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Plant Physiol, January 2001, Vol. 125, pp. 4-8
From Cot Curves to Genomics. How Gene Cloning Established New
Concepts in Plant Biology
Robert B.
Goldberg*
Department of Molecular, Cell, and Developmental Biology,
University of California, Los Angeles, California 90095-1606
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INTRODUCTION |
It is difficult to imagine carrying
out plant research without personal computers, the Internet, GenBank,
e-mail, cell phones, gene cloning, microchips, whole genomic sequences,
expressed sequence tags, RFLPs, PCR, knock-outs, Arabidopsis,
reverse genetics, transgenic plants, and molecular biology "kits"
that are ready-made to carry out almost any type of DNA manipulation
experiment imaginable. The plant world in 1975 was vastly different
from the one in which we, as plant scientists, operate in today. The
International Society of Plant Molecular Biologists did not exist.
International forums such as the Plant Molecular Biology Gordon
Conference, the Plant-Oriented Keystone Symposia, and the Plant
Molecular Biology Congress had not been established. One of the largest
gatherings of plant scientists occurred at the annual meetings of the
American Society of Plant Physiologists and seldom more than 20 or 30 scientists attended the nucleic acids section in which the most
exciting plant molecular biology results were presented. The "real
world" was different as well. The Vietnam War had just ended, the
Cold War with the Soviet Union raged on, the Berlin Wall split Europe
into East and West, and the world economy was in an inflationary spiral due to the emergence of the oil cartel that sent the prices of gasoline skyrocketing.
Genetic engineering had been "invented" by Stanley Cohen and
Herbert Boyer 2 years earlier (11) and was still limited to an elite
number of labs that understood bacterial genetics, had the plasmid
vectors for DNA cloning, and had access to the enzymes that we purchase
in cloning "kits" today. Procedures for cDNA cloning, creating
libraries of large eukaryotic genomes, and isolating structural genes
had not yet been published. Genetic engineering was as controversial
then as genetically modified organisms are today. The Asilomar
Conference took place in 1975, and scientists who wanted to use the
emerging tools of genetic engineering were required to follow strict,
self-imposed guidelines that specified the conditions under which DNA
manipulations could be carried out in the laboratory. Demonstrations
occurred across the globe forecasting that "monsters" would be
created by the new gene splicing techniques and one city (Cambridge,
MA) attempted to ban genetic engineering altogether. Nevertheless, it
was a magical time to be studying basic plant processes. For the first
time, there was a dream that one could finally "see" a plant gene
and begin to unravel the complexity of plant processes at the genome level.
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THE PRINCIPLES OF PLANT GENOME ORGANIZATION AND GENE REGULATION
WERE LAID DOWN IN THE PRECLONING ERA |
Plant genomes were investigated in the mid- to late-1970s by
quantitative DNA reassociation tools (i.e. Cot curves) that had their
origins in the 1960s when the principles of DNA denaturation and
renaturation were pioneered at the Carnegie Institution of Washington
by Roy Britten and his associates (7, 8) principles that are still
used today each time a gel blot or microchip experiment is carried out,
a primer Tm is calculated, or PCR conditions are punched into a
thermocycler. Plant genomes had been shown to contain repetitive DNA
sequences in the mid-1960s and were, therefore, considered to be
"eukaryotic-like" and similar to animal genomes in that respect (7, 8). In 1975, genome organization was the "code word" for those of
us who studied "genomics" and it was determined that plant genomes
had many families of repetitive sequences and that these repeats varied
in copy number and arrangement in the genome (17, 20). These repeats
were shown to be both scattered around the genome and localized in long
clusters and they were also shown to be flanked by complex single-copy
sequences (17, 20). Neither these repeats nor any flanking single-copy
DNA had been cloned or sequenced at this time. In fact, DNA sequencing procedures (29, 33) had not yet been invented and plant DNA sequences had not yet been cloned (3). However, the general concepts of plant
genome organization that were derived from DNA reassociation studies
have stood the test of time and have been illuminated in great detail
by a knowledge of the actual DNA sequences that span each Arabidopsis
chromosome (5).
During this same period, important principles of plant gene activity
were being established in global terms by the use of RNA-excess/DNA-RNA
hybridization techniques (i.e. Rot curves) with either cDNA or genomic
single-copy DNA probes (21, 22, 24, 25). The technique of subtraction
hybridization (or cascade hybridization as it was first referred to in
the literature) was established by Bill Timberlake in this era using
kinetically fractionated cDNA populations (36). Both cDNA and genomic
single-copy DNA subtraction procedures were used by many of us to
investigate developmental changes in plant mRNA populations (21, 22, 24, 25). Several important concepts emerged about higher plant cells in
this precloning population hybridization era. First, it became clear
that plants contained a complex set of nuclear RNAs and that only about
25% of this complexity was represented in the corresponding mRNA
population (21). Today, we know that the additional complexity in the
nuclear RNA represents primarily unprocessed introns in primary
transcripts. However, this was not understood at the time because plant
genes had not yet been cloned and sequenced, and introns had not yet been discovered in any eukaryotic gene. Second, it became clear that a
large number of genes were active in plant cells and that these genes
were highly regulated in the plant life cycle (21, 24, 25). Each plant
organ system was shown to have a unique set of active genes and it was
estimated that approximately 60,000 genes were required to program and
maintain the entire life cycle of the tobacco plant (24). This estimate
of the number of tobacco genes has stood the test of time for plants
with large genomes (i.e. corn) and, considering the "bluntness" of
the tools used and assumptions that had to be made (e.g. average mRNA
size), is not that far off from the 25,000 genes that has been shown by
sequencing to be present in the small Arabidopsis genome (5). Finally,
it was established that mRNA populations contained sequences with
varying degrees of prevalence and that both transcriptional and
posttranscriptional processes established the mRNA sequence sets
present in various plant organs and tissue types. By the end of the Cot
and Rot curve era (mid-1980s), it was clear that plant cells resembled
animal cells with respect to the number of genes and the complexity of
gene regulatory processes. It was not known, however, how any
individual gene was regulated or how sets of genes were co-expressed in
space and time.
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PLANT GENES CAN BE CLONED! |
By the end of the 1970s, exciting new procedures were developed by
Tom Maniatis and others to construct cDNA clones of specific eukaryotic
mRNAs and isolate the corresponding genes from the genome (26, 27). In
addition, techniques were devised to sequence DNA segments (29, 33),
visualize genes directly in the electron microscope in association with
their RNAs (i.e. R loops; 38), and detect specific DNA fragments and
mRNAs using DNA and RNA gel blots, respectively (1, 34). These
procedures established a new revolution in molecular biology because,
for the first time, the structures of individual genes could be studied
and their expression patterns, mechanisms of regulation, and
evolutionary origins analyzed. This was an exciting period and the most
surprising and startling observation made with the new DNA cloning
techniques was that the coding regions of eukaryotic genes were
interrupted by non-coding sequences (23)! New words, intron and exon,
were introduced into the molecular biology lexicon (19) and
posttranscriptional splicing mechanisms were hypothesized and studied (23).
Only a few plant scientists at that time had any experience with
bacterial genetics, the new recombinant DNA techniques, or access to
enzymes required for DNA cloning and manipulation. In fact, most of us
did not know a restriction enzyme from a ligase and had to learn from
"scratch" how to streak and grow bacterial cells in order to
attempt to clone plant DNA sequences! In the 1970s and 1980s (as well
as today) plant scientists were playing "catch-up" with their
animal counterparts and were competing for a meager pot of money. It
was during this time that Joe Key played a huge role in
establishing the U.S. Department of Agriculture Competitive Research
Grants Program after many years of fighting the U.S. Department of
Agriculture bureaucracy and Congress. This Program has made a major
impact over the past 25 years in keeping plant sciences in the
forefront of pioneering research.
Rumors began to circulate in the late 1970s that plant DNA could not be
cloned. One well-known plant molecular biologist (who will remain
anonymous) went from meeting to meeting like Paul Revere declaring that
plant DNA was "different" from animal or bacterial DNA and that it
could not be cloned! John Bedbrook and colleagues in Dick Flavell's
lab in Cambridge, England soon showed that this was not the case and
demonstrated directly that plant DNA could be cloned and replicated in
bacteria just like the DNA from other organisms (3). They reported
their results in 1979 at a meeting in Minneapolis and the era of plant
gene cloning began with the successful cloning of ribosomal DNA and
telomeric repeated sequences from wheat (3). A pioneering principle was establishedplant DNA was similar to that of all other organisms and
could be manipulated using the same enzymes, cells, and vector systems.
Soon thereafter, libraries of many plant genomes were constructed and,
in the early 1980s, were made available to plant scientists around the
world (16, 35). In addition, the first plant structural genes were
cloned, sequenced, and visualized in the electron microscope (16, 35).
These genes, encoding seed storage proteins (16, 35) and the small
subunit of ribulose bisphosphate carboxylase (4), were shown to contain
introns similar to those in animal genes, which supported the notion
that plant cells had genetic processes similar to those in animals. It
was also demonstrated that plant genes were located relatively close to
each other on plant chromosomes (approximately every 4-6 kb) and that
genes with different expression patterns were interspersed among each
other, implying that each functioned as an independent unit (16)a
suggestion that was verified during the post-transformation era (9, 31, 32).
During the same period, cDNA libraries were constructed for almost
every imaginable plant organ system and developmental state, and cDNA
clones representing prevalent plant mRNAs, such as those encoding seed
proteins, light-regulated proteins, hormone-induced proteins, and cell
wall proteins were identified. These cDNA clones were used to
demonstrate directly that both transcriptional and posttranscriptional
processes played a role in controlling plant gene expression, but that
the primary control for most plant genes was at the level of
transcription. In addition, the vast array of cDNAs that became
available and were sequenced and studied in the 1980s began to
illuminate a range of plant developmental, metabolic, and biochemical
processes. The age of understanding "how to make a plant" had begun.
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PICKING APART PLANT GENES |
As the new era of plant gene cloning began, another revolution was
occurring in several labs that were engaged in a fierce competitive
battle to be the first to transform plant cells. The laboratories of
Jeff Schell and Marc Van Montagu (Gent, Belgium), Rob Schilperoort
(Leiden, The Netherlands), Mary-Dell Chilton and Michael Bevan
(Washington University, St. Louis; Cambridge University, UK), and Rob
Fraley, Steve Rogers, and Rob Horsch (Monsanto, St. Louis) were
utilizing the new recombinant DNA techniques to construct
Agrobacterium tumafaciens T-DNA vectors that could be
used to introduce new genes into plant cells. In the mid-1970s, Mary-Dell Chilton had shown that A. tumafaciens T-DNA
was integrated into the chromosomes of plant cells (10), setting the
stage for the revolution in plant genetic engineering that continues to this day.
In 1983, the Gent, Monsanto, and Washington/Cambridge groups showed
independently that T-DNA vectors could be used to transfer bacterial
antibiotic resistance genes into plant cells and that these genes could
be expressed if engineered with the correct promoters (6, 12, 18). Much
to the surprise of everyone in the plant research world, a different
group, headed by Tim Hall, demonstrated that the phaseolin seed storage
protein gene from french beans could be transferred to sunflower cells
and be expressed (31). This now-famous (or infamous) "sunbean"
plant made the front page of the New York Times and was
proclaimed in Time to be a "glowing achievement... the
first time a gene from one plant had been inserted into the chromosomes
of an unrelated species and made to express itself." The sunbean
experiment was reported initially at the first University of California
(Los Angeles) Keystone Meeting on Plant Molecular Biology that I
organized in April of 1983 and was greeted at the time by a now-famous
plant cell biologist (who I will not name) as "nonsense!"
Nevertheless, it showed for the first time that gene cloning and
A. tumafaciens transformation techniques could be
combined to transfer foreign genes into plant cells and study their
function. The age of plant genetic engineering and gene manipulation
had begun!
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FROM PHENOTYPE TO GENE |
Throughout the 1980s and 1990s, many plant genes were cloned and
investigated in transformed cells in order to understand the mechanisms
regulating their expression. Numerous plant promoters were
characterized and DNA sequence elements programming transcription in
specific developmental states were uncovered. The prediction of earlier
experiments on the structure and organization of plant genes proved
correct and a major new concept emergedplant genes functioned as
independent units and contained regulatory regions that could program
their correct expression in foreign cell environments. These
experiments set the stage for engineering new crops with novel traits
that are produced at specific times during the plant life cycle (28).
A major switch in plant gene cloning occurred in the beginning of
the late 1980s and early 1990s. Many interesting genes that produced
novel phenotypes were being uncovered in corn and Arabidopsis using
genetic approaches that were being adopted rapidly by plant scientists.
Because their products were unknown and/or very rare, it was not
possible to use conventional cloning methods to isolate these genes.
Several pioneering procedures were invented that circumvented this
problem and enabled a wide range of plant genes to be cloned. First,
T-DNA was shown to act as a mutagen in plant cells and, as such, could
be used as a tag to identify and clone genes that specified novel
phenotypes (15). Ken Feldmann and his colleagues established a novel
seed transformation method to obtain large numbers of T-DNA transformed
Arabidopsis lines and this method was used to identify important plant
genes, such as those involved in the control of floral organ identity
and hormone perception (14, 15). In my opinion, this was one of the
most important advances in plant biology in the past 25 years because
it allowed, for the first time, a relatively simple way to clone plant
genes associated with fascinating mutant phenotypes. The availability
of Ken Feldmann's T-DNA lines caused numerous investigators (including
myself) to adopt Arabidopsis as a model system and opened up many new
problems in plant biology to investigation. It also paved the way to
the reverse genetics approaches in use todayidentifying mutant lines associated with randomly sequenced genes (30).
A second approach to cloning plant genes was also being developed at
the same time. During the 1980s, Nina Fedoroff and Sue Wessler cloned
the corn Ac and Ds transposable elements (13). This pioneering
experiment paved the way for using transposons to tag and capture novel
plant genes for which only a phenotype could be identified. The
transposon tagging and gene cloning procedure complemented the T-DNA
approach and led to the identification of many important new genes in
several plants including corn, snapdragon, and Arabidopsis (37). It
also became possible in the 1990s to use map-based cloning strategies
to identify and clone plant genesparticularly in Arabidopsis
because of its small genome size (2). With the completion of the
Arabidopsis Genome Project last fall, and the identification of 30,000 single nucleotide polymorphisms in the Arabidopsis genome,
map-based cloning of plant genes should permit the identification of
any gene for which there is a mutant phenotypeeven those induced by
chemical mutagens such as ethyl methane sulfonate.
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BACK TO THE FUTURE |
Looking back, in 1975 plant molecular biologists were asking
questions about the number of genes in plant chromosomes and how these
genes are regulated in development. We were using precloning tools of
DNA and RNA hybridization that gave precise answers, but which could
not focus in on specific genes. The questions addressed then are being
addressed once again today in the genomics age. In a sense, we have
come full circle in trying to understand how plant chromosomes are
constructed and how populations of genes are expressed in various
cells, tissues, and organs. We progressed from studying populations of
genes and mRNAs to investigating individual cloned genes and mRNAs to
using high throughput experiments with arrays of thousands of specific
genes in order to uncover the secrets of plant cells. Twenty years
after the cloning of the first plant DNA segments (3), the genomes of
Arabidopsis and rice have been sequenced and numerous expressed
sequence tag sequencing projects have uncovered tens of
thousands of mRNAs in a wide range of plants (5). It is remarkable that
the era of gene cloning is coming to an end. Nevertheless, the
challenges are no less daunting and are even more complex: What are the
functions of all plant genes and how is the information in plant
genomes utilized in order to program plant development from
fertilization to seed dormancy?
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FOOTNOTES |
*
E-mail bobg{at}ucla.edu; fax 310-825-8201.
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TOP
INTRODUCTION
THE PRINCIPLES OF PLANT...