Goldberg, Robert B. : Associate Professor, Biology, Los Angeles

Timberlake, William E. : Associate Professor, Bacteriology, Plant Growth Laboratory, Davis

Understanding mechanisms of gene expression William E . Timberlake , Associate Professor , Bacteriology , Plant Growth Laboratory , Davis , and Robert B . Goldberg , Associate Professor , Biology , Los Angeles ~ ~ Underlying the developing technology of plant genetic engineering is the knowledge R - LOOP MAPPING of that specific traits result from expression A NO INTRONS B INTRONS discrete regions of the DNA molecules pres - . ent in each cell . These regions comprise the familiar , basic units of biologicalinformation and heredity , the genes . Proteins , the mole - cules whose activities finally determine the form and function of the plant , are encoded of purine and pyri - by the linear arrangement DNA of individualgenes . midine bases in the In plant breeding , genes conferring desirable characteristics are transferred from one ge - netic background to another by â?? crossing . â?쳌 In contrast , the new approaches to plant im - provement do not depend entirelyon natural - ly occurringmechanisms for geneticrecombi - DNA nation . Rather , specific fragments of and , thus , specific genes , can be obtained from any organism , modified in the labora - tory as desired , and , in theory , introduced in - to any plant . D C The ultimate success of plant genetic engi - neering depends entirely on new information being made available by basic research in plant molecular biology . It is essentialthat we of elucidate the structure and organization important plant genes in order to understand the molecular processesthat control their or - derly expression . We must develop methods for identifying potentially useful plant genes DNA frag - at the molecular level , obtaining ments in which they are contained , and con - structively altering them outside the plant . We need to determine the mechanisms that of genes during coordinate the expression plant growth and development . Only then can efficient procedures be designed for in - troducing novel genetic information into Electron microscope visualization of soybean seed protein genes . Gene structures were investigated by a procedure known as R - loop mapping . Seed plants in a biologically relevant way . protein messenger RNA was reacted with recombinant DNA clones containing Although much remainsto be learned , and seed protein genes . The resulting DNAlRNA hybrid structure , called an R - loop , advances in plant molecularbiology have not was then visualized in the electron microscope . ( A ) shows an R - loop for a gene been as rapid as in bacterial , fungal , and ani - that lacks introns . ( B ) shows an R - loop for a gene with an intron . Actual seed a large body of infor - mal molecular biology , ( C ) , and protein gene structures shown are the lectin gene , which lacks an intron 11s storage protein gene , which has an intron ( D ) . Both genes are expressed in mation on plant gene structure , organization , soybean embryos and direct the synthesis of proteins present in the economically and expression has begun to accumulate . important dry seeds . The molecular organization of plants is ex - ceedingly complex . Their chromosomes , like 8 CALIFORNIA AGRICULTURE , AUGUST 1982 cumulate on the molecular mechanisms that relevant to plant genetic engineering . those of animals , contain enough genetic in - control the pattern of gene expression in formation to code for hundreds of thousands The structure of plant genes is variable . plant cells . The activitiesof genes may be reg - of the sequences of different proteins . Most Some appear to be similar to genes typical of ulated at any one of several biochemical present in the DNA exist in only single copies , lower eukaryotic organisms , such as fungi , in levels . For example , the soybean , like other and the vast majority of the genes are thought that theycomprisecontinuous protein coding crop plants , has a set of genes that codes for of DNA . However , a to occur in this type regions - that is , they are colinear with their seed storage proteins . These genes are trans - largeproportion of plant DNA consistsof se - messenger RNA products . Most , however , cribed into messenger RNAs that accumulate quencesthat arerepeated from a few to sever - of are more complex , as is characteristic to high levels in developing seeds , but their al thousand times in each nucleus . Some of genes in birds and mammals . In these cases , expression cannot be detected elsewhere in these reiterated DNA components - for ex - regions of the genes that code for protein , the soybean plant , even by extremely sensi - called exons , are interrupted by noncoding ample , those coding for certain specialized tive assay methods . Such results argue that protein products and for ribosomal RNA - sequences , termed introns . The cell modifies of the developmentally specific expression the RNA products of such genesin a reaction have known functions . The roles the others these genes is controlled at the level of trans - of the plant are currently only whereby the noncoding regions are excised play in the life cription , as is typical of lower organisms . In a matter for speculation . and the coding sequences spliced together to contrast , many of the genes that encode mes - can direct yield mature messenger RNAs that Estimates have now been made of the ac - RNAs occurring specificallyin one or - senger the synthesis of functional proteins . It has of a plantâ??s geneticinforma - tual proportion gan system are also transcriptionallyactivein tion that is transcribed into messenger RNA 75 percent of a typ - been estimatedthat about other organ systems . The organ - specific ex - ical plant gene consists of one or more introns . molecules , the nucleic acid sequences that of post - Recombinant DNA and DNA sequencing pression of these genes is the result of proteins . Ap - directly specifythe synthesis transcriptional control mechanisms . Some technologies have also allowed investigation proximately 11 percent of the unique DNA , RNAs present in the nucleus are selected for of DNA regions adjacent to plant genes . Se - or 5 percent of the entire genome , of tobacco quences required for the initiation transport to the cytoplasm , the site of cellular of RNA is expressed as messenger RNA in the sporo - protein synthesis ; others are rapidly broken synthesis , excisionof introns , and post - trans - 60 , OOOdif - phyte plant - equivalent to about of the polyadenylic acid down within the nucleus itself . criptional addition ferent genes needed to program and maintain We know virtually nothing about specific sequences normally found in messenger of the life cycle . Some of these this phase biochemical events that underly these regula - RNAs have been identified and are similar to genes are transcribed into messenger RNAs tory processes.Suchinformation isof seminal those observedin other types of organisms . It all of the organ systems of the present in importance to geneticengineering . For exam - has been discovered that plant genes having plant . It is thought that these genes code for of a ple , transfer into a plantâ??s chromosomes very different patterns of expression may be proteins whose activities are required for the gene conferring a desirable alteration in leaf closely associated with one another in the ge - lives of all cells , the â?? housekeeping â?쳌 genes . physiologycould kill theplant if thegenewere nome . In contrast , genes with closely related Many others , however , are expressed in only also expressed in the roots . . We have to ensure forms and functions may be dispersed one organ system . These genes presumably that genes introduced into plants in new ways throughout the genome . are responsible for the specialized functions will be expressed in the appropriate tissue and of Although the biological significance of the cells comprising the leaves , roots , at the proper time in development . these features of gene structure and organiza - stems , petals , anthers , and ovaries . tion is not entirely clear , such observations The actual physiological functions of the The study of plant gene expression has re - vast majority of plant genes are simply un - are yielding valuable clues to the processes cently been facilitated by application of re - known , even though they are certainly of controllingplant gene expressionand are sug - combinant DNA technology . Entire plant ge - substantial biological importance . The prob - gesting directions for future research . They nomes have been fragmented and introduced of most messen - lem is that the cellular levels are also providing the basis for modifying into bacterial viruses to form recombinant ger RNAs are exceedingly low , making them plant genes in the laboratory , adapting genes DNA sequence â?? libraries . â?쳌 Similarly , DNA difficult to identify and characterize . How - from other organismsor plant speciesfor use copies of messenger RNA species have been ever , many of the genes coding for rare - class in solving specificagricultural problems , and joined to bacterial plasmids and propagated of messenger RNAs are also expected to be constructing vectors to introduce novel ge - in bacteria . Individual recombinant DNA considerable agricultural significance . The clones containingplant genes of interest have netic information into plants . The following question is , how can such genes be more of specific reports present some examples been isolated from these collections and readily isolated and studied ? plant gene systems currently under intensive of gene analyzed , revealing several features Information is only now beginning to ac - U.C . research groups . examination by structure and organization that are clearly CALIFORNIAAGRICULTURE . AUGUST 1982 9