Phenylalanine ammonia-lyase in tobacco mosaic virus-infected hypersensitive tobacco. Density-labelling evidence of de novo synthesis.

A strong increase in phenylalanine ammonia-lyase (EC 4.3.1.5) activity occurs in tobacco mosaic virus-infected tobacco leaves developing necrotic local lesions. Comparison of physicochemical properties of the partially purified enzymes extracted from healthy and infected leaves showed that the hypersensitive reaction leads to an increase in the pool size of the same active enzyme molecules as those present in non-infected material. The molecular mechanism of enzyme formation was investigated by radiolabelling with [3H]leucine and by density labelling with 2H2O. Abnormal patterns of incorporation of radioactivity into all soluble proteins were found in infected leaves carrying local lesions. In contrast, uptake of deuterium into the amino acid pool was the same in healthy and infected leaves. Unstimulated phenylalanine ammonia-lyase was shown to be a long-lived enzyme (half-life: 25-35 h). Results of comparative density labelling experiments unequivocally demonstrated that the increased enzyme pool size arose from an increased rate of synthesis mediated by the hypersensitive reaction.

Fluorescent study of tobacco mosaic virus protein.

Protein fluorescence properties of tobacco mosaic virus [3 Trp residues per monomer (positions 17, 52, 152)] and of two tobacco mosaic virus mutants [green tomato atypical mosaic virus, 2 Trp (52, 152) and cucumber virus4, 1 Trp (unknown position)] have been studied. Emission spectra, fluorescence quantum yields and lifetimes were determined. Results showed that protein fluorescence is due to buried Trp only, except for the cucumber virus4 strain, in which Tyr also contributed to the emission. Comparison of the three strains showed that Trp 17 and Trp 52 have high fluorescence yields (phi17 = 0.29; phi52 = 0.37) whereas Trp 152 (probably present in cucumber virus4) is strongly quenched (phi152 = 0.035). An unusually efficient Tyr leads to Trp energy transfer was observed in tobacco mosaic virus protein, indicating that most of four Tyr residues are located near the highly fluorescent Trp.

Natural fluorescence properties of brome mosaic virus protein.

Some of the fluorescence properties of brome mosaic virus protein in different states of aggregation (dimer, capsid) have been studied, in particular the emission spectra, fluorescence quantum yields and lifetimes, as well as the effect of external quenchers and of temperature on the fluorescence. Brome mosaic virus protein, which contains two tryptophan and five tyrosine residues per monomer, displayed an unusual fluorescence spectrum maximum of 308 nm at pH 7.4 when excited at 280 nm. The emission maximum was shifted to 327 nm when excited at 295 nm. Analysis of the results showed that the tyrosine emission is characterized by a high value for the quantum yield (phi = 0.07), which is consistent with a location of most of these residues in helical regions of the protein, while the tryptophan emission is strongly quenched (phi=0.035). The effects of external quenchers suggested that two kinds of tryptophan residues might exist, one buried (phi=0.056) and one exposed, the quantum yield of the latter being particularly low (phi=0.014). The tryptophan fluorescence quenching is partially removed at pH 8.4 and totally eliminated after chemical (guanidinium chloride) or thermal denaturation of the protein. Formation of capsid induced an additional quenching of the exposed tryptophan residue while interaction with the RNA in the virus did not modify the emission parameters of the protein.

In vivo dimerization of cauliflower mosaic virus DNA can explain recombination.

Pairs of heterologous cauliflower mosaic virus (CaMV) genomes cloned in pBR322, one having a defective genome and both restricted at the same pBR322 cloning site, generate recombinant molecules in infected cells when co-inoculated on plants. Analysis of the restriction pattern of the isolated recombinant CaMV DNAs indicated that the intergenomic recombination may be explained by dimerization of two heterologous CaMV molecules and transcription into a hybrid 35S RNA responsible for replication of the recombinant genomes.

Physical map of DNA from a new cauliflower mosaic virus strain.

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.

Infectivities of native and cloned DNA of cauliflower mosaic virus.

Infectivity assays on turnips reveal that (i) cauliflower mosaic virus (CaMV) DNA, whether circular or linear, is as infectious as the complete virus; (ii) linear DNA obtained with restriction enzymes from the native CaMV DNA has the same specific infectivity as when first cloned in plasmid (pBR322) or bacteriophage (lambda gtWES) vectors and then restricted at the cloning site; (iii) in all cases studied mosaic symptoms are accompanied by virus production. DNA isolated from these viruses is again circular and possesses the three "gaps" characteristic of CaMV DNA. The cloned CaMV DNA, when linked to the vector DNA, is noninfectious or exhibits very low infectivity.

Expression of a putative plant viral gene in Escherichia coli.

A recombinant plasmid, pCB300, was constructed which carries a cauliflower mosaic virus (CaMV) DNA insert corresponding to nucleotides 1825-2280, including the coding sequence (1830-2219) of open reading frame III (ORF III). This CaMV DNA insert was fused with the amino-terminal portion of the beta-galactosidase gene. Transcription of the hybrid gene is controlled by the lac promoter, which is repressed in Escherichia coli strain JM103 and can be induced by isopropylthio-beta-D-galactoside (IPTG). When the promoter is derepressed, cells harboring the chimeric plasmid produce an Mr 16 000 fusion protein. This protein is immunodetected by antibodies raised against an amino terminal synthetic peptide of 19 amino acids corresponding to a sequence predicted from the nucleotide sequence of ORF III.

Hereditary ataxia and the sixth chromosome.

Possible linkage of the gene or genes for dominant hereditary ataxia and three genetic markers on the short arm of the sixth chromosome (HLA, properdin factor B [Bf], and glyoxalase I) was investigated in five families. Logarithmic odds (lod scores) were calculated for the linkages and found to be either inconclusive or in favor of nonlinkage. Caution is advised in the summing of lod scores for separate families because of the wide spectrum of clinical and anatomical manifestations of dominant hereditary ataxia. Three families with recessive hereditary ataxia were also studied. Identical haplotypes occurred in affected and unaffected siblings. It did not appear likely that the recessive genes of the parents were transmitted in linkage with the markers on the short arm of the sixth chromosome.

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.