The Bipartite Geminivirus Coat Protein Aids BR1 Function in Viral Movement by Affecting the Accumulation of Viral Single-Stranded DNA

The movement of bipartite geminiviruses such as squash leaf curl virus (SqLCV) requires the cooperative interaction of two essential virus-encoded movement proteins, BR1 and BL1. While the viral coat protein AR1 is not essential for systemic infection, genetic studies demonstrate that its presence masks the defective phenotype of certain BR1 missense mutants, thus suggesting that coat protein does interact with the viral movement pathway. To further examine the mechanism of this interaction, we have constructed alanine-scanning mutants of AR1 and studied them for the ability to mask the infectivity defects of appropriate BR1 mutants, for the ability to target to the nucleus and to bind viral single-stranded DNA (ssDNA) and multimerize, and for effects on the accumulation of replicated viral ssDNA. We identified a specific region of AR1 required for masking of appropriate BR1 mutants and showed that this same region of AR1 was also important for ssDNA binding and the accumulation of viral replicated ssDNA. This region of AR1 also overlapped that involved in multimerization of the coat protein. We also found that the accumulation in protoplasts of single-stranded forms of a recombinant plasmid that included the SqLCV replication origin but was too large to be encapsidated was dependent on the presence of AR1 but did not appear to require encapsidation. These findings extend our model for SqLCV movement, demonstrating that coat protein affects viral movement through its ability to induce the accumulation of replicated viral ssDNA genomes. They further suggested that encapsidation was not required for the AR1-dependent accumulation of viral ssDNA.     

Protein Synthesis Directed by the RNA from a Plant Virus in a Normal Animal Cell

RNA from tobacco mosaic virus can be translated inside oocytes of the frog Xenopus laevis. The main product is a polypeptide with a molecular weight of 140,000. There is no evidence for coat protein synthesis, and it is unlikely that the polypeptide that is made contains either a whole or a partial coat protein sequence.The picture of translation of tobacco mosaic virus RNA obtained using oocytes is very much simpler than that found using cell-free protein-synthesizing systems, in which a great many polypeptides are made under the direction of tobacco mosaic virus RNA. The reasons for this difference are discussed, and the relative merits of in vivo and in vitro protein-synthesizing systems are compared.     

A Cytochemical Study of DNA, RNA, and Protein in the Developing Maize Anther: II. Observations

Changes in acetic-alcohol fixable DNA, RNA, and protein werefollowed in the tapetum, sporogenous tissue, and spores of thedeveloping maize anther using standard cytochemical methodsand microdensitometry. In the tapetum, early nuclear divisionsoccur without prior DNA synthesis, giving a population of IC nuclei. Subsequent synthesis produces the equivalent of 34,000C amounts per pollen sac, 20 times more than is present in thespores before pollen mitosis. The main tapetal RNA synthesisis during the meiotic prophase, with a further period of accumulationin the interval, tetrad to young spores. In the meiocytes, theprincipal accumulation is in the early prophase, with no synthesisduring the meiotic divisions or through the tetrad period. Proteinaccumulation occurs in the tapetum up to mid-meiotic prophase;after this there is a pause, followed by further synthesis frommeiotic metaphase I to the final dissolution of the tissue.In the meiocytes, protein is accumulated through the early prophase;there is no synthesis during the meiotic mitoses or in the tetradperiod, but active accumula-tion occurs in the developing spores. The implications of these observations are discussed in relationto the function of the tapetum.     

Noise Genetics: Inferring Protein Function by Correlating Phenotype with Protein Levels and Localization in Individual Human Cells

To understand gene function, genetic analysis uses large perturbations such as gene deletion, knockdown or over-expression. Large perturbations have drawbacks: they move the cell far from its normal working point, and can thus be masked by off-target effects or compensation by other genes. Here, we offer a complementary approach, called noise genetics. We use natural cell-cell variations in protein level and localization, and correlate them to the natural variations of the phenotype of the same cells. Observing these variations is made possible by recent advances in dynamic proteomics that allow measuring proteins over time in individual living cells. Using motility of human cancer cells as a model system, and time-lapse microscopy on 566 fluorescently tagged proteins, we found 74 candidate motility genes whose level or localization strongly correlate with motility in individual cells. We recovered 30 known motility genes, and validated several novel ones by mild knockdown experiments. Noise genetics can complement standard genetics for a variety of phenotypes.