ABSTRACT
The full characterization of a novel insecticidal crystal protein, named Cry9Ca1 according to the revised nomenclature for Cry proteins, from Bacillus thuringiensis serovar tolworthi is reported. The crystal protein has 1,157 amino acids and a molecular mass of 129.8 kDa. It has the typical features of the Lepidoptera-active crystal proteins such as five conserved sequence blocks. Also, it is truncated upon trypsin digestion to a toxic fragment of 68.7 kDa by removal of 43 amino acids at the N terminus and the complete C-terminal half after conserved sequence block 5. The 68.7-kDa fragment is further degraded to a nontoxic 55-kDa fragment. The crystal protein has a fairly broad spectrum of activity against lepidopteran insects, including members of the families Pyralidae, Plutellidae, Sphingidae, and Noctuidae. A 50% lethal concentration of less than 100 ng/cm2 of diet agar was found for diamondback moth, European corn borer, cotton bollworm, and beet armyworm. It is the first insecticidal crystal protein with activity against cutworms. No activity was observed against some beetles, such as Colorado potato beetle. The protein recognizes a receptor different from that recognized by Cry1Ab5 in Ostrinia nubilalis and Plutella xylostella. In Spodoptera exigua and P. xylostella, it binds to a receptor which is also recognized by Cry1Cax but with a lower affinity. In these insects, Cry1Cax probably binds with a higher affinity to an additional receptor which is not recognized by Cry9Ca1. Elimination of a trypsin cleavage site which is responsible for the degradation to a nontoxic fragment did result in protease resistance but not in increased toxicity against O. nubilalis.
Subject(s)
Bacillus thuringiensis/chemistry , Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Endotoxins/chemistry , Insect Proteins , Lepidoptera , Amino Acid Sequence , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Bacterial Toxins/metabolism , Bacterial Toxins/toxicity , Base Sequence , Endotoxins/metabolism , Endotoxins/toxicity , Hemolysin Proteins , Larva , Molecular Sequence Data , Molecular Weight , Operon/genetics , Peptide Fragments/chemistry , Receptors, Cell Surface/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , TrypsinABSTRACT
The canine interferon-gamma (cIFN-gamma) chromosomal gene was isolated from a recombinant library, containing the entire dog genome, by screening with a rat IFN-gamma genomic probe. The structural organization of the cIFN-gamma gene closely resembles that of the human, murine, and rat IFN-gamma genes. It contains three intervening sequences and encodes a signal sequence of 23 amino acids followed by a mature protein of 143 amino acids. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the cIFN-gamma protein sequence with that of the corresponding murine, rat, human, and bovine proteins revealed a homology of 40%, 42%, 65%, and 76%, respectively. The cIFN-gamma gene was expressed under control of the simian virus 40 early promoter in Chinese hamster ovary (CHO) cells, deficient in dihydrofolate reductase (DHFR), after cotransformation with a plasmid containing the murine dhfr gene. Initial transformants with dhfr+ phenotype produced cIFN-gamma titers ranging from 10 to 10,000 laboratory units (LU)/ml of culture medium. A cIFN-gamma cDNA sequence was identified in a cDNA library constructed with partially purified RNA from a cIFN-gamma-producing CHO cell line. In vitro transcription of the cDNA and translation of the mRNA in Xenopus laevis oocytes resulted in secreted IFN-gamma with an antiviral activity specific for canine cells.
Subject(s)
Interferon-gamma/genetics , Recombinant Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Dogs , Gene Expression/genetics , Genetic Vectors/genetics , Genomic Library , Interferon-gamma/chemistry , Interferon-gamma/pharmacology , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Restriction Mapping , Tumor Cells, Cultured , Viral Plaque AssayABSTRACT
The nucleotide sequence of a novel insecticidal crystal protein(Cry)-encoding gene from a Bacillus thuringiensis serotype kurstaki isolate is described. The gene is related to the known coleopteran-active cryIII genes and encodes a CryIIID that is much more active against Colorado potato beetle than other CryIII.
