Promoter Cassette Research Articles

Polymorphism of gene cassette promoter variants of class 1 integron harbored in S. Choleraesuis and Typhimurium isolated from Taiwan

Polymorphism of gene cassette promoter variants of class 1 integron harbored in S. Choleraesuis and Typhimurium isolated from Taiwan

Distribution diversity and expression regulation of class 1 integron promoters in clinical isolates of Morganella morganii.

Morganella morganii is an emerging nosocomial opportunistic pathogen with increasing multidrug resistance. Antibiotic resistance, driven primarily by the horizontal transfer of resistance genes, has become a global health crisis. Integrons, mobile genetic elements, are now understood to facilitate the transfer of these genes, contributing to the rapid proliferation of resistant strains. Understanding the regulatory role of integrons in drug resistance gene expression is crucial for developing novel strategies to combat this pressing public health issue. To investigate the distribution of promoter types in the variable regions of class 1 integrons isolated from clinical isolates of M. morganii and their regulatory role in the expression of downstream drug resistance gene cassettes. Ninety seven clinical isolates of M. morganii were screened for the presence of class 1 integrons (intI1) using polymerase chain reaction (PCR). Gene cassettes within the variable regions of positive isolates were characterized, and the gene cassette promoter Pc variants and downstream auxiliary promoter P2 were identified. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was employed for homology analysis. Recombinant plasmids containing different variable region promoters and gene cassettes were constructed to evaluate drug resistance genes and integrase (intI1) expression levels using reverse transcription-quantitative PCR (RT-qPCR) and antimicrobial susceptibility testing. Of the clinical isolates, 28.9% (n = 28/97) were positive for class 1 integrons. 24.7% (n = 24/97) of these isolates carried gene cassettes encoding resistance to aminoglycosides and trimethoprim. Three Pc promoter types (PcH1, PcS, and PcW) were identified, while all P2 promoters were inactive with a 14-base pair spacing between the -35 and -10 regions. ERIC-PCR analysis classified the integron-positive strains into 6 genotypes, with high consistency in promoter types and gene cassettes within each genotype. RT-qPCR and antimicrobial susceptibility testing demonstrated that strong promoters significantly enhanced the expression of downstream drug resistance gene cassettes compared to weak promoters. Additionally, RT-qPCR revealed a negative correlation between intI1 expression and Pc promoter strength. Class 1 integrons are prevalent in M. morganii. The promoter types within these integrons are diverse, and promoter strength is closely linked to downstream gene cassette expression. Integron-positive strains exhibit high homology, suggesting horizontal gene transfer and dissemination in clinical settings.

Molecular characterization of class 1 integrons in carbapenem-resistant Enterobacterales isolates

ObjectiveCarbapenem-resistant Enterobacterales (CRE) infections result in higher treatment costs and mortality rates. Integrons play important roles in emergence and spread of antibiotic resistant genes. To get a better understand on the effects of integron on CRE resistance, distribution of common carbapenemase genes and class 1 integron in clinical CRE isolates were investigated. MethodCarbapenemase genes, including blaKPC, blaVIM, blaIMP, blaNDM, blaGES, blaVEB and blaOXA-23, were screened in 161 CRE isolates and subtypes of these genes were confirmed through sequence analysis. Class 1 integron was screened and common promoter and gene cassette arrays were determined by sequencing. The resistant rates to clinical commonly used antibiotics between integron positive and integron negative CRE isolates were compared. ResultsOf 161 CRE isolates, the most prevalent carbapenemase gene was blaKPC-2, which was detected in 139 isolates, including 99 Klebsiella pneumoniae. Class 1 integron was detected in 78 isolates. Twenty different gene cassettes, including two carbapenemase genes blaVEB-1 and blaIMP-4, and nine different gene cassette arrays, including blaVEB-1-aadB-arr-2-cmlA5-blaOXA-10-aadA1, aadB-catB8-blaOXA-10-aadA1-dfrA1-aacA4 and blaIMP-4-qacG-aacA4-catB3, were detected. Five types of common promoters were identified. Relative weak promoter PcH1 was the dominant type. Resistant rates of CRE isolates containing class 1 integrons to ceftazidime, amikacin, trimethoprim/sulfamethoxazole and gentamicin were higher than those without class 1 integrons (P < 0.05). ConclusionClass 1 integrons play important roles in the emergence and spread of CRE resistance. To the best of our knowledge, this is the first report of aadB-catB8-blaOXA-10-aadA1-dfrA1-aacA4 and blaIMP-4-qacG-aacA4-catB3 in the same Providencia rettgeri isolate and blaVEB-1-aadB-arr-2-cmlA5-blaOXA-10-aadA1 in P. rettgeri.

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

BackgroundMultiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis thaliana mutants. Low entry, reliable assembly of CRISPR/Cas9 vectors and efficient mutagenesis is necessary to enable a maximum of researchers to break through the genetic redundancy within plant multi-gene families and allow for a plethora of gene function studies that have been previously unachievable. It will also allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome.ResultsTo facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating mutations at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either a FastRed, FastGreen or FastCyan fluorescent seed selection cassette. FastRed and FastGreen binary vectors can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) from A. thaliana or the constitutive promoter from Petroselinum crispum (PcUBi4-2). Available enzymes are “classical” Cas9 codon-optimized for A. thaliana and a recently reported, intron-containing version of Cas9 codon-optimized for Zea mays, zCas9i. We observed the highest efficiency in producing knockout phenotypes by using intron-containing zCas9i driven under egg-cell specific pEC1.2 promoter. Finally, we introduced convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in some of the T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.ConclusionA rapid, simple and flexible CISPR/Cas9 cloning system was established that allows assembly of multi-guide RNA constructs in a robust and reproducible fashion, by avoiding generation of very big constructs. The system enables a flexible, fast and efficient screening of single or higher order A. thaliana mutants.

Endoplasmic reticulum chaperone BiP/GRP78 knockdown leads to autophagy and cell death of arginine vasopressin neurons in mice

The immunoglobulin heavy chain binding protein (BiP), also referred to as 78-kDa glucose-regulated protein (GRP78), is a pivotal endoplasmic reticulum (ER) chaperone which modulates the unfolded protein response under ER stress. Our previous studies showed that BiP is expressed in arginine vasopressin (AVP) neurons under non-stress conditions and that BiP expression is upregulated in proportion to the increased AVP expression under dehydration. To clarify the role of BiP in AVP neurons, we used a viral approach in combination with shRNA interference for BiP knockdown in mouse AVP neurons. Injection of a recombinant adeno-associated virus equipped with a mouse AVP promoter and BiP shRNA cassette provided specific BiP knockdown in AVP neurons of the supraoptic (SON) and paraventricular nuclei (PVN) in mice. AVP neuron-specific BiP knockdown led to ER stress and AVP neuronal loss in the SON and PVN, resulting in increased urine volume due to lack of AVP secretion. Immunoelectron microscopy of AVP neurons revealed that autophagy was activated through the process of AVP neuronal loss, whereas no obvious features characteristic of apoptosis were observed. Pharmacological inhibition of autophagy by chloroquine exacerbated the AVP neuronal loss due to BiP knockdown, indicating a protective role of autophagy in AVP neurons under ER stress. In summary, our results demonstrate that BiP is essential for the AVP neuron system.

Promoter activity of ORF-less gene cassettes isolated from the oral metagenome

Integrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation. In this study, the promoter activity of ORF-less GCs, previously recovered from the oral metagenome, was verified by cloning them upstream of a gusA reporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD. This is the first demonstration and detection of promoter activity of ORF-less GCs from Treponema bacteria and the development of an agar plate-based detection system will enable similar studies in other environments.

Polymorphisms of Gene Cassette Promoters of the Class 1 Integron in Clinical Proteus Isolates.

ObjectiveTo describe the polymorphisms of gene cassette promoters of the class 1 integron in clinical Proteus isolates and their relationship with antibiotic resistance.MethodsPolymorphisms of the gene cassette promoter in 153 strains of Proteus were analyzed by PCR and nucleotide sequencing. Variable regions of atypical class 1 integrons were detected by inverse PCR and nucleotide sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyze the phylogenetic relationships of class 1 integron-positive clinical Proteus isolates. Representative beta-lactamase genes (bla), including blaTEM,blaSHV,blaCTX-M-1,blaCTX-M-2,blaCTX-M-8,blaCTX-M-9,blaCTX-M-25 and blaOXA-1, and plasmid-mediated quinolone resistance (PMQR) genes including qnrA, qnrB, qnrC, qnrD, qnrS, oqxA, oqxB, qepA, and aac(6′)-Ib were also screened using PCR and sequence analysis.ResultsFifteen different gene cassette arrays and 20 different gene cassettes were detected in integron-positive strains. Of them, aadB-aadA2 (37/96) was the most common gene cassette array. Two of these gene cassette arrays (estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3) have not previously been reported. Three different Pc-P2 variants (PcS, PcWTGN-10, PcH1) were detected among the 96 Proteus strains, with PcH1 being the most common (49/96). Strains carrying the promoters PcS or PcWTGN-10 were more resistant to sulfamethoxazole, gentamicin and tobramycin than those carrying PcH1. Strains with weak promoter (PcH1) harbored significantly more intra- and extra-integron antibiotic resistance genes than isolates with strong promoter (PcWTGN-10). Further, among 153 isolates, representative beta-lactamase genes were detected in 70 isolates (blaTEM-1, 54; blaOXA-1, 40; blaCTX-M-3, 12; blaCTX-M-14, 12; blaCTX-M-65, 5; blaCTX-M-15, 2) and representative PMQR genes were detected in 87 isolates (qnrA, 6; qnrB, 3; qnrC, 5; qnrD, 46; qnrS, 5; oqxA, 7; aac(6′)-Ib, 13; aac(6′)-Ib-cr, 32).ConclusionTo the best of our knowledge, this study provides the first evidence for polymorphisms of the class 1 integron variable promoter in clinical Proteus isolates, which generally contain relatively strong promoters. Resistance genotypes showed a higher coincidence rate with the drug-resistant phenotype in strong-promoter-containing strains, resulting in an ability to confer strong resistance to antibiotics among host bacteria and a relatively limited ability to capture gene cassettes. Moreover, strains with relatively weak integron promoters can “afford” a heavier “extra-integron antibiotic resistance gene load”. Furthermore, the gene cassettes estX, psp and the gene cassette arrays estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 have been confirmed for the first time in clinical Proteus isolates. Beta-lactamase genes and PMQR were investigated, and blaTEM-1 and blaOXA-1 were the most common, with qnrD and aac (6′)-Ib-cr also being dominant.

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

BackgroundThe Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA.MethodsIn order to approach the natural conditions of infection and possible integration, we generated linear JcDV-gfp based molecules which were transfected into non permissive Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed.ResultsWe show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear “scrambled” whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter.ConclusionAltogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

Increasing oxygen availability for improving poly(3-hydroxybutyrate) production by Halomonas

Technologies enabling high-cell-density growth are required for economical industrial production of most biotechnological products. However, the key factor limiting cell density in bioreactors is the availability of oxygen during the late phases of fermentation. Although the expression of bacterial Vitreoscilla hemoglobin (VHb) is useful for enhanced oxygen availability, bacterial cell membrane makes efficient hemoglobin-oxygen contact a challenge. On the other hand, periplasmic spaces of Gram-negative microorganisms offer an excellent compartment for the intermittent storage of hemoglobin-bound oxygen. In this study, the cell growth was increased by a remarkable 100% using the twin-arginine translocase (Tat) pathway to export active VHb into the periplasm of Escherichia coli, Halomonas bluephagenesis TD01 and H. campaniensis LS21. Furthermore, eight low-oxygen-inducible vgb promoters were constructed in tandem to become a strong promoter cassette termed P8vgb, which better induces expression of both gene vgb encoding VHb and the PHB synthesis operon microaerobically. Both the P8vgb and VHb performed excellently in E. coli and two Halomonas spp., demonstrating their universal applicability for various organisms.

Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit.

The C-terminal extension region of SlGAD3 is likely involved in autoinhibition, and removing this domain increases GABA levels in tomato fruits. γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid with several health-promoting benefits. In many plants including tomato, GABA is synthesized via decarboxylation of glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), which generally contains a C-terminal autoinhibitory domain. We previously generated transgenic tomato plants in which tomato GAD3 (SlGAD3) was expressed using the 35S promoter/NOS terminator expression cassette (35S-SlGAD3-NOS), yielding a four- to fivefold increase in GABA levels in red-ripe fruits compared to the control. In this study, to further increase GABA accumulation in tomato fruits, we expressed SlGAD3 with (SlGAD3 OX ) or without (SlGAD3ΔC OX ) a putative autoinhibitory domain in tomato using the fruit ripening-specificE8 promoter and the Arabidopsis heat shock protein 18.2 (HSP) terminator. Although the GABA levels in SlGAD3 OX fruits were equivalent to those in 35S-SlGAD3-NOS fruits, GABA levels in SlGAD3ΔC OX fruits increased by 11- to 18-fold compared to control plants, indicating that removing the autoinhibitory domain increases GABA biosynthesis activity. Furthermore, the increased GABA levels were accompanied by a drastic reduction in glutamate and aspartate levels, indicating that enhanced GABA biosynthesis affects amino acid metabolism in ripe-fruits. Moreover, SlGAD3ΔC OX fruits exhibited an orange-ripe phenotype, which was associated with reduced levels of both carotenoid and mRNA transcripts of ethylene-responsive carotenogenic genes, suggesting that over activation of GAD influences ethylene sensitivity. Our strategy utilizing the E8 promoter and HSP terminator expression cassette, together with SlGAD3 C-terminal deletion, would facilitate the production of tomato fruits with increased GABA levels.

Abstract 2052: Efficient thermoregulation of heat-induced enzyme-prodrug therapy using recombinant E. coli containing a modified λpR promoter

Abstract Introduction: The λpR-cI857 transcriptional promoter cassette drives heat-induced protein expression in recombinant E. coli by inhibiting transcription below 37 °C (1). We have earlier reported on the development of magnetic nanoparticle hyperthermia (MNP) induced enzyme-prodrug therapy based on recombinant E. coli cells. The E. coli are designed to express cytosine deaminase (CD) under the transcriptional control of the λpR-cI857 promoter (2). CD converts non-toxic 5-fluorocytosine (5-FC) to the toxic 5-fluorouracil (5-FU). E. coli cells were co-encapsulated with MNP in immunoisolative alginate microcapsules and heated to 43 °C using an external alternating magnetic field (AMF). Using in vitro MTT assays, we demonstrated that the cytotoxicity of the heat activated E. coli in the presence of 5-FC, against 9L, MCF-7 and PC-3 tumor cells, was comparable to direct 5-FU treatment (2). However, we observed significant cytotoxicity at 37 °C resulting from basal CD expression. We now report on an improved strain of E. coli cells using a modified λpR promoter to minimize CD expression at 37 °C. Methods: A single base mutation was introduced in the λpR promoter during PCR amplification (1) from the pLDR20 vector. The modified promoter was ligated to the linearized pNEB206A vector and transformed into NEB-α cells using the USER™ cloning protocol (www.neb.com). NM522 cells containing the modified λpR-cI857 cassette and the CD gene were constructed and characterized following procedures established in our laboratory (2). The E. coli were then encapsulated in alginate microcapsules and heated at 43 °C to trigger CD expression. Cytotoxicity against CT26 colon cancer cells was evaluated using a MTT assay following the incubation of the cancer cells with the heated microcapsules and 5-FC (0.1 mM, 72 h, 37 °C). Results summary: We observed thermoselective CD expression and catalytic activity in E. coli heated at 43 °C. MTT cell viability assay shows that the cytotoxicity of the encapsulated, heat-activated E. coli cells against CT26 colon cancer cells was comparable to direct treatment with 5-FU. Importantly, the basal cytotoxicity at 37 °C was significantly lower than the E. coli cells activated at 43 ºC. We are extending our work to express CD periplasmically in endotoxin free hosts for improved catalytic efficiency and to eliminate cytotoxicity arising from bacterial lipopolysaccharides. We expect that the improved E. coli cells should facilitate de novo synthesis of 5-FU, on-demand and locally in the tumor, thereby reducing systemic toxicity and increasing therapeutic gain. Acknowledgement: This work is supported by a pilot grant from the Hitchcock Foundation.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

Replication-Competent Controlled Herpes Simplex Virus.

We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site ( attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-bla GES-1 /aacA4 gene cassette array, which harbours a fused gene cassette represented by bla GES-1 /aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of bla GES-1 /aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, bla GES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette bla GES-1 /aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than bla GES-1/ aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream bla GES-1/ aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-bla GES-1/ aacA4 free circular forms, but not to bla GES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

Identification of the avian RBP7 gene as a new adipose-specific gene and RBP7 promoter-driven GFP expression in adipose tissue of transgenic quail.

The discovery of an increasing number of new adipose-specific genes has significantly contributed to our understanding of adipose tissue biology and the etiology of obesity and its related diseases. In the present study, comparison of gene expression profiles among various tissues was performed by analysis of chicken microarray data, leading to identification of RBP7 as a novel adipose-specific gene in chicken. Adipose-specific expression of RBP7 in the avian species was further confirmed at the protein and mRNA levels. Examination of the transcription factor binding sites within the chicken RBP7 promoter by Matinspector software revealed potential binding sites for adipogenic transcription factors. This led to the hypothesis that the RBP7 promoter can be utilized to overexpress a transgene in adipose tissue in order to further investigate the function of a transgene in adipose tissue. Several lines of transgenic quail containing a green fluorescent protein (GFP) gene under the control of the RBP7 promoter were generated using lentivirus-mediated gene transfer. The GFP expression in transgenic quail was specific to adipose tissue and increased after adipocyte differentiation. This expression pattern was consistent with endogenous RBP7 expression, suggesting the RBP7 promoter is sufficient to overexpress a gene of interest in adipose tissue at later developmental stages. These findings will lead to the establishment of a novel RBP7 promoter cassette which can be utilized for overexpressing genes of interest in adipose tissue in vivo to study the function of genes in adipose tissue development and lipid metabolism.

Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme–prodrug therapy

Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme–prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions.

A Cassava vein mosaic virus promoter cassette induces high and stable gene expression in clonally propagated transgenic cassava (Manihot esculenta Crantz)

The study described a T-DNA vector with a Cassava vein mosaic virus promoter cassette (pCsVMV) and a kanamycin selectable marker gene driven by the 35S Cauliflower mosaic virus promoter with a view to stably express transgenes over repeated cycles of clonal propagation. A β-glucuronidase reporter gene under control of pCsVMV (pCsVMV-GUS) was introduced into the cassava landrace ‘Tokunbo’ via Agrobacterium-mediated genetic transformation. Transgenic tobacco plants (Nicotiana tabacum SR1) with the same gene construct were also produced. In tobacco, the pCsVMV-GUS was highly expressed in all tissues tested such as leaf, stem, petiole, and roots. In transgenic cassava, the pCsVMV-GUS gene was highly expressed in all tissues and most cell types of in vitro plants including leaf, stem, petiole, and fibrous roots. The pCsVMV-GUS gene was also highly expressed in these tissues as well as in tubers of greenhouse grown cassava. High and stable pCsVMV-GUS gene expression was maintained over 3 cycles of ratooning under greenhouse conditions, thus showing the absence of undesired gene silencing effects after repeated in vitro subculturing and vegetative propagation. From the high constitutive levels of GUS activity observed, the study concluded that the CsVMV promoter cassette was useful for high-level expression in cassava over repeated cycles of clonal propagation.

Polymorphism of gene cassette promoter variants of class 1 integron harbored in S. Choleraesuis and Typhimurium isolated from Taiwan.

Integrons, mobile genetic units, capture and incorporate antibiotic resistance gene cassette by site-specific recombination. Class 1 integrons are widespread and associated with dispersion of antibiotic resistance among Gram-negative bacteria. The expression of gene cassette in Class 1 can vary, based on the Pc promoter but seldom from another promoter hiding downstream of Pc, called P2. To probe distribution and prevalence of gene cassette promoter variants, we analyzed 169 S. Choleraesuis and 191 S. Typhimurium isolates from humans and animals, finding 95.27% occurrence of integrin among S. Choleraesuis, 83.25% among S. Typhimurium. PCR-RFLP analysis identified four promoters (PcS+P2, PcWTGN-10+P2, PcH1+P2, and PcWTGN-10+P2-GGG) in said integron-positive isolates; major types in S. Choleraesuis and S. Typhimurium were PcS+P2 and PcWTGN-10+P2, respectively. Likewise, β-galactosidase assay rated promoter strength of variants by transcriptional fusion constructs to show extended -10 promoter (TGn/-10 promoter) in Pc and three-nucleotide insertion (GGG) between -35 and -10 region of P2 improving promoter strength of gene cassette.

Analysis of the expression of &lt;I&gt;AtSb3&lt;/I&gt; gene containing MATH domains in &lt;I&gt;Arabidopsis thaliana&lt;/I&gt;

RT–PCR were used to investigate the expression of Atsb3 in Arabidopsis thaliana, and the vector with AtSb3gene promoter and GUS fusion expression cassette was constructed to obtain the transgenic lines of Arabidopsis thaliana,based on which the tissue localization of AtSb3 gene expression was investigated by histochemical staining. The results of RT–PCR showed AtSb3 gene was expressed only in root, not in the stem, leaf, flower and silique. The results of GUS staining showed that in seeds at early germinating stage,AtSb3 gene was only expressed in radicle. In cotyledon expansion period after seed germination, AtSb3 gene expression was only detected in root tip, not in other parts of the plant. In 4 to 5 leaf stage seedling,AtSb3 was not expressed in aboveground parts but in taproots and lateral roots. In the root tip, GUS staining was heavier in conjunction region of the root cap and meristematic zone, as well as in elongation zone and root hair, which indicated expression level of AtSb3 gene was higher in these parts; GUS staining in the region between meristematic zone and elongation zone was not observed.