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A Tool for Fundamental Plant Science Molecular biology ancT genetic engineering are aIreacly having a major impact on funciamental plant science. Gene cloning, gene transfer, anc! other new techniques are proving valuable research tools for probing gene structure, function, ancT plant clevelopment. This knowlecige, in turn, can be usec! in designing increasingly sophisticated methods to . . engineer 1mprovec . crops. Because of these recent advances, molecular biologists now know as much about the structure, organization, anc! expression of the plant genome as they clo about the animal genome, according to Robert GoIcI- berg of the University of California at Los Angeles. Nonetheless, in both areas, there is still much to learn. Far more is known about bacterial genes than the genes of either plants or animals. Bacteria are less com- plex than higher organisms anc! comparatively easier to stucly. A single- cellect bacterium contains some 5,000 genes, whereas the average plant genome is roughly 1,000 times larger, about the same size as the human genome. (The actual size of plant genomes vary greatly in size from one taxonomic group to the next.N To ciate onIv about a dozen plant genes ~ 1 , , , have been fully sequenced. As GoIciberg clescribecT, molecular biologists are learning that the or- ganization of the plant genome is also exceecTingly complex. Certain sequences of the DNA are repeater! huncTreds to thousands of times. No one knows the function of these reiterated sequences. Interspersec! among the cocTing regions, plant genes also contain se- quences of DNA that do not cocle for protein at all. These noncoding sequences are collect introns to distinguish them from the coding regions, called exons. "These introns seem to have 'popped' into the gene cluring evolutionary time, splitting the regions that code for proteins," GoIciberg explainecT. Introns interrupt the genetic cocle the instructions for pro- 27
28 Flowering Plants B irds Mammals Reptiles Amphibians Bony Fish . Cartilaginous Fish Echinoderms - Crustaceans _ I nsects Mollusks _ . Worms Molds Algae Fungi Gram-positive bacteria Gram-negative bacteria Mycoplasma GENETIC ENGINEERING OF PLANTS SIZE OF PLANT GENOMES 5X102 5x103 5x104 5x105 5X106 5x107 I . 1 ~ ~ , , l l l 1 I I r 1 '' l ! ! l 1 1 . ~ l =t 1 1 1 1 1 l ~ . . : . akb = 1,000 base pairs. . ~ Al .- .- ! .......... I.. 1 t l t 103 104 105 106 107 108 kb A comparison of the size of genomes. Plant genomes are 100 to 10,000 times larger than bacterial genomes. Courtesy of Robert Goldberg, Department of Biology, University of California at Los Angeles. teins and must somehow be removed before the gene can be ex- pressed. Molecular biologists have learned that genes possess their own mechanism for excising the introns; it is remarkably similar to methods developed by genetic engineers. The DNA is transcribed to RNA, then in a series of enzymatic cut-and-paste reactions, the introns are excised from the RNA. This processed strand of messenger RNA, minus the introns, can then be translated to protein. Because no one has yet dem- onstrated a function for introns, they have been jokingly referred to as junk DNA. If, however, they prove to have a role in gene expression, introns will present adclitional complexity and perhaps opportunities in . . . genetic engineering. Molecular biologists can determine how many genes are actually ex-
A TOOL FOR FUNDAMENTAL PLANT SCIENCE 29 TRANSLATION .- A ; - <~5 f(; Cytoplasm Messenger RNA Removal of introns. In plants and animals the DNA sequence of a gene that codes for a protein may be interrupted by noncoding stretches called introns. These introns must be removed before the gene can be expressed. The cells have a natural mechanism for excising introns. First, the DNA containing the introns is transcribed to RNA. Then enzymes within the cell nucleus cut the introns from the RNA and splice the coding sequences back together. The resulting messenger RNA then migrates to the cytoplasm to be translated into protein. pressed actively making protein at a given time in the life cycle of a plant. This is easily done because RNA can be used to identify the DNA from which it was transcribed. In tobacco, Goldberg reported, about 100,000 genes are active at a specific time in the life cycle of the plant. In other words, only about 5 percent of the DNA contained in the nucleus is used at one time to produce proteins. The function of the other 95 percent is unknown, though regulatory sequences are known to account for some of this DNA. The next question, Goldberg said, is the extent to which these genes are reguiatect in the plant: "Do all these organ systems have 100,000 genes that are on and active, or is there a differential gene expression that could contribute to the differentiated state of the particular cell?" In the tobacco plant, approximately 25,000 genes are on in each organ
35 30 25 20 15 X IL 1 ~ 5 o Cal 10 o :~. . . · - ::: : ::: , :.-: : :: :*: .-.-....... ~ . GENETIC ENGINEERING OF PLANTS 1~\ _ ; ·:.::- :- ::::: - ·: -:::- ~~ mN 0^ ~ ~0 Genes Expressed Only in One Organ Genes Expressed in Two or More Organs Housekeeping Genes Expressed in All Organs 30,000 25,000 20,000 15,000 1 0,000 5,000 In LL Ad UJ o LLJ m me Gene expression is under complex control in higher plants. Some genes are only ex- pressed in specific parts of the plant, while other genes are expressed in all cells of the plant. Courtesy of Robert Goldberg, Department of Biology, University~of California at Los Angeles. system, e.g., in the leaves, stems, or flowers. The most important fincI- ing, according to Goldberg, is that each organ system has a unique set of genes genes that are expressed only in that organ. For example, the petals and the leaves each contain approximately 7,000 specific genes. The ovaries and the anther contain approximately 10,000 specific genes. In addition, all the organs share some common genes, which Goldberg calls "housekeeping genes," that code for proteins necessary for all cells in the plant. In short, he saicl, plant genes are highly regulated. The regulatory circuitry is proving to be even more intricate than anticipated. Not only can genes be either active or silent, but there now
A TOOL FOR FUNDAMENTAL PLANT SCIENCE 31 seems to be an intermediate state. Some genes are switched on they are transcribed into messenger RNA but for some reason, the mes- senger RNA is never transported to the cytoplasm to the site of protein synthesis. Since no proteins are made, the genes are effectively off. Thus, in addition to the standard "on-off" switches, molecular biologists are searching for the regulatory mechanism that determines whether an active gene will actually be expressed. The central task in both molecular and developmental biology is to understand the regulatory controls responsible for differential gene expression during development to find the DNA sequences that in- struct a gene to be on in a leaf but off in a root. Only when these DNA sequences have been identified can molecular biologists use gene-splic- ing techniques with predictability to engineer new crop plants. Seed Protein Genes Elucidating the mechanism of these regulatory controls is not easy. Goldberg and his colleagues at UCT~A are tackling the problem by ex- amining the genes that code for storage seed proteins in the soybean plant. These proteins, contained in most plants, provide a source of nutrition for the plant when the seed germinates and begins growth. They are also an important food source for livestock and humans. Goldberg is studying the genes for these seed storage proteins for two reasons. First, he said, the genes are active at only a specific time in the plant's life cycle during embryogenesis. That makes them a useful system for examining the regulation of gene expression. Second, such research could have agricultural significance. Although seed stor- a~e Proteins in soybeans and other crons are important food sources. ~ 1 - -a - ~ r ~ r ~ ~ -- ~~~~~~~ .. . ~ . . . . . . . . . . . _ they are Descent In some amino acids essential to human nutrition. it that deficiency could be corrected, the nutritional value of these crops would be significantly enhanced. It might be possible to correct that deficiency through genetic engineering. There are two possible approaches. First, some of the storage proteins in a soybean contain more of the essential amino acids than do the other storage proteins. One approach would be to engineer the plant to pro- duce more of the protein that contains an abundance of essential amino acids. That involves changing gene regulation so that the expression of one gene is favored over the expression of another. The other approach would be to directly engineer the seed storage protein gene inserting more codes for essential amino acids. Ultimately, molecular biologists may attempt to construct a seed protein gene de nova. For any of these approaches, the molecular biologist must know exactly when during
32 GENETIC ENGINEERING OF PLANTS development the different storage protein genes are turned on, and where the switches are. Using molecular techniques, the UCLA researchers have been able to pinpoint exactly when during embryogenesis the seed protein genes are active. They have also confirmed, as expected, that the genes are not active in the adult plant. They have isolated a storage protein gene and are now studying its structure. They have found that it contains three introns, or noncoding sequences, anc! they have located the regulatory sequences responsible for excising those introns in the natural cut-and- paste reaction. They have also identified the gene's promoters and ter- minators the sequences responsible for starting and ending transcrip- tion. Yet they have not been able to identify the regulatory sequences responsible for turning the gene on and off during embryogenesis.
