ABSTRACT
Serine proteases constitute an important group of extra- and intracellular proteases in fungi. These enzymes are characterized by conserved regions around the active site residues, Asp, His and Ser. Based on this amino acid (aa) sequence conservation, we have used degenerate primer PCR to isolate subtilisin-specific genomic probes from Aspergillus niger, and cloned a gene, pepD, by screening a lambda genomic library using a PCR probe. The pepD gene contains three putative introns, which are 51-, 47- and 55-bp long and has an open reading frame coding for a protein which consists of 416 aa. The deduced aa sequence shows similarity to subtilisin-like proteases, in particular to fungal alkaline proteases. Signal sequence cleavage prediction indicates that the first 20 aa are probably removed upon transfer to the endoplasmic reticulum. The conservation of the pro-enzyme cleavage site in fungal alkaline proteases suggests that the mature protein is derived from this polypeptide via the removal of an additional 101 aa, resulting in a mature 30,294-Da enzyme consisting of 295 aa.
Subject(s)
Aspergillus niger/enzymology , Aspergillus niger/genetics , Endopeptidases/genetics , Genes, Fungal , Subtilisins/genetics , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , Conserved Sequence , DNA Primers , Endopeptidases/biosynthesis , Exons , Genomic Library , Introns , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Protein Sorting Signals/genetics , Restriction Mapping , Sequence Homology, Amino AcidABSTRACT
Cauliflower mosaic virus (CaMV) gene I encodes a 40-kDa protein, P1, which is thought to be involved in the cell-to-cell movement of the virus. In order to investigate its functioning, P1 was expressed in Saccharomyces cerevisiae transformed by an expression vector containing CaMV gene I. When produced in yeast, PI was 40 kDa in size and not N-glycosylated.
Subject(s)
Genes, Viral/genetics , Mosaic Viruses/genetics , Plants, Edible/microbiology , Viral Proteins/genetics , Blotting, Western , Cloning, Molecular , Gene Expression/genetics , Genetic Vectors/genetics , Glycosylation , Saccharomyces cerevisiae/geneticsABSTRACT
Antiserum was prepared against a synthetic peptide corresponding to the N-terminal 20 amino acids of the protein encoded by cauliflower mosaic virus (CaMV) open reading frame VII (ORF VII). This antiserum was used to detect the expression of CaMV ORF VII either in Saccharomyces cerevisiae transformed by an expression vector containing CaMV ORF VII or in CaMV-infected plants. Only in S. cerevisiae has a 14-kilodalton protein been detected.
Subject(s)
Brassica/microbiology , Genes, Viral , Mosaic Viruses/genetics , Saccharomyces cerevisiae/genetics , Brassica/genetics , Chromosome Mapping , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression , Immunoblotting , Plasmids , Viral Proteins/genetics , Viral Proteins/isolation & purificationABSTRACT
The interaction of the gene III product, P15, of cauliflower mosaic virus with different double-stranded DNA fragments of the viral genome was investigated. The results suggest that gene III product which showed DNA binding activity is a structural protein of the viral particle.
Subject(s)
DNA, Viral/metabolism , DNA-Binding Proteins/analysis , Genes, Viral , Plant Viruses/genetics , Viral Proteins/analysis , Amino Acid Sequence , Plant Viruses/analysis , Viral Structural Proteins/analysisABSTRACT
Gene I product of cauliflower mosaic virus was immunodetected in a cell-wall-enriched fraction from infected turnip leaves in addition to its detection in viroplasms and replication complexes. The immunoreaction was carried out with an antiserum raised against a 15 amino acid long synthetic peptide corresponding to the carboxy-terminus of potential gene I protein (P1). The presence of P1 in different subcellular fractions was investigated as a function of time during viral multiplication. At late infection times, P1 was found only in the cell-wall-enriched fraction.
Subject(s)
Cell Wall/analysis , Genes, Viral , Mosaic Viruses/genetics , Plants/microbiology , Viral Proteins/analysis , Mosaic Viruses/physiology , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Subcellular Fractions/analysis , Viral Proteins/physiology , Virus ReplicationABSTRACT
Antisera against the N-terminal and C-terminal parts of the potential ORF IV product were used to analyse extracts from CaMV-infected turnip leaves by immunoblotting. Polypeptides of 87, 83, 82, 60 and 57 kDa were detected. The origin of these proteins is discussed.
