Five retrotransposon families of rice (TosiTos5) have been reported previously. Here we report 15 new retrotransposon families of rice (Tos6-Tos2O). In contrast to yeast and Drosophila retrotransposons, all ofthe rice retrotransposons examined appear inactive (or almost inactive) under normal growth conditions. Three of the rice retrotransposons (ToslO, Tosl7, and Tosl9) are activated under tissue culture conditions. The most active one, Tosi 7, was studied in detail. The copy number of Tosl7 increased with prolonged culture period.

A series of chimeric promoters for higher-level expression of foreign genes in plants was constructed as fusions of a gene for beta-glucuronidase (GUS) with the terminator of a gene for nopaline synthase (nos) or of the cauliflower mosaic virus (CaMV) 35S transcript, and the strength of these promoters was assayed in transient and stable expression systems in tobacco and rice. As parts of these promoters, the CaMV 35S core promoter, three different 5'-upstream sequences of the 35S promoter, the first intron of a gene for phaseolin, and a 5'-untranslated sequence (omega sequence) of tobacco mosaic virus were used in various combinations. In tobacco and rice protoplasts, all three fragments of the 35S promoter (-419 to -90, -390 to -90 and -290 to -90, relative to the site of initiation of transcription), the intron, and the omega sequence effectively enhanced GUS activity. Some chimeric promoters allowed levels of GUS activity that were 20- to 70-fold higher than those obtained with the 35S promoter in pBI221. In tobacco protoplasts, the two longer fragments of the 35S promoter were more effective than the shortest fragment. In rice cells, by contrast, the shortest fragment was as effective as the two longer ones. The terminator of the 35S transcript was more effective than that of the nos gene for gene expression. In transgenic tobacco plants, a representative powerful promoter, as compared to the 35S promoter, allowed 10- and 50-fold higher levels of expression on average and at most, respectively, with no clear qualitative differences in tissue- and organ-specific patterns of expression. When the representative promoter was introduced into tobacco with a gene for luciferase, the autofluorescence of detached leaves after a supply of luciferin to petioles was great and was easily detectable by the naked eye in a dark room.

The complete nucleotide sequence of the tobacco retrotransposon Tto1, one of the few active retrotransposons of plants, was determined. The sequence analysis suggests that Tto1 carries all functions required for autonomous transposition through reverse transcription. Gene organization and the nature of the transcription product suggest that Tto1 uses a gene expression mechanism different from those employed by retroviruses and most retrotransposons to regulate Gag and Pol stoichiometry. Tto1 was introduced into rice to study its autonomous transposition in heterologous hosts. Transcription and transposition of Tto1 were observed in rice cells. To probe the autonomous transposition through reverse transcription, a modified Tto1 retrotransposon in which part of a reverse transcriptase gene was replaced with an intron-containing hygromycin resistance gene was constructed and introduced into rice cells. Loss of the intron was observed only when intact Tto1 was cotransfected. These results indicate that Tto1 can transpose autonomously through reverse transcription and that the host factors required for transposition are conserved among monocots (class Magnoliopsida; rice) and dicots (class Liliopsida; tobacco), which diverged approximately 200 million years ago. These findings are discussed in relation to the regulation and evolution of retrotransposons and the possible use of Tto1 as a molecular genetic tool.

Abstract.-It was proved that thejprotoplasts prepared from mesophyll of Nicotiana tabacum are infected by tobacco mosaic virus. The infection occurred when purified tobacco mosaic virus particles were added to a protoplast suspension in the presence of poly-L-ornithine. The virus multiplied in these protoplasts to a level of 106 virus particles per infected protoplast during 24 hours of incubation. The efficiency of infection was remarkably high, exceeding that by mechanical inoculation of tobacco leaves.Protoplasts can be prepared from plant cells by mechanical or enzymatic removal of cell walls." 2 These isolated naked cells should be far more suitable than conventional tissue materials for experiments of quantitative nature, since they can be used as a homogeneous suspension in liquid medium.3 The isolation of protoplasts from mesophyll was accomplished for the first time in this laboratory by a newly developed enzymatic technique." 4 Mesophyll protoplasts were prepared on a large scale by cellulase treatment of isolated mesophyll cells which had been obtained by digesting leaves with pectinase. The effectiveness of this technique prompted us to explore the potentialities of mesophyll protoplasts as a new experimental material for studying various activities of plant cells.Recently, RNA from tobacco mosaic virus (TMV) inoculated into tobacco mesophyll protoplasts was shown to give rise to a synchronous multiplication of this virus in these protoplasts.5 This finding indicated that the mesophyll protoplasts should be invaluable for studying plant virus multiplication at a cellular level. However, it is obvious that studies of the entire process of infection, including penetration and uncoating of infecting virus, are possible only when infection is caused by complete virus particles. In the following, we present evidence to show that complete particles of TMV infect tobacco mesophyll protoplasts and multiply within them.Materials and Methods.-Protoplasts of palisade parenchyma cells were prepared from mesophyll of healthy tobacco plants (Nicotiana tacum var. Bright Yellow) according to the procedures previously reported' with slight modifications.5 Figure 1 shows microscopical appearance of these protoplasts. The protoplasts were washed with and resuspended in 0.8 M mannitol at a concentration of 1 to 4 X 106/ml, as measured by a Coulter electronic counter.' For infection, purified TMV (OM, a common strain6) was dissolved to a concentration of 2 ttg/ml in 0.02 M potassium citrate buffer, pH 5.0, containing mannitol to 0.8 M and poly-L-ornithine (mol. wt. -130,000, Pilot Chemicals, Inc., Watertown, Mass.) to 2 ug/ml. After standing at 250C for 10 min, the TMV solution was added to an equal volume of the protoplast suspension, and the mixture was kept at 250C with occasional gentle swirling. After 60 min of infection, the protoplasts were separated from the inoculum virus by low speed centrifugation' and were washed several times with sterile 0.8 M mannitol containing CaCl2 to 0.1 mM. The washed protoplasts were...

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RNA of a common strain of tobacco mosaic virus was sequentially reconstituted first with protein of the same strain and then with protein of a tomato strain. The reconstituted particles were treated with specific antibodies against the two strain particles and were examined electron microscopically to locate the regions of rods encapsidated with the respective proteins. The results demonstrated that reconstitution starts at an internal region of the RNA about 830 nucleotides from an end that recent studies indicate to be the 3' terminus. Evidence was obtained to show that reconstitution proceeds toward the 5' terminus of the RNA until it is fully encapsidated. Encapsidation of the 3' distal portion of the RNA apparently occurs later and in a direction opposite to that of the encapsidation of the 5' distal portion. The process of tobacco mosaic virus (TMV) reconstitution from its RNA and protein has been extensively-studied since it was first demonstrated by Fraenkel-Conrat and Williams (1). The initial step of this process involves interaction of a particular region of the viral RNA with disk aggregates of coat protein subunits (2-4). Several laboratories proposed independently that the interaction takes place at or near the 5' terminus of RNA, being followed by unidirectional elongation of viral rods (2,5,6). As a presumed intermediate product, short helical rods with a tail of free RNA were found by electron microscopy (2,5,7,8).In view of the recent finding (9, 10) that the 5' terminus of TMV RNA is blocked by the sequence m7G5'ppp5'G, the hypothesis of polar reconstitution from the 5' terminus of RNA clearly needs reexamination. Because the distal m7G has free 2' and 3' hydroxy groups, both ends of TMV RNA are available for periodate oxidation. Consequently, our previous observation that no periodate-sensitive end group is encapsidated early during reconstitution (5) now suggests that neither end of the viral RNA is involved in the initiation of reconstitution. In fact, we showed that reconstitution occurs in the absence of the 5'-terminal blocking structure (11), and more recent studies with TMV RNA labeled at both ends indicated that the initiation site of reconstitution is located internally within the 3' half (12).In this paper we present direct evidence to show that reconstitution of TMV rods starts at an internal region on the viral RNA and proceeds bidirectionally. The evidence was provided by initiating the reconstitution of RNA of a common strain with homologous protein and subsequently completing the elongation with protein of a tomato strain, which is serologically distinguishable from the common strain. Virus rods thus reconstituted sequentially with two types of protein were examined by a technique that may be called "electron microscopic serology"; after the rods were treated with antibodies specific to each strain, binding of antibody molecules on the rods was located by electron microscopy. The technique has been successfully used to study the reconstitution of bacterial flagella (13)...

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