Identification of a novel plant virus promoter using a potyvirus infectious clone.

A putative promoter from the strawberry vein banding caulimovirus (SVBV) genome was identified by its ability to drive infection with full-length cDNA of the zucchini yellow mosaic RNA potyvirus (ZYMV). A high rate of infection was obtained with the cDNA under control of the SVBV promoter using particle bombardment technology. The SVBV promoter shows 60% homology to the cauliflower mosaic virus 35S promoter in the domain spanning the conserved motifs of CCACT (at -83) and the TATA box (at -31), to the transcription start. The 3'-end one-third of the putative promoter (328 bp) was sufficient to invoke full infectivity with the ZYMV clone, and drove transient reporter gene expression in Solanaceae and Cucurbitaceae transformed with a binary plant transformation vector. Stable expression of a reporter gene (GUS) under control of the truncated SVBV promoter was shown in transformed tobacco shoots in roots, leaves and stems.

Transgenic resistance to cucumber mosaic virus in tomato: blocking of long-distance movement of the virus in lines harboring a defective viral replicase gene.

ABSTRACT Tomato breeding lines were transformed with a defective replicase gene from RNA 2 of cucumber mosaic virus (CMV). A total of 63 transformants from five tomato genotypes were evaluated for resistance to CMV strains. The responses of R1 transgenic offspring fit into three categories: fully susceptible lines (44%), fully resistant lines (8%), and an intermediate-type mixture of susceptible and resistant seedlings in variable proportions (48%). Further characterization of the response of two highly resistant lines was performed by mechanical inoculation, aphid transmission, or grafting experiments. No virus was detected in noninoculated leaves from these lines, although a low level of virus accumulated initially in the inoculated leaf. The homozygous R2 plants and further generations that were evaluated (up to R5) showed resistance to the Fny-CMV strain, two Israeli isolates tentatively classified as subgroup IA, and K-CMV (a representative of subgroup IB). These lines were partially resistant to LS-CMV (a representative of subgroup II) when a high-virus-titer inoculum was used. Expression of the viral transgene was verified in these lines; however, the expected translation product was not detectable. In grafting experiments, we demonstrated that CMV virions were blocked in their ability to move from infected rootstocks of nontransformed tomato or tobacco into the transgenic scions. Interestingly, virions could not move through a transgenic intersection into the upper scion. These results provide an additional indication that replicase-mediated resistance affects long-distance movement.

Increases in Cytosolic Ca2+ in Parsley Mesophyll Cells Correlate with Leaf Senescence.

The ability to maintain the cytoplasmic Ca2+ concentration ([Ca2+]cyt) at homeostatic levels has been examined during leaf senescence in detached parsley (Petroselinum crispum) leaves. Fluorescence ratiometric imaging of mesophyll cells isolated from parsley leaves at various senescence stages and loaded with the Ca2+ indicator fura-2 has revealed a distinct elevation of [Ca2+]cyt, which was positively correlated with the progress of leaf senescence. This initial increase of [Ca2+]cyt, which was first observed in cells isolated from 3-d-senescent leaves, occurred 1 d before or in parallel with changes in two established senescence parameters, chlorophyll loss and lipid peroxidation. However, the [Ca2+]cyt elevation followed by 2 d the initial increase in the senescence-associated proteolysis. Whereas the [Ca2+]cyt of nonsenescent cells remained at the basal level, the elevated [Ca2+]cyt of the senescent cells was a long-lasting effect. Experimental retardation of senescence processes, achieved by pretreatment of detached leaves with the cytokinin benzyladenine, resulted in maintenance of homeostatic levels of [Ca2+]cyt in cells isolated from 3-d-senescent leaves. These observations demonstrate for the first time to our knowledge a correlation between elevated [Ca2+]cyt and the process of senescence in parsley leaves. Such senescence-associated elevation of [Ca2+]cyt, which presumably results from a loss of the cell's capability to extrude Ca2+, may serve as a signal inducing subsequent deteriorative processes.

Chromosome elimination in asymmetric somatic hybrids: effect of gamma dose and time in culture.

Mesophyll protoplasts of a kanamycin-resistant line of Nicotiana plumbaginifolia were gamma-irradiated and fused with mesophyll protoplasts of N. tabacum plants bearing the sulfur mutation. Hybrid calli were recovered by selection on media containing kanamycin. In one group of experiments, the degree of elimination of donor (N. plumbaginifolia) genetic material in the hybrid calli was assessed by dot-blot hybridization using a N. plumbaginifolia-specific repetitive-DNA sequence as a probe. The elimination of donor DNA was found to increase with increasing gamma dose for all doses tested (5-50 krad). Elimination of donor DNA was also found to continue in the calli for the first 12 months in culture. The degree of chromosome elimination was quite variable; for a 50-krad dose, some hybrids were recovered that retained less than 15% of the donor genome, whereas others retained nearly 50%. In a second set of experiments, the degree of donorchromosome elimination was assessed from the fraction of hybrid calli that exhibited complementation of the Su phenotype due to retention of a wild-type Su allele of the donor. When N. plumbaginifolia protoplasts were inactivated by treatment with iodoacetate, rather than gamma irradiation, all the hybrid calli were green. However, when the donor protoplasts were inactivated by irradiation, the fraction of hybrid calli that were able to complement the Su mutation decreased with increasing gamma dose; for a 50-krad dose only 40% of the hybrid calli were green. From these data, the degree of radiation-induced donor-chromosome elimination was calculated and was found to agree closely with that measured by dot-blot hybridization. We conclude that radiation-induced elimination of donor chromosomes increases with gamma dose and time in culture in N. tabacum (+)N. plumbaginifolia hybrids, but that donor-chromosome elimination is an inherently variable process.

Molecular tagging of the tobacco chromosome carrying the TMV-resistance gene (N gene) by Agrobacterium-mediated transformation.

The hypersensitive response of tobacco to inoculation with tobacco mosaic virus (TMV) is controlled by a single dominant gene, the N gene. As a first step in localizing and transferring the N gene, we have prepared a line of tobacco plants in which the kanamycin-resistance (Km(r)) gene is closely linked to the N gene. Nicotiana tabacum plants heterozygous for the N gene were transformed to Km(r) by Agrobacterium carrying pMON200. Eighty-nine independent transformed clones were regenerated and were backcrossed with nontransformed, TMV-sensitive plants. Progeny from these crosses were screened first for Km(r); then the Km(r) progeny were inoculated with TMV and scored for the hypersensitive response. Of the initial 89 clones, 68 appeared to have integrated a single functional Km(r) gene. Initial tests for TMV resistance indicated possible linkage between Km(r) and the N gene in 11 plants. With further testing, linkage has been established for two of these plant lines. In one of these lines, the two genes were 30-40 map units apart, and evidence of somatic instability in the linkage was obtained. However, in the second line, linkage between Km(r) and the N gene was tight, and recombination between the genes in this case was only 5%. Southern hybridization revealed that this plant contained only a single copy of the Km(r) gene. Linkage between Km(r) and the N gene in this plant line has been verified in each of two additional backcross generations.

Limited DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum tuberosum.

This paper describes the analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids. The hybrids were obtained by fusion of protoplasts of a cytoplasmic albino tomato genotype with leaf mesophyll protoplasts of a potato genotype carrying the ß-glucuronidase (GUS) gene of Escherichia coli. The potato protoplasts were either isolated from unirradiated plants or from plants irradiated with 50 or 500 Gy of γ-rays. Green calli were selected as putative fusion products. The hybridity of these calli was confirmed by isoenzyme analysis. All of the green calli tested contained a potato-specific chloroplast DNA restriction fragment, and most of the calli analysed were positive for ß-glucuronidase activity. In 72 of the hybrid calli we determined the percentage of potato nuclear DNA using species-specific probes. All of the tested green calli contained a considerable amount of potato genomic DNA, irrespective of the dose of irradiation of the potato parent. The limited degree of potato DNA elimination and the absence of true cybrids are discussed.

Atrial fibrillation and atrial enlargement in patients with mitral stenosis.

The present study was designed to assess the relative contribution of atrial fibrillation and left atrial pressure to changes in the size of the left and right atria in patients with mitral stenosis. The study included 155 subjects, 102 of whom underwent prospective echocardiography and Doppler cardiography, and 69 of whom underwent cardiac catheterization. The size of the atria was determined by two-dimensional echocardiography. There were no significant hemodynamic differences between patients with mitral stenosis who were in either sinus rhythm or atrial fibrillation. The left atrium was larger (p less than 0.001) in patients with mitral stenosis and atrial fibrillation (37.6 +/- 10.8 cm2) than in patients in sinus rhythm (27.8 +/- 7.7 cm2) or normal subjects (15 +/- 3.3 cm2). The size of the right atrium was larger (p less than 0.001) in patients with mitral stenosis and atrial fibrillation (21.7 +/- 5.2 cm2) than in patients in sinus rhythm (13.4 +/- 3.9 cm2) or normal subjects (13.8 +/- 3.7 cm2). Multiple regression analysis showed that the severity of mitral stenosis accounted for 38%, age for 7%, and atrial fibrillation for 11% of the change in the size of the left atrium. Atrial fibrillation accounted for 24%, age for 11, and mitral valve area for 3% of the change in the size of the right atrium. The analysis suggests that the onset of left atrial dilatation in mitral stenosis is the result of an early increase in left atrial pressure. Atrial fibrillation, which develops irrespective of the severity of the mitral stenosis, contributes to a further enlargement of the left and right atria.

Pericardial tamponade in sarcoidosis.

A woman with a three month history of progressive right heart failure was found to have sarcoid pericarditis complicated by pericardial tamponade. The pericardial fluid was serosanguineous, and numerous nodules were noted on the parietal and visceral pericardium. Non-caseating granulomas were found in biopsy specimens of the pericardium, lung and skin. Right-sided heart failure in sarcoidosis is usually attributed to cor pulmonale or primary myocardial sarcoid. Pericardial tamponade should be considered in patients who present with sarcoidosis complicated by right heart failure.

Time variation of mitral regurgitant flow in patients with dilated cardiomyopathy.

Angiographic results in patients with mitral regurgitation suggest that up to 50% of the regurgitant volume occurs during the preejection period. This contrasts markedly with the electromagnetic measurements of mitral regurgitant flow in anesthetized dogs, which suggest that only 5% of mitral regurgitant flow occurs during the preejection period. Therefore, we used two-dimensional and Doppler echocardiography to quantify mitral regurgitation during aortic ejection and in the preejection and postejection periods in eight patients with severe heart failure. Mitral regurgitant volume (RV) was calculated as the difference between total stroke volume (by two-dimensional echocardiography) and forward aortic flow (by pulsed Doppler). Regurgitant velocity (V) and time (RT) were measured by continuous-wave Doppler, and the mean regurgitant area (RAm) was calculated from the RT and mean regurgitant velocity (Vm): RAm = (RV/RT)/Vm. As a first approximation, the RA was assumed to be constant during systole, and the regurgitant volume during aortic ejection and during the preejection and postejection periods was calculated from: RVi = (Vmi) (RTi) (TAm), where Ti represents the duration of the appropriate period. Percentages of total regurgitant volume occurring during the preejection, ejection, and postejection periods were 13 +/- 4%, 79 +/- 5%, and 8 +/- 5%, respectively. Thus, in contrast to previously reported angiographic studies, mitral regurgitation occurs predominantly during the aortic ejection period. These results were not substantially changed by assuming a 20% reduction in effective regurgitant orifice area between the preejection and ejection periods and are consistent with data from chronically instrumented dogs with mitral regurgitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of milrinone on left ventricular performance and myocardial contractility in patients with severe heart failure.

The potent vasodilating properties of the bipyridine derivatives confounds the assessment of changes in contractility as measured by peak positive left ventricular dP/dt and explains why the positive inotropic action of these agents has not been consistently demonstrated in patients with ventricular dysfunction. Therefore we studied the individual dose-response effects of intravenous milrinone on myocardial contractility as measured by peak positive left ventricular dP/dt in the context of concurrent changes in ventricular filling pressure. Incremental doses of milrinone caused a dose-related increase in peak positive left ventricular dP/dt in six of 11 patients. Peak positive left ventricular dP/dt was unchanged in four patients and reduced in one patient, all of whom experienced a substantial fall in left ventricular filling pressure. Thus milrinone has a definite inotropic effect in addition to its potent vasodilating properties.

An in vitro Screening for Tomato Genotypes Exhibiting Efficient Shoot Regeneration.

Over 100 genotypes of tomato (Lycopersicon esculentum, wild relatives and suspected interspecific crosses) were assayed for shoot regeneration from in vitro cultured hypocotyl segments. Expiants incubated for 8 weeks in Murashige and Skoog medium supplemented with 2% glucose, 1 mg zeatin and 0.02 mg · l(-1) IAA, exhibited the best overall shoot regeneration. A wide variability in this response was observed, and approximately one-fourth (23/89) of the L. esculentum tested genotypes exhibited relatively high differentiation capabilities (>5 shoots per expiant).

Induction of high mitotic index in Petunia suspension cultures by sequential treatment with aphidicolin and colchicine.

Significantly higher than normal mitotic index (MI) values were induced in Petunia cell suspensions following treatments with colchicine, aphidicolin, drastic medium replacement, or a sequential application of aphidicolin and colchicine. This last treatment yielded the highest MI values: cells incubated with 30 µg/ml aphidicolin for 18 h, then cultured in drug-free medium for 8 h and finally exposed to 0.1% colchicine for 8 additional hours exhibited MI of 62.8% and 65.7% respectively, for the two cell lines in study.

Shoot regeneration in root cultures of Solanaceae.

Root cultures from several species of the Solanaceae were initiated and subcultured on Murashige and Skoog medium without growth regulators. Direct shoot regeneration was observed in four different species. The effect of several concentrations of auxin (IAA) and cytokinins (BAP, zeatin) on the number of shoots generated by two highly responsive species (Nicotiana exigua, N. debneyi) is described.

The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants.

The 2 M LiCl-soluble RNA fraction extracted from tobacco mosaic virus (TMV)-infected tobacco plants contains, in addition to the viral replicative form of 4 x 10(6) MW, three smaller double-stranded (ds) RNA species with apparent molecular weights (estimated by polyacrylamide gel electrophoresis, using ds RNAs as markers) of 2.25, 1.1, and 0.23 x 10(6). The synthesis of all four ds RNAs is insensitive to actinomycin D. They are completely RNase insensitive at high salt concentrations and are found both in directly inoculated and in apical tissues. In tissues incubated in the presence of 3H-uridine and actinomycin D, the three small ds RNAs accounted for 6 to 11.5% of the total radioactivity incorporated into viral ds RNA. On a molar basis, however, in apical leaves the smallest ds RNA was synthesized to almost the same level as the replicative form. By molecular hybridization, the three small ds RNAs have been shown to be of viral origin, and each contains sequences represented in the 5' end of complementary (negative strand) TMV RNA. Based on molecular weight data, none of the ds RNAs can be considered to be a ds form of the subgenomic TMV coat protein mRNA (the LMC), suggesting that it is not replicated independently. None of the small ds RNAs was found to be an endogenous product of the bound TMV RNA replicase.

Plant cell suspension cultures sustain long-term replication of potato spindle tuber viroid.

Cell suspension cultures were established from tomato plants (Lycopersicum esculentum cv. "Rutgers") infected with either a severe (TPS cell line) or a mild (TPM cell line) strain of potato spindle tuber viroid (PSTV) and from uninfected plants (TH cell line). Based on measurements of packed cell volume, the TPS line exhibited a slower growth rate than the TH line, and reached lower levels of total cell volume during the stationary phase. The TPM line was intermediate between the other two. All the lines were highly polyploid (over 72 chromosomes). PSTV was detected consistently by electrophoretic analysis in both the TPS and TPM lines after more than 11/2 years of subculturing. Newly synthesized PSTV in actively dividing cells was detectable after 1 hr of [3H]uridine labeling (ca. 0.1% of the radioactivity incorporated into total soluble RNA) and its proportion increased in subsequent samplings up to a steady state level of 0.24-0.28 or 0.31-0.39% for TPM and TPS, respectively. Analysis on two-dimensional gels of proteins synthesized in the TH and TPS cell lines following incorporation of radiolabeled amino acids detected neither quantitative nor qualitative changes resulting from maintenance of viroid in the cell line.

Degradation of [3H]thymidine by a pentosyltransferase (EC 2.4.2.4) in the plasma of man and different animals.

[3H]Thymidine is degraded by an enzyme (thymidine phosphorylase; EC 2.4.2.4) which we have identified in the plasma of man and some animals. The presence of this enzyme in plasma or sera used to supplement culture media may, under certain experimental conditions, limit the validity of measuring the uptake of radiolabeled thymidine as a means of defining DNA synthesis.