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J . Cell Sci. Suppl. 7, 123-138 (1987)Printed in Great Britain © The Company o f Biologists Limited 1987
123
ASPECTS OF THE A c /D s TRANSPOSABLE ELEMENT
SYSTEM IN MAIZE
W. J A M E S P E A C O C K , E L I Z A B E T H S. D E N N I S , E. J E A N
F I N N E G A N , T H O M A S A. P E T E R S O N a n d B R I A N H. T A Y L O R
CSIRO Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia
S U M M A R Y
Studies of the Ac (Activator) transposable element provided the data which led Barbara
McClintock to postulate that certain segments of chromosomes could transpose to different
locations in the genome. McClintock also recognized the existence of Ds (Dissociation) elements
which could transpose, but only in the presence of a trans-acting Ac element elsewhere in the
genome. D N A sequences corresponding to Ds and Ac have now been identified, and an
understanding of many of the properties of these transposable elements in the maize genome has
been acquired in recent years.
It is known that cryptic Ac elements and members of at least two families of Ds elements occur in
the genome of all maize lines examined. Ds elements also occur in Teosinte and the more distantly
related Tripsacum. We discuss the possible origin of these elements and consider the mechanism of
activation of cryptic Ac elements. A recent molecular analysis of a transition of an Ac-derived D i
element back to an active Ac element suggests one molecular mechanism by which changes in the
activity state of Ac may occur.
Distinctive phenotypes created by controlling elements within a target gene have been shown to
be governed by the properties of the insertion element and the position of the insertion within the
gene. Genetic effects include modulation of gene expression, alteration of gene products, instability
of mutant phenotypes, deletion and duplication of chromosome segments and the production of
chromosome rearrangements. We describe an example where a Ds insertion generates an additional
intron in the A dhl gene which reduces gene expression through mRNA instability. We also discuss
an Ac-dependent modulation of P gene activity in glume and pericarp tissues of maize which may
be attributed to an alteration either in patterns of gene expression or the developmental biology of
the flower.
The molecular consequences of Ac and Ds insertions and excisions are known at the DN A
sequence level but little is known of the mechanism of transposition. An initial approach has been
to analyse Ac transcription. Preliminary results showing transcription of a limited region of Ac are
discussed. The corresponding upstream regions have been linked to the coding region of
chloramphenicol acetyltransferase (CAT) and show promoter activity following electroporation
into tobacco protoplasts.
I N T R O D U C T I O N
Maize transposable elements have at times been considered as biological
anomalies, and those studying them as participating in fringe science. Clearly such
opinions are no longer tenable. Geneticists have long sought to identify and
characterize those factors which control and modify the coordinated expression of the
thousands of genes which are needed to produce an organism, the functional unit of
biological organization. In the late 1940s Barbara McClintock identified certain
factors in maize which seemed to control gene expression; she termed these factors
controlling elements.
124 W. J . Peacock and others
McClintock (1951, 1956) recognized two fundamental genetic properties of the
maize controlling elements which she studied. First, the elements are capable of
profoundly altering the expression of specific genes with which they are associated.
Association of a controlling element with a gene can partially or completely inhibit
expression, resulting in a range of possible activities from null to near normal levels.
Expression of the affected gene can be altered in developmental time or tissue
specificity, producing new patterns of gene expression. The second fundamental
property of McClintock’s elements is their ability to transpose to new genomic sites.
At these new sites, the elements can again affect gene control.
Presently there are over a dozen different controlling element systems known in
maize. Each system may comprise several families, with each family containing
perhaps 50 or more individual elements. Freeling (1984) has suggested that up to
50 % or more of the maize genome is or was transposable. McClintock identified and
studied two such systems, namely the Spm system and the Ac/Ds system. In this
paper we shall consider primarily the Ac/Ds system; many of its characteristics apply
to other systems.
The Ac/Ds system comprises two types of elements which are distinguished by
their transpositional capabilities. Ac (Activator) elements transpose and also enable
Ds elements to transpose. Ds (Dissociation) elements transpose only when an Ac element is present; they cannot transpose without Ac, nor do they promote
transposition of other Ds elements. McClintock found that a Ds element could be
derived from Ac, suggesting that at least some Ds elements are defective versions of
Ac.Individual Ac and Ds elements have now been isolated and characterized, and
their descriptions have provided molecular explanations for some of the genetic
phenomena. It is now known that Ac and Ds elements are discrete genetic units with
characteristic structure and DNA sequence; reversion of Ac or Ds-induced mutants
is correlated with excision of the element. The effect of Ac and Ds elements on gene
expression results from an interaction of element and gene in a manner which is a
function of the sequence of each and their relative positions. It is still unclear
whether McClintock’s elements participate significantly in developmental control of
gene expression. However, studies of these elements and the genes with which they
are associated are providing an incisive view of the molecular events underlying
development.
S E Q U E N C E C H A R A C T E R I S T I C S OF Ac A N D D s E L E M E N T S
A number of examples of both Ac and Ds elements have been analysed at the
molecular level. Most of these elements were recovered from mutant loci originally
described by McClintock, although several are of more recent origin. Two Ac elements, as well as a number of Ds elements, have been sequenced in their entirety.
The Ac elements which have been sequenced were both originally detected as
instabilities in the waxy locUs of maize and were designated Ac wx-m7 and Ac wx- m9. Ac wx-m7 was unusual in that it appeared to cycle between an Ac form and a Ds
Ac/Ds tratisposable elements 125
Open reading frame map of Ac (wx—m9)232
132 k H = >
120
H in dW lI
i>104
BamUl
0 0-5
102
•Met
1-0
91
<=
1-5
294
2-0 2-5 3-0
105
3-5
$
4-0
168
4-5
167
485 , 128
< = 3
Fig. 1. Open reading frame map of Ac wx-m9. Open reading frames were determined by
computer analysis of sequence data obtained by Pohlman et al. (1984a,b). Only open
reading frames in excess of 75 codons are shown. The position of the first methionine
codon in each open reading frame is indicated by an asterisk. The 10 base pair terminal
inverted repeats are indicated by short arrows at the ends of the element.
form, while Ac wx-m9 continued to function as an Ac. Ac wx-m9 was cloned first
using a waxy gene probe, followed by Ac wx-m7, which was identified by
hybridization to both waxy DNA and a probe containing DNA from a Ds insertion
(Fedoroff et al. 1983; Behrens et al. 1984). The sequences of these elements were
found to be nearly identical (Pohlman et al. 1984a,b ; Muller-Neumann et al. 1984)
suggesting that the cycling phenotype of Ac wx-m7 was not sequence specific.
Indeed, a cycling derivative of Ac wx-m9 was discovered by Schwartz (Schwartz &
Dennis, 1986) and is described later in this review.
The Ac elements cloned from the waxy gene are 4563 bp in length and are
bounded by 10 bp indirect repeats, with an additional noncomplementary base on the
termini. The Ac insertions are flanked by 8 bp duplications of host DNA which were
apparently generated when the Ac elements entered the target DNA. Computer
analysis of the Ac sequence reveals the presence of a number of large open reading
frames, with those in excess of 75 codons shown in Fig. 1. At the present time the
significance of these open reading frames has not been established, although
sequence analysis of a Ds element which arose directly from /\c wx-m9 indicates that
a 194 bp deletion within the 485 codon ORF is sufficient to block Ac function
(Pohlman et al. 1984«). This result suggests that at least part of the 485 codon ORF
is translated into protein which is part of a trans-acting transposase or a modulator
thereof.
The internal sequence of Ac contains a number of direct and inverted repeats, the
functions of which are unknown. Electron micrographs of a segment of cloned Ac
wx-m7 DNA heteroduplexed to a cloned segment of the wx gene appeared to show a
stem structure of approximately 150 bp involving the ends of the Ac element
(Behrens et al. 1984). Subsequent computer analysis of the sequence of the Ac wx-
126 W. J . Peacock and others
m7 termini (Muller-Neumann et al. 1984) indicated that such a structure was
possible, although evidence indicating a role for this structure in vivo is lacking.
As mentioned previously, Ds elements can be generated by internal deletions of
Ac. Examples of this type of Ds include Ds wx-m6 (Pohlman et al. 1984a) and sh- m6233 (Week et al. 1984). sh-m6233 is a complex insertion consisting of one Ac deletion-derived Ds inserted in reverse orientation into a second, identical Ds. This
structure is also present in what appears to be an even more complex insertion in sh- m5933. In this locus, the double Ds insertion is repeated in deleted form at the
opposite end of a 30 kb insertion. Revertants of sh-m5933 have lost the 30 kb insert,
indicating that Ds elements can interact to move large segments of DNA (Courage-
Tebbe et al. 1983). Hybridization of the terminal regions of Ac to maize genomic
DN A indicates the presence of a large number of Ac-related sequences (Pohlman
et al. 1984a). Similar hybridizations with central regions of Ac revealed a much
lower multiplicity of sequences. This suggests that many of the hybridizing
sequences are Ac deletion-derived Ds elements, although other origins of these
sequences are possible.
A second class of Ds element is typified by the D sl element isolated from Adhl- Fm335 (Peacock et al. 1983; Sutton et al. 1984). D sl is inserted into the transcribed
leader sequence of the alcohol dehydrogenase 1 (A d h l) gene of maize, resulting in a
mutant phenotype with low ADH activity (Osterman & Schwartz, 1981). The insert
is 405 bp and does not hybridize to the Ac wx-m9 clone. Sequence analysis revealed
an 11 bp inverted repeat at the ends of the element, 10 bp of which are identical to
that of Ac, with an additional complementary nucleotide on the outermost ends. Like
Ac and the Ac-derived Ds elements, this element produces an 8 bp duplication of
host D N A upon insertion. It is genetically a Ds element in that there is a high
frequency of reversion to full ADH activity only when an Ac element is present in the
genome (Osterman & Schwartz, 1981). With the exception of the termini the
sequence of D sl is unrelated to Ac, suggesting that the 11 bp repeats are sufficient for
Ac-induced transposition.
Analysis of revertants of this D sl element provided the first data on the molecular
consequences of excision of this family of transposable elements (Peacock et al. 1983;
Sachs et al. 1983; Sutton et al. 1984). Schwartz obtained a number of reversions of
the mutant to normal levels of ADH activity which were cloned and sequenced across
the original site of insertion. In these revertants the 8 bp duplication is generally
retained, although in at least one instance a perfect excision has occurred (Dennis
et al. 1986). A common observation is that nucleotides directly adjacent to the site of
insertion have been deleted or changed to the complementary nucleotides (Sachs
et al. 1983; Pohlman et al. 1984a,b \ Week et al. 1984; Dennis et al. 1986). Models
which have been proposed to explain this observation suggest that because these
alterations do not always occur and are variable in nature, they are likely to arise
during DNA repair following excision (Peacock et al. 1984; Saedler & Nevers,
1985). Larger deletions are also associated with Ac/Ds excision (McClintock, 1950)
and may involve recombination with related sequences away from the site of
Ac/Ds transposable elements 111
insertion (Dennis et al. 1986) or another copy of the transposable element (Courage-
Tebbe et al. 1983).
The propensity of the Ds element sh-m5933 for generating chromosomal
rearrangements and breakages may be a consequence of its particular structure
(Dóring et al. 1984). Most Ac and Ds elements do not have a high frequency of
chromosomal breakage associated with their insertion/excision cycles. The occur
rence of several elements of one particular family in different loci within the genome
could lead to chromosomal rearrangements by recombination between them. This
would be comparable to the translocation-generating events analysed by Mikus &
Petes (1982). In Drosophila it has been proposed that hot spots of chromosomal
rearrangement may be due to recombination between members of a dispersed family
of transposable elements (Engels & Preston, 1981).
F A M I L I E S OF D s E L E M E N T S I N M A I Z E A N D R E L A T E D S P E C I E S
There are a number of Ac and Ds related segments in the maize genome. Using
D sl as a probe, Sutton et al. (1984) reported 40—50 bands in Southern hybridiz
ations. Fedoroff et al. (1983) reported a similar number of bands for the Ac-related
Ds family. These do not cross hybridize with the D sl segments. Segments which
hybridize at high stringency to the D sl element have also been found in Teosinte, the
immediate wild precursor of maize, and in Tripsacum dactyloides, a more distantly
related species (Gerlach et al. 1987).
Gerlach et al. (1986) cloned a number of these related segments and have shown
that in all cases the cloned fragments contained a D sl sequence of approximately
400 bp, with at least 90 °lo sequence homology to D s l . All but one of these segments
have the 11 bp inverted repeat termini characteristic of D sl and are bounded by
direct 8 bp repeats. The remaining element, D slO l, had a 10 bp inverted repeat at its
termini and was flanked by a duplication of 6bp rather than 8 bp. This suggests that
the length of the inverted repeat may influence the length of the staggered cut in the
genomic target. Other members of the D sl family have been cloned by Wessler et al. (1986) from the wx-m l mutation and by Schiefelbein & Nelson (personal communi
cation) from the bz-wm mutation. There is no obvious consensus sequence for the
target sites.
The elements from Tripsacum have all the features of the maize sequences, and
members of the maize family are at least as diverged from each other as they are from
the Tripsacum elements. It seems reasonable to assume that all D sl sequences trace
back to a single element at some time in the past. We cannot make a distinction
between the alternatives of horizontal transfer of the elements, between maize,
Teosinte and Tripsacum or a vertical evolution from a common ancestor of the three
genera. If we make the assumption that the nucleotide substitution rate in these
elements is comparable to the neutral rates of nucleotide substitution which apply to
animal pseudogenes (5X 10-9 substitutions per site per year, Li, 1983) then the D sl elements duplicated and diverged from a common sequence between 8 and 25 million
years ago. It seems that these elements have been resident in the Maydeae genomes
for a long period of time.
D s l G E N E R A T E S A N A D D I T I O N A L I N T R O N I N T H E A d h l G E N E
Maize geneticists have reported a wide range of mutant phenotypes induced with
controlling elements and some of these effects are now understood at the molecular
level. An interesting example is the Adhl-Fm335 mutant in which the D sl element is
inserted into the transcribed leader sequence of the Adhl gene (Peacock et al. 1983;
Sutton et al. 1984). In a homozygous Adhl-Fm335 stock the amount of ADH1
enzyme is decreased to about 10% of the normal level but the specific activity
(Osterman & Schwartz, 1981) and temperature stability of the enzyme remain the
same (Sachs, unpublished results). How does the insertion of the D sl element in the
upstream region cause this mutant phenotype?
Northern hybridization analyses show that the length of the Adh 1 -specific mRNA
in the mutant is approximately the same as it is in the progenitor allele (Gerlach et al.1982); however, the amount of the mRNA is 100-fold lower. Under anaerobic
conditions the mutant plant shows a 20-fold increase in ADH1 enzyme activity and
an increase in Adhl -specific RNA of 20- to 50-fold. The kinetics of induction of Adhl mRNA levels are identical to the progenitor Adhl-F plant.
The transcribed regions of Adhl-Fm335 and AdhlF were compared directly in SI
mapping experiments using probes derived from the 5' region of the gene (Peacock
et al. 1984; Dennis el al. 1987). When the probe was prepared from the progenitor
allele, RNA from both the mutant and progenitor protected exactly the same length
fragment, but with a much weaker signal in the mutant. This indicates that all the
sequences present in the progenitor mRNA are also present in the Ds mutant mRNA
and that the transcription start site is exactly the same as in the progenitor. When the
mutant mRNA is used to protect a probe synthesized from the 5' region of the
mutant gene two fragments are seen. The first fragment extends 3' from the site of
insertion of the Ds element to the first exon-intron boundary, and the second
fragment, approximately 66 bp long, corresponds to a segment extending from the
start of transcription to a point 12 bases inside the D sl element. These results
indicate that only 12 bp of the Ds insertion are present in the mRNA of the mutant
gene and the remainder of the element is processed as an intron from the transcript
(Dennis et al. 1987). The intron donor sequence is 12bp from the D sl 5' terminus
and the intron acceptor site is at the junction between the 3' end of the D sl segment
and the Adhl leader sequence.
The processing of the D sl element from the mRNA does not in itself explain the
low level of messenger activity in the mutant. Run-on transcription experiments have
shown that the mutant has approximately the same rate of transcription as the
progenitor allele (L. Beach, personal communication), suggesting the low steady-
state level of mRNA is due to instability of the RNA in the mutant relative to the
progenitor allele.
128 W. J . Peacock and others
Ac/Ds transposable elements 129
The behaviour of the Ds element as an intron implies that sequences around the
donor and acceptor splice sites should resemble the consensus sequences seen for
splice sites in both plants and animals. They do, and moreover the sequence
TCCTAAC occurs 30bp before the 3' splice site. This sequence is identical to the
consensus lariat acceptor sequence (Ruskin et al. 1984) of introns and is located in
the correct position.
We conclude that the D sl element in the Adhl-Fm335 gene has all the necessary
sequence attributes to be spliced as an intron and that although transcription occurs
at a normal rate the RNA is less stable. This provides an example of how a
transposable element can introduce a new intron into a gene.
C R Y P T I C A c E L E M E N T S A N D T H E I R A C T I V A T I O N
One of the most perplexing problems with regard to transposable elements in
maize is their origin. Southern analyses with internal central probes of Ac support
the supposition that complete or near complete Ac segments may be present in maize
genomes, even though there is no detectable active Ac element (Fedoroff et al.1983). Other transposable element systems have been identified following genetic
trauma events. Peterson (1953) found the En (Spm) element in seed which had been
exposed to the Bikini atomic bomb blast and, more recently, Rhoades & Dempsey
(1982) described new transposable element systems in stocks exhibiting chromosome
breakage phenomena. It has been hypothesized that potentially active transposable
elements may be triggered into activity by conditions of genomic stress (McClintock,
1984).
An analysis which provides one possible explanation for the activation of cryptic
Ac elements has been carried out by Schwartz & Dennis (1986). They compared
three alleles of wx-m9; wx-m9 Ac, a Ds derivative (wxm9-Ds-cy) and revertants of
the derivative to an active Ac form (wx-m9 AcR). They cloned the waxy locus from
each derivative and showed that the Ac element was present in precisely the same
position in each and that there were no differences in restriction enzyme patterns of
the elements. However, when they examined the genomic organization of the
elements there were marked differences in restriction patterns with enzymes for
which the recognition sites are sensitive to methylation. The active Ac element is
methylated only at the left end, in contrast to the inactive (Ds) derivative which is
methylated at all H pall sites that occur throughout the elements. This particular Ds derivative reverts to an active Ac state, and in a number of revertants certain H pall sites at the right hand end of the element were no longer methylated. Different
revertants had different H pall sites unmethylated but all are grouped in the region
of the putative transposase gene promoter (see later). Although demethylation is
associated with reversion to the active Ac state, it is not complete and a number of the
H pall sites and all of the P vu ll sites remain methylated. It seems that particular
regions in the promoter segment are active or inactive depending upon their state of
130 W. 7- Peacock and others
méthylation. Dellaporta & Chomet (1985) described a change from an active to an
inactive state of the Ac element in the wx-m7 allele which was paralleled by a change
in digestibility of the P vu ll sites in the element. In this case, the méthylation state of
the H pall sites was not tested, nor were revertants isolated.
A N E F F E C T OF Ac O N E X P R E S S I O N OF A D E V E L O P M E N T A L L Y R E G U L A T E D
G E N E ?
One of the most interesting aspects of the effects of transposable elements on maize
gene expression is a phenomenon termed ‘presetting’ by McClintock. McClintock
(1964) observed the presetting phenomenon in her studies of two derivatives of the
a-m2 allele. In these a-m2 derivatives, the A gene, which is required for anthocyanin
pigmentation, is associated with the receptor of Spm ; when an active Spm is present
in the same nucleus, a variegated pigmentation pattern results. Interestingly,
McClintock recorded examples in which variegation could occur in the absence of an
active Sp m ; this variegation required the previous exposure of the a-m2 derivative
alleles to an active Spm element. McClintock concluded that the previous exposure
to the active Spm element had preset the a-m2 derivative alleles to produce a
variegated pigmentation pattern. The preset pattern was found infrequently, and it
was usually not heritable. However, examples of transmission of the preset pattern
through two generations were reported (McClintock, 1964, 1965).
McClintock (1967) reported other patterns of gene expression which she inter
preted as presetting. In the c2-m2 allele, the C2 gene, which is required for plant and
kernel pigmentation, is controlled by the Spm element. Plants carrying c2-m2 produce ears with large sectors of coloured cob tissue indicative of early reversion of
c2-m2 to the active C2 allele. However, kernels within those sectors had aleurone
tissue with the variegated pattern of unchanged c2-m2. In this case, it seemed that
the Spm element at c2-m2 had preset the locus at an early stage in development to
produce certain patterns of expression later in development; a wild type (C2) pattern
in the cob tissue, and a variegated (c2-m2) pattern in the aleurone tissue.
Schwartz (1982) has proposed a similar presetting of the maize P locus involving
Ac. The P gene controls the accumulation of a red pigment produced in the pericarp
and cob tissues (Styles & Ceska, 1977). The pericarp, which is the outer layer of the
mature maize kernel, is formed by outgrowth of the ovary wall and hence is maternal
tissue. The dominant P-RR allele specifies red pigmentation in both pericarp and
cob, while the recessive P-WW allele conditions white pericarp and cob. Pigmen
tation in cob and pericarp can be independently controlled according to the allelic
state of P. For example, the allele P-WR specifies white pericarp and red cob, while
P-RW determines red pericarp and white cob.
The P -W allele, which conditions variegated pericarp and cob, was shown by
Brink & Nilan (1952) to constitute an insertion of the transposable element Mp in the
P-RR gene. [Mp (Modulator) is functionally and molecularly equivalent to Ac and
Ac/Ds transposable elements 131
will be referred to as Ac from hereon.] As a consequence of its insertion in P -W , Ac suppresses P-RR expression. Transpositions which remove Ac from P -W restore P expression, resulting in red sectors of varying size on a colourless background.
Emerson (1917), and later Schwartz (1982), noted that on ears carrying P -W , pigmentation in two regions of the pericarp, the crown and the gown, could be
independently controlled. The crown comprises the region of silk attachment at the
top of the kernel, while the gown constitutes the sides of the kernel and that portion
of the top not including the crown. Ears carrying P -W often have large sectors of
kernels with pigmented crowns (dark crown kernels); the gowns of these kernels are
usually variegated, but may be fully red or white. Multi-kernel dark crown sectors are
invariably associated with a precisely coincident sector of pigmented cob. Although
these patterns of pigmented crown and cob occur in clonally-derived sectors, they are
not heritable (Emerson, 1917; Schwartz, 1982). These patterns resemble the
patterns of c2-m2 expression in cob and pericarp observed by McClintock (1967).
In order to explain the occurrence of non-heritable crown/cob pigmentation
patterns, Emerson (1917) proposed that the pericarp derives from two distinct cell
lineages which are set apart early in embryo development. One lineage of epidermal
origin gives rise to the pericarp crown and the floral parts of the cob. A second
subepidermal lineage gives rise to the pericarp gown and the megaspore mother cells.
Mutations of P -W to P-RR can occur independently in either cell lineage. In the
epidermal lineage, these mutations result in sectors of dark crown kernels over a
coloured cob sector. However, since this epidermal lineage does not contribute to the
megasporocytes, these mutations are somatic only and thus are not heritable.
Mutations of P -W to P-RR in cells of the subepidermal lineage produce sectors of
kernels with coloured pericarp gowns. Since this lineage also gives rise to the germ
cells, these mutational events are heritable. At Emerson’s suggestion, Randolph
(1926) performed a cytological study of the distribution of pigment in the pericarp
crown and gown in dark crown P -W kernels. However, Randolph found no
restriction of pigment to epidermal or subepidermal layers as predicted by Emerson’s
model.
In the light of the negative evidence reported by Randolph, Schwartz (1982)
proposed an alternative hypothesis for the non-heritable crown/cob pigmentation
patterns on P -W ears. Schwartz suggested that these patterns result from a
presetting of the P -W allele at an early stage of development to produce a particular
tissue-specific pattern of expression later in development.
We were encouraged by Schwartz to explore the molecular basis of the preset
patterns of P expression. Since the Ac element had been cloned previously (Fedoroff
et al. 1983), we used Ac as a probe to isolate a DNA clone derived from the P -W allele (Peterson & Schwartz, 1986). We found that the P -W clone contained an entire
Ac element with a restriction map identical to Ac elements previously isolated from
the maize waxy locus. Interestingly, the Ac at P -W was not bounded by short direct
repeats of maize genomic DNA. Identical results have been obtained by Lechelt
et al. (1986).
132 W. jf. Peacock and others
We then examined the structure of the P gene in P-RR revertants using as a probe
a sequence flanking the Ac element in the P - W clone. Southern blotting showed that
the Ac element had excised from the P locus in the P-RR revertant allele. This
finding confirmed our identification of the cloned P -W allele, and also indicated that
the 8 base pair flanking repeats are not necessary for excision of Ac.We initiated the presetting studies by examining a P allele derived from kernels
within a sector of mutant tissue on a P-W /P -W R ear. Kernels within this sector had
crowns which were completely pigmented, lightly pigmented, or unpigmented. The
gowns of the kernels within this sector were largely unpigmented, in marked contrast
to the variegated gowns on kernels outside the mutant sector. Any contribution of the
P -W allele to cob pigmentation was masked by expression of the P-WR allele which
specifies red cob. Plants grown from kernels within the mutant sector produced ears
with completely colourless pericarp and cob; the crown pigmentation pattern was not
inherited. Further genetic analyses showed that the progenitor P -W allele had
mutated to a P-WW allele and had lost the Ac element.
We examined the molecular structure of the derivative P-WW allele using as probe
a DN A fragment located 3 kb from the Ac element in the P -W allele. This probe
detects RNA transcripts found specifically in the maize pericarp and cob tissue, and
not in the embryo and endosperm, suggesting that the probe represents a part of the
P structural gene. In Southern analyses, comparing the progenitor P -W and the
derivative P-WW allele we found that the P-WW allele had a deletion which removed
sequences homologous to the probe. The deletion may have resulted from imprecise
excision of Ac. These results show that the embryos (and presumably the pericarp
gown) of the kernels within the original mutant sector carried the P-WW deletion
allele. We suggest that the pericarp crown tissue carried a P -W allele, and that,
within this crown tissue, reversions to P-RR by excision of Ac produced the dark
‘crown pigmentation seen in the original mutant sector.
We suggest that these data fit the hypothesis originally proposed by Emerson
(1917) and supported recently by Greenblatt (1985). An even more important
question is whether the crown/ cob and gown tissues develop from the same cell
lineage, or from two lineages separated early in development. In our experiments, if
the gown and crown cells were derived from the same ancestral cell which suffered
the P gene deletion, we would expect that the sector should consist of completely
white kernels. The occurrence of kernels with pigmented crowns within the P-WW sector is consistent with the notion that the crown/cob and gown tissues are derived
from different cell lineages, since a deletion of the P gene occurring within the gown
lineage would not affect P gene expression in the crown/cob lineage. Clearly,
additional data are required to test this inference. However, if valid, this cell-lineage
explanation of the non-heritable dark crown pigmentation pattern might also apply to
the previously reported patterns of expression of the P -W and c2-m2 alleles which
were attributed to genetic presetting. These data do not bear on the original
observations of presetting at the a-m2 allele, but they emphasize the interaction of
transposable element, gene and development in determining a particular phenotype.
Ac/Ds transposable elements 133
A c AS A G E N E T I C T O O L I N H E T E R O L O G O U S S P E C I E S
There is evidence that Ac can catalyse its own transposition in a heterologous
species by a mechanism of excision that is probably identical to that used in maize
(Baker et al. 1986). An Ac element (Ac wx-m9) with short flanking segments of waxy DN A was introduced into tobacco cells via a T-DNA vector. In almost half of the
transformed calli analysed, Ac had transposed, creating an ‘empty site’ within the wx DN A segment. Sequencing showed that the excision event was of the type observed
in Ac/Ds revertants in maize, i.e. with small deletions and/or base changes adjacent
to the site of the original insertion. Parallel experiments using a Ds element derived
from Ac wx-m9 (Ds wx-m9) did not result in excision or transposition of Ds, implying that Ac encoded function(s) were responsible for the transpositions seen in
tobacco cells.
In maize, Ac has already been used to isolate genes in which an Ac insertion has
resulted in an altered phenotype (Fedoroff et al. 1984). In this procedure, the gene
of interest is ‘tagged’ by an Ac or Ds insertion, then identified by hybridization to an
Ac probe. One problem with this approach is the difficulty of associating changes in
mobility of any Ac hybridizing band with the mutation because of the high number
of copies of Ac and its derivatives in the maize genome. In a heterologous system,
where there is no background of Ac related sequences, transposon tagging of genes
and the correlation of a mutant phenotype with an alteration in the pattern of bands
hybridizing to Ac may be simplified. The mutant allele could then be used to clone
the corresponding wild type gene.
The ability of transposable elements to jump from one region of DNA to another
suggests that they might be harnessed for use as transformation vectors. Such a
system has already been developed using P elements in Drosophila (Spradling &
Rubin, 1982). The demonstration of normal excision and integration of Ac in the
tobacco genome indicates that Ac could potentially be of value as a vector to facilitate
the integration of defined DNA segments into a range of plant genomes. In its
simplest form, the gene to be transferred would be placed with a selectable marker
within the Ac element. Following transfer of the engineered Ac into plant cells,
either by a direct transfer method or via a Ti plasmid vector, the transposase
function of the Ac element would excise the Ac segment from its carrier plasmid and
reinsert it into host plant DNA. A complication of this approach is that the gene to be
transferred must not be integrated into the Ac element at a site which disrupts the
transposase.
A second approach which avoids this problem is to place the DNA to be
transferred within an Ac or Ds element and to rely on an intact Ac element cloned on
a separate plasmid to produce the transposase activity in trans. We have investigated
the feasibility of this approach for the stable transformation of tobacco protoplasts.
When a kanamycin resistance marker inserted into the D sl transposable element was
electroporated into tobacco protoplasts with or without Ac provided in trans, no
substantial Ac-dependent increase in the number of kanamycin resistant colonies was
134 W. J . Peacock and others
obtained. This result suggests that additional modifications to the system may be
necessary in order to produce useful levels of transformation enhancement.
One modification which may be effective is the replacement of weak endogenous
Ac promoters with much stronger promoters, such as the Cauliflower Mosaic virus
(CaMV) 35S promoter. A prime candidate for such a replacement is the region
upstream of the 167/168 codon ORF (Fig. 1), since this segment appears to have
weak promoter activity and is just upstream of a transcribed region (see next section).
We have replaced this region with the CaMV 35S promoter and have detected
enhanced transcription of the downstream region, however data are not yet available
regarding the effect of this modification on functional activity.
The tobacco system also offers the prospect of being able to analyse the function of
Ac by in vitro manipulation of Ac, followed by the introduction of the mutated Ac and subsequent analysis of its expression and function.
T R A N S C R I P T I O N OF Ac
Kunze & Starlinger (1986) have reported three different size classes of poly A
RNA that hybridized to an Ac probe in Northern analysis. Only the largest transcript
(3-5 kb) appears to be Ac specific - the smaller transcripts may be derived from
transcription of Ds elements inserted into transcriptionally active DNA, since these
are observed in both Ac containing and Ac free plants. Kunze and Starlinger suggest
that the single large transcript from Ac is initiated about 150 bp from one inverted
repeat and terminates at a position 264 bp from the other end, although cDNA clones
isolated thus far lack the 5' terminal 500 bp. Comparison of these cDNA clones with
the genomic Ac sequence indicates the presence of four introns comprising 650 bases
in the primary transcript.
Computer analysis of the sequence of the Ac element (Pohlman et al. 1984a,b) shows that in one orientation there are nine open reading frames in excess of 75
codons, which together encompass 3-5 kilobases of DNA; in the opposite orientation
there are five such ORFs, encompassing only 1-3kb of DNA (Fig. 1). The ORFs
observed, if transcribed, could represent complete coding regions of proteins, or
could be spliced to form larger ORFs by the removal of introns. We therefore
directed our initial efforts to obtaining evidence for Ac transcription of the 5' region
of the strand carrying the longest ORFs.
Two approaches were taken in the analysis of this region. Segments of Ac DNA
located upstream of both the 167/168 codon ORF and the 485 codon ORF were
positioned upstream of a chloramphenicol acetyltransferase gene in a CAT ex
pression vector and transferred into Nicotiana plumbaginifolia protoplasts by
electroporation. The vector also carried a region of the octopine synthase gene which
enhances the expression of known promoter segments (Ellis et al. 1987). After 24 h
incubation, the protoplasts were assayed for CAT activity. Low levels of promoter
orientation-dependent CAT activity were detected for both the 167/168 upstream
region and the 485 codon upstream region.
Ac/Ds transposable elements 135
The second approach was to determine whether the 167/168 codon ORF and the
485 codon ORF regions are transcribed. Probe DNA complementary to the possible
transcripts associated with these regions was prepared and used in SI mapping of
total RNA and poly A RNA isolated from Ac+ or Ac~ seedlings. The seedlings were
grown from seeds, taken from several independent cobs, which had been separated
into Ac+ and Ac~ on the basis of variegation in aleurone colour. As shown in Fig. 2,
probes la and lb initiate at the same point 693 bases 3' of the BamW\ site and differ in
length by 110 bases (800 bases and 690 bases, respectively).
Two protected fragments were observed when either one of these probes was used.
The sizes of the protected fragments were estimated to be 510 and 570 bases in length
and are identical for both probes, indicating that the region between theBamHI and
Pvul sites is not transcribed. The intensity of the two bands was approximately
102
294
84
<==]
485
105
C = 3168
< ..... " I167
128
Probe lb
Probe 2 Probe la
Fig. 2. Map of Ac transposable element showing location of probes used in Si analysis.
Restriction site coordinates and open reading frames (see also Fig. 1) were determined
from sequence data of Pohlman et al. (1984a,b). Probe DN A was prepared by primer
extension of segments of Ac cloned into single strand vectors (Yanisch-Perron et al. 1985). Probes la and lb were initiated from a 19 base primer and digested with either BamHl or
Pvul . Probe 2 was initiated from a site on the vector adjacent to the Hind i l l site and
digested w ithP tu ill. Uniformly labelled probes were hybridized to RNA, treated with Si
nuclease, and analysed on polyacrylamide sequencing gels.
136 W. J . Peacock and others
equal and they were much more intense in RNA derived from Ac-containing
seedlings. The same bands were seen when either total or poly A RNA was used to
protect the radioactive probe. The appearance of these bands in Ac~ material may
arise from a small number of Ac+ seeds due to misclassification of the Ac-containing
seed. In addition, the possibility that the transcripts observed arise from Ac-derived
Ds elements present in the maize genome cannot be excluded.
The shorter fragment is consistent with an intron splice junction located at
nucleotide 4226, approximately 500 bp from the probe initiation site. A consensus
intron acceptor site is located at this position (Mount, 1982). Other intron-like
characteristics of the upstream region include a potential lariat sequence (Ruskin
et al. 1984) located at nucleotides 4292-4299 and three potential donor splice sites
located at 4351-4361. Translation could begin at the ATG codon located a short
distance upstream. Alternate initiation, splicing, or termination, could account for
the 570 base protected fragment.
Probe 2 (Fig. 2) covers the region from Hind.Hl to P v u ll, a distance of 468 bases,
all of which lies within the 485 codon ORF. A fragment of approximately 470 bases is
protected by poly A RNA isolated from Aocontaining seedlings. A 194 bp deletion 3'
of this H in d lll site within the ORF has been shown to block autonomous
transposition in maize (Fedoroff et al. 1983). Together these results suggest that the
485 codon ORF is transcribed and translated into part or all of a protein essential for
transposition.
It is anticipated that nuclease Sj mapping with different probes should enable us to
define more exactly the regions of Ac that are represented as RNA. Northern analysis
should provide information on the size and number of transcripts while cDNA
cloning and sequencing will precisely define the intron/exon boundaries. Our
interest lies in the mechanism of Ac/Ds transposition and in the nature of protein
DNA interaction in the transposition and expression of Ac. One approach to this
problem is the generation of Ac encoded proteins in vitro for use in DNA binding
studies.
C O N C L U D I N G R E M A R K S
Although we now have a molecular description of several Ac/Ds transposable
elements and the events accompanying their insertion and excision, little is known
about their movement and how it is regulated. Our approach to this question will be
to try to characterize the RNA and protein products of these elements, as well as the
regulatory sequences which control their expression. In this way we hope to address
some of the remaining questions about Ac activity, e.g. whether there is developmen
tal control of Ac movement, whether genomic stress causes Ac activation, how
methylation/demethylation might function to regulate Ac movement, and what is
the basis for the inverse dosage effect of Ac.The study of transposable elements at the genetic and molecular levels has already
yielded valuable insight into how these elements function and interact with their
target genes. We anticipate that continued study of these elements at the molecular
Ac/Ds transposable elements 137
level will be of practical benefit in exploiting these elements for gene tagging and
mutagenesis, and as potential vectors for gene transfer.
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