ABSTRACT
Two monoclonal antibodies, E3cD8 and E3cA10, were generated to the EBNA 3c nuclear protein from the B95.8 isolate of Epstein-Barr virus (EBV). Both antibodies efficiently precipitate EBNA 3c from B95.8-transformed lymphoblastoid cell lines, and E3cA10 also detects EBNA 3c on Western blots. Whereas E3cD8 reacts with all 11 Type-1 isolates of EBV tested, and E3cA10 reacts with 14 of 17 Type-1 isolates, neither antibody detects the EBNA 3c protein encoded by Type-2 isolates. E3cD8 recognizes a peptide sequence (PA/PPQAPYQGY) in a repeat region of the B95.8 EBNA 3c coding sequence which is not present in the prototype Type-2 AG876 sequence. The E3cA10 antibody epitope has been mapped to the minimal five amino acid B95.8 peptide, WAPSV, which has an alanine to valine substitution in the AG876 virus isolate. This substitution was also found in three Type-1 EBV isolates that expressed EBNA 3c proteins not detected by E3cA10. In immunoprecipitation studies E3cA10 additionally coprecipitated the EBNA 2 protein from Type-1 isolates of EBV. The possibility of a direct interaction between EBNA 2 and EBNA 3c was ruled out by the demonstration that the antibody precipitated EBNA 2 from the Raji cell line which carries a virus with a deleted EBNA 3c gene. Since the WAPSV epitope identified in EBNA 3c is not present in EBNA 2, and no EBNA 2 linear peptide reactivity was detected in ELISA, it seems likely that E3cA10 recognizes a conformational epitope on EBNA 2. However, from the present data we cannot exclude the possibility that the antibody reacts with a cellular protein that physically associates with EBNA 2.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , DNA-Binding Proteins/immunology , DNA-Binding Proteins/isolation & purification , Herpesvirus 4, Human/immunology , Amino Acid Sequence , Antibodies, Viral/immunology , Antibody Specificity , Base Sequence , Cell Line, Transformed , DNA, Viral/analysis , Electrophoresis, Polyacrylamide Gel , Epitopes/genetics , Epitopes/immunology , Epstein-Barr Virus Nuclear Antigens , Herpesvirus 4, Human/chemistry , Herpesvirus 4, Human/growth & development , Herpesvirus 4, Human/isolation & purification , Molecular Sequence Data , Oligopeptides/immunology , Precipitin Tests/methods , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sequence Analysis, DNA , Sequence Deletion/physiologySubject(s)
Alkaline Phosphatase/metabolism , Molecular Biology/methods , Alkaline Phosphatase/antagonists & inhibitors , Animals , Cations , Cattle , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Escherichia coli/enzymology , Hydrogen-Ion Concentration/drug effects , Substrate Specificity/drug effects , Sulfhydryl Reagents/pharmacology , TemperatureSubject(s)
Molecular Biology/methods , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , Animals , Biocatalysis/drug effects , Cations , DNA/metabolism , Enzyme Activators/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Hydrogen-Ion Concentration/drug effects , Polynucleotide 5'-Hydroxyl-Kinase/antagonists & inhibitors , Substrate Specificity/drug effects , Sulfhydryl Reagents/pharmacology , TemperatureABSTRACT
The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.
Subject(s)
Genetic Vectors , Plasmids , Rhizobium/genetics , Cloning, Molecular , DNA, Bacterial , DNA, Recombinant , Electrophoresis, Agar GelABSTRACT
Gene expression during the ripening of tomato fruit was investigated by cDNA cloning and hybrid-select translation. A cDNA library was prepared from poly(A)-containing mRNA from ripe tomato fruit and sreened by differential hybridization. 146 ripening-related cDNA clones were found. Eleven groups and eight unique clones have been identified so far. The sizes of the cloned cDNA inserts were determined and type-members for seven groups were used in hybrid selection experiments. Six of the seven clones encode translation products corresponding to six ripening related polypeptides detected previously by in vitro translation of total cytoplasmic RNA (14). One cDNA group codes for a Mr 48 000 protein that was identified as polygalacturonase on the basis of immunoprecipitation with specific antiserum raised against tomato polygalacturonase. re]19840918 rv]19850613 ac]19850618.
ABSTRACT
Breakdown of chlorophylls in attached senescing sycamore leaves held in darkness was significantly less over a 14-d period than that occurring in leaves exposed to natural light. Chlorophyll a declined more rapidly than chlorophyll b in both situations, the stability of the latter being particularly increased in darkness. The differences between dark-maintained leaves and those exposed to light with respect to soluble protein, cytoplasmic RNA, and free amino-nitrogen were much less marked. The data indicate that chlorophyll loss during senescence is, at least in part, the result of a direct photochemical degradation of the pigment.
