An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata (L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone. In in vitro assays, the threonine (Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of cross-resistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R/S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas. Received: 26 October 1999 / Accepted: 28 December 1999     

运动发酵单胞菌ZM4、CP4菌株基因工程选择标记的研究

尽管质粒和选择标记的使用作为基因工程最基本的一环而为人们所熟知,但对一些特殊菌种（菌株）或研究很少的菌种（菌株）的基因工程操作来说,质粒和选择标记可能仍然是一个并未完全解决的问题,因而需要不断提高认识、不断改进。运动发酵单胞菌Zymomonasmobilis具有突出的产醇性能,但其多种内源质粒和多种抗性的特点,增加了其基因工程操作时质粒和选择标记选用的难度。本研究在测定四个抗生素即Ap、Cm、Te、Km对典型菌株ZM4、CP4的最低生长抑制浓度的基础上,初步确定了这两个菌株基因工程操作时的四个抗生素使用浓度依次分别为300、100、25、350μg／mL（ZM4）和500、100、25、250μg]mL（CP4）;并进一步通过穿梭载体pZB21、宽宿主载体pBBR1MCS-2和整合载体pBR328-ldhR—cml—ldhL的转化,初步分析和证明了这些选择标记和在相应抗生素浓度下的效果：首先,对每一个选择标记基因来说,前述抗生素浓度是适于携带此选择标记基因的质粒的转化筛选和相应转化子培养的;其次,在前述抗生素浓度下,综合筛选平板阳性率和转化效率、培养物菌体形态异常程度等指标,四个选择标记基因中,以Cm和Tc抗性标记基因效果最好,Km抗性标记基因居中,Ap抗性标记基因最差。这些结果为ZM4、CP4基因工程遗传改造用抗性标记基因、质粒、抗生素的选择及转化系统的完善奠定了基础。     

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large‐scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods.     

Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase

Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their structure-activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.     

Olfactory marker protein (OMP) exhibits a beta-clam fold in solution: implications for target peptide interaction and olfactory signal transduction

Olfactory marker protein (OMP) is a ubiquitous, cytoplasmic protein found in mature olfactory receptor neurons of all vertebrates. Electrophysiological and behavioral studies demonstrate that it is a modulator of the olfactory signal transduction pathway. Here, we demonstrate that the solution structure of OMP, as determined by NMR studies, is a single globular domain protein comprised of eight beta-strands forming two beta-sheets oriented orthogonally to one another, thus exhibiting a "beta-clam" or "beta-sandwich" fold: beta-sheet 1 is comprised of beta3-beta8-beta1-beta2 and beta-sheet 2 contains beta6-beta5-beta4-beta7. Insertions include two, long alpha-helices located on opposite sides of the beta-clam and three flexible loops. The juxtaposition of beta-strands beta6-beta5-beta4-beta7-beta2-beta1-beta8-beta3 forms a continuously curved surface and encloses one side of the beta-clam. The "cleft" formed by the two beta-sheets is opposite to the closed end of the beta-clam. Using a peptide titration series, we have identified this cleft as the binding surface for a peptide derived from the Bex1 protein. The highly conserved Omega-loop structure adjacent to the Bex1 peptide-binding surface found in OMP may be the site of additional OMP-protein interactions related to its role in modulating olfactory signal transduction. Thus, the interaction between the OMP and Bex1 proteins could facilitate the interaction between OMP and other components of the olfactory signaling pathway.     

Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation

Genomic and cDNA clones of the acetolactate synthase (ALS) gene of Chlamydomonas reinhardtii have been isolated from a mutant, c85-20 (Hartnett et al., 1987), that is resistant to high concentrations of sulfometuron methyl (SMM) and related sulfonylurea herbicides. Comparison of the ALS gene sequences from the wild-type and the SMM resistant (SMMr) strains revealed two amino acid differences in the mature enzyme, a lysine to threonine change at position 257 (K257T) and a leucine to valine change at position 294 (L294V). Transformation of wild-type C. reinhardtii with the mutant ALS gene produced no transformants with ability to grow in the presence of a minimum toxic concentration of SMM (3 microm). Substitution of the ALS promoter with the promoter of the C. reinhardtii Rubisco small subunit gene (RbcS2) permitted recovery of SMMr colonies. In vitro mutagenesis of the wild-type ALS gene to produce various combinations of mutations (K257T, L294V and W580L) indicated that the K257T mutation was necessary and sufficient to confer the SMMr phenotype. Optimum transformation rates were obtained with two constructs (pJK7 and pRP-ALS) in which all introns in the coding region were present. Rates of transformation with construct pJK7 were approximately 2.5 x 10-4 transformants/cell (i.e. one transformant for each of 4000 initial cells) using electroporation and 8.5 x 10-6 transformants/cell using the glass bead vortexing method. These results suggest that pJK7 and pRP-ALS can serve as important additional dominant selectable markers for the genetic transformation of C. reinhardtii.     

Green fluorescent protein as a visual selection marker for papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.) transformation

Chemical-based selection for plant transformation is associated with a number of real and perceived problems that might be avoided through visual selection. We have used green fluorescent protein (GFP), as a visual selectable marker to produce transformed papaya (Carica papaya) plants following microprojectile bombardment of embryogenic callus. GFP selection reduced the selection time from 3 months on a geneticin (G418) antibiotic-containing medium to 3–4 weeks. Moreover, GFP selection increased the number of transformed papaya plants by five-to eightfold compared to selection in the presence of antibiotics. Overall, the use of GFP for selecting transgenic papaya lines improved our throughput for transformation by 15- to 24-fold while avoiding the drawbacks associated with the use of antibiotic resistance-based selection markers.Abbreviations BA: Benzyladenine - 2, 4-D: 2,4-Dichlorophenoxyacetic acid - GFP: Green fluorescent protein - IBA: Indole-3-butyric acid - NAA: -Naphthaleneacetic acid - MS: Murashige and Skoog plant culture mediumCommunicated by R.J. Rose     

Peptide deformylase is a potential target for anti-Helicobacter pylori drugs: reverse docking, enzymatic assay, and X-ray crystallography validation

Colonization of human stomach by the bacterium Helicobacter pylori is a major causative factor for gastrointestinal illnesses and gastric cancer. However, the discovery of anti-H. pylori agents is a difficult task due to lack of mature protein targets. Therefore, identifying new molecular targets for developing new drugs against H. pylori is obviously necessary. In this study, the in-house potential drug target database (PDTD, http://www.dddc.ac.cn/tarfisdock/) was searched by the reverse docking approach using an active natural product (compound 1) discovered by anti-H. pylori screening as a probe. Homology search revealed that, among the 15 candidates discovered by reverse docking, only diaminopimelate decarboxylase (DC) and peptide deformylase (PDF) have homologous proteins in the genome of H. pylori. Enzymatic assay demonstrated compound 1 and its derivative compound 2 are the potent inhibitors against H. pylori PDF (HpPDF) with IC50 values of 10.8 and 1.25 microM, respectively. X-ray crystal structures of HpPDF and the complexes of HpPDF with 1 and 2 were determined for the first time, indicating that these two inhibitors bind well with HpPDF binding pocket. All these results indicate that HpPDF is a potential target for screening new anti-H. pylori agents. In addition, compounds 1 and 2 were predicted to bind to HpPDF with relatively high selectivity, suggesting they can be used as leads for developing new anti-H. pylori agents. The results demonstrated that our strategy, reverse docking in conjunction with bioassay and structural biology, is effective and can be used as a complementary approach of functional genomics and chemical biology in target identification.     

Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

Summary We have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. Unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. Furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the absence of virus infection and viral gene expression. In this study, we have expressed a novel, selectable marker gene, puromycin acetyltransferase, under the control of the Drosophila melanogaster hsp70 promoter or under the control of the AcMNPV ie-1 promoter which is active in Spodoptera frugiperda cells in the absence of virus infection. In addition, we have constructed expression vectors which coexpress two genes from separate promoters, the pac gene which confers resistance to puromycin and a baculovirus gene which inhibits apoptosis, derived from Orygia pseudotsugata nuclear polyhedrosis virus. Both genes were expressed in stable populations of S. frugiperda cells in the absence of continuous drug selection.     

A novel plant nuclear gene encoding chloroplast ribosomal protein S9 has a transit peptide related to that of rice chloroplast ribosomal protein L12

We have cloned a novel nuclear gene for a ribosomal protein of rice and Arabidopsis that is like the bacterial ribosomal protein S9. To determine the subcellular localization of the gene product, we fused the N-terminal region and green fluorescent protein and expressed it transiently in rice seedlings. Localized fluorescence was detectable only in chloroplasts, indicating that this nuclear gene encodes chloroplast ribosomal protein S9. The N-terminal region of rice ribosomal protein S9 was found to have a high sequence similarity to the transit peptide region of the rice chloroplast ribosomal protein L12, suggesting that these transit peptides have a common lineage.     

A site-directed mutagenesis interrogation of the carboxy-terminal end of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker

We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1, and three new site-directed mutants, omr1-5, omr1-7, and omr1-8with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the carboxy-terminal (C-term) allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog -O-methylthreonine (OMT) confirmed the desensitization to feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7, appeared to influence one of the two separate Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile-binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile-binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile-binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5, omr1-7, and omr1-8 had a normal phenotype, undistinguishable from wild type. Therefore, alleles omr1-5, omr1-7, and omr1-8, proved to be ideal as environmentally-friendly, dominant, selectable markers for plant transformation.     

Purification and evaluation of a novel antioxidant peptide from corn protein hydrolysate

In the present study, corn protein hydrolysate (CPH) with antioxidant activity was obtained by enzymatic hydrolysis. Corn gluten meal (CGM) was hydrolyzed using two proteases (Alcalase and Protamex) to produce the antioxidant peptide. Extrusion and starch removal of corn protein were used as pretreatment procedures before proteolysis. Hydrolysis by Alcalase has more remarkable digesting efficiency on corn protein than that by Protamex. Therefore, the hydrolysate catalyzed by Alcalase was fractionated by ultrafiltration, and peptide with the highest antioxidant activity was purified from <6 kDa molecular weight fraction. The amino acid sequence of the novel peptide was Gln-Gln-Pro-Gln-Pro-Trp as identified by a quadrupole time-of-flight mass spectrometer (Q-TOF2), with molecular weight of 782.34 Da, which was matched to γ-zein f (50–55). The new peptide was further synthesized by Fmoc solid-phase method. It showed scavenging activity against DPPH, ABTS, and hydroxyl free radicals in dose dependent manner with EC₅₀ values of 0.95, 0.0112 and 4.43 mg/mL, respectively. It also exhibited notable reducing power of 0.54 at 2.0 mg/mL, but showed weaker Fe²⁺-chelating capacity with EC₅₀ value of 6.27 mg/mL. These results suggest that the hexapeptide is a potential natural antioxidant that can be used as drug or functional food ingredient.     

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5-end and the long 3-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.     

Model of Alzheimer's disease amyloid-beta peptide based on a RNA binding protein

Although Alzheimer's Abeta peptide has been shown to form beta-sheet structure, a high-resolution molecular structure is still unavailable to date. A search for a sequence neighbor using Abeta(10-42) as the query in the Protein Data-Bank (PDB) revealed that an RNA binding protein, AF-Sm1 from Archaeoglobus fulgidus (PDB entry: 1i4k chain Z), shared 36% identical residues. Using AF-Sm1 as a template, we built a molecular model of Abeta(10-42) by applying comparative modeling methods. The model of Abeta(10-42) contains an antiparallel beta-sheet formed by residues 16-23 and 32-41. Hydrophobic surface constituted by residues 17-20 (LVFF) separates distinctly charged regions. Residues that interact with RNA in the AF-Sm1 crystal structure were found to be conserved in Abeta. Using a native gel we demonstrate for the first time that RNA can interact with Abeta and selectively retard the formation of fibrils or higher-order oligomers. We hypothesize that in a similar fashion to AF-Sm1, RNA interacts with Abeta in the beta-hairpin (beta-turn-beta) structure and prevents fibril formation.     

An improved method for Beauveria bassiana transformation using phosphinothricin acetlytransferase and green fluorescent protein fusion gene as a selectable and visible marker

An improved transformation method for the biocontrol agent, Beauveria bassiana, was developed. For convenience of transformation selection and detection, the coding regions of the genes for phosphinothricin acetyltransferase and green fluorescent protein were fused and an expression vector, pBFT, carrying this fusion was constructed. Under optimum conditions, over 60 transformants microg(-1) plasmid DNA were obtained. B. bassiana conidia frozen 1 month at -80 degrees C were fully competent for transformation. The method was significantly less laborious and more rapid than current methods for B. bassiana. The bar::egfp provides a selectable and visible marker which may expedite future genetic engineering of this fungus.