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166

Controlling gene expression in transgenics
Daniel R Gallie
The repertoire of cis-regulatory elements has increased
to a level of sophistication that offers considerable spatial
and temporal control over transgene expression. Recent
advances made with transgenes have revealed that the
control of their expression is also influenced by factors that
range from transgene copy number and arrangement to
nuclear architecture and chromosomal location. These factors
must now be included with the standard considerations of
transcriptional and translational enhancers of gene expression
during transgene design.

Addresses
Department of Biochemistry, University of California Riverside,
CA 92521-0129, USA; e-mail: drgallie@citrus.ucr.edu
Current Opinion in Plant Biology 1998, 1:166–172
http://biomednet.com/elecref/1369526600100166
 Current Biology Ltd ISSN 1369-5266

Abbreviations
CP
coat protein
dsRNA
double-stranded RNA
MAR
matrix attachment region
PVX
potato virus X
RdRp
RNA-dependent RNA polymerase
TEV
tobacco etch virus
UAR
upstream activator region

Introduction
The first phase of transgenic technology in the 1980’s
focused on the isolation of genes and developing gene
transfer protocols. Once these initial goals had been

reached, the emphasis changed from simple gene transfer
to one of ever increasing control over the spatial or temporal control of gene expression. Transgenic technology is
also being used to control endogenous gene expression as a
means to generate functional gene knockouts. This review
will highlight some of the more intriguing and innovative
paths that transgenic technology has traveled in recent
months.

The influence of MARs and insertion site on
transgene expression
Matrix attachment regions (MARs) are AT-rich (>70%)
chromosomal DNA regions attached to the nuclear matrix
that often flank actively expressed genes [1–3] and affect
expression through influencing chromatin structure. MARs
are 1 kb long and are separated by loop domains ranging
from 5–200 kb [4]. A MAR flanking a transgene can
increase expression 4–140-fold in plants (reviewed in
[5•]), perhaps by maintaining the chromosomal region
containing the transgene in an open configuration to
facilitate communication between an enhancer and a

promoter [6].

Analysis of insertion sites following direct gene transfer
in rice revealed massive rearrangements of genomic DNA
at the site and in flanking regions of the transgene
[7]. In contrast, Agrobacterium-mediated T-DNA insertion
typically does not involve genomic rearrangements and
usually only a small number of T-DNA insertions (low
copy number) are made. The insertions also exhibit a less
complex expression pattern than those made using direct
gene transfer [8], although an analysis of transgene stability
in Nicotiana tabacum and N. plumbaginifolia indicated that
Agrobacterium-introduced T-DNA loci were destabilized
during in vitro culture [9]. Stable transgene expression was
observed when a simple T-DNA insertion was flanked on
one side by a MAR in gene-rich regions that are close to
the telomere [10•]. In contrast, low or unstable expression
was associated with rearranged, multiple or incomplete
copies of the T-DNA flanked by prokaryotic vector
sequences that insert into paracentromeric or intercalary

locations within a chromosome (i.e. regions distant from
telomeres) [10•]. Although stably expressed loci may
not completely escape the repression of expression
associated with gene silencing [10•], MARs may reduce its
severity [5•].

Gene silencing
Awareness of gene silencing arose only after the advent of
transgenic plant production on a wide scale. Since then,
much work has been done to elucidate what now appears
to be several mechanisms that lead to gene silencing.
Silencing is divided into two categories: homology-dependent gene silencing and post-transcriptional gene silencing
(or co-suppression). The former arises when a host senses
the presence of numerous copies of identical or highly
related genes and responds by methylating each copy of
the gene, thus resulting in their transcriptional repression.
This may be a manifestation of a defense mechanism that
evolved in plants in response to transposable elements as
a means to repress their further introgression throughout
the genome [11]. See the review by Matzke and Matzke

(this issue, pp 142–148) for a more detailed discussion of
this type of silencing.
Co-suppression requires transgene expression [12•] and
increases with the transcriptional state and copy number
of the transgene [13•,14••]. For example, when a gene
encoding chalcone synthase (Chs) was inserted under the
control of a 35S promoter containing multiple copies of the
35S upstream activator region (UAR) more co-suppression
was observed than when only one copy of the UAR was
used [14••]. Nonsense codon-mediated destabilization of
Chs mRNA reduced co-suppression [14••]. These data support an RNA threshold model in which co-suppression is
triggered by a high level of transgene RNA (Figure 1). The
threshold may be either a direct detection of sense RNA

Controlling gene expression in transgenics Gallie

167

may not be required to initiate gene silencing [17].
Further work revealed that this silencing resulted from

multimeric transgene/T-DNA loci arranged as inverted
repeats in which silencing sequences are proximal to
the center were more effective than when the repeats
were distal to the center [18•]. Read through from
upstream genes in complex transgene loci proceeding
through polyadenylation sequences which inefficiently
terminate transcription [19•] may account for some cases
of co-suppression.

or an indirect effect, resulting from synthesis of transgene
antisense RNA by endogenous RNA-dependent RNA
polymerase (RdRp) activity or aberrant transgene RNA
(i.e. unspliced, truncated, poly(A)–RNA, double-stranded
(ds) RNA or modified RNA). Although these results were
obtained with simple transgene insertions, different results
were observed for complex transgene arrangements, such
as inverted repeats. In the latter case, a higher frequency
of co-suppression requiring a lower level of transcription
was observed [14••,15,16•], data which strongly support an
aberrant RNA model (Figure 1).

DNA methylation may also play a role in the production
of aberrant RNAs. Cytosine methylation within the 3′
flanking region of a gene was found to correlate with

Earlier work demonstrating that a promoterless transgene
can trigger co-suppression suggested that transcription

Figure 1

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5'

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+ (sense)

Infecting
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Cytoplasm

+_ (antisense)

Viral RdRp

Viral RdRp
(c)
Transgene-mediated 5'
5'

viral resistance
5'

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Com y T-DNA
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5'

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+
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Sequence-specific
viral resistance

} Genomic
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Viral RdRp ++
+

} Subgenomic
RNAs

5'

3'
5'
(a)
Aberrant RNA
model
3'
5'
5'

3'

5'

(b)
RNA threshold
model
dsRNA
nucleases

5'

AAAAA-3'

3'
Endogenous
RdRp

dsRNA
activated
kinases

dsRNA
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5'
dsRNA
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AAAAA-3'

5'

5'
5'
Antisense
RNAs

Current Opinion in Plant Biology

Potential mechanisms proposed to explain co-suppression. Single copy insertion of a T-DNA rarely leads to co-suppression. Multiple copies
of T-DNA in a simple arrangement or having undergone complex rearrangement are shown in the nucleus integrated into chromosomal
sites. Following transcription, the RNAs are transported to the cytoplasm, although a nuclear co-suppression mechanism may occur in some
cases. (a) Aberrant RNA model: transgene RNA from a complex rearranged T-DNA locus may form extended regions of double-stranded
RNA (dsRNA) which may be degraded by active dsRNA nucleases or may be recognized by dsRNA-activated kinases which in turn activate
dsRNA nucleases. (b) RNA threshold model: high levels of transgene RNA from simple multicopy T-DNA loci may form localized regions of
dsRNA which may trigger degradation by dsRNA nucleases. Alternatively, the high level of RNA may serve as a template for endogenous
RNA-dependent RNA polymerase (RdRp) to produce antisense RNAs which form dsRNA with the template and form a target for dsRNA
nucleases. (c) Transgene-mediated viral resistance: replication of sense viral RNA by viral RdRp results in the production of antisense RNA
followed by synthesis of sense genomic and subgenomic RNAs. Expression of viral transgene RNA in combination with replicating antisense viral
RNA may trigger the activation of dsRNA nucleases and the repression of further viral replication. This transgene-mediated defense mechanism
may employ a natural viral resistance mechanism that has been observed in response to infection by some viruses, such as nepoviruses. The
dsRNA-nuclease-mediated degradation may require active translation of the target RNA. Ribosome-associated dsRNA nuclease has been
recently identified and characterized in rye [55].

168

Plant biotechnology

co-suppression and the accumulation of unproductive
RNAs [20,21]. Co-suppression may be influenced by
environmental conditions [22,23] and appears to be
elicited more easily by certain sequences, for example,
nontranslatable viral genes (N or NSM) of tomato spotted
wilt virus [24•], the GUS 3′ coding region [21], or the
3′ flanking region of neomycin phosphotransferase II (nptII )
[20], suggesting that specific cleavage sites or secondary
structure may be necessary for efficient co-suppression.
Moreover, co-suppression often resets following meiotic
division and does not reappear until a few weeks
after germination [22,25•,26,27], perhaps as a result of
a change in the amount or activity of the machinery
required for the perception or implementation of the
co-suppressed state during seed development. Expression
is not reactivated, however, when plantlets regenerate
through mitotic division, that is to say, in vitro from
co-suppressed leaf explants [25•].
Although aberrant or antisense RNAs produced as a
consequence of high transgene expression may initially
induce co-suppression, how these RNAs continue to exert
a direct effect once the level of sense RNA, upon which
they may depend for their continued production, has
dropped following the onset of co-suppression remains
an outstanding question. Modification (e.g. methylation
of the coding or 3′ flanking regions) of the silenced
gene may increase aberrant RNA production, although
the reactivation of expression of post-transcriptional gene
silencing following meiotic division is quite unlike the
persistence of homology-dependent gene silencing (i.e.
methylation of the promoter and 5′ flanking region resulting in transcriptional repression) following meiotic division
and transgene segregation, suggesting that methylation
may not play a significant role in post-transcriptional
gene silencing. It is more likely that the detection of
aberrant RNAs, for example through intramolecular or
intermolecular basepairing, may result in cellular changes
that sensitize the cell to aberrant RNA in order that the
co-suppressed state can persist following the reduction of
the original signal.
A diffusible signal from tobacco stock co-suppressed with
a nitrate reductase (Nia2) transgene triggered co-suppression in a grafted, non-suppressed Nia transgene scion even
when the two were separated by 30 cm of stock of a
nontarget wild-type tobacco [28••]. This systemic acquired
silencing required active transcription from the transgene
in the target scion, was specific to the transgene in the
target, was observed when the stock and scion (recipient of
the graft) were both transgenic for either nitrite reductase
(Nii) or gus, and did not affect wild-type scions [28••].
Replicating viruses can also trigger co-suppression [29•]
(Figure 1). The presence of a viral transgene can result
in transgene-mediated resistance to the virus, even if the
infecting virus contains only a 60 nucleotide region of
homology with the viral transgene [30•]. Two types of

RNA-mediated resistance to viral pathogens have been
described, ‘immunity’ and ‘recovery’, and are probably
highly related. Tobacco containing four or six copies of
a nontranslatable tobacco etch viral (TEV) coat protein
(CP) gene triggered co-suppression of the viral coat protein
transgene in these immune plants prior to TEV infection,
although 20–28 days of growth following germination was
required to manifest both co-suppression and immunity
[31••], observations that suggest that perception and/or
implementation of the co-suppression mechanism may be
under developmental control. The one or two copies of
the TEV CP transgene present in the plants exhibiting
a recovery phenotype were insufficient to trigger co-suppression prior to TEV infection and the plants remained
susceptible until replicating TEV triggered co-suppression
and viral inhibition in new leaves [31••]. Transgene RNA
degradation intermediates in co-suppressed tissue were
present on polysomes as poly(A)– RNAs with intact 5′
ends and polyadenylated 3′ fragments [31••], suggesting a cytoplasmic location for co-suppression. Similar
poly(A)– RNAs were observed with Chs transgene mRNA
[32•] although polysome association was not examined.
Transgene-mediated viral resistance (Figure 1) may be a
manifestation of a natural virus defense mechanism: only
infected and adjacent Nicotiana leaves inoculated with
nepovirus exhibited viral symptoms whereas leaves that
subsequently developed had reduced viral levels and were
resistant to further infection with similar viruses [33••].
One prediction from viral-mediated co-suppression is that
wild-type viral RNAs, in which newly synthesized viral
RNAs are packaged by the CP, might serve as weaker
inducers of co-suppression than would CP null mutants
that no longer encapsidate the viral RNA. Such results
were obtained with a CP-deficient potato virus X (PVX)
transgenic construct in which the CP gene was replaced by
the gus gene, resulting in a greater difference between the
highest to lowest levels of GUS expression in transgenic
plants compared to transgenics in which GUS expression
was driven by a 35S promoter [34•]. These observations
suggest that co-suppression was triggered in some plants
perhaps as a consequence of a higher level of unpackaged
viral RNA accumulating earlier in the infection cycle.
A post-transcriptional silencing mechanism in Neurospora
crassa that is transgene-mediated is known as quelling.
Quelling defective (qde) mutants [35••] fail to quell
transgenes suggesting that the qde gene product is not
transgene specific. As high levels of transgene expression
do not cause quelling, qde genes may function by sensing
aberrant RNAs or by targeting and degrading the native
RNA [35••].

Cis-acting regulatory elements
New enhancers and promoters providing tissue-specific
[36,37], developmental [38], environmental [39] and
growth-regulator-controlled or hormonal [40,41,42•] regulation continue to be identified, increasing the repertoire

Controlling gene expression in transgenics Gallie

available for tailoring transgene expression to specific
needs. Cis-regulatory elements are increasingly turning
up in locations other than the 5′ flanking region of
a gene (Figure 2). Although an intron often increases
expression post-transcriptionally, introns can also contain
other regulatory information. For example, a 3.8 kb
intragenic region containing the large second intron of
AGAMOUS (AG) is required for the correct spatial and
temporal expression of AG in Arabidopsis flowers [43••].
The Me1 gene from Flaveria bidentis requires an apparent
enhancer-like element in the 3′ flanking region for
high-level expression in leaves [44], whereas the spinach
PsaD gene requires its intron for correct plastid- and
light-dependent regulation [45].

The 5′-leader and coding region of the pea plastocyanin
(PetE ) gene is required for proper light- mediated
regulation [46] and is similar to that observed for pea
ferredoxin (Fed-1) mRNA [47]. In contrast, the 5′ leader
of the spinach PetE gene is required for full promoter
activity and is not involved in light-dependent regulation
[48]. A light-responsive element has also been reported
within the coding region of the tobacco psaDb gene
[49]. These examples, in addition to previously reported

169

cases, illustrate the diversity of locations and functions of
cis-acting regulatory elements.

Transgenic dominant mutants as an
alternative to gene knockout
Although gene knockouts are easily performed in yeast
and are possible in mice, plants have not yet proven to
be amenable to targeted chromosomal deletions. Antisense
or co-suppressed approaches are currently the most widely
used, species-independent means to repress specific gene
expression. To these must now be added the use of
dominant transgene mutants. The introduction of the
Arabidopsis ethylene receptor mutant, etr1-1, previously
shown to inhibit ethylene perception in Arabidopsis [50],
into tomato and petunia resulted in significant delays in
fruit ripening, flower senescence and abscission, demonstrating the interspecies effectiveness of this dominant
mutant [51••].
The floury2 maize seed, characterized by a soft, starchy
endosperm, reduced levels of zein seed storage protein,
and an elevated lysine content are a consequence of an
alanine-to-valine substitution in the signal peptide of the
24 kDa precursor to a 22 kDa zein that prevents correct

Figure 2

Transcriptional enhancers
mRNA stability elements
Translational
Transcriptional
enhancers
enhancers

Intron-mediated
enhancement

Transcriptional
enhancers

5' leader

MAR

Promoter

mRNA stability
elements
Transcriptional Transcriptional
enhancers
enhancers

3' UTR

Transcriptional
enhancers

mRNA stability
elements

Poly(A)
site

MAR

Transgene

Exon sequence

Intron sequence

Transcriptional
start site
Current Opinion in Plant Biology

Potential cis-acting elements that can contribute to the transcriptional or post-transcriptional regulation of transgene expression. The transgene is
shown integrated into the genome near a matrix-attachment region (MAR) in the 5′ and/or 3′ flanking sequence. Transcriptional enhancers may
be present in the 5′ and/or 3′ flanking regions (i.e. downstream of the site of polyadenylation), immediately downstream of the transcriptional
start site, or within intron sequences. The presence of an intron can also enhance transgene expression post-transcriptionally. Translational
enhancers can be present in either the 5′ leader and/or 3′ untranslated regions (UTRs) of the transcribed sequence. Elements that influence
transgene mRNA stability can be present in either the 5′ leader and/or 3′ UTRs regions or within the coding region. Sequences within the
coding region (e.g. secondary structure or a high incidence of rare codons) that cause translational pausing can affect the rate of protein
synthesis.

170

Plant biotechnology

processing and results in the inappropriate accumulation
of the mutant zein protein in the endoplasmic reticulum.
Introduction of the mutant transgene into wild-type maize
resulted in the floury2 phenotype [52]. This dominant
mutant approach may prove to be useful in altering seed
storage protein characteristics not only in maize but in
other cereals.

tivated cellular factors in plants exhibiting co-suppression
or systemic acquired silencing may reveal how these
phenomena may be implemented. Ironically, the recent
discovery of co-suppression (quelling) in fungi and the
isolation of quelling-deficient mutants in Neurospora [35••]
may represent the most promising developments in
unraveling the decade-long quest to uncover the basis of
co-suppression in plants.

Conclusions
From this brief overview of some recent studies in
transgene expression, it is clear that a wealth of regulatory
mechanisms exists in plants and many may yet be
discovered. Those interested in transgene design must
now be aware of the many types and locations of cis-acting
regulatory elements as well as how and where integration
may affect transgene expression. In contrast to the
complex and, in some instances, contradictory observations
made in earlier work, reports within the past year have
begun to reveal that several different mechanisms may
be contributing to gene silencing and that these may be
manifestations of defense strategies that have evolved in
response to transposons and viral invasion. The presence
of such natural defense mechanisms raises questions of
how expression from multigene families escapes the same
repression observed during co-suppression. Members of
many multigene families, however, are expressed in a
tissue-specific manner so that not all are actively expressed
in the way that multicopy transgenes are. Expression from
multigene family members that are scattered throughout
the genome does argue against a DNA-pairing-mediated
means of producing aberrant RNAs that might function as
the trigger for post-transcriptional gene silencing.
Several predictions can be made from the evidence
suggesting that co-suppression requires the participation of
dsRNA nuclease activity. Viral transgenic plants infected
by a coat protein deficient, homologous virus might be
expected to ‘recover’ faster, with a greater frequency, or
to a greater extent than with the wild-type virus. The
possible role of dsRNA in triggering co-suppression and
the diffusible signal observed in grafting experiments
with Nia2, Nii, and gus transgenic plants raises the
possibility of dsRNA detection, dsRNA induction of
protein activities, and the propagation of the signal
throughout the plant. dsRNA-activated proteins have been
well characterized in virally infected animal cells, in which
interferon serves as the induction signal for uninfected
cells [53]. A dsRNA-activated kinase has been identified
in barley [54]; it appears to be the plant homolog of
the mammalian PKR (double-stranded-RNA-dependent
kinase), a dsRNA-activated kinase that phosphorylates and
represses the activity of the eukaryotic initiation factor
2, resulting in translational repression. The 2′,5′-oligoadenylate synthase, another mammalian dsRNA-activated
and interferon-induced enzyme, generates 2′-5′-linked
oligoadenylates that specifically activate RNase L [53].
A dsRNA nuclease associated with ribosomes has been
recently identified in rye [55]. Examination of dsRNA-ac-

References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:

• of special interest
•• of outstanding interest
1.

Schoffl F, Schroder G, Kliem M, Rieping M: An SAR sequence
containing 395 bp DNA fragment mediates enhanced, genedosage-correlated expression of a chimaeric heat shock gene
in transgenic tobacco plants. Transgenic Res 1993, 2:93-100.

2.

Van der Geest AHM, Hall GE, Spiker S, Hall TC: The β-phaseolin
gene is flanked by matrix attachment regions. Plant J 1994,
6:413-423.

3.

Chinn AM, Comai L: The heat shock cognate 80 gene of tomato
is flanked by matrix attachment regions. Plant Mol Biol 1996,
32:959-968.

4.

Pienta KJ, Getzenberg RH, Coffey DS: Cell structure and DNA
organization. Crit Rev Euk Gene Express 1991, 1:355-385.

Spiker S, Thompson WF: Nuclear matrix attachment regions
and transgene expression in plants. Plant Physiol 1996, 110:1521.
A thorough review that describes the effect and function of nuclear matrix
attachment regions in plants and relates this information to how similar sequences function in animals and yeast.

5.
•

6.

Van der Geest AHM, Hall TC: The β-phaseolin 5′ matrix
attachment region acts as an enhancer facilitator. Plant Mol
Biol 1996, 33:553-557.

7.

Takano M, Egawa H, Ikeda J-E, Wakasa K: The structures of
integration sites in transgenic rice. Plant J 1997, 11:353-361.

8.

Pawlowski WP, Somers DA: Transgene inheritance in plants
genetically engineered by microprojectile bombardment. Mol
Biotech 1996, 6:17-30.

9.

Risseeuw E, Franke-van Dijk MEI, Hooykaas PJJ: Gene targeting
and instability of Agrobacterium T-DNA loci in the plant
genome. Plant J 1997, 11:717-728.

10.
•

Iglesias VA, Moscone EA, Papp I, Neuhuber F, Michalowski
S, Phelan T, Spiker S, Matzke M, Matzke AJM: Molecular and
cytogenetic analyses of stably and unstably expressed
transgene loci in tobacco. Plant Cell 1997, 9:1251-1264.
A study investigating the effect of the chromosomal position of the insertion
site on gene silencing. The authors show that stably expressed transgene
loci integrate as simple T-DNA arrangements near AT-rich regions that bind
to nuclear matrices in vitro which may also function as matrix attachment regions in vivo. Stably expressed transgene loci also integrated into gene-rich
regions near the telomeres of chromosomes. Unstably expressed loci were
those that had undergone rearrangements and had integrated into regions
distant from telomeres.
11.

Bennetzen JL: The Mutator transposable element system of
maize. Curr Top Microbiol Immunol 1996, 24:195-229.

12.
•

English JJ, Davenport GF, Elmayan T, Vaucheret H, Baulcombe DC:
Requirement of sense transcription for homology-dependent
virus resistance and trans-inactivation. Plant J 1997, 12:597603.
This is an analysis of the requirement for active transgene transcription in
transgene-mediated viral resistance. The authors show that the presence of
a transcriptionally silenced transgene locus was incapable of mediating resistance to an infecting virus engineered to contain a copy of the transgene.
13.
•

´ e´ JI Jr, Elmayan T: A
Vaucheret H, Nussaume, Palauqui J-C, Quiller
transcriptionally active state is required for post-transcriptional
silencing (cosuppression) of nitrate reductase host genes and
transgenes. Plant Cell 1997, 9:1495-1504.

Controlling gene expression in transgenics Gallie

A carefully controlled analysis which shows a positive correlation between
the copy number of a transgene or host gene and the frequency or onset of
co-suppression. Data are also presented that demonstrate that the frequency
or onset of co-suppression is reduced in the absence of transcription from
the host gene and is abolished if transcription from the transgene is impeded.
Que Q, Wang H-Y, English JJ, Jorgensen RA: The frequency
and degree of cosuppression by sense chalcone synthase
transgenes are dependent on transgene promoter strength
and are reduced by premature nonsense codons in the
transgene coding sequence. Plant Cell 1997, 9:1357-1368.
This thorough analysis correlates transgene RNA level with the frequency
and degree of co-suppression. An increase in the number of enhancers introduced upstream of a 35S promoter resulted in increased co-suppression.
In a separate approach, the authors used the presence of nonsense codons
to reduce the steady state level of transgene mRNA. When present early in
a transcript, nonsense codons destabilize mRNA. Introduction of premature
stop codons resulted in not only reduced transgene RNA levels but also a
reduced level of co-suppression.
14.
••

15.

Cluster PD, O’Dell M, Metzlaff M, Flavell RB: Details of T-DNA
structural organization from a transgenic Petunia population
exhibiting co-suppression. Plant Mol Biol 1996, 32:1197-1203.

16.
•

Jorgensen RA, Cluster PD, English J, Que Q, Napoli CA:
Chalcone synthase cosuppression phenotypes in petunia
flowers: comparison of sense vs. antisense constructs and
single-copy vs. complex T-DNA sequences. Plant Mol Biol
1996, 31:957-973.
The authors present a thorough analysis of co-suppression of chalcone synthase expression in petunia. They demonstrate that co-suppression is determined by the repetitiveness and organization pattern of the transgene
and that the genomic sequence surrounding the integration site has little
influence on the co-suppression. Analysis of transgenics carrying either a
single copy or multiple dispersed copies of the transgene suggested that
co-suppression correlates with transgene dosage.
17.

Van Blokland R, Van der Geest N, Mol JNM, Kooter JM:
Transgene-mediated suppression of chalcone synthase
expression in Petunia hybrida results from an increase in RNA
turnover. Plant J 1994, 6:861-877.

18.
•

Stam M, de Bruin R, Kenter S, van der Hoorn RAL, van Blokland
R, Mol JNM, Kooter JM: Post-transcriptional silencing of
chalcone synthase in Petunia by inverted transgene repeats.
Plant J 1997, 12:63-82.
A follow up analysis of promoterless transgene silencing in which co-suppression was correlated with T-DNA rearrangements following integration.
The authors demonstrate that insertion of inverted transgene repeats is a
potent trigger of co-suppression, particularly when the inverted repeats are
proximal to the center of the inserted DNA.
Thompson AJ, Myatt SC: Tetracycline-dependent activation of
an upstream promoter reveals transcriptional interference
between tandem genes within T-DNA in tomato. Plant Mol Biol
1997, 34:687-692.
This paper demonstrates that some polyadenylation regulatory regions that
are in common use may not result in the efficient termination of transcription.
This can lead to transcriptional interference between tandem genes within
a T-DNA. The authors also point out that such interference could occur
with other T-DNA constructs near promoters subject to regulation. Such
transcriptional read through might contribute to triggering co-suppression
for those T-DNAs that integrate in complex rearrangements.
19.
•

20.

Van Houdt H, Ingelbrecht I, Van Montagu M, Depicker A: Posttranscriptional silencing of a neomycin phosphotransferase II
transgene correlates with the accumulation of unproductive
RNAs and with increased cytosine methylation of 3′ flanking
regions. Plant J 1997, 12:379-392.

21.

English JJ, Mueller E, Baulcombe DC: Suppression of virus
accumulation in transgenic plants exhibiting silencing of
nuclear genes. Plant Cell 1996, 8:179-188.

22.

´ e´ JJP, Charles
Palauqui J-C, Elmayan T, Dorlhac de Borne F, Cret
C, Vaucheret H: Frequencies, timing, and spatial patterns of
co-suppression of nitrate reductase and nitrite reductase in
transgenic tobacco plants. Plant Physiol 1996, 112:1447-1456.

23.

Cerutti H, Johnson AM, Gillham NW, Boynton JE: Epigenetic
silencing of a foreign gene in nuclear transformants of
Chlamydomonas. Plant Cell 1997, 9:925-945.

24.
•

Prins M, de Oliveira Resende R, Anker C, van Schepen A, de
Haan P, Goldbach R: Engineered RNA-mediated resistance
to tomato spotted wilt virus is sequence specific. Mol PlantMicrobe Inter 1996, 9:416-418.
This work characterizes transgene-mediated plant resistance to viruses of a
negative sense virus. The authors show that the transgene probably targets
the corresponding viral mRNA and not the replicating viral genome.

171

Balandin T, Castresana C: Silencing of a β-1,3-glucanase
transgene is overcome during seed formation. Plant Mol Biol
1997, 34:125-137
The authors demonstrate that the silenced state of a co-suppressed transgene is maintained throughout vegetative growth and floral development but
transgene expression is restored in developing seeds. In contrast, expression was not restored in plantlets regenerated in vitro from leaf explants,
suggesting that gene silencing is reversed following meiotic but not mitotic
cell division.
25.
•

26.

Hart CM, Fisher B, Neuhaus J-M, Meins F: Regulated inactivation
of homologous gene expression in transgenic Nicotiana
sylvestris plants containing a defense-related tobacco chitinase
gene. Mol Gen Genet 1992, 235:179-188.

27.

Boerjan W, Bauw G, Van Montagu M, Inze D: Distinct
phenotypes generated by overexpression and suppression
of S-adenosyl-L-methionine synthetase reveal developmental
patterns of gene silencing in tobacco. Plant Cell 1994, 6:14011414.

Palauqui J-C, Elmayan T, Pollien J-M, Vaucheret H: Systemic
acquired silencing: transgene- specific post-transcriptional
silencing is transmitted by grafting from silenced stocks to
non-silenced scions. EMBO J 1997, 16:4738-4745.
Data are presented demonstrating that co-suppression may involve a diffusible signal. Stock that exhibited Nia2-transgene-mediated co-suppression
triggered co-suppression in a grafted, nonsuppressed Nia2 transgene scion
even when the two were separated by 30 cm of stem of a nontarget wild-type
tobacco. Systemic acquired silencing required active transcription from the
transgene in the target and was specific to the transgene in the target scion.
Transmission was unidirectional from stock to scion.

28.
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29.
Baulcombe DC: Mechanisms of pathogen-derived resistance to
•
viruses in transgenic plants. Plant Cell 1996, 8:1833-1844.
An excellent, recent review of the different types of pathogen-derived resistance to viruses, protection mediated by viral proteins, sense- and antisensemediated resistance, and the evidence supporting the various models.
Sijen T, Wellink J, Hiriart J-B, van Kammen A: RNA-mediated virus
resistance: role of repeated transgenes and delineation of
targeted regions. Plant Cell 1996, 8:2277-2294.
The authors show that an infecting virus containing as little as a 60
nucleotide region of homology with a transgene is sufficient to trigger
transgene-mediated viral resistance. The arrangement of the transgene in
the genome and perhaps the extent to which the transgene’s coding region
is methylated may be important in determining the strength of the resistance.
As similar fates of the recombinant viral genomes were observed, whether
sequences of a sense or antisense orientation relative to the transgene were
used, a similar mechanism may be used in both cases.

30.
•

31.
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Tanzer MM, Thompson WF, Law MD, Wernsman EA, Uknes S:
Characterization of post-transcriptionally suppressed
transgene expression that confers resistance to tobacco etch
virus infection in tobacco. Plant Cell 1997, 9:1411-1423.
The authors show that the observed ‘immunity’ and ‘recovery’ resulting from
transgene-mediated viral resistance are probably manifestations of the same
phenomenon. Tobacco containing four or six copies of a nontranslatable
tobacco etch viral (TEV) coat protein (CP) gene triggered co-suppression
in immune plants prior to TEV infection, although 20–28 days of growth
following germination was required to manifest both co-suppression and
immunity as the silenced transgene underwent meiotic resetting. Prior to the
onset of the silenced state, these plants were susceptible to TEV infection.
The presence of only one or two copies of the TEV CP transgene was insufficient to trigger co-suppression prior to TEV infection and such transgenics
were susceptible until the multiplying TEV triggered co-suppression. This
co-suppression, in turn, led to suppression of TEV replication in new leaf
growth resulting in the ‘recovery’ observation. Transgene RNA degradation
intermediates were present on polysomes as poly(A)– RNAs with intact 5′
ends and polyadenylated 3′ fragments in co-suppressed tissue, suggesting
that the RNAs were targeted for destruction following their transport to the
cytoplasm and their recruitment onto polysomes.
Metzlaff M, O’Dell M, Cluster PD, Flavell RB: RNA-mediated RNA
degradation and chalcone synthase A silencing in petunia. Cell
1997, 88:845-854.
Fragments chsA mRNA were examined in silenced petunia flowers. Both
poly(A)+ and poly(A)– mRNA fragments were observed. A region within the
3′ end of chsA mRNA was more resistant to degradation than was the remainder of the mRNA, suggesting that degradation may be initiated through
endonucleolytic cleavage.
32.
•

Ratcliff F, Harrison BD, Baulcombe DC: A similarity between viral
defense and gene silencing in plants. Science 1997, 276:15581560.
In this paper, the authors present evidence demonstrating the similarity between natural nepovirus-induced recovery and transgene-induced gene silencing. Plants which had recovered from nepovirus infection were only resistant to infection by an unrelated virus if a portion of nepoviral sequence

33.
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Plant biotechnology

had been introduced into the second infecting viral genome, demonstrating
that virus resistance is sequence specific. This type of virus-mediated resistance may induce a resistance mechanism similar to that observed with
transgene-induced co-suppression.
Angell SM, Baulcombe DC: Consistent gene silencing in
transgenic plants expressing a replicating potato virus X RNA.
EMBO J 1997, 16:3675-3684.
This work demonstrates that active viral replication is required to trigger gene
silencing. No gene silencing was observed in transgenic plants expressing a
potato virus X (PVX) that is unable to replicate. Plants with a PVX transgene
carrying a copy of the gus gene (PVX/GUS) in place of the coat protein gene
(CP) exhibited a greater difference between the highest and lowest levels
of GUS expression than did plants with a PVX transgene carrying a copy of
the gus gene in addition to the coat protein gene, suggesting that lack of
encapsidation of the PVX/GUS RNA functioned as a more potent trigger of
co-suppression.

34.
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Cogoni C, Macino G: Isolation of quelling-defective (qde)
mutants impaired in post-transcriptional transgene-induced
gene silencing in Neurospora crassa. Proc Natl Acad Sci USA
1997, 94:10233-10238.
A genetic approach to determining the genetic basis for co-suppression,
referred to as quelling in fungi. The results with quelling defective (qde)
mutants suggest that the qde gene product is not transgene specific during
quelling. Quelling was not caused by high levels of transgene expression,
suggesting that qde gene function may be to sense the production of aberrant sense RNA and to target or degrade the native RNA.
35.
••

36.

37.

38.

Hong HP, Ross JHE, Gerster JL, Rigas S, Datla RSS, Hatzopoulos
P, Scoles G, Keller W, Murphy DJ, Robert LS: Promoter
sequences from two different Brassica napus tapetal oleosinlike genes direct tapetal expression of β-glucuronidase in
transgenic Brassica plants. Plant Mol Biol 1997, 34:549-555.
Van der Geest AHM, Hall TC: A 68 bp element of the βphaseolin promoter functions as a seed-specific enhancer.
Plant Mol Biol 1996, 32:579-588.
Beaudoin N, Rothstein SJ: Developmental regulation of two
tomato lipoxygenase promoters in transgenic tobacco and
tomato. Plant Mol Biol 1997, 33:835-846.

39.

Fordham-Skelton AP, Lilley C, Urwin PE, Robinson NJ: GUS
expression in Arabidopsis directed by 5′ regions of the pea
metallothionein-like gene PsMTA. Plant Mol Biol 1997, 34:659668.

40.

Shah J, Klessig DF: Identification of salicylic acid-responsive
element in the promoter of the tobacco pathogenesis-related
β-1,3-glucanase gene, PR-2d. Plant J 1996, 10:1089-1101.

41.

Rouster J, Leah R, Mundy J, Cameron-Mills V: Identification of
a methyl jasmonate-responsive region in the promoter of a
lipoxygenase 1 gene expressed in barley grain. Plant J 1997,
11:513-523.

Aoyama T, Chua N-H: A glucocorticoid-mediated transcriptional
induction system in transgenic plants. Plant J 1997, 11:605612.
A description of a tightly controlled, transcriptional induction system for use
in plants. A chimeric transcription factor containing the yeast GAL4 DNAbinding domain, the herpes viral protein VP16 trans-activating domain, and
the rat glucocorticoid receptor domain functions as the regulatory factor that
is activated by dexamethasone, a strong synthetic glucocorticoid. A 35S
promoter containing six tandem copies of the GAL4 upstream acting sequence is used to drive regulated expression from the transgene of interest.
Induction levels correlated with the concentration of dexamethasone used
and maximum expression was obtained within four hours of induction. Three
to four days were required after dexamethasone was removed to return to
pre-induction levels of expression.

regulation are located intragenically. Plant Cell 1997, 9:355365.
An important demonstration that transcriptional regulatory information is not
confined to the 5′ and 3′ flanking regions of a gene but can also be found
within the transcribed region. A 3.8 kb intragenic sequence within AGAMOUS (AG) was found to be necessary for its proper spatial and temporal expression during flower development. This intragenic sequence was
required to respond to the negative regulators of AG (apetala2, leunig and
curly leaf) and to the positive regulator, LEAFY.
44.

Marshall JS, Stubbs JD, Chitty JA, Surin B, Taylor WC:
Expression of the C4 Me1 gene from Flaveria bidentis requires
an interaction between 5′ and 3′ sequences. Plant Cell 1997,
9:1515-1525.

45.

Bolle C, Herrmann RG, Oelmuller
¨
R: Intron sequences are
involved in the plastid- and light-dependent expression of the
spinach PsaD gene. Plant J 1996, 10:919-924.

46.

Helliwell CA, Webster CI, Gray JC: Light-regulated expression of
the pea plastocyanin gene is mediated by elements within the
transcribed region of the gene. Plant J 1997, 12:499-506.

47.

Dickey LF, Nguyen T-T, Allen GC, Thompson WF: Light
modulation of ferredoxin mRNA abundance requires an open
reading frame. Plant Cell 1994, 6:1171-1176.

48.

Bolle C, Herrmann RG, Oelmuller
¨
R: Different sequences for
5′-untranslated leaders of nuclear genes for plastid proteins
affect the expression of the β-glucuronidase gene. Plant Mol
Biol 1996, 32:861-868.

49.

Yamamoto Y, Konda Y, Kato A, Tsuji H, Obokata J: Lightresponsive elements of the tobacco PSI-D gene are located
both upstream and within the transcribed region. Plant J 1997,
12:255-265.

50.

Bleecker AB, Estelle MA, Somerville C, Kende H: Insensitivity
to ethylene conferred by a dominant mutation in Arabidopsis
thaliana. Science 1988, 241:1086-1089.

51.
••

Wilkinson JQ, Lanahan MB, Clark DG, Bleecker AB, Chang C,
Meyerowitz EM, Klee HJ: A dominant mutant receptor from
Arabidopsis confers ethylene insensitivity in heterologous
plants. Nat Biotechnol 1997, 15:444-447.
In this work, an introduced Arabidopsis etr1-1 mutant receptor gene confers dominant ethylene insensitivity on tomato and petunia as it had in Arabidopsis. Expression from etr1-1 resulted in delayed fruit ripening, flower
senescence, and flower abscission. The effect was specifically on reducing
ethylene perception and not on reducing ethylene synthesis, as ethylene
evolution following pollination in etr1-1 transgenic petunia exceeded that of
the control.
52.

Coleman CE, Clore AM, Ranch JP, Higgins R, Lopes MA,
Larkins BA: Expression of a mutant α-zein creates the floury2
phenotype in transgenic maize. Proc Natl Acad Sci USA 1997,
94:7094-7097.

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Mathews MB, Sonenberg N, Hershey JWB: Interactions between
viruses and the cellular machinery for protein synthesis. In
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Hershey JWB. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press; 1996:505-548.

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Langland JO, Langland LA, Browning KS, Roth DA:
Phosphorylation of plant eukaryotic initiation factor-2 by the
plant-encoded double-stranded RNA-dependent protein kinase,
pPKR, and inhibition of protein synthesis in vitro. J Biol Chem
1996, 271:4539-4544.

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Siwecka MA: Purification and some properties of a novel
dsRNA degrading nuclease bound to rye germ ribosomes. Acta
Biochimica Polonica 1997, 44:61-68.

42.
•

43.
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Sieburth LE, Meyerowitz EM: Molecular dissection of the
AGAMOUS control region shows that cis elements for spatial