To test whether this difference is due to an artifact of our extraction procedure, we have tried to denature both extracts with a number of techniques; however, leaf polypeptides never broke down to such small components. This result, although preliminary, is the first evidence of a difference in DNA associated proteins from two plant organs and as such, is worth further investigation. Our technical approach is to isolate proteins which bind specifically with the T-DNA regulatory sequences of the root-specific and the leaf-specific T-DNA genes. First, proteins are isolated on the basis of their general DNA-binding capacity. To this effect, protein extracts from each plant organ are run on DNA-cellulose affinity columns to fractionate proteins which show general DNA-binding capacities. The test we use for visualizing DNA-binding proteins is a modified version of the technique published by Bowen et al., 1980: proteins are transferred to nitrocellulose filters and probed with nick-translated labelled DNA. The DNA sequences we are using for these experiments are sub-clones containing the leaf-specific and the root- specific promoters of the pRiA4 T -DNA. The goal is to show that there is indeed a specificity in the recognition of each of these sequences by the two types of extracts and to identify what proteins are’ involved. The difficulty that will be encountered in this part of the project is in defining conditions that will enhance the sequence specificity of the DNA-binding proteins. This has been a major difficulty in the identification of sequence-specific recognition factors. Retarded electrophoretic migration of DNA complexed to proteins has proven to be a useful method in detecting specific over non-specific binding of proteins to isolated sequences: it has recently been used to identify specific transcription factors in SV 40 infected cells, and in O2 induced cytochrome expression in yeast. We are now testing several approaches in order to determine the general and sequence specific DNA-binding properties of the Tobacco root and leaf proteins.
These include investigating factors influencing the formation of DNA-protein complexes in solution,ebb flow tray such as salt conditions, length of reaction and subsequent washes, and competition experiments with random sequence DNA such as calf-thymus DNA. If necessary, the DNA-cellulose column fractionation will be more elaborate to yield protein species in significantly purified form so as to reduce the background of non-specific binding. DNA-protein complex formation will also. be tested using smaller probes i.e purified promoter sequences from the plasmid that bear the leaf and root pRi T-DNA regulated genes. We hope thus to discriminate between sequence specificity and general binding affinities of the proteins now being isolated. This approach will focus on the other aspect of tissue-specificity i.e a study of the DNA sequences that interact with cellular factors to control the activity of specialized genes. It is also the most straightforward part of the project proposed. We would like to define what the DNA sequences are that interact with the proteins that are being isolated-in 1-. This we will determine by DNA foot-printing experiments: DNA-protein complexes will be formed -using purified protein fractions from the root or leaf extracts and purified restriction fragments containing sequences of the pRiA4 T-DNA for the root and leaf-specific promoters. It will thus be possible to identify the precise positions of the binding-sites of the tissue-specific proteins in the 5’upstream regions of the two genes under consideration. . Foot printing experiments are difficult if the proteins required to complex with the DNA are in low abundance. Hence, this experiment depends on previous work to attain a semi-purification of the proteins required. The initial aim of this work will be to introduce the plasmids we have from the Ri TDNA which contain genes expressed preferentially in different tissues, into protoplasts from these tissues. We will study the expression of the genes on these plasmids at the RNA and protein level.· The DNA will be introduced into the protoplasts either by transfection or by electroporation, both techniques are presently being used in our lab. The assays in this case will be short term expression assays in which the RNA from the protoplasts which carry the plasmids ,. will be isolated within 48 hr of introduction of the DNA.
The RNA encoded by the gene driven from the plasmid promoter will be quantified. To study expression at the level of protein the promoters will be isolated and linked to in vitro to the gene coding for the chloramphenicol acetyltransferase enzyme . CAT activity will be measured after introducing the DNA into protoplasts. A large part of this work will be directed at maintaining the tissue specific expression of the rotoplast by adjusting the growth conditions during the course of the experiment. We will modulate the culture media by adjusting the ratio of auxin to cytokinin and determine the effect on tissue specific expression. Also, we will investigate the involvement of oligosaccharins in determining the tissue specific expression of these plasmids. After the transient expression experiments are underway, plants will be regenerated containing these modified Ri T-DNAs. Protoplasts will be isolated from root and leaf tissues of these plants, and nuclei will immediately be tested for transcriptional activity, using the technique of Ackerman et al., CAT activity will be measured in parallel, as it is easily detectable in small amounts of material. By addition to root-nuclei of extracts from leaf tissues and harvesting of the subequent transcription complexes, it is hoped that it will be possible to activate the silent root-specific promoter . This assay will be used to test the factors isolated in the DNA binding work described above for tissue-specific activators. The overall goal of this program is to develop an understanding of the processes involved in hydrocarbon production in plants. Specifically, we are interested in the mechanisms and control of isoprenoid biosynthesis. Acquiring this basic information will be necessary before we can genetically manipulate plants to increase hydrocarbon yields. We use the latex isolated from laticifer cells of the Euphorbia lathyris plant for our biosynthetic studies. These cells are the site of both the biosynthesis and the storage of large quantities of sterols . We are currently examining the final steps of sterol synthesis, the epoxidation and cyclization of squalene, to determine if more than one squalene cyclase is ,.- involved in sterol synthesis. We are also attempting to identify the organelle involved in the conversion of mevalonic acid to sterols. A secondary interest is the’ synthesis of sesquiterpenes which are a better candidate for direct use as fuel than are the triterpenoids.
The manipulation of a plant towards increased sesquiterpene production at the expense of sterol product is thus a long-term goal of our research. This requires an understanding of the processes involved, specifically the initial cyclization of farnesyl pyrophosphate ,flood and drain tray the branch point at which sesquiterpene synthesis diverts from the general isoprenoid biosynthetic pathway. It may be possible to increase the hydrocarbon content of plants by changing the environment around these plants. We have found that by increasing the day length from about 12 hours to 16. hours and by maintaining a constant day/night temperature region, we observed about a 9-fold increase in the activity of 3-hydrox-3-methyglutaryl-Coenzyme A Reductase . Since this enzyme plays a key role in the synthesis of isoprenoids, it is possible that hydrocarbon production is sensitive to environmental controls. Controlled changes in environmental conditions could restrict growth while having only small effects on the photosynthetic rate . If applied when the plant approached maturity, this could result in an increase in assimilates available for partitioning into isoprenoid biosynthesis. We also hope to identify the internal controls of carbon allocation, since they also could be manipulated to increase plant hydrocarbon production. To investigate these posiblities we are observing the effects of three different environmental variables on hydrocarbon production: salinity stress, water stress .and nitrogen deficiency. After establishing the growth conditions necessary to grow E. lathyris hydroponically, we completed the preliminary study of salinity stress. Extractions of the hydrocarbons and sugars will be performed on the water-stressed plants. However, experiments utilizing nutrient stress have not been successful to this point as low levels of nitrogen cause rapid senescence and death. The effects of salinity on growth were determined from changes in shoot length, total fresh weight and root and shoot dry weights. Changes in the photosynthetic apparatus were determined from chlorophyll content, thylakoid proton gradient formation~ and in vivo fluorescnece patterns. Changes in the levels of energy-rich compounds were determined by heptane and methanol extractions of the dried plant. Only the shoot portion of the plant was used for this extraction as this is what would be available for harvest. The preliminary results indicate that salinities of 50 mM NaCI and greater affect both growth and photosynthesis, though the reduction in growth is sharper than the reduction in photosynthesis. Results of extraction experiments with plant components also indicate that increased salinity causes an increase in carbon allocation to the heptane and methanol fractions, with most of the increase occurring in the carbohydrate-rich methanol fraction. Salinization also caused a 3-fold increase in HMGR activity. To obtain a more comprehensive understanding of carbon flow through E. [athyris, we performed some preliminary carbon-labeling experiments. Now that we have established that it is possible to use isotope labeling to monitor latex production and follow carbon translocation in E. [athyris, this technique will be used further in stress experiments. trlterpenols, and their fatty acid esters.
To understand the bio-synthetic processes involved in the production of sterols. it is necessary to elucidate the structures of these compounds. We have previously identified four of the six major triterpenols as cyloartenol, 24-methylenecyc1oartenol, lanosterol and 24-methylenelanosterol. With the results obtained from 3H-NMR, chemical shift reagents, 13C-NMR and optical rotation we have identified the fifth compound as eupha-7,24- dienol. Wehav~ made a preliminary identification of the sixth compound as euphol on the basis of its behavior in gas chromatography. In most plant systems the initial product of squalene cyclization is cycloartenol and not lanosterol as found in animals and fungi.’ However, since lanosterol is a major component of E. [athyris latex, we are attempting to determine if lanosterol is a product of cyc1oartenol, produced via separate, parallel pathway, or is a precursor of cyc1oartenol. To determine if one of the first two mechanisms is responsible for squalene cyclization, we have synthesized deuterium-labeled MVA to be used as a susbtrate to follow its latex-catalyzed conversion to lanosterol. . One of the major difficulties in this analysis is the high level of endogenous sterols .present in the latex. These make the small amount of newly synthesized deuterated compounds difficult to detect by gas chromatography-mass spectrometry. Attempts at delipidation by extraction with organic solvents such as butanol and diisopI:opyl ether greatly reduced the level of triterpenols but also. destroyed all bioysnthetic activity. Purification by rate sedimentation and isopycnic centrifugation has reduced the lipid level to some extent, but we are still trying to find the proper analytical conditions that will allow us to employ the GC-MS to observe the deuterium label. By choosing special sequences for synthetic DNA oligomers we have been able to form stable “sticky end” dimers in solution from DNA hairpin with dangling ends. The sequences are chosen with a central section which is not complementary to any other in the molecule, which is flanked by a self-complementary sequence. Such sequences have been shown previously to lead to hairpin formation. We have modified these sequences to include a self complementary dangling tail, which allows two such molecules to associate in solution. We have observed the imino proton spectra from these molecules to confirm that such a dimer is indeed the stable form in solution. The imino protons have been assigned using nuclear Overhauser effect difference spectra, and confirm that the dimer is essentially a single continuous double helix in solution. On each of the backbone strands, however, there is a break and a missing phosphate. We have now collected complete two dimensional NOE data sets of two different sequences. In these spectra, when collected at sufficiently short mixing times, the cross peak intensities reflect the inverse sixth power of distances between protons.