Development of intron-containing barnase gene (barnase-int) encoding a toxic protein to facilitate its cloning in bacterial cells

Developing gene constructs with the barnase gene and propagating them in Escherichia coli or Agrobacterium tumefaciens is difficult as unintended leaky expression leads to the death of the bacterial cells. In the present work, we circumvented this problem by developing intron containing barnase genes. We tested the use of two different introns viz., (i) the first intron (190 bp) of the catalase gene of castor bean and (ii) the twelfth intron (92 bp) of Pyruvate ortho-phosphate dikinase 2 (PPDK2) gene from cotton. Due to the absence of splicing in bacterial cells the unintended expression of the barnase protein was blocked allowing easy development and propagation of constructs. Further, we demonstrated that the intron introduced in the barnase gene was efficiently spliced out in the tapetum tissue of the anther in transgenic tobacco lines leading to male-sterility.     

Field trial detects incomplete barstar attenuation of vegetative cytotoxicity in Populus trees containing a poplar LEAFY promoter::barnase sterility transgene

We tested the efficacy of an attenuation system developed to preclude the deleterious effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5′ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar driven by either the CaMV 35S basal promoter +5 to −72 fragment (35SBP), 35SBP fused to the TMV omega element (35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar-attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ratios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenuated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and barstar:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenuation. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees.     

Inducible,tightly regulated and non-leaky neuronal gene expression in mice

The Tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgene expression in mice with over 500 lines created to date. Although this system has been optimized over the years, it still has limitations such as residual transgene expression when turned off, referred to as leakiness. Here, we present a series of new Tet-OFF transgenic mice based on the second generation tetracycline-responsive transactivator system. The tTA-Advanced (tTA2^S) is expressed under control of the neuron-specific Thy1.2 promoter (Thy-OFF), to regulate expression in the mouse brain. In addition, we generated a lacZ reporter line, utilizing the P _tight Tet-responsive promoter (P _tight–lacZ), to test our system. Two Thy-OFF transgenic lines displaying two distinct patterns of expression were selected. Oral doxycycline treatment of Thy-OFF/P _tight–lacZ mice demonstrated tight transgene regulation with no leak expression. These new Thy-OFF mice are valuable for studies in a broad range of neurodegenerative diseases such as Alzheimer’s disease and related forms of dementia, where control of transgene expression is critical to understanding mechanisms underlying the disease. Furthermore, P _tight–lacZ reporter mice may be widely applicable.     

A novel strategy for regulated expression of a cytotoxic gene.

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.     

The production of male-sterile wheat plants through split barnase expression is promoted by the insertion of introns and flexible peptide linkers

The successful use of transgenic plants depends on the strong and stable expression of the heterologous genes. In this study, three introns (PSK7-i1 and PSK7-i3 from Petunia and UBQ10-i1 from Arabidopsis) were tested for their ability to enhance the tapetum-specific expression of a split barnase transgene. We also analyzed the effects of introducing multiple copies of flexible peptide linkers that bridged the fusion domains of the assembled protein. The barnase fragments were assembled into a functional cytotoxin via intein-mediated trans-splicing, thus leading to male sterility through pollen ablation. A total of 14 constructs carrying different combinations of introns and peptide linkers were transformed into wheat plants. The resulting populations (between 41 and 301 independent plants for each construct) were assayed for trait formation. Depending on which construct was used, there was an increase of up to fivefold in the proportion of plants exhibiting male sterility compared to the populations harboring unmodified constructs. Furthermore, the average barnase copy number in the plants displaying male sterility could be reduced. The metabolic profiles of male-sterile transgenic plants and non-transgenic plants were compared using gas chromatography–mass spectrometry. The profiles generated from leaf tissues displayed no differences, thus corroborating the anther specificity of barnase expression. The technical advances achieved in this study may be a valuable contribution for future improvement of transgenic crop systems.     

The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L.

Male-sterile lines were generated in oilseed mustard (Brassica juncea) with a cytotoxic gene (barnase) in conjunction with either of two tapetum-specific promoters, TA29 and A9. Several transformation vectors based on different promoter and marker gene combinations were developed and tested for their efficacy in generating agronomically viable male-sterile lines. Use of strong constitutive promoters (e.g. CaMV 35S or its double-enhancer variant) to express the marker gene (bar) in barnase constructs generated male-sterile plants at an extremely low frequency with most plants showing abnormalities in vegetative morphology, poor female fertility, low seed germination frequencies and/or distortion in segregation ratios of transgenes. Such abnormalities were considerably reduced on using weaker promoters (e.g. nos) to drive the marker gene (nptII) in barnase constructs and could therefore be attributed to leaky expression of the barnase gene under enhancing effects of strong constitutive promoters. We show that the use of a Spacer DNA fragment between the barnase gene (driven by a tapetum-specific promoter) and the CaMV 35S promoter-driven bar gene insulates tissue-specific expression of the barnase gene over all developmental stages of transgenic plants and significantly enhances recovery of agronomically viable male-sterile lines. All TA29-barnase male-sterile lines containing the Spacer DNA fragment exhibited normal morphology, growth and seed set on backcrossing as observed for wild-type plants. Around 75% of single-copy events tested further also showed proper segregation of the marker gene/male-sterile phenotype among backcross progeny. Constructs based on the use of Spacer DNA fragments as insulators could be successfully used to alleviate limitations associated with transformation of plant systems using cytotoxic genes for development of agronomically viable male-sterile lines in crop plants and for cell/tissue ablation studies in general.     

Intein‐mediated protein assembly in transgenic wheat: production of active barnase and acetolactate synthase from split genes

Engineering traits by the assembly of non‐functional gene products is a promising tool for modern plant biotechnology. In this article, we describe the establishment of male sterility and herbicide resistance in wheat (Triticum aestivum) by complementing inactive precursor protein fragments through a split intein system. N‐ and C‐terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from the Synechocystis sp. gene DnaB and delivered into the wheat genome via biolistic particle bombardment. Both barnase fragments were expressed under the control of a tapetum‐specific promoter. High efficiency of the split barnase system was achieved by introducing GGGGS linkers between the fusion domains of the assembled protein. Depending on the vector version that was transformed, up to 51% of primary transformed plants produced sterile pollen. In the F₁ progeny, the male‐sterile phenotype segregated with both barnase gene fragments. Expression of the cytotoxic barnase in the tapetum did not apparently affect the vegetative phenotype and remained stable under increased temperatures. In addition, the reconstitution of sulphonylurea resistance was achieved by DnaE intein‐mediated assembly of a mutated acetolactate synthase (ALS) protein from rice. The impacts of the technical advances revealed in this study on the concepts for trait control, transgene containment and hybrid breeding are discussed.     

Stringent doxycycline dependent control of CRE recombinase in vivo

The strategy of modulating gene activities in vivo via CRE/loxP recombination would greatly profit from subjecting the recombination event to an independent and stringent temporal control. Here, we describe a transgenic mouse line, LC-1, where the expression of the cre and luciferase gene is tightly controlled by the Tet system. Using the R26R mouse line as indicator for CRE activity, and mouse lines expressing tetracycline controlled transactivators (tTA/rtTA) in various tissues, we show that; (i) in the non-induced state CRE recombinase is tightly controlled throughout the development and adulthood of an animal; (ii) upon induction, efficient recombination occurs in the adult animal in all tissues where tTA/rtTA is present, including hepatocytes, kidney cells, neurons and T lymphocytes; and (iii) no position effect appears to be caused by the LC-1 locus. Moreover, using the novel rTA^LAP-1 mouse line, we show that in hepatocytes, complete deletion of the loxP-flanked insert in R26R animals is achieved less than 48 h after induction. Thus, the LC-1 mouse appears suitable for exploiting two rapidly increasing collections of mouse lines of which one provides tTA/rtTA in specific cell types/tissues, and the other a variety of loxP-flanked genes.     

Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice

Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 μg/ml in milk.     

The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.     

Identification and characterization of the promoter of a gene expressing mainly in the tapetum tissue of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Anther and tapetum-specific genes are important for understanding male gametophyte development, as well as for their use in the development of barnase/barstar-gene based male sterility and restorer system for hybrid seed production. An essential component of the system is the availability of tapetum-specific promoters. In the present study, anther-specific genes were identified in cotton using microarray-based differential expression analysis, some of which show expression specific to the anthers at a stage where tapetum tissue was fully developed. Validation of the identified genes using RT-PCR and in situ hybridization identified one novel gene (AEG—Anther Expressing Gene) encoding a putative lipid binding protein as having a tapetum-specific expression. Further, three paralogs of the gene were identified in the cotton genome out of which the gene AEG1 (Anther Expressing Gene1) was found to express in the tapetum layer. Analysis of transgenic plants developed in cotton using 1.5 Kb promoter region of the of AEG1 gene fused upstream to the reporter gene β-glucuronidase revealed a broad window of expression of the AEG1 promoter in the tapetum tissue from the tetrad stage of anthers till the degeneration of the tapetum cells. Low levels of expression were also observed in the root tissues. Expression was not observed in the stem and leaves. The broad window of expression of AEG1 promoter in the tapetum tissue makes it a suitable candidate for the expression of the barstar gene for effective fertility restoration in the barnase/barstar system.     

Doxycyline regulation in a single retroviral vector by an autoregulatory loop facilitates controlled gene expression in liver cells

The tetracycline system has limitations in liver cells, such as toxic effects and low controllability. We generated different retroviral vectors for controlled gene expression in liver cells, in which the regulatory elements were arranged in different patterns. Only the organization of the tetracycline system in an autoregulatory loop in the sense orientation results in high retroviral titres and in tight regulation of gene expression in highly differentiated hepatoma cells. Because of the toxicity of the transactivator tTA, it was impossible to establish doxycycline-dependent stable HepG2 cell lines. To avoid sequelching-related toxicity in liver cells, we replaced tTA with new non-toxic transactivators. By using tTA2, tTA3 and tTA4, we observed tight doxycycline-dependent gene expression in 23, 49 and 45% of the isolated clones. The tTA4 vector was used to transduce hepatocytes of mice in vivo. Tight doxycycline-controllable gene regulation was also observed in the liver of mice, confirming our hypothesis that retroviral vectors with autoregulatory loops of the tetracycline system facilitate inducible gene expression in the liver in vivo. Our new retroviral vector system allows rapid isolation of controllable clones in a very high yield and should make the tetracycline system more applicable to liver-derived cells and in liver gene therapy in vivo.     

Production and Purification of the Extracellular Ribonuclease of Bacillus Amyloliquefaciens (Barnase) and its Intracellular Inhibitor (Barstar) II. Barstar

Procedures are reported for the extraction and purification of barstar, the intracellular inhibitor of barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, (a strain formerly erroneously classified as B. subtilis, strain H), from frozen acidified cells obtained during the isolation of barnase¹. Methods include affinity chromatography on agarose to which barnase is covalently bound. Experiments relating to the purity, molecular weight, and amino acid composition of the product are reported.     

Analysis of activity driven by upstream regulatory modules (URM) of tapetum specific genes TA29 and A9 at ectopic locations in tobacco transgenics

TA29 and A9 are genes from Nicotiana tabacum and Arabidopsis thaliana respectively, which express in a tapetum specific manner. The upstream regulatory modules (URMs; i.e. the promoter and the 5′UTR) of these genes have been used in development of male sterile and restorer lines expressing the barnase and barstar genes for hybrid seed production. While initial studies show that these URMs drive the expression in a tapetum specific manner, there are no recordings of unintended (leaky) expression driven by these URMs at ectopic locations due to position effect in developed transgenic lines. The information on leaky expression driven by tissue specific URMs is important for their use in developing transgenic plants. The present study records the leaky activity of both these URMs in transgenic tobacco lines using β-glucuronidase as a reporter gene. Leaky activity was observed in about one-fourth of the lines developed with TA29. Most interestingly in these lines, the leaky expression of the reporter gene was observed to be restricted to the meristematic tip region of the roots and at the leaf gap from where leaf trace diverges from stem bundles. Such a restricted and unique pattern of leaky activity of a tissue specific promoter or a URM has never been reported before, including the URM of the A9 gene analyzed in the present study. This observation suggests the presence of cryptic cis-elements within the URM of TA29 gene that can possibly activate it in meristematic tissue when integrated at certain ectopic locations. The URM of the A9 gene was also observed to show leaky activity. However, there was no unique pattern as observed with that of TA29. Further, in the study we also show that while the smaller (290 bp) length of TA29 URM can be used to drive the expression of barnase gene to develop male sterile lines, it adversely affects the regeneration of transgenic tobacco lines due to leaky expression. This adverse effect is significantly reduced when the full length (1.5 kb) URM of the TA29 gene is used.     

Artemisinin derivatives induce iron-dependent cell death (ferroptosis) in tumor cells

BackgroundApoptosis and other forms of cell death have been intensively investigated in the past years to explain the mode of action of synthetic anticancer drugs and natural products. Recently, a new form of cell death emerged, which was termed ferroptosis, because it depends on intracellular iron. Here, the role of genes involved in iron metabolism and homeostasis for the cytotoxicity of ten artemisinin derivatives have been systematically investigated.Material and methodsLog₁₀IC₅₀ values of 10 artemisinin derivatives (artesunate, artemether, arteether, artenimol, artemisitene, arteanuin B, another monomeric artemisinin derivative and three artemisinin dimer molecules) were correlated to the microarray-based mRNA expression of 30 iron-related genes in 60 cell lines of the National Cancer Institute (NCI, USA) as determined in 218 different microarray hybridization experiments. The effect of desferoxamine and ferrostatin-1 on the cytotoxicity of artenimol of CCRF-CEM cells was determined by resazurin assays. The mRNA expression of TFRC was exemplarily validated by immunohistochemical detection of transferrin receptor protein expression.ResultsThe mRNA expression of 20 genes represented by 59 different cDNA clones significantly correlated to the log₁₀IC₅₀ values for the artemisinins, including genes encoding transferrin (TF), transferrin receptors 1 and 2 (TFRC, TFR2), cerulopasmin (CP), lactoferrin (LTF) and others. The ferroptosis inhibitor ferrostatin-1 and the iron chelator deferoxamine led to a significantly reduced cytotoxicity of artenimol, indicating ferroptosis as cell death mode.ConclusionThe numerous iron-related genes, whose expression correlated with the response to artemisinin derivatives speak in factor for the relevance of iron for the cytotoxic activity of these compounds. Treatment with ferroptosis-inducing agents such as artemisinin derivatives represents an attractive strategy for cancer therapy. Pre-therapeutic determination of iron-related genes may indicate tumor sensitivity to artemisinins. Ferroptosis induced by artemisinin-type drugs deserve further investigation for individualized tumor therapy.     

No association of TF gene polymorphisms with hepatitis B virus Clearance and hepatocellular carcinoma occurrence in a Korean population

Development of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The transferrin (TF) gene encodes a blood plasma protein that delivers iron ion in the body. The iron uptake level has been shown to be different in HCC tumor regions, indicating a possible association between iron uptake level and HCC. To investigate whether genetic polymorphisms of TF are related with HBV clearance and/or HCC occurrence, we sequenced genomes of 24 individuals and detected 37 variants. Subsequently, eight single nucleotide polymorphisms (SNPs) in TF including 4 in the promoter region, 1 in 5′UTR and 3 in coding regions were selected and genotyped in 1,101 Korean subjects including 428 spontaneously recovered (SR) patients as controls and 673 chronic carriers (CC) as cases. Results of logistic analyses adjusted for age and gender, however, revealed no significant associations of polymorphisms and haplotypes in the TF gene with HBV clearance and HCC occurrence (P > 0.05). Since age of HBV infection is a risk factor in progression to HCC, further Cox proportional regression analysis for age of HCC as a relative hazard was performed; but no association between TF polymorphisms and onset age of HCC was found (P > 0.05). Although TF gene polymorphisms have been previously reported to be associated with various diseases, our findings indicate that genetic variations of the TF gene do not influence HBV clearance and HCC occurrence in a Korean population.