The presence of modified ribonucleotides derived from adenosine, guanosine, cytidine, and uridine is a hallmark of almost all cellular RNA, and especially tRNA. The objective of this chapter is to describe a few simple methods that can be used to identify the presence or absence of a modified nucleotide in tRNA and to reveal the enzymatic activity of particular tRNA-modifying enzymes in vitro and in vivo. The procedures are based on analysis of prelabeled or postlabeled nucleotides (mainly with [(32)P] but also with [(35)S],[(14)C] or [(3)H]) generated after complete digestion with selected nucleases of modified tRNA isolated from cells or incubated in vitro with modifying enzyme(s). Nucleotides of the tRNA digests are separated by two-dimensional (2D) thin-layer chromatography on cellulose plates (TLC), which allows establishment of base composition and identification of the nearest neighbor nucleotide of a given modified nucleotide in the tRNA sequence. This chapter provides useful maps for identification of migration of approximately 70 modified nucleotides on TLC plates by use of two different chromatographic systems. The methods require only a few micrograms of purified tRNA and can be run at low cost in any laboratory.

The Balbiani ring (BR) genes in the midge Chironomus, a genus belonging to Diptera, code for large secretory proteins, used to construct the larval tube. The 15-23-kb long core block in each gene consists of an array of tandemly arranged approximately 200-bp long repeat units, where a single repeat unit is composed of a constant and a subrepeat region. In order to investigate the evolutionary fate of highly repetitive coding DNA, the BR1gamma core block in Chironomus pallidivittatus was characterized and compared to the orthologous core block in the sibling species Chironomus tentans. We find that the 75-100 repeat units in the BR1gamma core block have evolved in an unusual fashion. In all repeat units the constant regions display an expected high degree of homology between the two species, 94% at the nucleotide level. In contrast, the subrepeat regions in all repeat units have diverged concertedly, both as to length, number and sequence of the subrepeats. The observed changes in all repeat units of the core block probably have occurred after speciation of C. pallidivittatus and C. tentans. These findings demonstrate that a tandemly reiterated coding sequence can rapidly and concertedly convert into a related sequence, much in the same way as has been described for satellite DNA.

Tobacco etch virus, a plant potyvirus, expresses its RNA genome as a large polyprotein precursor which undergoes extensive proteolytic processing to yield seven or more mature products. Two of these products, proteins with apparent molecular weights of 49,000 and 54,000 (49K and 54K proteins), aggregate in the form of crystalline inclusions within the nuclei of infected cells. Cell-free translation of synthetic transcripts was used to map the genes for these two products on the viral genome and to express an enzymatically active protein. The 49K protein was determined to be a viral protease responsible for several cleavages of the polyprotein, including its own autocatalytic excision. Analyses of products expressed from the 49K protein genes which were altered by deletion revealed that only the carboxyl-terminal half was required for proteolytic activity.

The distribution of transcripts encoding hydroxyproline-rich glycoproteins in hypocotyls of Phaseolus vulgaris L. infected with Colletotrichum lindemuthianum was examined by in situ hybridization to tissue sections. The expression of hypersensitive resistance in an incompatible interaction was accompanied by a massive early accumulation of transcripts in the epidermal, cortical, and perivascular parenchymal tissues immediately adjacent to the inoculation site. In a compatible interaction, there was no accumulation of transcripts in the epidermal and cortical tissues even though fungal hyphae ramified throughout these tissues. However, transcripts accumulated at a later stage in the perivascular tissue directly below the site of infection and in tissue several millimeters from the inoculation site. Thus, there is a spatial and tissue-specific counterpart to the differential timing of transcript accumulation in incompatible versus compatible interactions (AM Showalter, JN Bell, CL Cramer, JA Bailey, CJ Lamb [1985] Proc Natl Acad Sci USA 82: 6551-6555). These differences in the spatial distribution and tissue specificity of transcript accumulation imply the differential induction of signaling systems involved in race:cultivar-specific interactions.

2S albumin seed storage proteins undergo a complex series of posttranslational proteolytic cleavages. In order to determine if this process is correctly carried out in transgenic plants, the gene AT2S1 encoding an Arabidopsis thaliana 2S albumin isoform has been expressed in transgenic tobacco. Initial experiments using a reporter gene demonstrated that the AT2S1 promoter directs seed specific expression in both transgenic tobacco and Brassica napus plants. The entire AT2S1 gene was then transferred into tobacco plants, where it showed a tissue specific and developmentally regulated expression. Arabidopsis 2S albumin accumulates up to 0.1% of the total high-salt extractable seed protein. Protein sequencing demonstrated that the amino termini of the two Arabidopsis 2S albumin subunits were correctly processed, suggesting that the protease(s) necessary for posttranslational processing of 2S albumin precursors may display common specificities among different dicot plant species. Immunocytochemical studies showed that the Arabidopsis 2S albumin is localized in the protein body matrix of tobacco endosperm and embryo. Correct processing and targeting of the 2S albumin in transgenic plants suggests that modified versions could be expressed, allowing the study of 2S albumin processing and in particular the possible roles of the processed fragments in protein stability and/or targeting.

The catalase activity, CAT-2 and CAT-3 isozyme protein levels, and the steady-state mRNA levels for each of the three catalase genes were determined in the scutellum, root, epicotyl, and leaf of the developing maize (Zea mays L.) seedling. Catalase activity was highest in the scutellum, with 10-fold lower enzyme activity in the leaf and epicotyl. Very low levels of catalase activity were found in the root. The highest levels of CAT-2 protein were found in the scutellum, with about 10-fold lower levels in the green leaf. CAT-2 protein was present in trace amounts early in root development and no CAT-2 protein was detected in the epicotyl. Shortly after germination, CAT-3 protein was present at high levels in both the epicotyl and green leaf. With development, the amount of CAT-3 protein decreased slowly in the epicotyl and rapidly in the green leaf. Low levels of this isozyme were detected in the scutellum and root. The Cat1 transcript accumulated to low levels in all four tissues during the 14 day developmental period. High levels of the Cat2 transcript were found in the scutellum, with moderate levels of the mRNA in the green leaf. The Cat2 transcript levels were very low in the root and epicotyl. While the Cat3 mRNA level in the scutellum was low, high levels of the Cat3 transcript were detected in the root, epicotyl, and leaf. There was a positive correlation between the accumulation of a catalase isozyme and its transcript, indicating that the tissue specificity of maize catalase gene expression was regulated pretranslationally.

The relationship between tubulin gene expression and cell elongation was explored in developing internodes of Glycine max (L.) Merr., using light as a variable to alter the rate of elongation. First internodes of etiolated seedlings elongated two to three times more rapidly than did those of seedlings growing under a 12 hour diurnal light/dark cycle. Furthermore, light slowed or completely halted internode elongation in the etiolated seedlings, depending upon the age of the seedlings at the time of the light treatment. Steady state levels of beta-tubulin mRNA were determined in Northern blots and by solution hybridization of poly(A)(+)RNA with a probe derived from the coding region of a previously characterized soybean beta-tubulin gene.(MJ Guiltinan, DP Ma, RF Barker, MM Bustos, RJ Cyr, R Yadegari, DE Fosket [1987] Plant Mol Biol 10: 171-184). Internodes of light-grown seedlings exhibited levels of beta-tubulin mRNA that differed by a factor of three, and varied concomitantly with the elongation rate. Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of beta-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of beta-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA.

Effects of red and blue light at irradiances from 1.6 to 28.3 micromolar per square meter per second on chloroplast pigments, light-harvesting pigment-proteins associated with photosystem II, and the corresponding mRNA were evaluated in maize (Zea mays L.) plants (OP Golden Bantum) grown for 14 days under 14 hours light/10 hours dark cycles. Accumulation of pigments, pigment-proteins, and mRNA was less in blue than in red light of equal irradiance. The difference between blue and red light, however, varied as a function of irradiance level, and the pattern of this variation suggests irradiance-controlled activation/deactivation (switching) of blue-light receptor. The maximum reduction in blue light of mRNA and proteins associated with light-harvesting complex occurs at lower irradiance levels than the maximum reduction of chlorophylls a and b.

A variety of expression systems and selection régimes have been developed to transform plants such as tobacco, petunia, and tomato. We investigated several of these to determine whether the promoters and selectable markers used in dicotyledonous plants are suitable for selecting transformed rice callus. We compared transient expression driven by constitutive and regulated promoters in rice (Oryza sativa) protoplasts and found that the 2' promoter of the octopine T-DNA is approximately 3 to 4 times more efficient than the CAMV 35S promoter, 10 times more efficient than the nos promoter and the 1' promoter, and more than 100 times better than two other regulated plant promoters. Similar results were obtained in tobacco (Nicotiana tabacum) protoplasts with the exception that the nos promoter was expressed nearly 10 times better in rice. Further studies demonstrated that rice callus growth is sensitive to low concentrations of methotrexate, phosphinothricin, and bleomycin, and to moderate concentrations of G418 and hygromycin, but is only partially inhibited by relatively high concentrations of kanamycin. Finally, we tested the ability of stably introduced resistance genes to protect callus against some of the selective agents. Genes that inactivated phosphinothricin or G418 permitted transformed calli to grow almost unimpeded on toxic concentrations of these selective agents. However, a gene conferring resistance to methotrexate could not be used to select for activily growing transformants. Southern analysis of the transformed cell lines demonstrated that 50% of the transformants contained a single plasmid copy and that nearly all integrated copies showed rearrangements. These results on the use of selectable markers in rice should facilitate efforts to obtain transformants of this important grain.

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.

We have examined the expression of acyl carrier protein (ACP) mRNA levels and ACP activity in leaves, where fatty acids function primarily in membrane synthesis, and in developing soybean seeds, where fatty acids are primarily used for oil storage. An RNA probe transcribed from a synthetic spinach ACP-I gene hybridized on Northern blots to ACP mRNA from both seed and leaf tissue from soybean, spinach, and rapeseed. In each species, the ACP transcript from leaf was slightly larger than that from seed. Both the amounts of ACP protein and the levels of ACP mRNA were substantially higher in young leaf tissue of spinach and soybean when compared to mature leaf tissue. Light-grown spinach leaves also contained higher ACP activity and accumulated more ACP mRNA than dark-grown leaves. ACP mRNA levels measured in developing soybean seeds peaked at 20 days after flowering then decreased 10-fold by 70 days after flowering. In each tissue, the developmental changes in ACP protein levels can be accounted for by changes in ACP mRNA abundance. Comparison of the relative prevalence of mRNA and protein for ACP and lectin in soybean seeds suggests a major difference in mRNA translational efficiency and/or protein stability for these two proteins.

Gas vesicle-deficient mutants of Halobacterium halobium arise spontaneously at high frequency (about 1%). The mutants are readily detected, forming translucent colonies on agar plates in contrast to opaque wild-type colonies. To investigate the mechanism of this mutation, we recently cloned a plasmid-encoded gas vesicle protein gene, gvpA, from H. halobium. In the wild-type NRC-1 strain the gvpA gene is encoded by a multicopy plasmid of approximately 150 kilobase pairs (kb). We have now characterized 18 gas vesicle-deficient mutants and 4 revertants by phenotypic and Southern hybridization analyses. Our results indicate that the mutants fall into three major classes. Class I mutants are partially gas vesicle-deficient (Vac(delta-)) and unstable, giving rise to completely gas vesicle-deficient (Vac(-)) derivatives and Vac(+) revertants at frequencies of 1-5%. The restriction map of the gvpA gene region in class I mutants is unchanged but the gene copy number is reduced compared to the Vac(+) strains. Class II mutants can be either Vac(delta-) or completely Vac(-) but are relatively stable. They contain insertion sequences within or upstream of the gvpA gene. A Vac(-) class II mutant, R1, contains the 1.3-kb insertion sequence, ISH3, within the gvpA gene, whereas four Vac(delta-) class II mutants contain other insertion sequences upstream of the gene. Class III mutants are stable Vac(-) derivatives of either the wild-type or class I mutants and have no detectable copies of the gvpA gene. Based on these results, we discuss the mechanisms of gas vesicle mutations in H. halobium.

The mobility properties of the Drosophila melanogaster P element in drosophilid and nondrosophilid species has been determined using a P-element mobility assay that is conducted transiently in insect embryos. P elements are mobilizable in all drosophilids tested, including species outside the genus Drosophila but not in the related Tephritidae (order: Diptera), although the P-element gene necessary for mobility, transposase, is transcribed. These results show that without modifications P elements will not serve as general insect gene vectors and suggest that nonconserved host-encoded factors participate in the transposition of P elements. Our methods will be generally useful for analyzing the cis- and trans-acting factors required for P-element mobility in vivo and could be used to analyze the mobility properties of other transposable elements in insects.

Antisense nopaline synthase (nos)(D-nopaline synthase; EC 1.5.1.19) RNA is stably expressed from the cauliflower mosaic virus 35S promoter in transformed tobacco plants. The expression of a previously introduced wild-type nos gene is inhibited by the antisense RNA, with less nos enzyme activity detected (by a factor of 8-50) depending on the tissue analyzed. The steady-state levels of nos mRNA are reduced in the presence of the antisense RNA, implying that mRNA degradation is probably the main mode of action for the decrease in expression in this system. The antisense RNA-expressing gene and its inhibition of nos expression are shown to be heritable, demonstrating that it is a potentially useful method for the modification of phenotype.

An in vitro system was developed that results in the self-assembly of subunit precursors into complexes that resemble those found naturally in the endoplasmic reticulum. Subunits of glycinin, the predominant seed protein of soybeans, were synthesized from modified cDNAs using a combination of the SP6 transcription and the rabbit reticulocyte translation systems. Subunits produced from plasmid constructions that encoded either Gy4 or Gy5 gene products, but modified such that their signal sequences were absent, self-assembled into trimers equivalent in size to those precursors found in the endoplasmic reticulum. In contrast, proteins synthesized in vitro from Gy4 constructs failed to self-assemble when the signal sequence was left intact (e.g., preproglycinin) or when the coding sequence was modified to remove 27 amino acids from an internal hydrophobic region, which is highly conserved among the glycinin subunits. Various hybrid subunits were also produced by trading portions of Gy4 and Gy5 cDNAs and all self-assembled in our system. The in vitro assembly system provides an opportunity to study the self-assembly of precursors and to probe for regions important for assembly. It will also be helpful in attempts to engineer beneficial nutritional changes into this important food protein.

Viroids are single-stranded, covalently closed circular RNA pathogens that can be isolated from certain higher plants afflicted with specific diseases. Their small size (246-375 nucleotides; M(r) 0.8-1.3 x 10(5)) and ability to replicate autonomously make viroids a unique model system in which to study the relationships between the structure of an RNA and its biological function. The demonstrated infectivity of certain cloned viroid cDNAs allows the use of site-specific mutagenesis techniques to probe structure-function relationships suggested by comparative sequence analysis. Several site-specific mutations that disrupt base pairing in either the native structure or secondary hairpin I destroyed the ability of potato spindle tuber viroid cDNA to initiate infection. Alterations in the terminal loops of the native structure also abolished cDNA infectivity. One pseudorevertant, a mutant cDNA containing compensating changes that restore base pairing in the native structure, was marginally infectious; a second pseudorevertant in which base pairing was restored within the stem of secondary hairpin I was not infectious. The behavior of these mutants dramatically demonstrates the effect of remarkably small structural changes on viroid infectivity and emphasizes the importance of the conserved rod-like native structure for viroid function.

The satellite RNA of tobacco ringspot virus depends upon tobacco ringspot virus for its replication and source of coat protein. The satellite RNA reduces virus accumulation and the severity of virus-induced symptoms. Repetitive sequence, dimeric, and higher forms of the satellite RNA are known to autolytically process to form biologically active monomeric RNA of 359 nucleotide residues [Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R.& Bruening, G.(1986) Science 231, 1577-1580], with a 5'-hydroxyl and a 2',3'-cyclic phosphodiester as the new terminal groups. We show here that transcripts of full-length and truncated DNA clones of the satellite RNA sequence also process in a nonenzymic reaction. One such transcript was an RNA that has about one-fourth of the satellite RNA sequence, representing the 3'-terminal and 5'-terminal portions of monomeric RNA joined in the junction that is cleaved in dimeric RNA. This RNA autolytically processed more efficiently than molecules with a larger proportion of the satellite RNA nucleotide sequence.

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.

In French bean (Phaseolus vulgarisL.), the glycine-rich wall protein GRP 1.8 is specifically synthesized in protoxylem tracheary elements of the vascular system. A 494 bp upstream promoter fragment of the gene encoding GRP 1.8 was isolated and translationally fused to the beta-glucuronidase reporter gene. Transgenic tobacco plants containing this construct expressed the gene in vascular tissue of roots, stems, leaves and flowers. The gene was developmentally expressed during differentiation of both primary and secondary vascular tissue and was also rapidly induced (in < 30 min) after excision-wounding of young stems. This wound response is more rapid than in bean hypocotyls, indicating possible differences between the activation mechanism for glycine-rich protein gene expression in wounded bean and tobacco. Only a subset of cells were found to participate in the wound response. In young stems, the GRP wound induction was localized in pith parenchyma cells adjacent to the wound surface, where vessel regeneration is known to occur. Thus, a promoter fragment of 494 bp, including 427 bp upstream from the transcription start site, contains information for tissue-specific and wound-induced gene regulation. The cell-type specificity of expression suggests that the GRP 1.8 promoter is regulated by very specific developmental and environmental signals.

A chimeric oat phytochrome structural gene with an uninterrupted coding region was constructed for expression of the monocot protein in transgenic plants. The structural gene was placed under the transcriptional control of either a light-regulated oat phytochrome promoter or the constitutively active cauliflower mosaic virus 35S promoter. These genes were then introduced into Nicotiana tabacum and N.plumbaginifolia. None of the regenerated plants showed expression of oat phytochrome RNA when transcription was controlled by the oat promoter. In contrast, RNA was obtained in plants when the structural gene was functionally linked to the 35S promoter. Transformants expressing oat phytochrome RNA produced a full length 124-kd polypeptide that was recognized by oat-specific anti-phytochrome monoclonal antibodies. The oat protein was a substrate for chromophore addition in tobacco as judged by its red/far-red photoreversible sensitivity to trypsin degradation. Production of oat phytochrome in transgenic plants gave rise to increased phytochrome spectral activity in both light- and dark-grown plants. This increased phytochrome content resulted in phenotypic changes in transformed plants, including semi-dwarfism, darker green leaves, increased tillering and reduced apical dominance. The possible significance of expressing a biologically active phytochrome in transgenic plants is discussed.