In vitro translation of tomato bushy stunt virus RNA.

In vitro translation of tomato bushy stunt (TBSV)-RNA in a rabbit reticulocyte system resulted in synthesis of five proteins P 18, P 25, P 34, P 35, and P 40. The P 40 protein was identified as the viral coat protein. Fractionation of TBSV-RNA and subsequent translation provided evidence for the existence of discrete subgenomic RNAs.

The molecular biology of caulimoviruses.

The molecular biology of the caulimoviruses has already inspired quite a few review articles: this review is limited to a general description of the type-member of this group, namely cauliflower mosaic virus. Details are presented of major results obtained on the organization and function at the molecular level of caulimoviruses and cauliflower mosaic virus in particular.

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.

Incidence of specific anosmia in Northern Germany.

Thresholds for the perception of 6 primary odorants were tested in a sample of 153 unrelated healthy individuals including 101 males and 52 females. Using some special precautions and directions for preparing aqueous solutions of primary odorants, screening for specific anosmia was found to be a practicable method with reliable results. The observed perception thresholds showed a bimodal distribution. Individuals with higher values probably are specific anosmics. Relatively lower frequencies of anosmia observed in this sample, as compared to reported values in Caucasians of the USA, are probably due to genetic differences. The pheromone character of some odorants and their possible genetic relevance is discussed.

A simple gene-expression system for the small subunit of ribulose bisphosphate carboxylase in leaves ofNicotiana sylvestris.

InNicotiana sylvestris only four transcripts coding for the small subunit of RUBISCO are present in leaves. They are very closely related as they are identical in the nucleotide sequence of the non-coding regions and show only three silent point differences in the region coding for the mature peptide.The main difference among these four transcripts lies in the length of the non-coding regions. Half of the SmRNA population as confirmed by direct RNA sequencing has an additional nucleotide sequence in the leader region. Two cDNAs have an additional nucleotide sequence at the end of the 3' non-coding region. Based on these criteria the transcripts were classified into two groups:.group I has a 73-nucleotide-long leader sequence and the nucleotides T, A and C at position 327, 432 and 519 in the coding region..group II has a 60-nucleotide-long leader sequence and the nucleotides C, G and T at these positions in the coding region.The two cDNAs showing a difference in the length of the 3' non-coding region belong to group II.The study of all these transcripts argues for the possibility that only two families of genes are expressed in leaves ofN. sylvestris.

Dermatoglyphic findings in patients with fragile X-chromosome.

Finger- and palmar prints of hemi- and heterozygote fragile X-patients with mental retardation (10 males and 5 females) were compared to dermatoglyphic findings in 20 mentally retarded patients (10 males and 10 females) without fragile X and to 200 healthy unrelated persons (100 males and 100 females). Characteristic whorls and double-loops with high ridge-counts on finger-tips and a pronounced transversal course of palmar ridges were restricted to males with fragile X. Female carriers of fragile X showed, corresponding to male patients, some abnormalities of the digital- and palmar ridge-pattern. Contrary to males, in carriers as well as in mentally retarded females without fragile X, fingerprints with low ridge-counts were found. Common to all mentally retarded patients, but more pronounced in males with fragile X, abnormal palmar creases and hand-measurements were observed. These findings probably are related to prenatal retarded growth of the length of the palma and of the middle-finger.

A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa.

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.

Detection in vivo of a new gene product (gene III) of cauliflower mosaic virus.

Cauliflower mosaic virus DNA contains six major open reading frames (ORFs). As only the mRNA corresponding to the transcription of gene VI and its translation product have been isolated, the identification in infected plants of products corresponding to the five other putative genes remains to be established. The present paper reports the detection of an ORF III product by means of antibodies raised against an NH(2)-terminal synthetic peptide of 19 amino acids corresponding to a sequence predicted from the nucleotide sequence of ORF III. The detection of this gene product raises the question of the mechanism of its expression.

A second cauliflower mosaic virus gene product influences the structure of the viral inclusion body.

We have used electron microscopy of thin sections and experiments on isolated viroplasms to compare the properties of four strains of cauliflower mosaic virus (CaMV), three of which were partially or completely deleted in open reading frame (ORF) II. Our results confirm that this gene is required for aphid transmissibility and show that the product of ORF II influences the firmness with which virions are held within the viroplasm. Analysis of the proteins in the viroplasms showed that a mutant with a partial deletion in ORF II produced a protein smaller than the normal ORF product. This smaller protein was non-functional with respect both to aphid transmissibility and properties of the viroplasms.

Electron microscopic studies of the different topological forms of the cauliflower mosaic virus DNA: knotted encapsidated DNA and nuclear minichromosome.

Cauliflower mosaic virus (CaMV) DNA exists under different topological forms in infected plants. First, the population of encapsidated CaMV DNA molecules appears heterogeneous when analysed by gel electrophoresis. The electron microscopic study reported here reveals that CaMV virion DNA contains simple and multiple topological knots. Second, a supercoiled DNA form never found in virions exists as a chromatin-like nucleoprotein complex with nucleosome subunits in the nuclei of infected leaves. The compaction ratio of the minichromosomes is compatible with the nucleosomal structure, the number of nucleosomes (41.0 +/- 2.5) is in keeping with the length of the viral genome.

Expression of the gene coding for the small subunit of ribulosebisphosphate carboxylase during differentiation of tobacco plant protoplasts.

A hybridization probe was used to study the regulation of expression of the gene coding for the small subunit of ribulose 1,5-bisphosphate carboxylase, during functional differentiation of protoplasts. A library of cDNA from poly(A)-containing RNA extracted from specially treated tobacco leaves was constructed in the plasmid pBR322 by blunt-end ligation. This library was screened by colony hybridization with 32P-labelled cDNA prepared from mRNA coding for the precursor of the small subunit. A positive colony was identified containing recombinant plasmids with a nucleotide sequence homologous to this mRNA. These plasmids, bound to diazobenzyloxymethylated cellulose paper, were then used as a hybridization probe. The results showed unambiguously that the small subunit was not transcribed in protoplasts but was transcribed in undifferentiated white and chlorophyll-containing green callus cultures derived from protoplasts. The discrepancy between these results and those obtained with classical techniques is discussed.

In vivo recombination of cauliflower mosaic virus DNA.

LIGATION AND RECOMBINATION OF THE DNA OF CAULIFLOWER MOSAIC VIRUS (CAMV) IS DEMONSTRATED BY THE FOLLOWING EXPERIMENTS: (i) Ligation: Different noninfectious fragments of the CaMV genome (obtained after insertion into plasmid pBR322 followed by enzymatic excision) regained infectivity when mixtures of them were used to inoculate their host. The symptom appearance was delayed by comparison with a typical CaMV infection, and only the newly formed leaves were affected. (ii) Recombination: Pairs of noninfectious recombinant full-length CaMV genomes (integrated into pBR322 at different restriction endonuclease sites) regained infectivity upon simultaneous inoculation of a sensitive host. The symptomatology of the resulting infection was indistinguishable from that of a typical CaMV infection. We show that progeny DNA had the same characteristics (size, structure, restriction endonuclease digestion pattern) as bona fide CaMV DNA, and that the vector pBR322 had been completely eliminated. A cloned tandem dimer of CaMV DNA with a partial deletion similarly was infectious in the plant assays. This system should be useful to study the expression of mutant genomes, thus allowing characterization of the CaMV genes.

Free cauliflower mosaic virus supercoiled DNA in infected plants.

Evidence is presented indicating that free viral DNA exists in Cauliflower mosaic virus-infected leaves. Among the different viral DNA forms detected in total DNA extracts is a supercoiled form. We discuss its possible role in the replication process.

The tRNA-like structure of turnip yellow mosaic virus RNA: structural organization of the last 159 nucleotides from the 3' OH terminus.

The secondary structure of the isolated tRNA-like sequence (n=159) present at the 3' OH terminus of turnip yellow mosaic virus RNA has been established from partial nuclease digestion with S1 nuclease and T1, CL(3), and Naja oxiana RNases. The fragment folds into a 6-armed structure with two main domains. The first domain, of loose structure and nearest the 5' OH terminus, is composed of one large arm which extends into the coat protein cistron. The second, more compact domain, is composed of the five other arms and most probably contains the structure recognized by valyl-tRNA synthetase. In this domain three successive arms strikingly resemble the T[unk], anticodon, and D arms found in tRNA. Near the amino-acid accepting terminus, however, there is a new stem and loop region not found in standard tRNA. This secondary structure is compatible with a L-shaped three-dimensional organization in which the corner of the L and the anticodon-containing limb are similar to, and the amino-acid accepting region different from, that in tRNA. Ethylnitrosourea accessibility studies have shown similar tertiary structure features in the T[unk] loop of tRNA and in the homologous region of the viral RNA.

Identification by immunoprecipitation of cauliflower mosaic virus in vitro major translation product with a specific serum against viroplasm protein.

A highly specific antiserum was prepared against purified cauliflower mosaic virus viroplasm-protein (VmP). A virus specific in vitro major translation product (TPmaj), encoded by the 19S poly(A) RNA fraction from cauliflower mosaic virus infected turnip leaves, was recognized by this antiserum. The N-terminal sequence of TPmaj corresponds to the sequence following the first in-phase initiation codon in gene VI of the cauliflower mosaic virus genome. Both VmP and TPmaj have blocked termini and probably start from the same AUG codon.