SUPERIOR REGENERATION AND AGROBACTERIUM INFECTABILITY OF BROCCOLI AND CAULIFLOWER TISSUES AND THE IDENTIFICATION OF A PROCEDURE FOR THE GENERATION OF TRANSGENIC PLANTS.

Broccoli and cauliflower are among the most regeneratively intractable genotypes found in the brassicaceae. To develop a method for transfer of the gene encoding S-adenosylmethionine hydrolase (SAMase) into inbred broccoli and cauliflower germplasm, we investigated the morphogenic competence and Agrobacterium susceptibility of a wide range of tissues of varied source. Appropriately controlled expression of the SAMase gene should, theoretically, reduce the plant's capacity for ethylene biosynthesis and extend the post harvest shelf life of the flower head. Through examination of the in vitro response of a wide range of tissues we identified procedures which support caulogenesis from 100% of explants, each producing more than 30 shoots which readily convert to plantlets. Studies with several wild type and disarmed Agrobacterium strains, and utilization of the binary vector system and appropriate marker and reporter genes, led to the identification of methods for high frequency T-DNA transfer to explant tissues and the flow frequency of transgenic plants containing SAMase gene.

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Tolerance to, and Uptake and Degradation of 2,4,6-Trinitrotoluene (TNT) are Enhanced by the Expression of a Bacterial Nitroreductase Gene in Arabidopsis thaliana

Zeitschrift für Naturforschung C ◽

10.1515/znc-2005-3-412 ◽

2005 ◽

Vol 60 (3-4) ◽

pp. 272-278 ◽

Cited By ~ 34

Author(s):

Mami Kurumata ◽

Misa Takahashi ◽

Atsushi Sakamoto ◽

Juan L. Ramos ◽

Ales Nepovim ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Transgenic Plants ◽

Degradation Products ◽

Plant Genome ◽

Pcr Analysis ◽

Wild Type ◽

In Planta ◽

Gene Encoding ◽

Plant Body ◽

Wide Range

Abstract Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C. 1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The obtained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the noil-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mᴍ TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when they were floated on TNT solution. HPLC analysis showed that the peak due to TNT taken up into plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indicates that the expression of bacterial NTR improved the capability of plants to degrade TNT.

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Receiving of genetic-modified wheat plants with heterolous ornitin-δ-aminotransferase gene

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v22.952 ◽

2018 ◽

Vol 22 ◽

pp. 222-227

Author(s):

O. M. Honcharuk ◽

O. V. Dubrovna

Keyword(s):

Triticum Aestivum ◽

Transgenic Plants ◽

Bread Wheat ◽

Binary Vector ◽

Triticum Aestivum L ◽

Callus Cultures ◽

Pcr Analysis ◽

Genetic Construct ◽

Agrobacterium Mediated Transformation

Aim. Receiving of genetically modified plants of bread wheat with heterologous ornithine‑δ‑aminotransferase gene. Methods. Agrobacterium-mediated transformation of callus cultures in vitro, PCR-analysis. Results. By Agrobacterium-mediated transformation of the morphogenic calluses of bread wheat (Triticum aestivum L.) using the AGLO strain containing the binary vector pBi-OAT with the target ornithine-δ-aminotransferase (oat) and selective neomycinphosphotransferase II (nptII), transgenic plants-regenerators have been obtained. Conclusions. As a result of the genetic transformation of Zimoyarka variety, 12 wheat regenerants were obtained in the genome which revealed a complete integration of the genetic construct containing the oat and nptII transgenes. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, ornithine‑δ‑aminotransferase gene, PCR-analysis.

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Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits

PLoS Pathogens ◽

10.1371/journal.ppat.1009116 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009116

Author(s):

Allison N. Dammann ◽

Anna B. Chamby ◽

Andrew J. Catomeris ◽

Kyle M. Davidson ◽

Hervé Tettelin ◽

...

Keyword(s):

Transcription Factor ◽

Amniotic Fluid ◽

Deletion Strain ◽

Growth Defect ◽

Wild Type ◽

Human Amniotic Fluid ◽

Wide Range ◽

Pregnant Mice ◽

Group B

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

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Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2523-2535.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2523-2535

Author(s):

J H Hegemann ◽

J H Shero ◽

G Cottarel ◽

P Philippsen ◽

P Hieter

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Chromosome ◽

Mutational Analysis ◽

Point Mutations ◽

Vector System ◽

Chromosome Loss ◽

Wild Type ◽

Sequence Elements ◽

Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

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Targeting mutant p53-expressing tumours with a T cell receptor-like antibody specific for a wild-type antigen

Nature Communications ◽

10.1038/s41467-019-13305-z ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Lionel Low ◽

Angeline Goh ◽

Joanna Koh ◽

Samantha Lim ◽

Cheng-I Wang

Keyword(s):

Cell Receptor ◽

Class I ◽

Human Antibody ◽

Mutant P53 ◽

Wild Type ◽

Class I Mhc ◽

Type Antigen ◽

Wide Range

AbstractAccumulation of mutant p53 proteins is frequently found in a wide range of cancers. While conventional antibodies fail to target intracellular proteins, proteosomal degradation results in the presentation of p53-derived peptides on the tumour cell surface by class I molecules of the major histocompatibility complex (MHC). Elevated levels of such p53-derived peptide-MHCs on tumour cells potentially differentiate them from healthy tissues. Here, we report the engineering of an affinity-matured human antibody, P1C1TM, specific for the unmutated p53125-134 peptide in complex with the HLA-A24 class I MHC molecule. We show that P1C1TM distinguishes between mutant and wild-type p53 expressing HLA-A24+ cells, and mediates antibody dependent cellular cytotoxicity of mutant p53 expressing cells in vitro. Furthermore, we show that cytotoxic PNU-159682-P1C1TM drug conjugates specifically inhibit growth of mutant p53 expressing cells in vitro and in vivo. Hence, p53-associated peptide-MHCs are attractive targets for the immunotherapy against mutant p53 expressing tumours.

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Deficiency of the Stress Kinase P38α Results in Embryonic Lethality

Journal of Experimental Medicine ◽

10.1084/jem.191.5.859 ◽

2000 ◽

Vol 191 (5) ◽

pp. 859-870 ◽

Cited By ~ 207

Author(s):

Melanie Allen ◽

Linne Svensson ◽

Marsha Roach ◽

John Hambor ◽

John McNeish ◽

...

Keyword(s):

Map Kinase ◽

Es Cells ◽

Interleukin 1 ◽

Embryonic Stem ◽

Wild Type ◽

Antiinflammatory Drugs ◽

Gene Encoding ◽

Significant Difference ◽

Induced Apoptosis

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38α. Mice null for the p38α allele die during embryonic development. p38α1/− embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38α−/− ES cells lacked p38α protein and failed to activate MAP kinase–activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38α1/+ ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38α, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38α−/− ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38α1/+ but not p38α−/− IL-1R–positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38α1/+ and p38α−/− ES cells. Therefore, these studies demonstrate that p38α is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38α does not serve an indispensable role in apoptosis.

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The Flagellar Motility ofChlamydomonas pf25 Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0630 ◽

2006 ◽

Vol 17 (1) ◽

pp. 227-238 ◽

Cited By ~ 23

Author(s):

Chun Yang ◽

Pinfen Yang

Keyword(s):

Regulatory Proteins ◽

Flagellar Motility ◽

Wild Type ◽

Dependent Protein Kinase ◽

Wide Range ◽

Anchoring Protein ◽

Radial Spokes ◽

Dependent Protein ◽

Cilia And Flagella

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas.

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Disruption of the ech42 (Endochitinase-Encoding) Gene Affects Biocontrol Activity in Trichoderma harzianum P1

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.5.419 ◽

1999 ◽

Vol 12 (5) ◽

pp. 419-429 ◽

Cited By ~ 84

Author(s):

S. L. Woo ◽

B. Donzelli ◽

F. Scala ◽

R. Mach ◽

G. E. Harman ◽

...

Keyword(s):

Antifungal Activity ◽

Trichoderma Harzianum ◽

Pythium Ultimum ◽

Single Copy ◽

Wild Type ◽

Gene Encoding ◽

Culture Filtrates ◽

In Vitro Antifungal Activity ◽

Better Than

The biocontrol strain P1 of Trichoderma harzianum was genetically modified by targeted disruption of the single-copy ech42 gene encoding for the secreted 42-kDa endochitinase (CHIT42). Stable mutants in which ech42 was interrupted, and unable to produce CHIT42, were obtained and characterized. These mutants lacked the ech42 transcript, the protein, and endochitinase activity in culture filtrates, and they were unable to clear a medium containing colloidal chitin. Other chitinolytic and glucanolytic enzymes expressed during mycoparasitism were not affected by the disruption of ech42. The disrupted mutant D11 grew and sporulated similarly to the wild type. In vitro antifungal activity of the ech42 disruptant culture filtrates against Botrytis cinerea and Rhizoctonia solani was reduced about 40%, compared with wild type; antifungal activity was fully restored by adding an equivalent amount of CHIT42 as secreted by P1. The mutant exhibited the same biocontrol effect against Pythium ultimum as strain P1, but the antagonism against B. cinerea on bean leaves by the mutant was significantly reduced (33% less biocontrol), compared with strain P1. Conversely, the endochitinase-deficient mutant performed better than the wild type (16% improvement of survival) in biocontrol experiments in soil infested with the soilborne fungus R. solani. These results indicate that the antagonistic interaction between the T. harzianum strain and various fungal hosts is based on different mechanisms.

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RepB C-terminus mutation of an ori pRi vector affects plasmid copy number inAgrobacteriumand transgene copy number in plants

10.1101/365320 ◽

2018 ◽

Author(s):

Zarir Vaghchhipawala ◽

Sharon Radke ◽

Ervin Nagy ◽

Mary L. Russell ◽

Susan Johnson ◽

...

Keyword(s):

Transgenic Plants ◽

Copy Number ◽

Binary Vector ◽

Single Copy ◽

Plasmid Copy Number ◽

Plant Production ◽

Silent Mutation ◽

Wild Type ◽

Plasmid Copy ◽

C Terminus

AbstractA nativerepABCreplication origin, ori pRi, was previously reported as a single copy plasmid inAgrobacterium tumefaciensand can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors forAgrobacterium-mediatedtransformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::RirepABCoperon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed thatAgrobacteriumcells with the RepBY299Hmutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299Hmutation on transformation and quality plant production, the RepBY299Hmutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299Hmutation inAgrobacteriumcells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

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O-acetylation controls the glycosylation of bacterial serine-rich repeat glycoproteins.

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016116 ◽

2020 ◽

pp. jbc.RA120.016116

Author(s):

Ravin Seepersaud ◽

Alexander C. Anderson ◽

Barbara A. Bensing ◽

Biswa P Choudhury ◽

Anthony J. Clarke ◽

...

Keyword(s):

Linkage Analysis ◽

Regulatory Mechanism ◽

Human Platelets ◽

Bacterial Virulence ◽

Wild Type ◽

Post Translational Modification ◽

Gram Positive Bacteria ◽

Wide Range ◽

Platelet Binding

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec (aSec) system. While all aSec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin GspB. Since these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues, and that O-acetylation prevented Glc deposition. Whereas streptococci expressing non-acetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to wild-type levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, since O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

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