteins and classes of RNA. The protocol requires ∼5 d to complete, excluding the time required for high-throughput sequencing and bioinformatic analyses.

Possible Role for the Phoshorylated Tail in Retaining CSQ2 to Specific Sites Within the Secretory Pathway

Each of the two calsequestrin (CSQ) genes expresses muscle-type specificity; CSQ1 is present only in skeletal muscle, while CSQ2 is found in cardiac tissue and slow-type skeletal muscle. The two forms of the protein share about 60% identity and have similar 3D structures. Yet, differences in distribution of negative surface charges lead to differences in intracellular polymerization and localization. A second difference in structure is the CSQ2-specific C-terminal extension, a tail that contains a cluster of protein kinase CK2-sensitive serines. In this study, we examined the subcellular localization of CSQ1 and CSQ2 following their acute overexpression in adult rat cardiomyocytes, using isoform specific antibodies. The native rat CSQ2 showed the expected junctional SR distribution, co-staining with ryanodine receptor. Canine and human CSQ2 localized within 48 h to the same puncta as the native protein, with subtle differences in distribution. In contrast to all CSQ2 proteins, CSQ1 was not observed in jSR, rather accumulating in punctae near the cell surface. To test whether the C-terminal 23-mer extension on CSQ2 (cT) underlied this difference we constructed a chimeric cDNA encoding CSQ1-cT. This protein accumulated in yet a third pattern, distinct from both the transverse jSR localization (CSQ2 compartment) and the subsarcolemmal accumulation (CSQ1 compartment). CSQ1-cT appeared to assemble in an array of punctae aligned longitudinally across the myoplasm. We conclude that CSQ2, following its perinuclear biosynthesis, encounters junctional SR sites where it can accumulate by polymerization. In contrast, CSQ1 proceeds to cortical sites along microtubules to concentrate near the cell surface. Thus, the cardiac-specific cT may interact with an upstream sorting protein, suggesting a possible mechanism of CSQ retention beyond its polymerization. Studies in cultured non-muscle cells support the hypothesis for cT-dependent CSQ retention.

In vitro and in vivo evidence for differences in the protease activity of two arabis mosaic nepovirus isolates and their impact on the infectivity of chimeric cDNA clones

Regulated processing of nepovirus polyproteins allows the release of mature proteins and intermediate polyproteins. Infectious cDNA clones of the mild NW isolate of arabis mosaic virus (ArMV) and chimeric clones incorporating RNA1 segments of Lv, a severe isolate, were generated. Clones containing the Lv X2–NTB cleavage site were not infectious unless the Lv protease was present. The Lv and NW X2–NTB cleavage sites differ at positions P6, P4 and P2. In vitro, processing at the X2–NTB site was undetectable or reduced in chimeric polyproteins containing the Lv X2–NTB site and the NW protease but was restored when both the Lv protease and X2–NTB site were present. In contrast, cleavage at this site was increased in polyproteins that contained the NW X2–NTB site and the Lv protease. These results show that the ArMV-Lv protease has greater activity and is active on a greater range of cleavage sites than that of ArMV-NW.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

BackgroundValuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous.ResultsWe present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells.ConclusionsWe have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.

Endosperm-specific expression of human acid beta-glucosidase in a waxy rice

BackgroundThe deficiency of human acid beta-glucosidase (hGCase) causes Gaucher disease, a rare genetically-inherited disorder currently treated by enzyme replacement therapy using recombinant CHO-derived GCase. In an attempt to provide an alternative and more efficient production system, a chimeric cDNA coding for hGCase operatively linked to the signal peptide of rice glutelin 4 (GluB4) was put under the control of the GluB4 endosperm-specific promoter and inserted into the genome of a waxy rice.ResultsMolecular, immunological and biochemical analyses showed that recombinant hGCase, targeted to the protein storage vacuoles of rice endosperm cells, is equivalent to the native protein and has a glycosylation pattern compatible with direct therapeutic use. Compared to a previous study carried out on transgenic tobacco seeds, enzyme contents per unit of biomass were drastically increased; in addition, differently from what observed in tobacco, rice seed viability was unaffected by hGCase even at the highest production level. Transgenic seed polishing combined with a pretreatment of seed flour greatly facilitated hGCase extraction and purification with an industrially-scalable procedure.ConclusionsThis study opens up the possibility to efficiently produce in the rice seed pharmaceutical compounds which are available in limited amounts or completely excluded from clinical practice due to the inadequacy of their production systems.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-5-34) contains supplementary material, which is available to authorized users.