Tissue-specific expression of the TMV coat protein in transgenic tobacco plants affects the level of coat protein-mediated virus protection.

Transgenic tobacco plants were produced that express a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) under the control of the promoter from a ribulose bisphosphate carboxylase small subunit (rbcS) gene. Plant lines expressing comparable levels of CP from the rbcS and cauliflower mosaic virus 35S promoters were compared for resistance to TMV. In whole plant assays the 35S:CP constructs gave higher resistance than the rbcS:CP constructs. On the other hand, leaf mesophyll protoplasts isolated from both plant lines were equally resistant to infection by TMV. This indicated that the difference in resistance between the lines in the whole plant assay reflects differences at the level of short- and/or long-distance spread of TMV. Therefore, we propose that the difference in tissue-specific expression between the 35S and rbcS promoters accounts for greater resistance in the plant lines that express the 35S:CP chimeric genes.

Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences.

Transgenic tobacco plants which express a chimeric gene encoding the tobacco mosaic virus (TMV) coat protein (CP) and the TMV 3' untranslated region are protected against infection by TMV. In this study chimeric genes that encode the sequences representing the TMV CP subgenomic RNA, but do not produce protein (because of removal of the initiation codon), and RNA that lacks the tRNA-like sequence of the TMV 3' end were expressed in transgenic plants. Only plants that accumulated CP, regardless of the presence of absence of the 3' end of TMV-RNA, were protected against infection by TMV. The results indicate that the CP per se, rather than TMV RNA, is responsible for the resistance to infection by TMV. Furthermore, the degree of protection is dependent upon the level of accumulated CP.

Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA.

Transgenic tobacco plants that express RNA sequences complementary to the tobacco mosaic virus (TMV) coat protein (CP) coding sequence with or without the tRNA-like structure at the 3' end of the TMV RNA were produced. Progeny of self-pollinated plants were challenged with TMV to determine their resistance to infection. Plants that expressed RNA sequences complementary to the CP coding region and the 3' untranslated region, including the tRNA-like sequences, were protected from infection by TMV at low levels of inoculum. However, plants that expressed RNA complementary to the CP coding sequence alone were not protected from infection. These results indicate that sequences complementary to the terminal 117 nucleotides of TMV, which include a putative replicase binding site, are responsible for the protection. However, the level of protection in these plants was considerably less than in transgenic plants that expressed the TMV CP gene and accumulated CP. Since the mechanisms of protection in the two systems are different, it may be possible to increase protection by introducing both sequences into transgenic plants.

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants.

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.

Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants.

We have compared the level of expression of the Cauliflower Mosaic Virus 35S promoter and the nopaline synthase promoter when fused to a common reporter gene. A cassette containing the neomycin phosphotransferase (type II) coding sequence followed by the nopaline synthase 3' nontranslated region was used for transcriptional and translational evaluation of the two different promoters. These chimeric genes were introduced into petunia plants and the copy number of the gene, the steady state level of NPTII transcript and the levels of NPTII enzyme activity were determined. In this paper, we report that the NPT II transcript levels are on the average 30 fold higher in plants containing CaMV 35S promoter and leader sequences than in plants containing the same reporter gene but nopaline synthase promoter and leader sequences. Similarly, plants containing the CaMV 35S promoter had an average of 110 fold higher levels of NPTII enzyme activity than those containing the nopaline synthase promoter. The significance of these results for expression of foreign genes in plants is discussed. In addition, we describe the construction of a convenient plant expression cassette vector (pMON316) which utilizes the CaMV 35S promoter.

Engineering herbicide tolerance in transgenic plants.

The herbicide glyphosate is a potent inhibitor of the enzyme 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in higher plants. A complementary DNA (cDNA) clone encoding EPSP synthase was isolated from a complementary DNA library of a glyphosate-tolerant Petunia hybrida cell line (MP4-G) that overproduces the enzyme. This cell line was shown to overproduce EPSP synthase messenger RNA as a result of a 20-fold amplification of the gene. A chimeric EPSP synthase gene was constructed with the use of the cauliflower mosaic virus 35S promoter to attain high level expression of EPSP synthase and introduced into petunia cells. Transformed petunia cells as well as regenerated transgenic plants were tolerant to glyphosate.

Inheritance of functional foreign genes in plants.

Morphologically normal plants were regenerated from Nicotiana plumbaginifolia cells transformed with an Agrobacterium tumefaciens strain containing a tumor-inducing plasmid with a chimeric gene for kanamycin resistance. The presence of the chimeric gene in regenerated plants was demonstrated by Southern hybridization analysis, and its expression in plant tissues was confirmed by the ability of leaf segments to form callus on media containing kanamycin at concentrations that were normally inhibitory. Progeny derived from several transformed plants inherited the foreign gene in a Mendelian manner.

In vitro transformation of petunia cells by an improved method of co-cultivation with A. tumefaciens strains.

A method (termed co-cultivation) for transforming plant cells in vitro with A. tumefaciens strains, which was originally developed by Marton et al. (1978) Nature 277: 129-131, has been modified by the incorporation of a novel feeder plate culture system and been extended to use with petunia protoplasts. Using efficient cell plating and selection conditions for phytohormone-independent growth, large numbers of independent transformed calli can be obtained efficiently (∼10(-1)) and in less than 3 weeks following protoplast isolation. Southern hybridization analysis has confirmed that the majority of the resulting in vitro transformants contain a single copy of full length T-DNA.The high efficiency of this procedure allows simple screening to identify plant cells transformed by Ti plasmids attenuated by deletion of internal T-DNA regions. Results are presented that demonstrate the co-cultivation method can be used in conjunction with short term assays for monitoring plant gene expression.

Introduction of cloned human papillomavirus genomes into mouse cells and expression at the RNA level.

The entire DNA genomes of five different human papillomaviruses (HPVs) were cloned into the BamHI site of pBR322 (HPV-1a, HPV-3, HPV-4, and HPV-9) or the EcoRI site of pBR325 (HPV-2), using as starting materials virus preparations isolated from papillomas of individual patients. Under stringent hybridization conditions (Tm-28 degrees), the five cloned HPVs exhibited less than 10% homology with one another. To establish model cell systems that may be useful for the identification of HPV genes and HPV gene products, mouse thymidine kinase negative (tk-) cells were cotransformed to the tk+ phenotype with the herpesvirus thymidine kinase gene and each of the five HPV cloned DNAs (either as intact recombinants or excised HPV DNA without removal of pBR). In most tk+ cell clones, a complex pattern of multiple high molecular weight inserts of HPV DNA were present in high copy number. Most of the HPV DNA sequences in the cotransformed cells were not present as unit-length episomal viral DNA. Analyses of the integration pattern (DNA blot) and RNA expression (RNA blot) of several HPV-1a and HPV-3 transformed cell lines suggest that some copies of the viral genome are integrated in a similar manner in different cell lines leading to the expression of identical viral RNA-containing species. Two of the cell lines transformed by the intact HPV-1a/pBR322 recombinant synthesized substantial amounts of four discrete viral polyadenylated cytoplasmic RNA species of 1.9, 3.2, 3.8, and 4.5 kb. Two cell lines transformed by the intact HPV-3/pBR322 recombinant synthesized 4-5 polyadenylated cytoplasmic viral RNA species ranging from 0.8 to 4.6 kb. The analysis shows that each viral RNA species appears to be a hybrid RNA molecule containing both HPV and pBR322 sequences. Based on these findings and the molecular organization of the HPV-1a genome (O. Danos, M. Katinka, and M. Yaniv (1982). EMBO J. 1, 231-237), it is possible that transcription of each of the HPV-1a RNA species is initiated using the HPV early promoter and terminated in pBR322.

Expression of bacterial genes in plant cells.

Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.

Isolation of a human papillomavirus from a patient with epidermodysplasia verruciformis: presence of related viral DNA genomes in human urogenital tumors.

The DNA genome of a human papillomavirus (HPV), tentatively designated HPV-EV, was molecularly cloned from hand to leg lesions of a patient with epidermodysplasia verruciformis, a chronic skin disease associated with a 30% risk of developing cancer. Using stringent hybridization conditions, we observed less than 5% homology between HPV-EV and the cloned genomes of HPV-1, HPV-4, HPV-5, and HPV-5a. HPV-EV DNA showed approximately 6% homology with HPV-2 and 36% homology with HPV-3. These data suggest that HPV-EV is partially related to HPV-3. Using 32P-labeled cloned HPV-EV as probe in Southern blot hybridization experiments, we detected HPV-EV-related DNA in the carcinoma in situ (Bowenoid lesion) of the vulva of the patient from which HPV-EV was isolated. HPV-EV-related DNA was detected in 2 of 10 vulva carcinomas and in 2 of 31 cervical carcinomas. Related DNA sequences were found in papillomas from each of two patients with condyloma acuminata (anogenital warts), which is of interest considering that condylomas have been reported to convert occasionally to carcinomas. The positive vulva DNAs were also probed with other cloned HPV DNAs: HPV-1, HPV-4, and HPV-5a-related sequences were not detected; HPV-3 and HPV-2 DNA probes detected strong and weak DNA bands, respectively, of the same size as found with HPV-EV. The HPV DNA sequences were present in the positive tumors mainly as free viral DNA molecules; no evidence for integration into cellular DNA was found. The emerging biological picture with papillomaviruses is that cells transformed by these viruses are maintained in a transformed state by free episomal genomes. Thus, our findings are consistent with the idea, but by no means establish, that HPVs play a role in human cancer by a similar mechanism.

Determination of coumarin and umbelliferone mixtures in whole blood by spectrophotofluorometry.

A spectrophotofluorometric method is described for the quantitative analysis of coumarin, umbelliferone, and mixtures thereof in whole blood. The two drugs were selectively isolated from blood by solvent extraction. Analysis of the isolated coumarin was based on the measurement of the fluorophore at activation and emission wavelengths of 361 and 491 nm, respectively. The fluorophore was obtained by irradiating an alkaline methanolic solution of the drug with UV light. A linear relationship between fluorescence and concentration existed over the concentration range of 0.02-0.2 mug of coumarin/ml. A mean recovery value of 94.8% was obtained from whole blood. The isolated umbelliferone was determined according to established methods at activation and emission wavelengths of 370 and 450 nm, respectively, and the limit of detection was 10 times more sensitive than previously reported. A linearity response was obtained between 1 and 10 ng of umbelliferone/ml. Good recovery data for mixtures of coumarin and umbelliferone in whole blood were obtained.

Pharmacokinetics of coumarin upon i.v. administration in man.

The disposition pharmacokinetics of coumarin has been studied after i.v. administration in man, and might be described best by an open two-compartment model. Three dose sizes have been studied, 0.125, 0.2 and 0.25 mg/kg. The mean biological half-life was found to be 1.81, 1.45 and 1.49 h, respectively, for the three dose sizes. Coumarin is extensively distributed. The volume of the central compartment apparently comprises the extracellular and intracellular fluid. The study indicates dose-dependent (non-linear) pharmacokinetics with respect to distribution.