Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cellto-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides doser to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.Positive-sense RNA viruses have evolved numerous strategies for gene regulation. One common strategy is the use of subgenomic mRNAs, transcribed from genomic RNA, for the expression of internal open reading frames (ORFs). In this manner the expression of multiple genes residing on a common genomic RNA can be independently regulated. One such virus that utilizes this strategy is the type member of the tobamovirus group, tobacco mosaic virus (TMV).The genome of TMV resides on a single strand of positivesense RNA, 6395 nucleotides (nt) in length, and encodes at least four proteins (1). The TMV genome is organized such that two 5'-coterminal ORFs encoding 126-kDa and 18&-kDa replicase proteins are translated from the genomic RNA, while an internal ORF encoding the 30-kDa cell-to-cellmovement protein and a 3'-proximal ORF encoding the coat protein (structural) are translated from respective subgenomic mRNAs (2-5). A third subgenomic mRNA has been detected for an additional internal ORF, within the 183-kDa ORF, encoding a putative 54-kDa protein that has not yet been detected in infected plants (6). The TMV subgenomic mRNAs are transcribed from negative-sense genomic RNA and share a common 3' terminus.Of particular interest is that genes of TMV expressed via subgenomic mRNAs are independently regulated, both quantitatively and temporally. The 30-kDa pro...

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by TnS mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T74i10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.The 39-kb IncW plasmid pSa, originally isolated from Shigella flexneri (49), confers resistance to streptomycin, chloramphenicol, kanamycin, spectinomycin, and sulfonamides (22) and is capable of self-mobilization and replication in a wide range of gram-negative bacteria (45). An interesting trait of pSa is its ability to completely abolish the oncogenic activity of Agrobacterium tumefaciens (32). The plasmid also prevents hairy-root-forming ability of the related species A. rhizogenes (47). As long as pSa is present in the bacterium, oncogenic suppression by pSa is absolute. The presence of pSa does not cause instability of the oncogenic Ti plasmid, and oncogenicity is regained when the strain is cured of pSa (15). Also, pSa does not block conjugative transfer of the Ti plasmid, and when the Ti plasmid is transferred from a pSa-bearing strain to a recipient strain, oncogenicity is conferred on the recipient (15).To identify the region of pSa that confers oncogenic suppressive (OS) activity Zaitlin (54) tested several subclones of pSa and located the OS activity to a 7.6-kb fragment cloned in pUCD111. The number of loci and location of these loci which conferred OS activity were not defined, however. Furthermore, the function of the products of the OS locus remained unknown. We have shown, however, that OS activity is unrelated to bacterial auxin production (24), which was proposed by Chernin et al. to be involved in tumorigenesis (8). Also, induction of the virulence genes of the Ti plasmid and T-DNA border cleavage do occur in the presence of pSa (9...