Sequencing from plant tissue, isolation

ACC 71 synthase, i. e. (S)-adenosylmethionine methylthioadenosine lyase (EC 4.4.1.14), has been purified from several plant tissues [116]. Recently, ACC synthase cDNA clones have been isolated and sequenced from wounded fruit tissues of tomato, winter squash, zucchini, ripening apple and tomato fruit. Using the polymerase chain reaction (PCR), four different ACC synthase gene fragments were obtained by amplification of cDNA derived from mRNA of tomato... 

This classical cloning strategy has proven to be successful in many cases, but it is time-consuming and laborious. Purification of a native enzyme to homogeneity is difficult, and any undesired contaminant protein can be mistakenly subjected to sequencing. Cloning efforts from plant material expressing low levels of the desired enzyme are often unsuccessful. Plant tissues that are known to be enriched in the desired activity provide the best results. Furthermore, the enzymatic activity must be maintained in vitro as the desired enzyme is diluted aud purified. If accessory proteins or cofactors are required for stability or fimctioual activity, the purified protein will become inactive as it is isolated from the crude cell extract. A robust in vitro assay with correct substrates is also required, and this may not be possible if the substrates are unavailable or if the enzymatic products are unstable. 

A special type of furanoacetylenes (e.g. 70-78) has been isolated from leaves and edible parts of broad bean (Vtctafaba) and/or lentil (Lens culinaris) (Table 5.1 and Figure 5.9) infected by fungi such as Botrytis cinerea and B. fabae. These furanoacetylenes are considered as phytoalexins as they do not seem to be present in healthy plant tissue (Mansfield et al. 1980 Robeson and Harborne 1980), although they may in fact be present in minute amounts. From incorporation studies with [ C] -acetate precursors (Cain and Porter 1979 Al-Douri and Dewick 1986) it has been shown that the furanoacetylenes (e.g. 70-78) are most likely biosynthesized from the Cig -acetylene dehydrocrepenynic acid followed by two -oxidation sequences at the carboxyl end leading to the Cn-acetylene 107. Further dehydrogenation, oxidation and isomerization steps then finally lead to the furanoacetylenes 70-78 as illustrated in Figure 5.10. 

Fig. 4. Primer extension analysis of rRNAs isolated from four plant species treated with dimethyl sulfate. Sequencing lanes are indicated as A, T, G, and C. A plus sign (+) over the lane denotes RNA prepared from DMS-treated tissues a minus (—) denotes RNA isolated from mock-treated tissues. The nucleotide numbers are the coordinates at which primer extension reactions terminate (see Figs- 3 and 5A). The position of the modified base in the sequence of the 18 S rRNA is determined by comparing novel bands in the DMS-reacted RNA with the sequencing ladder and adding one nucleotide (see Fig. 2 for details). Various regions of the RNA are shown for the different plant species. Several sites of chain termination arising from nascent structure are also evident.

The methodology required for successful modification of plant metabolism has been perfected for some plant species and for specific enzymes. This methodology includes transformation of plant cells or tissues regeneration of a healthy plant from the transformed cells isolation of the gene encoding the enzyme of interest identification of promoter sequences for tissue-specific expression and targetting of the protein into the cellular compartment desired. 

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