花椰菜花叶病毒(CaMV)的基因表达调控

花椰菜花叶病毒（CaMV）是一种具有重要经济价值和生物学意义的植物双链DNA病毒,它有7个开放阅读框（ORF）,其中6个呆各自编码一种蛋白产物。35S启动子区域含有3个转录因子专一的结合位点;RNA多腺苷化位点具有AAUAAA特征序列,它和其上游序列对35SRNA的加工和翻译有影响作用;下游ORFI在转录激活因子存在时可被翻译。由此表明,CaMV的表达调控表现在不同调控机制工作的基础之上。     

高灵敏度电化学发光PCR方法检测花椰菜花叶病毒35 S启动子

大多数转基因植物中使用花椰菜花叶病毒(cauliflower mosaic virus,CaMV)35 S作为启动子,因此可通过检测该启动子来判断植物样品中是否含有转基因成分。实验将高灵敏度电化学发光PCR方法用于检测转基因烟草中的CaMV35 S启动子,将该启动子的PCR产物与生物素标记的探针杂交,可以起到特异性筛选产物的作用;与发光标记物——三联吡啶钌标记的探针杂交,从而实现电化学发光检测。两种探针同时与待测样品的PCR产物进行杂交,进一步对样品进行特异性筛选,从而提高了检测的准确性,避免了假阳性结果的产生。实验结果表明:该法可以准确的区分待测样品中是否含有35 S启动子,从而区别转基因烟草和非转基因烟草。电化学发光PCR方法灵敏度高,可靠性强,操作简便,结果准确,有望成为一种高效的转基因植物检测方法。     

Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.     

A new model for the tertiary structure of 5S ribonucleic acid in plants

Chemical, enzymatic and physicochemical methods of a structural analysis of 5S rRNAs in lupine, wheat germ, and other plants led us to propose a new three-dimensional model of these molecules The main features of the model are tertiary interactions between the β- and γ-domains of the molecule, specifically nucleotides (34)CCCA(37) in loop C and nucleotides (85)GGGU(88) in loop D. In addition we propose tertiary base-pairing in A100-U53 between loops B and E. We have confirmed this model by NMR spectroscopy and by chemical modification with diethylpyrocarbonate. Our results are consistent with the proposed model and are also applicable to all eukaryotic 5S rRNAs. Our model is clearly differentiated from others by intramolecular tertiary hydrogen bonds between the two domains.     

Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA

We have previously shown that stable base-pairing at a translational initiation site in Escherichia coli can inhibit translation by competing with the binding of ribosomes. When the base-pairing is not too strong, this competition is won by the ribosomes, resulting in efficient translation from a structured ribosome binding site (RBS). We now re-examine these results in the light of RNA folding kinetics and find that the window during which a folded RBS is open is generally much too short to recruit a 30S ribosomal subunit from the cytoplasm. We argue that to achieve efficient expression, a 30S subunit must already be in contact with the mRNA while this is still folded, to shift into place as soon as the structure opens. Single-stranded regions flanking the structure may constitute a standby site, to which the 30S subunit can attach non-specifically. We propose a steady-state kinetic model for the early steps of translational initiation and use this to examine various quantitative aspects of standby binding. The kinetic model provides an explanation of why the earlier equilibrium competition model predicted implausibly high 30S-mRNA affinities. Because all RNA is structured to some degree, standby binding is probably a general feature of translational initiation.     

Agrobacterium mediated transformation of Petunia hybrida with yeast Δ-9 fatty acid desaturase

Petunia var. Ultra Blue was transformed with a delta-9 fatty acid desaturase gene from Saccharomyces Cerevisiae using Agrobacterium tumefaciens. Transformed plants were verified at the genomic level. Gas chromatographic analysis revealed an increase in the level of monounsaturated 16:1 and 18:1 fatty acids in transformed leaves. These fatty acids were found at very low levels (less than 2%) in control plants. Despite the changes in lipid composition, phenotypic difference were not apparent.Abbreviations ACP = acyl carrier protein - LB = luria bertania medium - CaMV = cauliflower mosaic virus     

A microassay for detection of DNA and RNA in small numbers of plant cells

Summary A microtechnique for the detection of DNA or RNA in small numbers of plant cells (1–50) has been developed using cauliflower mosaic virus (CaMV) infection of turnip as a model system. Both DNA and RNA extracted from 10 mesophyll protoplasts from CaMV-infected plants can be detected by hybridization using a radioactive probe made from cloned CaMV DNA (pCaMV10). No hybridization above background was detected in extracts of protoplasts from uninfected plants. At least 0.15 pg (11 000 molecules) of purified pCaMV10 DNA can be detected. This method is superior to existing macro techniques for nucleic acid detection as smaller amounts of tissue are required and the detection is approximately 100-fold more sensitive. re]19850326 rv]19850530 ac]19850611     

A bipartite system for the constitutive and inducible expression of high levels of foreign proteins in plants

We have developed combined transgene/virus vector systems for the expression of heterologous proteins in plants. The systems are based on the bipartite RNA plant virus, cowpea mosaic virus (CPMV), and involve the amplification of integrated copies of either full-length or deleted versions of RNA-2 carrying a foreign gene. In the case of plants transgenic for full-length versions of RNA-2 carrying the green fluorescent protein (GFP), amplification can be achieved by supplying RNA-1 either exogenously or by crossing. This allows either inducible or constitutive expression of the foreign gene and results in an infection that can be passaged to further plants. Replication of deleted versions of RNA-2 harbouring GFP requires the presence of both RNA-1 and a suppressor of gene silencing, a function which we show can be supplied by HcPro from potato virus Y. Replication of the deleted versions of RNA-2 can be achieved by supplying the suppressor and RNA-1 either exogenously or by crossing, showing that this system can also be used in an inducible and constitutive format. The use of deleted forms of RNA-2 has the advantage that no infectious virus is produced, providing an effective method of biocontainment. The CPMV-based systems have advantages over existing plant expression systems in terms of the expression levels obtainable and the simplicity and flexibility of use, and should be of great practical benefit in the development of plants as bioreactors.     

A Distinct Subpopulation of Cauliflower mosaic virus (CaMV) in South Iran

Cauliflower mosaic virus (CaMV) with a high incidence and widespread distribution on Brassica crops in Iran reduces the yield and quality of these crops. The complete sequences of three open reading frames (ORFs) 2, 4 and 6 coding for aphid transmission (AT), coat protein (CP) and inclusion body protein/translation transactivator (TAV) genes, respectively, were determined for two Iranian CaMV isolates from Kerman (south Iran). They induced latent or mild mottle (L/MMo) infection in Brassica oleracea var. capitata so are considered as the (L/MMo) biotype. Clear recombination breakpoints were detected between ORF2 and ORF6 in two Kerman isolates using concatenate fragments. Phylogenetic analysis revealed three Iranian CaMV subpopulations in which the two Kerman isolates in the new subgroup C were added to the two previously reported Iranian subpopulations A (central and west Iran) and B (north‐east Iran). Also three regions of pairwise identity were detected which representing: 97.1–100, 93.8–97.1 and 90.6–93.8% for subgroups A, C and B, respectively. Our analysis showed the high variability of Iranian CaMV population and provided valuable new information for understanding the diversity and evolution of caulimoviruses. Furthermore, star phylogeny was found in the subgroup C with overall lack of nt diversity and high haplotype diversity as evidence of a recent population expansion after a genetic bottleneck although this may have been modified subsequently by clinal genetic drift. The appearance of new genetic types demonstrates a high potential of risks and should be considered in the planning of efficient control programmes.     

Location of Template on the Human Ribosome as Revealed from Data on Cross-Linking with Reactive mRNA Analogs

In this review we summarize data on the location of template on the human ribosome that we obtained from cross-linking (affinity labeling) experiments using reactive mRNA analogs. Types of mRNA analogs, model complexes of these analogs with 80S ribosomes, and methods for analysis of the ribosomal components (proteins and rRNA nucleotides) cross-linked with the mRNA analogs are reviewed. From analysis of the cross-linking data, we suggest a scheme for the arrangement of mRNA on the human ribosome and compare the organization of the mRNA binding center on human and Escherichia coli ribosomes.     

A Novel Satellite RNA of Cucumber Mosaic Virus Induces Unique Line-pattern Mosaic Symptoms in Tobacco

The CMV(YW) isolate of cucumber mosaic virus (CMV) induced unique line‐pattern mosaic symptoms in systemically infected leaves of tobacco (Nicotiana tabacum cv. Ky57). By northern hybridization analysis using cDNA to CMV(Y) satellite RNA as a probe, it was confirmed that CMV(YW) contained a satellite RNA. which was designated sat‐YW RNA; this was 388 nucleotides in length and did not have either a conserved domain that induces necrosis in tomato or chlorosis in tobacco. CMV(YW) free of sat‐YW RNA. which was isolated by the single lesion isolation method using Chenopodium amaranticolor, did not induce the unique line‐pattern mosaic symptom. Furthermore, the sat‐YW RNA‐mediated line‐pattern mosaic symptom was also induced by in vitro transcribed infectious sat‐YW RNA in tobaccos infected with either CMV(YW) or CMV(Y) genomic RNA. These results clearly demonstrated that sat‐YW RNA induces the unique line‐pattern mosaic symptom on CMV‐infected tobaccos.     

Alterations of the conformation of the RNAs of alfalfa mosaic virus upon binding of a few coat protein molecules

Structural changes in the single-stranded genome RNAs (RNAs 1, 2 and 3) and the subgenomic coat protein messenger (RNA 4) of alfalfa mosaic virus upon addition of a few coat protein molecules of the virus were investigated by measuring the fluorescent intensity of bound ethidium bromide and by circular dichroism. No effect could be observed in the case of the genome RNAs. However, in RNA 4, which is of much less complexity than the genome RNAs, a reduction of the ethidium bromide binding by 30% was found, whereas the positive molar ellipticity at 265 nm was reduced by 9% upon binding of the coat protein. Both changes point to a reduction of the ordered structure of the RNA. Since the protein is known to bind first at the 3′-terminus of RNA 4 and probably also of the genome RNAs, the conformational changes observed could be those thought to be necessary for replicase recognition in this positive-stranded RNA virus which needs the coat protein for starting an infection cycle.     

Polyethyleneimine derivative for nucleic acid model

Water-soluble polyethyleneimine (PE) derivatives containing nucleic acid bases and hydrophilic amino acids such as homoserine (Hse) and serine were prepared by the activated ester method as nucleic acid models. From spectroscopic measurements, the polymers were found to interact with DNA accompanied by an induction of conformational change. Hypochromicity in UV spectra indicated that a stable polymer complex was formed between poly (A) with PEI-Hse-Ura by complementary hydrogen bonding with equimolar nucleic base units (adenine∶uracil=1∶1). The induced conformation of DNA by the interaction with the polymer containing uracil and homoserine (PEI-Hse-Ura) was concluded to be a super triple helical structure. The formation of the polymer complex, DNA:PEI-Hse-Ura, was found to be affected by the presence of metal ions such as Ca²⁺ and Cu²⁺.     

RNA viruses as vectors for the expression of heterologous proteins

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.     

A novel function for a ubiquitous plant enzyme pectin methylesterase: the enhancer of RNA silencing

Co-agroinjection of Nicotiana benthamiana leaves with the pectin methylesterase (proPME) gene and the TMV:GFP vector resulted in a stimulation of virus-induced RNA silencing (inhibition of GFP production, virus RNA degradation, stimulation of siRNAs production). Conversely, co-expression of TMV:GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced the GFP transgene-induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm and activation of siRNAs and miRNAs production. It was hypothesized that DCL1 relocated to the cytoplasm may use as substrates both miRNA precursor and viral RNA. The capacity for enhancing the RNA silencing is a novel function for the polyfunctional PME.     

The effect of Pymetrozine, a feeding inhibitor of Homoptera, in preventing transmission of cauliflower mosaic caulimovirus by the aphid species Myzus persicae (Sulzer)

Mature turnip plants, mechanically infected as seedlings with the semi-persistent, aphid transmitted caulimovirus, cauliflower mosaic (CaMV), were treated by spraying with either a solution of Pymetrozine plus adjuvant oil, adjuvant oil or water only. At the same time turnip seedlings were sprayed for each of the three treatments. Two h after spraying, Myzus persicae were caged onto an infected turnip plant for each of the three treatments. Twenty four h later, groups of 20 aphids were transferred from the infected plants, to seedlings from each of the three treatments. After 24 h, these were removed and seedlings were later recorded for infection. This acquisition/transmission assay was repeated at 3, 7, 14 and 21 days from treatment. Only aphids exposed to the Pymetrozine treated source plants were shown to move off the plant and failed to transmit CaMV effectively to treated or control seedlings during the 0 and 3 day assays. The majority soon died when transferred to test seedlings. Progressively, more aphids were found to survive and transmit CaMV during the 7 day and 14 day assays. By 21 days no significant effect could be recorded between treatments and controls. Aphids transferred from control treated source plants to Pymetrozine treated seedlings were able to transmit CaMV within all the assays, although higher mortality was recorded in the day 0 assessment when compared to those transferred to control treated seedlings. We conclude from this trial, that a single foliar treatment of 100 mg litre¹ Pymetrozine to CaMV infected turnip plants, effectively reduces the vectoring capability of M. persicae, that feed on these plants, for up to 7 days. However, Pymetrozine failed to stop virus transmission to treated seedlings from the ingress of viruliferous aphids. Pymetrozine was not shown to cause any phytotoxic responses to plants used in this trial.     

Two amino acids near the N‐terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity

Cucumber mosaic virus (CMV) 2b suppresses RNA silencing primarily through the binding of double‐stranded RNA (dsRNA) of varying sizes. However, the biologically active form of 2b remains elusive. Here, we demonstrate that the single and double alanine substitution mutants in the N‐terminal 15th leucine and 18th methionine of CMV 2b exhibit drastically attenuated virulence in wild‐type plants, but are efficiently rescued in mutant plants defective in RNA‐dependent RNA polymerase 6 (RDR6) and Dicer‐like 4 (DCL4). Moreover, the transgenic plants of 2b, but not 2blm (L15A/M18A), rescue the high infectivity of CMV‐Δ2b through the suppression of antiviral silencing. L15A, M18A or both weaken 2b suppressor activity on local and systemic transgene silencing. In contrast with the high affinity of 2b to short and long dsRNAs, 2blm is significantly compromised in 21‐bp duplex small interfering RNA (siRNA) binding ability, but maintains a strong affinity for long dsRNAs. In cross‐linking assays, 2b can form dimers, tetramers and oligomers after treatment with glutaraldehyde, whereas 2blm only forms dimers, rather than tetramers and oligomers, in vitro. Together, these findings suggest that L15 and M18 of CMV 2b are required for high affinity to ds‐siRNAs and oligomerization activity, which are essential for the suppression activity of 2b on antiviral silencing.     

A structural model for the assembly of the 30S subunit of the ribosome

The order in which proteins bind to 16S rRNA, the assembly map, was determined by Nomura and co-workers in the early 1970s. The assembly map shows the dependencies of binding of successive proteins but fails to address the relationship of these dependencies to the three-dimensional folding of the ribosome. Here, using molecular mechanics techniques, we rationalize the order of protein binding in terms of ribosomal folding. We determined the specific contacts between the ribosomal proteins and 16S rRNA from a crystal structure of the 30S subunit (1FJG). We then used these contacts as restraints in a rigid body Monte-Carlo simulation with reduced-representation models of the RNA and proteins. Proteins were added sequentially to the RNA in the order that they appear in the assembly map. Our results show that proteins nucleate the folding of the head, platform, and body domains, but they do not strongly restrict the orientations of the domains relative to one another. We also examined the contributions of individual proteins to the formation of binding sites for sequential proteins in the assembly process. Binding sites for the primary binding proteins are generally more ordered in the naked RNA than those for other proteins. Furthermore, we examined one pathway in the assembly map and found that the addition of early binding proteins helps to organize the RNA around the binding sites of proteins that bind later. It appears that the order of assembly depends on the degree of pre-organization of each protein's binding site at a given stage of assembly, and the impact that the binding of each protein has on the organization of the remaining unoccupied binding sites.