Cucumber mosaic virus-induced RNA replicase: Solubilization and partial purification of the particulate enzyme

Gill, Dalip S.; Kumarasamy, Ramasamy; Symons, Robert H.

doi:10.1016/0042-6822(81)90131-8

Cited by 26 publications

(8 citation statements)

References 18 publications

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“…1B). After further steps, the CMV replicase, first described as consisting of two polypeptide chains of Mr 100,000 and 110,000 by Symons and coworkers (21)(22)(23), and obtained nearly pure by Takanami and Fraenkel-Conrat (18), has now been obtained free from contaminating proteins but apparently associated with a third, short peptide chain (Fig. 2) 2.…”

Section: Resultsmentioning

confidence: 99%

RNA-directed RNA polymerases from healthy and from virus-infected cucumber.

Khan¹,

Hiriyanna²,

Chávez³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Much work has been done on the isolation, purification, and characterization of the RNA-directed RNA polymerase (EC 2.7.7.48) of cucumber mosaic virus (CMV)-infected cucumbers. Uninfected plants were reported to have no such enzyme, but we recently detected low levels of the activity in cucumber. Since tobacco and cowpea contain such an enzyme that is variably increased in amount by various virus (as well as viroid) infections, we assumed that this would also be the case upon CMV infection of cucumber. However, further purification and characterization of the RNA-directed RNA polymerases from healthy and from infected cucumber suggests that these are different enzymes. The presumed CMV replicase was obtained pure and consists of a major polypeptide of Mr 100,000 and minor components of Mr 110,000 and about 10,000. The Km is 5 pM ([3H]GTP) when tobacco mosaic virus RNA is used as template.The role of plant RNA-directed RNA polymerases (EC 2.7.7.48) in the replication ofRNA viruses remains uncertain. The RNA of cowpea mosaic virus, a virus resembling picornaviruses (1), appears to become replicated without participation of the cowpea RNA-directed RNA polymerase, although the amount of the enzyme is greatly increased by that virus infection (2-4). Several studies of viral replication complexes-particulate aggregates containing several proteins, membranous structures, and RNA-have given conflicting results in terms of containing the host enzyme and/or one or several viral gene products (5)(6)(7). In barley infected with brome mosaic virus (BMV), evidence for a virus-specific component in viral RNA replication was reported (5, 8). However, in tobacco no evidence was found for any role of a protein encoded by the infecting tobacco mosaic virus (TMV) in viral RNA replication (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).The tobacco enzyme, as well as those of other plants, have molecular weights of 130,000-140,000. In contrast, the presumed viral RNA replicases have approximate molecular weights of 180,000 plus possibly 130,000 (TMV and turnip yellow mosaic virus) or 110,000 and possibly 70,000 [BMV and cucumber mosaic virus (CMV)]. Yet the molecular weight of the largest gene product of tobacco necrosis virus is only about 65,000. The need for both host and viral components for viral RNA replication is quite likely. This has been observed to be the case with the RNA bacteriophages and picornaviruses. Our present concern has been with cucumber, a plant reported to lack RNA-directed RNA polymerase, and the replication of CMV in this plant. We have found small amounts ofRNA-directed RNA polymerase in cucumber, partially characterized it, and compared it with the enzyme, produced in greater amount and obtained in pure form, from CMV-infected cucumber. It appears that the presumed CMV replicase has a molecular weight of about 100,000 and 110,000, whereas that of the cucumber enzyme is about 140,000. The earlier report that the Mr 100,000 enzyme differs in peptide pattern from the viral gene product corresponding ...

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Section: Resultsmentioning

confidence: 99%

RNA-directed RNA polymerases from healthy and from virus-infected cucumber.

Khan¹,

Hiriyanna²,

Chávez³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The buffer system of Laemmli [3 I] was modified as described previously [39]. Fluorographs were prepared by exposing Kodak XRP-1 X-ray film to the dried gel at --70°C [11].…”

Section: Sodium Dodecyl-sulphate-polyacrylamide Gel Electrophoresis (mentioning

confidence: 99%

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

1982

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Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA3). GA3 greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably α-amylase, and decreasing others. α-Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA3. There was the expected increase in α-amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA3 caused many changes, particularly in the level of mRNA for α-amylase. In vitro synthesized α-amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for α-amylase began to increase 2-4 h after GA3 was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of α-amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA3 on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA3 reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for α-amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA3 and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of α-amylase, possibly also by changing the efficiency of translation of its mRNA.

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“…Protein samples were electrophoresed on SDS-polyacrylamide gradient minigels (15% to 30% [w/v] acrylamide) with equal numbers of TCA-insoluble counts loaded in each track. The gels were fluorographed after soaking in 20% (w/v) naphthalene and 0.5% (w/v) 2,5-diphenyloxazole in dimethylsulfoxide (Gill et al, 1981). Total protein from mature seeds was electrophoresed on the same type of SDS-polyacrylamide gradient minigel and protein was visualized by silver staining using a kit (BioRad, Hercules, CA).…”

Section: So 4 Labeling and Sds-pagementioning

confidence: 99%

Limits to Sulfur Accumulation in Transgenic Lupin Seeds Expressing a Foreign Sulfur-Rich Protein

Tabe

Droux

2002

Plant Physiology

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The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met-and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds.

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Cucumber mosaic virus-induced RNA replicase: Solubilization and partial purification of the particulate enzyme

Cited by 26 publications

References 18 publications

RNA-directed RNA polymerases from healthy and from virus-infected cucumber.

RNA-directed RNA polymerases from healthy and from virus-infected cucumber.

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Limits to Sulfur Accumulation in Transgenic Lupin Seeds Expressing a Foreign Sulfur-Rich Protein

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