ABSTRACT
Ever since the initial discovery of the molecules and genes involved in disease resistance in plants, attempts have been made to engineer durable disease resistance in economically important crop plants. Unfortunately, many of these attempts have failed, owing to the complexity of disease-resistance signalling and the sheer diversity of infection mechanisms that different pathogens use. Although disease-resistant transgenic plants or seeds are not yet available commercially, future product development seems likely as our current level of understanding of pathogenesis and plant defence improves.
Subject(s)
Genetic Engineering , Plant Diseases , Plants/genetics , Plants/immunology , Plants, Genetically ModifiedABSTRACT
Plant disease control is entering an exciting period during which transgenic plants showing improved resistance to pathogenic viruses, bacteria, fungi and insects are being developed. This review summarizes the first successful attempts to engineer fungal resistance in crops, and highlights two promising approaches. Biotechnology provides the promise of new integrated disease management strategies that combine modern fungicides and transgenic crops to provide effective disease control for modern agriculture.
Subject(s)
Genes, Plant , Plant Diseases/genetics , Biotechnology/methods , Genetic Engineering/methods , Immunity, Innate/geneticsABSTRACT
Streptomyces lividans, S. coelicolor and S. griseofuscus were examined for the presence of the enzymes of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). All three species were shown to possess Enzyme I, HPr and fructose-specific Enzyme II (IIFru) activities. In S. lividans and S. coelicolor, all three PTS enzymes were fructose-inducible, but in S. griseofuscus the system was expressed constitutively. These organisms apparently lack the HPr(Ser) kinase and HPr(Ser-P) phosphatase that characterize low-GC Gram-positive bacteria.
Subject(s)
Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Streptomyces/enzymology , Bacillus subtilis/enzymology , Fructose/metabolism , Genetic Complementation Test , Hydrogen-Ion Concentration , Kinetics , Phosphorylation , Species Specificity , Streptomyces/geneticsABSTRACT
We used a DNA-protein interaction screening method to isolate a cDNA, Erg-3, whose product binds to a site, designated pi, present in the immunoglobulin (Ig) heavy-chain gene enhancer. Erg-3 is an alternatively spliced product of the erg gene and contains an Ets DNA-binding domain. Fli-1 and PU.1, related Ets proteins, also bind to the same site. In addition, PU.1 binds to a second site, designated microB, in the Ig heavy-chain enhancer. We demonstrate that the pi binding site is crucial for Ig heavy-chain gene enhancer function. In addition, we show that Erg-3 and Fli.1, but not PU.1, can activate a reporter construct containing a multimer of protein-binding sites, synergistically with helix-loop-helix protein E12. We discuss how combinatorial interactions between members of the helix-loop-helix and Ets families may account for the tissue specificity of these proteins.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation , Genes, Immunoglobulin , Immunoglobulin Heavy Chains/biosynthesis , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary/isolation & purification , DNA, Complementary/metabolism , Deoxyribonuclease I , Glutathione Transferase/biosynthesis , HeLa Cells , Humans , Immunoglobulin Heavy Chains/genetics , Molecular Sequence Data , Proto-Oncogene Proteins c-ets , Recombinant Fusion Proteins/biosynthesis , Restriction Mapping , Transcription, Genetic , TransfectionABSTRACT
The Adenovirus DNA binding protein (DBP) imposes a regular, rigid and extended conformation on single stranded DNA (ssDNA) and removes secondary structure. Here we show that DBP promotes renaturation of complementary single DNA strands. Enhancement of intermolecular renaturation is sequence independent, can be observed over a broad range of ionic conditions and occurs only when the DNA strands are completely covered with DBP. When one strand of DNA is covered with DBP and its complementary strand with T4 gene 32 protein, renaturation is still enhanced compared to protein-free DNA, indicating that the structures of both protein-DNA complexes are compatible for renaturation. In contrast to promoting intermolecular renaturation, DBP strongly inhibits intramolecular renaturation required for the formation of a panhandle from an ssDNA molecule with an inverted terminal repeat. We explain this by the rigidity of an ssDNA-DBP complex. These results will be discussed in view of the crystal structure of DBP that has recently been determined.
Subject(s)
Adenoviridae/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Nucleic Acid Renaturation , Bacteriophage T4/metabolism , Base Sequence , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/isolation & purification , Kinetics , Models, Structural , Nucleic Acid Conformation , Osmolar Concentration , Protein Binding , Viral Proteins/metabolismABSTRACT
The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.
Subject(s)
Adenoviruses, Human/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , DNA/metabolism , Adenoviruses, Human/genetics , Circular Dichroism , DNA/genetics , DNA/ultrastructure , DNA-Binding Proteins/ultrastructure , Free Radicals , HeLa Cells , Humans , Hydroxides/metabolism , Hydroxyl Radical , Microscopy, Electron , Protein Binding , Protein Conformation , Restriction MappingABSTRACT
Initiation of Adenovirus DNA replication in vitro requires the presence of three viral proteins (pTP, pol, DBP) and two cellular transcription factors, NFI and Oct-1, that stimulate replication more than 100-fold. NFI assists in binding and positioning of the DNA polymerase in the origin whereas Oct-1 changes the structure of origin DNA. Optimal templates contain, in addition to origin sequences, the covalently bound viral terminal protein (TP). This terminal protein stimulates the template activity over 20 fold compared to protein-free templates. To study the way in which TP exerts its function in vitro we devised a novel method to isolate and label a short origin containing fragment in which the TP was bound in a functional form. This fragment replicated very efficiently and could be used for studying the binding of other replication proteins. Employing alpha-chymotrypsin digestion we show that for enhancement of replication in vitro only a small part of TP is required.
Subject(s)
Adenoviruses, Human/physiology , DNA Replication/physiology , Replicon/physiology , Viral Proteins/physiology , Adenoviruses, Human/genetics , Anion Exchange Resins , Chymotrypsin , Deoxyribonucleases, Type II Site-Specific , Nucleoproteins/isolation & purification , Peptide Fragments/isolation & purification , Structure-Activity Relationship , Viral Proteins/isolation & purificationABSTRACT
Nuclear Factor IV (NFIV) is a heterodimeric DNA-binding protein from HeLa cells, recognizing molecular ends and is identical to the autoantigenic target Ku. We have identified the two NFIV/Ku subunits, by comigration, in the 2D-gel database of transformed human amnion cell (AMA) proteins. We observed that the large subunit of NFIV/Ku consists of at least 3 charge variants that correspond to SSP IEFs 5705 (81.2 kDa, pI 5.74), 6707 (81.2 kDa, pI 5.67) and 6706 (81.9 kDa, pI 5.60) in the AMA catalogue. The relative amounts of the 2 major variants (IEFs 5705 and 6707) was dependent on the state of cell proliferation. Inhibition of DNA-synthesis by hydroxyurea also changed the relative levels of the variants, whereas aphidicolin or a thymidine block had no effect. These results suggest a possible role for NFIV/Ku in DNA replication.
Subject(s)
Antigens, Nuclear , DNA Helicases , DNA-Binding Proteins/chemistry , Databases, Factual , Nuclear Proteins/chemistry , Amnion/chemistry , Cell Division , Cell Line, Transformed , DNA-Binding Proteins/analysis , Electrophoresis, Gel, Two-Dimensional , Humans , Ku Autoantigen , Nuclear Proteins/analysis , Sequence Homology, Nucleic AcidABSTRACT
We have isolated a cDNA encoding the 84-kD subunit of NFIV. Tryptic peptide sequences were identified within the coding sequences, confirming its proper identity. The primary sequence of the protein is identical to that of the large subunit of the Ku autoantigen. A missing NFIV peptide sequence was identified within the sequence of the small subunit of Ku. In addition, the proteins are identical in immunological aspects. We suggest that the Ku and NFIV proteins are identical. This connection adds new biochemical data to our knowledge of the Ku autoantigen.
Subject(s)
Antigens, Nuclear , Antigens, Surface/chemistry , DNA Helicases , DNA-Binding Proteins/chemistry , Nuclear Proteins/chemistry , Amino Acid Sequence , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Base Sequence , DNA/analysis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Ku Autoantigen , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/immunology , Peptide Mapping , Precipitin Tests , RabbitsABSTRACT
The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI.
Subject(s)
Adenoviruses, Human/metabolism , CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/metabolism , DNA/metabolism , Transcription Factors , Adenoviruses, Human/genetics , DNA Replication , DNA, Viral/genetics , DNA, Viral/metabolism , Deoxyribonuclease I , HeLa Cells/metabolism , Humans , Kinetics , Macromolecular Substances , NFI Transcription Factors , Nuclear Proteins , Protein Binding , Substrate Specificity , Y-Box-Binding Protein 1ABSTRACT
Initiation of transcription on mitochondrial DNA of Saccharomyces cerevisiae was studied in an in vitro system with a mtRNA polymerase fraction reconstituted from separately purified components and with DNA templates containing the promoter of the gene coding for large rRNA. The effect of various point mutations in this promoter region was quantitated in assays containing a wildtype promoter in equimolar amount as internal control. Despite the strong conservation around the position at which RNA initiation occurs (ATATAAGTApuTA, initiation nucleotide underlined), none of the single point mutations abolished transcription-initiation completely. Some reduce the efficiency of initiation to 10-20% compared to the wild type promoter, while others have a much less pronounced effect. A change of the A at position +4 into a G even results in a promoter up mutation. Remarkably, alteration of the A at position +1 into a G or a T affects the efficiency of initiation only slightly and initiation is maintained at the same position.
