5231
Development 126, 5231-5243 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV0254
Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes
unregulated cell division in floral meristems
Steven E. Jacobsen1,2,*, Mark P. Running1,‡ and Elliot M. Meyerowitz1
1Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA
2Department of Molecular Cellular and Developmental Biology, UCLA, Los Angeles, CA 90095-1606,
USA
‡Present
address: USDA-ARS Plant Gene Expression Center, 800 Buchanan St., University of California Berkeley, Albany CA 94710, USA
*Author for correspondence (e-mail: jacobsen@ucla.edu)
Accepted 22 September; published on WWW 9 November 1999
SUMMARY
Arabidopsis thaliana floral meristems are determinate
structures that produce a defined number of organs, after
which cell division ceases. A new recessive mutant, carpel
factory (caf), converts the floral meristems to an
indeterminate state. They produce extra whorls of stamens,
and an indefinite number of carpels. Thus, CAF appears to
suppress cell division in floral meristems. The function of
CAF is partially redundant with the function of the
CLAVATA (CLV) and SUPERMAN (SUP) genes, as caf clv
and caf sup double mutants show dramatically enhanced
floral meristem over-proliferation. caf mutant plants
also show other defects, including absence of axillary
inflorescence meristems, and abnormally shaped leaves and
floral organs. The CAF gene was cloned and found to
encode a putative protein of 1909 amino acids containing
an N-terminal DExH/DEAD-box type RNA helicase
domain attached to a C-terminal RNaseIII-like domain. A
very similar protein of unknown function is encoded by a
fungal and an animal genome. Helicase proteins are
involved in a number of processes, including specific mRNA
localization and mRNA splicing. RNase III proteins are
involved in the processing of rRNA and some mRNA
molecules. Thus CAF may act through some type of RNA
processing event(s). CAF gives rise to two major transcripts
of 2.5 and 6.2 kb. In situ hybridization experiments show
that CAF RNA is expressed throughout all shoot tissues.
INTRODUCTION
meristem. In particular, LEAFY (LFY) and APETALA1 (AP1)
mutations produce some loss of floral meristem identity and a
partial loss of determinacy, and double mutant combinations of
LFY and AP1 lead to a greatly enhanced loss (Huala and
Sussex, 1992; Weigel et al., 1992; Bowman et al., 1993). In
addition, several mutations affecting apical meristem structure
affect floral meristem structure in the same way, with clv1,
clv2, and clv3 mutations resulting in over-proliferation and
partial loss of determinacy in the floral meristem, and
mutations in WUS and STM resulting in a loss of organs,
especially in the inner whorls.
Other genes have been shown to affect floral meristem
determinacy while not affecting apical meristem development.
Mutations in SUPERMAN (SUP) lead to increased number of
stamens and carpels, while AGAMOUS (AG) mutants show
many extra whorls of sepals and petals (Bowman et al., 1991,
1992; Schultz et al., 1991).
Most of these genes have been cloned; some are putative
transcription factors while others seem to be involved in cell
to cell signaling processes. AG and AP1 encode MADS-Box
type transcription factors (Yanofsky et al., 1990; Mandel et al.,
1992), SUP encodes a zinc-finger protein (Sakai et al., 1995),
STM and WUS encode homeodomain proteins (Long et al.,
Shoot apical meristems of angiosperms are the ultimate source
of plant aerial structures. These meristems initiate leaves,
axillary meristems, and flower meristems, while continually
maintaining a pool of undifferentiated cells (Steeves and
Sussex, 1989). In Arabidopsis thaliana the shoot apical
meristem is indeterminate, with floral meristem formation
continuing until senescence. Several genes affecting shoot
meristem function have been described in Arabidopsis.
Mutations in the CLAVATA (CLV) genes (CLV1, CLV2 and
CLV3) lead to an over-proliferation of meristem cells (Clark et
al., 1993, 1995; Alvarez and Smyth, 1994; Kayes and Clark,
1998; Fletcher et al., 1999), while mutations in the
SHOOTMERISTEMLESS (STM), and WUSCHEL (WUS) loci
cause defects in meristem maintenance (Barton and Poethig,
1993; Laux et al., 1996).
The Arabidopsis floral meristem differs from the shoot
apical meristem both in the type of organs produced (floral
meristems produce sepals, petals, stamens and carpels) and in
its determinate nature. Several genes have been shown to affect
floral meristem identity, and mutations in these genes lead to
a partial conversion of the flower meristem into a shoot
Key words: Flower development, Meristem determinacy, RNaseIII,
Helicase, Arabidopsis thaliana, carpel factory (caf)
5232 S. E. Jacobsen, M. P. Running and E. M. Meyerowitz
1996; Mayer et al., 1998), LFY encodes a new type of
transcription factor (Parcy et al., 1998), CLV1 encodes a
transmembrane leucine-rich repeat receptor kinase (Clark et
al., 1997), and CLV3 may encode its ligand (Fletcher et al.,
1999). Though it seems likely that these genes act through
regulation of downstream genes involved in the control of cell
division and differentiation functions, none of these target
genes nor the specific mechanisms involved are known.
We have identified the CARPEL FACTORY (CAF) gene,
which plays a role in floral meristem determinacy. caf mutant
flowers show extra stamens and carpels, reminiscent of sup
mutants, but also show organ morphogenesis defects. The CAF
gene encodes a protein with similarities to both DExH/DEADbox type RNA helicases and RNaseIII proteins, suggesting a
mechanism for control of floral meristem proliferation. The
Schizosaccharomyces pombe and Caenorhabditis elegans
genomes both contain a gene coding for a protein highly
similar to that encoded by CAF, indicating that CAF-type
RNAse III-helicase proteins may play a role in many
eukaryotic organisms.
MATERIALS AND METHODS
Mutant isolation and analysis
The caf mutant was isolated in a screen of mutants generated by
Agrobacterium tumefaciens-mediated transformation of Arabidopsis
thaliana seeds of ecotype Wassilewskija (Feldmann, 1992).
Phenotypic and genetic analysis of caf was carried out both in the
Wassilewskija background and in a line of caf that had been
backcrossed to the Ler ecotype five times. The phenotype was
similar in both backgrounds. The caf phenotype did not seem to be
qualitatively different when grown at 16°C, 23°C, or 29°C. All of
the reported analyses of this mutant was carried out at 23°C. Plants
were sown in a 1:1:1 mixture of vermiculite:perlite:soil, grown
under constant illumination, and watered periodically with a dilute
solution of Miracle Grow (20:20:20) plant fertilizer. Scanning
electron microscopy (SEM) was performed as described by Bowman
et al. (1989). Confocal laser scanning microscopy was performed as
described by Running et al. (1995). Negatives and slides were
scanned and digitized using Nikon Coolscan slide scanner.
Brightness, contrast and color balance were adjusted using Adobe
Photoshop 3.0 and figures were printed using a Kodak 8300 digital
printer.
CAF gene cloning
Genomic DNA was extracted from plants homozygous for caf,
partially digested with Sau3A1, partially filled with dGTP and dATP,
cloned into the partially filled XhoI site of lambdaGem-11 (Promega),
and packaged with Gigapack II Gold packaging extracts (Stratagene).
The cDNA library used was constructed from floral mRNA (Weigel
et al., 1992). The longest cDNA (cDNA8) contained 2558 bp of
sequence corresponding to the 3′ half of the full cDNA sequence.
Three separate rounds of 5′ PCR-RACE were performed using a kit
(GIBCO-BRL) as outlined in the manufacturer's instructions.
A CAF-containing cosmid clone (CosA) was obtained by
screening a Wassilewskija genomic DNA library constructed in a
pOCA18 derivative which confers hygromycin resistance to
transgenic plants (obtained from the Arabidopsis Biological
Resource Center; Olszewski et al., 1988; Schulz et al., 1995). The
CosA clone was transformed into the Agrobacterium tumefaciens
strain ASE, and whole plants were transformed using the vacuuminfiltration method (Bechtold et. al., 1993). Transgenic plants were
then crossed to caf mutant plants, and F3 populations of these
crosses were selected for lines which were homozygous for
kanamycin resistance (and hence were homozygous for the caf
mutation), and which were segregating for hygromycin resistance
(3:1, hygromycin resistant:hygromycin sensitive). Within these
populations, all of the hygromycin-resistant plants showed a wildtype phenotype (an example is shown in Fig. 7B) while all of the
hygromycin-sensitive plants showed the caf mutant phenotype,
showing that CosA complements caf. Since CosA contained not only
the CAF gene, but also an adjacent pyrophosphatase gene whose 3′
end is very close to the T-DNA, there remained a remote possibility
that the pyrophosphatase gene was also affected by the T-DNA, and
that this could contribute to the caf phenotype. To rule out this
possibility, we showed by RNA blot analysis that the size and
abundance of a 1 kb transcript homologous to the pyrophosphatase
gene was unaffected in the caf mutant. Furthermore, DNA sequence
analysis showed that the pyrophosphatase gene in the caf mutant has
a sequence which is identical to that of wild-type WS plants. Thus,
neither the structure nor expression of the pyrophosphatase gene are
affected in caf.
For mapping, an EcoRI restriction fragment length polymorphism
(RFLP) detected with CosA was mapped in a Ler × Columbia
mapping population (Chang et al., 1988). For secondary confirmation,
a HincII RFLP contained within the helicase domain of the CAF gene
was mapped in a Niederzenz X Columbia mapping population. A
similar map position was obtained.
Southern blot analysis showed that the T-DNA insertion at the CAF
locus is complex, consisting of multiple tandem copies of the T-DNA.
The junction between the CAF gene and the T-DNA was determined
by cloning and sequencing a 6.5 kb EcoRI fragment of genomic DNA
from a lambda clone isolated from the caf genomic library. This clone
contained a region of CAF corresponding the RNaseIII-like domain,
up to amino acid 1836 of CAF (see insertion site shown in Fig. 7A),
adjacent to 493 bp of sequence homologous to the right border of the
T-DNA followed by 1684 bp of sequence homologous to the vector
pBR322, followed by the Trn903, OCS3′, and Trn5-1′-2′ portions of
the T-DNA (Feldmann, 1992).
CAF RNA expression
To ensure that our DNA probes were specific for the CAF gene, we
tested two probes corresponding to the 5′ and 3′ ends of CAF, on
Southern blots. The N-terminal helicase probe was a 1809 bp
PCR product corresponding to positions 475-2283 in the 5815 bp
cDNA. The primers used to generate this product were JP237
(5′AATAGGAAACGTACTCGTAATT) and JP236 (5′AATGTATGCCAGCACCGTCTT). The C-terminal RNaseIII probe was a
1838 bp PCR product corresponding to positions 3930-5767 in
the cDNA. The primers used to generate this were JP121
(5′GTTTCTTCCACCTGAACTA) and JP131 (5′CTACATCTCGTTGAAGAGAGTA). These probes were hybridized to filters
containing genomic DNA cut with the restriction enzymes EcoRI,
XbaI, BglII, HindIII, HincII, or EcoRV. Only the fragments predicted
from the CAF genomic sequence were detected. These Southern blots
and the northern blots shown in Fig. 9 were prepared, hybridized, and
washed under high stringency conditions as previously described
(Chang et al., 1988).
In situ hybridization experiments were carried out using sense or
antisense RNA probes synthesized with T7 or T3 polymerase from
cDNA8 plasmid DNA that had been linearized with EcoRI or XhoI.
The remainder of the in situ procedure was done according to Drews
et al. (1991), with modifications by Sakai et al. (1995). These in situ
hybridization experiments were carried out twice independently, with
similar results. One of the experiments was carried out both in the
presence and absence of 500 µg/ml polyuridylic acid (Pharmacia
Biotech, product number 27-4440), to ensure that the poly(A) tail on
cDNA8 was not causing an increase in the background hybridization.
No difference in signal was observed between the poly(U) plus or
minus treatments.
carpel factory in floral meristem determination 5233
RESULTS
carpel factory floral determinacy defects
During a screen of T-DNA mutagenized lines (Feldmann,
1992) for mutants with abnormal flower development, we
found a recessive mutant with indeterminate flowers. The
mutant gene was named CARPEL FACTORY (CAF). caf
flowers show a lack of floral determinacy that is evident in both
the third and fourth whorls. Wild-type Arabidopsis flowers
produce four organ types, each occupying a separate whorl or
concentric ring (Fig. 1A). The first whorl consists of four
sepals, the second whorl contains four petals, the third whorl
has six stamens, and the fourth or inner whorl is composed of
two carpels which later fuse to form the gynoecium. caf flowers
contain a roughly normal number of sepals, petals, and stamens
in the first three whorls (Fig. 1B). However, most caf mutant
flowers contain one or two extra stamens interior to the third
whorl stamens (Fig. 1C). Occasionally, flowers contain
multiple whorls of stamens (up to 40 stamens/flower have been
observed) (Fig. 1D). These defects are reminiscent of the sup
mutant phenotype (Schultz et al., 1991; Bowman et al., 1992).
The caf gynoecium is severely affected. While wild-type
flowers produce a gynoecium consisting of two fused carpels
(Fig. 1E), caf flowers typically consist of a gynophore (a region
of internode elongation between the third and fourth whorl),
and several sterile unfused carpels (Fig. 1F). Usually,
additional growth can be seen interior to the first whorl of
carpels, forming structures which resemble carpels, carpelloid
filaments, or filaments with no obvious floral character (Fig.
1G). Occasionally, staminoid structures are also present
amongst the carpels (Fig. 1G). These ectopic organs are
produced at the flanks of a visible group of undifferentiated
cells in the center of the caf floral meristem (Fig. 1H-J).
Scanning electron microscopy of floral meristems shows that
the region of cells in the fourth whorl is larger in caf flowers
than in wild type (Fig. 2). Whereas wild-type floral meristems
terminate in the development of two carpels (Fig. 2A), caf
Fig. 1. carpel factory flowers produce extra stamens and carpels.
(A) A wild-type Arabidopsis flower containing four sepals (Se), four
petals (Pe), six stamens (St), and a gynoecium (Gy) composed of two
fused carpels. (B) A typical caf flower containing four sepals, four
petals and six stamens in the first three floral whorls. (C) A caf
flower with two sepals and two petals removed to reveal the stamens.
Red arrow points to a stamen interior to the third whorl stamens.
(D) A caf flower with all sepals and petals removed to reveal four
whorls of stamens. This extreme stamen number phenotype is
observed in about 1% of the flowers. (E) A wild-type gynoecium
consisting to two fused carpels. White arrow shows the abscission
zone of the senesced organs of the first three whorls. (F) A caf
mutant gynoecium showing a gynophore (red arrow) and three sterile
unfused carpels. White arrow shows the attachment of the organs of
the first three whorls (G) An older caf flower in which the organs in
the first three whorls have senesced and abscised. The gynoecium
consists of a gynophore (white arrow), and many carpels and
filamentous organs. Red arrow shows a stamen produced amongst the
carpels. Blue arrow shows a filamentous organ. (H) A caf flower with
an exposed meristem (red arrow). (I) Cross section of a wild-type
flower stained with toluidine blue. Two sepals, two anthers, and the
central gynoecium are visible. (J) Cross section of a caf flower
stained with toluidine blue. Two sepals and three anthers are visible
as well as a gynophore (g) and a proliferating floral meristem (m) in
the center of the flower.
fourth whorl cells continue to proliferate and give rise to
additional primordia on the flanks of an indeterminate
meristem (Fig. 2B,C).
carpel factory leaf and floral morphogenesis defects
caf affects the shape of most plant organs. As early as 9 days after
development, caf seedlings can be recognized by the thinner
5234 S. E. Jacobsen, M. P. Running and E. M. Meyerowitz
Fig. 2. Wild-type and caf fourth whorl
development. The three panels show a wild-type
(wt) floral meristem at stage 7 (Smyth et al., 1990),
which has terminated in the development of two
carpel primordia (A), and two caf floral meristems
(B,C) showing excess cells in the center of the
flower after the initiation of carpel primordia. In all
three panels, the white arrow indicates the center of
the flower, and the black arrow, a stamen. Bar in A
(for A-C) is 10 µm.
shape of the cotyledons and rosette leaves and a decrease
in the overall size of the seedling (Fig. 3A,B). Mature
rosette and cauline leaves are also thinner than wild type
(Fig. 3C). Secondary meristems, which normally arise from
the axils of rosette leaves and cauline leaves, are almost
always absent in caf plants (Fig. 3D-G). Decapitating plants
does not induce the development of these meristems,
suggesting that these meristems are missing or nonfunctional and not merely inactive. Roughly 10% of the
leaves show severe defects. Some leaves are reduced to
filamentous structures which resemble the petiole or midrib
tissue, but which lack leaf blade character (Fig. 3H).
Intermediate leaves are also produced that have patches of
blade tissue on the adaxial surface of a filamentous structure
resembling the midrib (Fig. 3I). SEM analysis confirms that
these filamentous structures have surface features similar to
wild-type petioles or midribs (lack of stomata and cells in
regular files) (not shown).
The first formed flowers are sometimes highly reduced
(Fig. 3J). Some consist of floral pedicels and very few
sepal-like organs, while others are reduced to filamentous
structures. The reduced flowers are primarily observed
within the first 20 flowers.
Some of the caf morphological defects can be traced to
early defects in the development of meristems. Scanning
electron microscope (SEM) analysis of 14-day-old wildtype and caf plants revealed that a small percentage of the
developing caf floral meristems were smaller than wildtype ones (Fig. 4A,B). These smaller primordia probably
correspond to the reduced flowers observed on more
mature plants. Flowers produced by 30-day-old plants
(after elongation of the inflorescence stem) showed fewer
defects than those from 14-day-old plants (Fig. 4C,D). We
also noted a slight increase in the size of the caf apical
meristem relative to wild type (Fig. 4A-D). This size
difference was confirmed by confocal analysis (Fig. 4E,F).
caf sepals develop in roughly the correct number and
position, but are narrower than wild-type sepals (Fig.
1A,B). SEM analysis shows that the sepals are narrower
than in wild type at the earliest stages of development (Fig.
4C,D). Though most caf petals are similar to wild-type
petals (Fig. 1A,B), occasionally they are reduced to
filamentous structures (not shown). All stamens contain a
reduced number of pollen sacs. Approximately 80% of
stamens have an anther consisting of two pollen sacs
instead of the wild-type number of four, while the
remaining 20% of stamens consist of a filament with no
Fig. 3. Morphological defects of caf mutants. Nine-day-old wild-type (A)
and caf (B) seedlings photographed at the same magnification. (C) Mature
rosette leaves from wild type (bottom) and caf (top). Rosettes of wild type
(D) and caf (E). White arrows show the axil of a rosette leaf and a stem. An
axillary meristem has developed in this position in wild type but not in caf.
Wild-type (F) and caf (G) cauline leaves showing an axillary meristem in
wild type but not in caf. (H) Filament on a caf plant in a position normally
occupied by a cauline leaf. (I) caf cauline leaf showing two patches of blade
tissue on the adaxial surface of a filamentous structure resembling the
midrib. The arrow shows the midrib region lacking the leaf blade.
(J) Reduced flowers produced during the early development of caf
inflorescences. White arrow shows a flower consisting of a pedicel and one
filamentous organ.
carpel factory in floral meristem determination 5235
Fig. 4. Apical and floral meristem structure in wild type and caf. In
14-day-old plants, wild-type (wt) meristems (A) are producing
normal flowers, while some caf meristems (B) are producing highly
reduced flowers. Arrows indicate an example of a caf primordium
which has not initiated floral organ primordia, but which occupies a
position where in wild type, sepal primordia have already developed.
Apical meristems from 30-day-old plants of wild type (C) and caf
(D). Black bar in A (for A-D) is 100 µm. (E,F) Confocal laser
scanning microscopy reveals that in 14-day-old plants, caf
inflorescence meristems are slightly larger than wild-type meristems.
While wild-type meristems were 75±0 µm in diameter, caf meristems
were 84.6±3.0 µm (numbers are mean ± standard error where n=5).
Bar in E (for E and F) is 50 µm.
anther at all (Fig. 5A-C). Normal anthers containing 4 pollen
sacs have not been observed. Some stamen primordia are much
smaller than normal (see Fig. 2B). These smaller primordia may
give rise to the antherless stamens observed in mature flowers.
The structures produced in the fourth whorl of caf flowers are
usually carpelloid or filamentous. The carpelloid organs range
in phenotype from relatively normal but unfused carpels (Fig.
5D) to filamentous organs tipped with stigmatic tissue (Fig. 5E).
While caf pollen is functional, caf plants are female sterile.
Genetic interactions of caf with mutations affecting
floral meristem patterning
We constructed double mutants of caf with several mutations
affecting floral organ identity. apetala2 (ap2) mutations affect
the identity of the first and second whorl organs. The ap2-1
mutation converts the first whorl organs to leaves and the second
whorl organs to staminoid petals (Bowman et al., 1989). The
ap2-1 caf double mutant has a phenotype similar to that of caf
Fig. 5. Floral organ defects of caf mutants. (A) A wild-type stamen
with four pollen sacs (right), a reduced caf stamen with two pollen
sacs (middle), and an antherless caf stamen (left). (B) A cross section
of a wild-type anther and (C) cross section of a caf anther containing
two pollen sacs, both at the same magnification. (D) A caf flower
bearing two relatively normal but unfused carpels. (E) A caf flower
containing filamentous organs tipped with stigmatic tissue. Bars in D
and E, 100 µm.
single mutants, except that it has a floral organ identity
phenotype similar to that of ap2-1 mutants (not shown).
pistillata (pi) mutations affect the identity of the second and
third whorl organs, converting the petals and stamens to sepals
and carpels (Bowman et al., 1989). The pi caf double mutant
shows an additive phenotype, exhibiting morphological
characteristics and meristem over-proliferation defects of caf
mutants but the floral organ identity phenotype of pi mutants
(not shown). ag mutations affect the identity of the third and
fourth whorl organs and cause floral indeterminacy, such that
ag flowers produce sepals in the first whorl, petals in the second
and third whorls, then a new flower showing the repeating
pattern (sepals, petals, petals)n (Fig. 6A; Bowman et al., 1989).
The ag caf double mutant has a phenotype similar to caf single
mutants throughout most of the plant. However, interior to the
first whorl sepals ag-1 caf double mutant flowers contain only
petals (Fig. 6B). This phenotype is reminiscent of sup ag double
mutant flowers (Bowman et al., 1992; Schultz et al., 1991),
which also contain only petals in the inner whorls. In summary,
the double mutants of caf with the floral homeotic mutants ap21, pi-1, and ag-1 show a largely additive phenotype; that is, they
display the floral meristem over-proliferation phenotype
5236 S. E. Jacobsen, M. P. Running and E. M. Meyerowitz
characteristics of the caf mutant, but also show floral organ
misspecification in a manner similar to that seen in the floral
homeotic single mutants.
We also combined the caf mutation with other mutations that
affect floral organ number. sup mutant flowers display extra cell
proliferation in the developing third whorl, resulting in the
production of an excess number of stamens (Fig. 6C; Bowman
et al., 1992; Schultz et al., 1991). sup-1 caf double mutant
flowers show greatly enhanced gynophore development and
floral indeterminacy (Fig. 6D). After producing the organs in
the first three whorls (arrow), double mutant flowers produce
many staminoid, carpelloid, and leaf-like organs. Thus, SUP
and CAF appear to be partially redundant in their control of
floral determinacy. The clv3-2 mutant has an enlarged apical
meristem and enlarged floral meristems that produce an excess
number of organs in all of the floral whorls (Fig. 6E; Clark
et al., 1995). Additionally, clv3-2 plants often produce
extra whorls of carpels interior to the fourth whorl (Clark
et al., 1995). clv3-2 and caf exhibit an essentially additive
interaction with respect to vegetative development. clv3-2
caf plants have enlarged apical meristems (similar to clv32 single mutant plants) and produce few secondary
meristems (similar to caf single mutants) (not shown).
However, clv3-2 caf flowers exhibit an enhanced floral
indeterminacy phenotype, producing a bouquet of
stamens, carpels, and filaments (Fig. 6F).
CAF cloning
caf was isolated from a screen of Agrobacterium T-DNA
insertion mutants (Feldmann, 1992). After 3 back crosses
of caf to wild-type Landsberg erecta (Ler) plants, a single
kanamycin resistance locus cosegregated with the caf
phenotype, suggesting that CAF was tagged with a T-DNA
element. To isolate plant sequences flanking the T-DNA, we
generated a caf genomic lambda library and screened for
clones containing homology to the right border of the TDNA. A clone was isolated that contained homology both
to the right border and to plant genomic DNA. Southern
blot analysis showed that this plant DNA cosegregated with
the caf phenotype. Plant DNA flanking the T-DNA insertion
was used to probe a cDNA library, and twelve hybridizing
clones were found. One of these clones (cDNA8; 2.5 kb in
length) was sequenced, and found to contain a long ORF,
followed by a 76 base pair 3′ untranslated region, and a short
poly(A) tail. PCR analysis showed that of the other 11
cDNA clones, three had 5′ ends which were at a very similar
position, while eight were somewhat shorter. To determine
the 5′ end of the CAF RNA, we performed 5′ RACE and
found additional sequences that extended the ORF. Two
additional rounds of RACE were performed using primers
progressively closer to the 5′ end of the full length RNA.
The resulting predicted RNA of 5815 nucleotides
(Accession number, AF187317) contains an ORF of 1909
amino acids (Figure 7A). An in-frame stop codon in the
genomic sequence nine nucleotides upstream of the
predicted RNA sequence suggests that the first methionine
shown in Fig. 7 is the start of translation (see Accession
number AC007323 for the complete genomic sequence).
A cosmid clone (CosA) with homology to CAF was
isolated and transformed into wild-type plants, and the
resulting transgenic plants were crossed to caf mutant
plants. This cosmid fully complemented the caf mutant floral
phenotype (Fig. 7B), as well as all other caf phenotypes (not
shown). To determine the genomic sequence of CAF, three
adjacent EcoRI fragments were subcloned from CosA and
sequenced. These fragments contained 8603 bp of genomic
sequence comprising the entire CAF coding region and
sequences upstream and downstream. The sequence predicts
that the CAF coding sequence is interrupted by 19 relatively
small introns. One of the fragments contained the junction
between the putative CAF promoter and the cosmid cloning
vector. The distance from the vector junction to the 5′ end of
the predicted RNA sequence was 675 bp. Since this cosmid
complements caf, this suggests that all of the relevant 5′
promoter sequences are contained within this 675 bp region.
48 bp downstream from the 3′ end of the CAF cDNA sequence
Fig. 6. agamous-1 caf, superman-1 caf, and clavata3-2 caf double mutants.
(A) An ag-1 single mutant flower showing sepals (red arrow) in the inner
whorls. (B) An ag-1 caf double mutant flower showing only petals in the
inner whorls. (C) A superman-1 mutant flower. (D) A sup-1 caf double
mutant flower showing greatly enhanced gynophore development and floral
indeterminacy. After producing the organs in the first three whorls (arrow),
double mutant flowers produce many staminoid, carpelloid, and filamentous
organs. (E) clv3-2 single mutant flowers, showing extra organs in all
whorls. (F) clv3-2 caf flowers exhibiting enhanced floral indeterminacy.
Arrow shows the position of the first three whorls.
carpel factory in floral meristem determination 5237
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NGVSDTNSQINEVTVTPQVIAKETVKENGLQKNGGKRDEFSKEEGDKDRKRARVCSYQSERSNLSGRGHVNNFRE
150
GDRFMNRKRTRNWDEAGNNKKKRECNNYRRDGRDREVRGYWERDKVGSNELVYRSGTWEADHERDVKKVSGGNRE
225
CDVKAEENKSKPEERKEKVVEEQARRYQLDVLEQAKAKNTIAFLETGAGKTLIAILLIKSVHKDLMSQNRKMLSV
300
FLVPKVPLVYQQAEVIRNQTCFQVGHYCGEMGQDFWDSRRWQREFESKQVLVMTAQILLNILRHSIIRMETIDLL
375
ILDECHHAVKKHPYSLVMSEFYHTTPKDKRPAIFGMTASPVNLKGVSSQVDCAIKIRNLETKLDSTVCTIKDRKE
450
LEKHVPMPSEIVVEYDKAATMWSLHETIKQMIAAVEEAAQASSRKSKWQFMGARDAGAKDELRQVYGVSERTESD
525
GAANLIHKLRAINYTLAELGQWCAYKVGQSFLSALQSDERVNFQVDVKFQESYLSEVVSLLQCELLEGAAAEKVA
600
AEVGKPENGNAHDEMEEGELPDDPVVSGGEHVDEVIGAAVADGKVTPKVQSLIKLLLKYQHTADFRAIVFVERVV
675
AALVLPKVFAELPSLSFIRCASMIGHNNSQEMKSSQMQDTISKFRDGHVTLLVATSVAEEGLDIRQCNVVMRFDL
750
AKTVLAYIQSRGRARKPGSDYILMVERGNVSHAAFLRNARNSEETLRKEAIERTDLSHLKDTSRLISIDAVPGTV
825
YKVEATGAMVSLNSAVGLVHFYCSQLPGDRYAILRPEFSMEKHEKPGGHTEYSCRLQLPCNAPFEILEGPVCSSM
900
RLAQQAVCLAACKKLHEMGAFTDMLLPDKGSGQDAEKADQDDEGEPVPGTARHREFYPEGVADVLKGEWVSSGKE
975
VCESSKLFHLYMHNVRCVDFGSSKDPFLSEVSEFAILFGNELDAEVLSMSMDLYVARAMITKASLAFKGSLDITE
1050
NQLSSLKKFHVRLMSIVLDVDVEPSTTPWDPAKAYLFVPVTDNTSMEPIKGINWELVEKITKTTAWDNPLQRARP
1125
DVYLGTNERTLGGDRREYGFGKLRHNIVFGQKSHPTYGIRGAVASFDVVRASGLLPVRDAFEKEVEEDLSKGKLM
1200
MADGCMVAEDLIGKIVTAAHSGKRFYVDSICYDMSAETSFPRKEGYLGPLEYNTYADYYKQKYGVDLNCKQQPLI
1275
KGRGVSYCKNLLSPRFEQSGESETVLDKTYYVFLPPELCVVHPLSGSLIRGAQRLPSIMRRVESMLLAVQLKNLI
1350
SYPIPTSKILEALTAASCQETFCYERAELLGDAYLKWVVSRFLFLKYPQKHEGQLTRMRQQMVSNMVLYQFALVK
1425
GLQSYIQADRFAPSRWSAPGVPPVFDEDTKDGGSSFFDEEQKPVSEENSDVFEDGEMEDGELEGDLSSYRVLSSK
1500
TLADVVEALIGVYYVEGGKIAANHLMKWIGIHVEDDPDEVDGTLKNVNVPESVLKSIDFVGLERALKYEFKEKGL
1575
LVEAITHASRPSSGVSCYQRLEFVGDAVLDHLITRHLFFTYTSLPPGRLTDLRAAAVNNENFARVAVKHKLHLYL
1650
RHGSSALEKQIREFVKEVQTESSKPGFNSFGLGDCKAPKVLGDIVESIAGAIFLDSGKDTTAAWKVFQPLLQPMV
1725
TPETLPMHPVRELQERCQQQAEGLEYKASRSGNTATVEVFIDGVQVGVAQNPQKKMAQKLAARNALAALKEKEIA
1800
LIAEIDPG(stop)
T-DNA
caf-1
ESKEKHINNGNAGEDQGENENGNKKNGHQPFTRQTLNDICLRKNWPMPSYRCVKEGGPAHAKRFTFGVRVNTSDR
1875
GWTDECIGEPMPSVKKAKDSAAVLLLELLNKTFS
1909
C
Chromosome 1
Dist
cM
Marker
Id
Name
(1)
an
(252)
CAF
(2)
(3)
(4)
(5)
(6)
(7)
(237)
(8)
(225)
(9)
lbAt488
g4715
lbAt322
lPhAra1
pCITd91
lbAt241
P450-48
lbAt333
lAt59
lbAt219
6.8
8.1
1.2
2.0
1.1
2.5
2.1
0.3
3.4
2.5
1.2
Fig. 7. The CARPEL FACTORY gene. (A) Predicted amino acid sequence of the CAF protein. The two underlined regions are putative nuclear
localization sequences. Also shown is the position of the T-DNA insertion in the caf-1 mutant. Above the insertion site is the predicted open
reading frame extension conferred by the T-DNA. (B) Flower from a caf homozygote which contains a transgenic copy of the wild-type CAF
gene and exhibits a wild-type phenotype. (C) Map position of the CAF gene as deduced by RFLP mapping of an EcoRI restriction site present
in CosA. CAF maps on the top of chromosome 1 distal to lambdaAt488 (see Materials and Methods). Map shows the distance between markers
in centimorgans (cM) and the log likelihood value (LOD; Chang et al., 1988). GenBank accession no. AF187317.
was a sequence identical to a 3′ EST clone with homology to
pyrophosphatase (EST Z29202), suggesting that there are only
47 bp between the 3′ end of the CAF gene and the 3′ end of
the adjacent gene. Using restriction fragment length
polymorphism mapping, we mapped the CAF gene to the top
of chromosome one (Fig. 7C).
The T-DNA insertion in the caf mutant lies in the 19th exon
of the CAF gene, and thus separates most of the CAF gene from
part of the 19th exon, the 19th intron, and the 20th exon. This
truncates the predicted CAF protein at amino acid number
1836, replacing the last 73 amino acids of CAF with eight
amino acids from the T-DNA sequence (Fig. 7A).
Similarity of CAF with helicase and RNAse III like
sequences
Analysis of the predicted CAF sequence with PSORT (Nakai
and Kanehisa, 1992) suggests that CAF is a nuclear protein,
which contains two putative bipartite nuclear localization
signals (underlined in Fig. 7A). The sequence of CAF shows
extensive similarity along most of its length to a predicted
protein of 1374 amino acids in Schizosaccharomyces pombe
(C8A4.08C) and a predicted protein of 1822 amino acids in
Caenorhabditis elegans (K12H4.8), both of unknown function.
Relative to these proteins, CAF contains an N-terminal
extension of 238 amino acids which contains a high content of
charged residues (Fig. 8A). In the N-terminal region, these
three predicted proteins show similarity to each other (an
average of 34% identity) and to a number of DExH and DEAD
box type RNA and DNA helicase proteins belonging to
helicase superfamily II (Fig. 8B; Gorbalenya et al., 1989;
Gorbalenya and Koonin, 1993). The database entries most
similar to these helicase domains were three predicted proteins
from three different completely sequenced archaebacterial
species, one predicted protein from Saccharomyces cerevisiae,
and two predicted proteins from C. elegans, all of unknown
function. These sequences represent a distinct family within
helicase superfamily II, being much more similar to each other
than to other proteins of the DExH and DEAD type [so named
5238 S. E. Jacobsen, M. P. Running and E. M. Meyerowitz
A
238
541
566
CAF
1909 aa
478
S. pombe
C8 A4 .0 8 C
1374 aa
507
C. elegans
K1 2 H4 .8
1822 aa
B
Vas
Eif
Afu
Mja
Mth
Sce
Caf
Spo
Ce1
Ce2
Ce3
D
R
H
H
H
S
E
D
R
Q
N
N
G
P
P
P
F
R
I
V
R
P
V
I
L
L
L
Y
K
S
R
I
D
421
N
Y
I
I
I
V
E
S
A
H
A
K
A
K
K
K
Y
K
F
D
R
V
S
Y
E
P
P
P
V
L
L
Q
V
G
G
N
K
E
T
V
L
Q
R
T
F
F
T
T
K
N
E
P
C
Q
E
K
E
I
L
I
Y
E
Q
F
L
L
I
K
E
E
E
E
Q
L
N
C
V
T
S
R
R
R
R
R
R
R
R
R
P
A
M
L
T
D
R
K
D
N
T
I
I
Y
Y
Y
Y
Y
Y
Y
Y
Y
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
K
Q
I
Q
Q
Y
L
Q
V
E
E
C
R
S
I
L
T
D
D
E
E
E
S
A
I
I
L
I
V
V
L
L
L
I
I
A
A
A
V
L
Y
L
C
V
P
L
A
A
A
H
E
N
D
Q
Q
V
P
T
N
D
K
Q
I
K
V
P
I
C
A
A
V
S
A
A
A
A
A
S
I
L
L
I
L
L
L
147
64
139
149
86
161
64
*
G
G
G
G
G
G
G
G
G
G
G
G
**
K T/S
KTA
KTA
KTT
KTA
KTV
KTF
KTL
KTL
KTF
KTV
KTE
207
786
P
P
R
A
A
V
A
L
P
L
L
174
S
K
T
K
R
F
K
S
T
Q
E
G
G
K
K
K
Q
A
K
K
G
G
R
Y
G
K
Q
K
K
K
D
D
N
K
N
N
N
N
N
N
N
L
V
T
T
S
T
T
T
T
T
C
*
+
M
I
L
L
M
L
I
L
I
I
V
A
A
V
C
I
C
A
L
V
V
I
C
Q
V
V
V
A
F
V
Q
T
V
A
A
I
L
A
I
L
M
L
A
A
Q
Q
P
S
P
P
E
R
G
P
P
T
S
T
T
T
T
T
T
T
T
T
*
G
G
G
G
G
A
G
G
G
G
G
G
S
T
L
L
L
M
A
A
S
S
S
Fig. 8. Similarity of CAF with helicase and RNAse III like sequences.
(A) Schematic of the domain structure of CAF and two similar predicted
sequences from S. pombe (C8A4.08C; Accession no. Q09884) and C.
elegans (K12H4.8; Accession no. P34529). Hatched box denotes an Nterminal CAF sequence with a high content of charged residues that is
not found in the other two proteins. Stippled boxes denote the
ATPase/Helicase domains. Vertically hatched boxes denote the RNaseIIIlike domains, which are duplicated in all three predicted proteins. Black
boxes denote the double stranded RNA binding domains, which are
76
A
T
I
I
V
I
I
L
I
I
V
F
F
A
A
A
A
A
A
A
A
A
L
A
A
I
V
S
I
V
V
A
I
+
L
I
L
L
L
T
L
K
L
N
Y
P
S
V
V
V
V
L
L
L
I
A
I
I
I
I
A
M
I
I
L
I
A
L
L
A
A
A
L
K
K
K
K
L
S
Q
S
G
E
N
Q
E
E
K
K
Q
R
I
R
Y
S
K
H
H
L
I
L
L
L
F
V
L
Y
F
I
L
L
T
R
R
H
E
G
E
E
E
N
K
K
W
K
E
V
S
E
D
D
Q
Q
R
R
L
I
L
-
M
L
F
-
S
I
A
-
P
Q
Q
P
-
H
E
L
-
E
E
S
D
S
T
L
L
N
Q
S
S
E
D
L
G
E
Q
L
L
Y
T
N
E
G
G
G
G
K
E
K
R
K
R
H
K
K
K
R
A
D
D
G
K
K
K
R
R
P
P
T
G
G
S
A
M
K
A
F
S
Q
Q
K
K
K
K
L
I
F
K
R
V
A
V
V
V
I
S
S
A
V
V
L
L
L
L
I
V
V
L
V
*
+
I
V
F
I
I
F
F
F
F
F
L
*
+
V
L
L
L
L
T
L
L
V
M
L
S
A
A
A
S
A
V
V
V
T
V
R
K
I
I
S
V
D
N
C
T
S
*
+
F
M
L
L
L
C
L
L
V
I
M
*
+
V
F
V
L
I
L
L
I
L
I
M
*
+
V
V
V
I
V
V
I
I
I
F
I
F
L
K
N
G
Q
Q
H
D
N
E
G
V
I
I
T
L
T
H
G
S
D
E
D
P
C
P
S
V
M
Y
Y
E
E
L
S
F
A
F
Y
Y
L
M
D
A
D
-
K
G
K
T
Q
Q
K
K
H
E
I
A
I
I
C
T
V
V
V
T
V
G
S
V
I
T
A
G
G
G
Q
N
I
C
S
A
S
I
H
M
Q
I
G
H
L
L
L
L
Y
F
V
I
F
A
S
T
T
L
C
Y
H
Q
H
V
C
G
G
G
D
G
G
G
G
G
Y
I
E
K
S
K
E
E
Q
-
G
G
V
I
I
M
L
T
-
G
G
P
Q
K
S
G
S
S
-
T
T
P
P
P
R
Q
I
S
S
S
S
N
E
K
E
K
D
E
G
D
E
F
V
K
K
E
N
F
M
L
S
R
R
R
R
R
R
W
S
W
S
H
A
D
E
D
V
Q
E
S
S
S
N
V
R
Q
K
N
G
E
Q
R
L
E
V
T
C
K
W
L
Q
P
G
I
L
Q
T
C
T
R
T
Q
K
A
K
E
R
N
D
R
R
T
V
I
I
I
L
I
I
V
I
I
*
+
L
V
L
L
L
L
F
F
L
I
I
E
E
E
E
E
I
Q
N
D
L
L
R
I
F
I
F
V
E
M
L
A
G
G
A
G
G
G
G
G
****
+T/SA/GT/S
FSATF
LSATM
MTASP
LTASP
LTASP
LTATP
MTASP
MTASP
LTASL
LTASL
LTASL
R
M
W
Y
W
W
E
L
M
I
V
E
E
K
I
A
S
K
K
Q
I
G
A
K
K
K
N
R
S
A
C
P
A
G
S
K
K
Y
H
K
T
H
H
R
K
Q
R
Q
N
H
D
H
I
V
V
I
S
F
V
V
V
V
V
A
G
S
A
A
A
M
I
I
A
M
T
T
T
T
T
T
T
T
T
T
T
P
P
P
P
P
P
A
A
A
P
P
G
G
Q
Q
Q
Q
Q
D
Q
Q
Q
*
o
T
T
T
S
S
T
K
K
K
N
K
R
R
K
R
K
R
V
V
V
S
I
E
E
P
P
P
P
P
P
N
M
P
*
+
L
L
L
L
L
L
L
L
L
I
L
A
A
V
V
A
V
V
V
V
L
V
I
Q
E
E
I
A
Y
F
E
N
G
*
o
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
+
I
I
H
H
H
Q
Q
Q
Q
Q
Q
F
A
Y
E
I
A
A
A
A
K
N
R
N
E
K
E
E
I
A
D
E
Q
F
R
S
A
V
Y
H
S
R
A
K
L
L
F
C
I
I
I
I
F
R
V
K
K
R
L
R
R
E
S
L
K
V
R
Q
E
G
N
S
V
S
K
F
M
V
V
F
I
Y
Y
A
A
L
L
M
T
L
C
M
M
A
T
A
A
A
A
A
T
A
L
L
V
T
V
T
V
I
L
V
A
D
S
G
G
G
G
K
G
G
K
K
M
R
S
-
G
G
K
N
Q
N
N
F
F
H
D
H
S
H
S
K
P
A
P
P
P
E
D
Y
Y
Y
Y
Y
D
Q
V
M
I
L
R
Y
I
Domain Ia
Domain I
Vas
Eif
Afu
Mja
Mth
Sce
Caf
Spo
Ce1
Ce2
Ce3
P
P
P
P
P
P
P
N
E
P
P
R
R
V
V
T
V
I
L
C
M
I
L
V
V
I
V
V
L
F
L
I
L
+
F
M
I
F
L
L
M
o
D
N
N
-
E
S
S
S
A
S
V
F
I
V
I
V
P
I
E
L
L
G
A
T
V
A
T
M
M
K
Q
S
P
N
K
o
G
K
E
E
T
E
S
E
D
D
E
K
Q
K
K
C
E
V
G
L
N
D
N
Q
Q
C
L
L
V
M
A
Y
M
R
L
I
H
E
N
N
K
R
Q
Y
D
I
L
L
I
I
R
Y
K
A
Y
N
Q
K
G
N
L
W
L
L
M
E
E
E
C
L
T
Q
S
A
A
K
L
A
S
L
L
F
M
M
I
D
D
D
D
N
V
A
K
P
D
H
L
L
S
S
S
S
S
T
K
V
I
T
F
F
V
I
V
V
V
A
I
V
I
L
M
I
C
C
V
C
H
E
T
K
R
Q
E
E
N
T
V
T
R
N
D
N
N
N
N
I
V
A
K
Q
Y
P
L
L
L
L
K
S
S
D
S
V
I
G
G
F
D
D
E
D
N
S
S
R
I
I
M
I
R
L
L
I
V
I
E
E
N
S
K
N
V
E
D
A
L
A
H
E
K
E
E
S
E
E
I
V
I
V
V
I
L
L
L
L
L
G
K
E
E
V
E
E
A
S
R
N
I
K
V
V
V
I
K
D
K
G
E
V
E
R
R
K
R
H
Y
Y
Y
H
G
E
T
T
T
T
V
F
G
S
V
G
L
E
E
E
E
P
C
A
P
G
A
T
W
D
G
E
M
L
K
I
K
C
L
D
D
D
S
P
P
P
V
P
S
E
S
E
P
M
S
E
Y
P
D
D
G
D
D
D
D
E
E
E
D
D
V
I
V
V
V
I
I
S
V
K
S
K
R
A
K
R
V
V
Y
V
V
V
o
Q
Q
P
P
P
K
V
V
I
L
E
T
F
Y
Y
Y
Y
E
M
I
L
L
+
I
Y
V
I
L
M
Y
Y
C
C
C
Y
I
G
A
K
K
E
L
E
-
H
-
R
-
N
-
S
-
L
L
E
E
E
E
D
Q
N
N
S
N
D
S
D
D
D
D
E
V
L
I
I
L
S
R
L
I
L
K
R
A
R
R
Q
T
R
G
G
G
G
S
G
A
D
E
F
Y
R
R
R
V
I
F
Y
Q
R
F
P
L
V
L
P
M
I
I
L
V
E
K
E
D
R
K
E
N
E
S
S
D
Y
D
E
D
D
T
D
D
T
D
T
I
V
F
V
I
I
L
M
F
F
*
D/H
ADR
ADE
AHR
AHH
CHR
AHR
CHH
CHH
CHH
CHN
VHK
P
P
G
G
G
A
G
S
V
L
L
L
I
C
-
T
S
S
N
D
D
R
S
K
F
Y
*
E
E
E
E
E
E
E
E
E
E
E
E
*
o
E
D
D
D
D
D
N
T
K
K
D
D
N
L
D
N
N
I
L
I
I
Y
L
I
L
L
F
L
*
D
D
D
D
D
D
D
D
D
D
D
D
V
I
V
F
I
F
L
I
V
I
S
L
F
L
Y
L
V
I
R
R
A
A
A
R
H
R
H
L
N
*
+
L
L
F
A
F
I
L
F
F
F
F
V
I
I
I
I
V
V
V
V
L
V
Domain II
Vas
Eif
Afu
Mja
Mth
Sce
Caf
Spo
Ce1
Ce2
Ce3
R
D
S
A
R
S
N
I
I
L
R
S
N
Q
M
F
V
I
I
I
M
T
Q
M
M
M
M
V
H
S
N
V
R
Q
N
D
S
S
E
D
D
E
E
Y
H
Y
S
F
F
Y
Y
W
A
A
S
A
Y
Y
H
K
Y
Y
Y
Y
H
H
Y
Y
V
A
V
T
T
R
L
E
F
F
F
N
T
A
K
K
I
V
L
V
P
K
N
-
A
A
A
V
K
A
M
-
K
K
S
K
D
V
K
G
-
E
K
N
F
L
L
N
-
Y
F
Y
I
S
L
M
-
V
L
I
D
K
K
P
-
T
K
R
Q
R
K
K
E
-
M
L
T
N
F
H
D
G
-
R
N
A
K
A
N
F
K
H
-
P
S
K
D
R
S
K
T
P
S
-
E
N
K
K
H
S
R
L
V
L
-
H
T
P
C
P
Y
P
P
P
P
P
Q
Q
L
H
L
R
A
R
R
Q
Q
Q
L
E
D
D
E
K
K
K
G
D
+
M
V
I
V
I
V
V
A
K
Q
R
E
R
K
K
G
G
V
D
G
Domain III
Vas
Eif
Afu
Mja
Mth
Sce
Caf
Spo
Ce1
Ce2
Ce3
E
N
K
K
P
K
R
P
V
V
K
V
I
R
S
A
N
E
R
K
K
K
D
S
K
T
K
R
I
L
I
K
K
N
D
D
E
E
E
I
E
E
A
K
F
G
N
E
W
P
W
K
A
L
E
P
I
W
I
I
V
I
T
V
I
I
L
K
K
K
R
K
E
M
V
G
G
R
Y
L
V
I
V
V
W
P
C
M
D
A
D
D
D
R
P
S
P
L
F
Y
K
T
I
L
M
L
L
S
G
T
I
R
L
P
P
T
L
H
D
I
N
E
S
C
E
N
P
L
E
S
P
R
R
K
D
E
E
E
E
T
I
N
L
Y
L
L
M
F
L
I
I
I
F
L
I
Y
K
K
E
E
K
K
D
N
E
E
E
R
D
D
Q
K
T
H
I
T
V
A
I
I
M
V
A
L
L
K
L
R
I
I
I
H
S
T
E
K
E
E
A
E
T
G
E
R
L
L
Q
A
I
L
K
Q
L
I
L
L
V
C
F
M
F
A
I
K
N
R
G
E
E
D
M
L
D
T
E
E
K
M
E
E
E
Q
E
G
Q
C
A
V
A
A
T
T
E
E
I
L
L
V
A
L
V
V
L
K
K
K
K
Q
Q
A
E
A
I
E
N
P
A
G
F
G
S
R
R
R
V
S
C
V
L
M
F
L
L
L
S
K
N
I
S
K
K
K
Q
R
L
T
R
K
R
I
M
Q
K
I
T
T
S
S
S
T
A
T
K
E
L
G
W
F
R
R
------------------------Q (20 aa)
----H---N---N----
E
P
E
N
R
D
H
T
T
L
I
I
E
D
G
-
L
L
L
A
S
L
I
-
R
K
K
I
D
D
E
-
E
D
N
E
G
P
Q
P
L
A
L
L
A
R
R
P
G
G
G
A
R
R
N
W
V
V
I
N
P
Q
M
I
I
I
Y
L
I
I
I
E
N
D
E
I
K
E
N
V
S
T
E
H
D
T
T
P
I
I
C
K
S
T
F
E
A
S
D
L
L
E
K
N
D
P
R
R
K
R
K
S
V
V
S
A
A
T
D
N
S
T
G
Q
I
V
T
F
Q
K
K
K
I
N
K
R
R
P
R
T
K
N
Y
T
A
P
K
D
E
D
A
T
A
V
D
N
L
L
L
F
L
L
F
S
Y
L
I
L
K
A
A
R
S
E
A
E
K
A
E
E
Q
F
Y
L
L
A
M
L
L
L
Y
Q
N
R
Q
G
G
D
D
E
N
G
Q
Q
P
P
S
A
K
R
S
W
W
P
L
L
L
V
C
A
A
L
Q
Q
Q
A
A
D
Q
A
N
K
Y
W
K
G
E
R
I
K
R
E
-
A
I
I
V
T
H
-
A
F
A
A
G
A
A
-
S
S
R
N
Q
Q
G
-
S
Y
S
P
S
V
Y
-
Q
D
T
T
F
W
Q
-
S
E
S
I
L
E
N
-
S
E
P
P
S
K
W
-
E
V
P
E
A
E
V
I
I
K
R
G
L
L
C
I
A
I
Q
G
N
Q
C
K
S
K
Q
E
F
Y
Y
W
D
I
M
L
E
E
R
R
E
I
N
N
A
L
A
N
R
K
L
K
L
I
I
F
V
S
V
L
S
K
S
F
N
Q
S
N
Vas
Eif
Afu
Mja
Mth
Sce
Caf
Spo
Ce1
Ce2
Ce3
I
V
L
I
F
V
G
V
L
C
L
L
Q
T
L
T
P
A
S
A
Q
V
I
P
S
E
E
E
S
L
D
D
D
F
K
I
A
C
L
V
M
K
R
W
K
G
T
E
N
F
L
L
T
S
Q
R
G
Q
E
F
T
T
S
L
R
W
Y
W
N
T
A
L
F
M
I
-
S
G
A
I
-
E
E
K
N
-
M
L
I
N
V
Q
T
E
-
K
K
N
N
V
V
S
A
-
L
L
V
V
S
W
M
L
-
Q
M
E
G
L
L
I
D
K
H
H
H
Q
L
Y
T
V
Y
A
A
A
M
Q
L
I
L
M
V
K
L
L
C
V
K
K
K
E
E
E
K
E
D
R
E
V
L
L
L
R
L
F
L
C
Y
I
L
L
L
L
V
L
F
L
E
E
E
K
E
E
E
C
E
T
S
T
I
G
T
P
T
A
Q
Q
Q
Y
L
R
G
G
G
G
S
G
V
K
I
I
Y
I
K
S
R
R
N
V
A
V
P
T
I
D
V
F
L
F
A
K
E
N
L
K
L
H
F
A
R
M
F
M
S
N
Q
N
A
L
K
H
P
Y
Y
Y
Y
E
K
K
P
A
L
I
L
F
K
K
I
E
M
R
N
L
Q
V
K
K
V
V
K
K
R
N
A
A
S
A
A
L
L
L
K
A
I
L
F
V
S
K
C
E
E
N
K
R
M
E
T
V
L
A
Y
Y
E
Q
K
E
G
G
L
L
M
A
R
K
F
K
K
R
K
E
T
T
T
T
P
Q
P
D
K
S
T
E
L
Y
E
A
K
K
N
S
M
I
G
Y
G
D
E
-
G
N
N
D
Y
-
S
L
A
E
R
-
K
K
K
K
H
E
T
-
A
S
A
K
D
L
P
-
A
A
A
S
E
A
N
-
K
K
K
T
M
I
F
-
S
S
G
N
E
D
T
-
I
I
L
K
E
R
V
-
V
V
L
I
L
N
-
G
N
A
A
K
M
-
D
D
D
A
I
I
-
P
E
P
E
F
R
-
I
K
D
F
V
I
-
F
V
F
Y
G
E
W
-
K
R
T
Y
E
D
E
-
K
E
R
H
L
W
R
-
A
A
A
P
P
K
Y
-
V
V
M
I
D
N
H
-
I
N
H
L
D
N
N
-
A
L
L
K
P
K
Q
-
L
L
T
N
V
Y
L
-
S
M
R
I
V
S
V
-
K
K
R
K
S
D
G
-
A
N
G
N
T
-
M
Q
G
G
G
-
M
C
E
P
S
G
C
S
S
E
H
R
A
K
K
D
G
N
V
I
E
L
Y
D
N
N
L
E
P
E
S
V
M
S
D
I
I
H
P
G
S
S
K
A
P
K
E
F
A
V
T
T
V
V
V
V
V
F
V
V
E
E
E
G
A
D
Q
E
H
H
H
H
D
S
Y
Y
P
P
P
G
G
T
S
K
K
K
K
K
D
T
L
L
L
L
V
V
V
F
Vas
Eif
Afu
Mja
Mth
Sce
Caf
Spo
Ce1
Ce2
Ce3
E
G
D
Q
T
T
P
I
K
K
R
C
P
D
Q
K
L
V
L
V
K
K
R
D
K
V
M
R
V
V
V
H
E
D
E
D
Q
F
I
D
I
M
I
E
S
K
R
I
T
L
V
L
L
L
L
L
V
L
K
K
K
M
I
L
F
E
K
E
N
R
D
K
E
E
Q
Q
Q
I
E
F
L
L
I
N
T
F
L
L
F
L
L
L
L
I
E
E
Q
E
-
T
-
F
-
N
-
P
-
E
-
F
-
Q
-
K
-
E
-
K
R
-
L
A
M
-
K
T
K
-
Y
Y
L
-
K
K
K
K
Q
R
E
-
R
H
K
K
Q
Q
N
N
G
G
T
S
A
R
S
P
K
D
S
A
D
E
A
V
D
D
E
D
D
S
H
D
E
S
E
A
S
F
V
L
S
P
R
R
R
R
R
R
S
R
E
T
A
V
I
I
V
A
T
A
T
I
I
V
I
I
I
I
I
V
I
I
V
V
I
V
I
V
I
V
I
I
I
L
*
F/Y
FV
FI
FT
FA
FT
FT
FV
FV
FV
FV
RL
E
N
N
Q
Q
E
E
E
D
R
K
T/S
TK
TR
YR
YR
FR
LR
RV
RK
QR
TR
KY
o
R
R
D
D
D
E
V
A
Y
Y
T
G
K
S
T
T
S
A
T
I
E
H
A
V
A
V
L
A
A
A
A
A
Q
o
D
D
E
E
E
L
L
F
Y
T
S
F
W
M
K
E
E
V
T
S
I
V
L
L
L
I
I
I
L
L
L
L
P
A
T
V
V
Y
V
P
S
L
N
H
S
E
K
K
L
K
Q
F
K
F
V
L
M
V
F
L
M
I
F
F
M
L
G
S
H
N
N
Q
D
R
N
N
E
A
E
L
R
S
H
S
Y
K
R
L
L
C
V
I
N
G
E
D
S
T
K
A
K
E
E
P
Q
R
D
S
E
Q
W
L
M
E
L
V
P
M
-
K
L
-
A
M
F
G
-
E
K
K
I
-
L
T
F
K
-
P
L
V
S
-
S
N
N
E
G
L
L
P
W
Y
L
N
E
D
S
P
D
-
G
G
Q
F
N
Y
-
F
I
I
I
I
I
V
-
P
K
N
R
R
R
V
-
V
A
A
P
C
A
G
-
A
I
V
H
A
H
A
M
V
K
R
K
I
S
S
S
S
L
F
F
F
F
M
F
G
G
G
V
I
Y
I
I
I
R
L
T
G
G
G
G
G
G
N
N
N
Q
Q
Q
Q
H
H
L
K
K
A
A
N
A
N
G
A
S
Q
S
N
S
R
N
P
S
T
G
R
K
R
A
S
S
S
A
A
D
E
S
K
Q
D
D
S
-
N
G
E
E
Q
S
S
-
D
G
E
G
G
Q
A
-
F
E
G
D
-
-- -- -- -- -- D (49
-- F- L- I- V- -
-
-
*
+
I
V
I
I
I
I
I
V
V
V
V
K
Q
P
P
P
G
R
P
K
P
T
N
Q
S
S
S
E
Q
S
Q
E
A
I
V
T
V
V
V
C
C
C
C
C
*
o
K
S
D
N
D
D
N
N
N
S
N
H
L
L
Y
L
L
V
L
L
L
L
*
+
V
V
V
I
V
I
V
V
V
V
I
*
+
I
I
V
I
V
I
M
I
I
I
I
N
N
F
F
M
C
R
R
K
K
K
Y
Y
Y
Y
Y
Y
F
F
F
Y
Y
D
D
E
E
E
D
D
N
D
N
N
M
L
L
I
R
Y
C
P
P
A
P
P
T
A
C
P
S
S
T
V
V
V
T
K
R
L
A
S
o
K
N
P
P
P
S
T
T
D
T
G
I
R
S
S
S
S
N
S
D
E
E
E
E
P
V
V
M
E
A
I
I
I
I
L
T
R
I
I
o
D
N
R
R
R
K
A
Q
S
A
Q
Y
Y
A
F
M
N
Y
Y
Y
H
L
V
I
I
I
I
I
I
V
V
V
V
*
H/Q
HR
HR
QR
QR
QR
QR
QS
QS
QS
QR
QQ
I
I
K
R
R
M
R
R
K
R
R
*
G
G
G
G
G
G
G
G
G
G
G
G
*
R
R
R
R
R
R
R
R
R
R
R
R
T
G
T
A
T
T
A
A
A
G
A
G
G
G
M
G
G
-
*
R
C
R
R
R
R
R
R
R
R
R
R
V
F
-
G
G
G
G
K
K
K
A
R
A
A
N
R
R
E
R
R
P
M
A
L
K
N
K
E
G
K
D
G
A
G
N
N
G
G
G
G
G
G
S
S
S
S
S
R
V
R
K
R
K
D
K
R
E
R
A
A
I
V
M
I
Y
F
Y
C
S
T
I
V
Y
V
V
I
L
V
V
V
S
N
V
V
V
L
L
I
I
L
L
F
M
L
L
L
L
M
F
T
I
L
F
V
V
I
I
F
V
L
V
T
S
D
T
T
A
T
S
E
N
E
N
V
P
E
K
K
E
S
R
T
E
S
K
E
E
G
G
K
N
G
E
K
I
S
Domain IV
Vas
Eif
Afu
Mja
Mth
Sce
Caf
Spo
Ce1
Ce2
Ce3
F
F
S
-
P
T
S
-
T
V
E
-
T
S
E
-
S
A
A
-
I
M
Q
-
H
H
I
-
G
G
K
K
K
S
-
D
D
G
G
G
G
S
H
S
Q
R
M
M
M
L
M
M
M
A
Q
L
D
R
S
T
N
K
T
S
T
Q
Q
Q
Q
Q
Q
S
F
K
S
S
K
K
K
K
K
S
R
K
Q
Q
Q
E
E
E
Q
M
Q
R
R
K
E
R
R
Q
Q
Q
Q
M
Q
Q
Q
Q
E
D
I
I
R
K
Q
K
T
M
Q
Q
V
E
E
D
E
D
D
E
E
L
A
I
T
A
I
V
T
T
V
K
T
L
M
I
I
I
I
I
L
L
L
L
R
R
D
E
K
H
S
H
R
K
D
D
E
K
R
S
N
K
K
R
M
K
F
F
F
F
F
F
F
F
F
F
F
*
o
K
R
R
K
R
K
R
K
H
A
N
N
S
R
K
M
K
D
T
R
D
N
G
G
G
E
G
G
G
G
N
G
G
o
S
S
V
G
N
E
H
K
E
E
R
M
S
Y
H
Y
V
Y
I
I
L
K
R
K
S
D
N
T
N
N
R
K
*
+
V
V
V
V
V
V
L
V
C
I
V
*
+
L
L
L
L
L
L
L
L
L
L
I
*
+
I
I
V
V
L
V
V
I
I
V
V
**
T/S o
ATS
TTD
ATS
STS
STS
CTS
ATS
ATA
ATS
STS
ATS
*
+
V
L
V
V
V
I
V
V
V
V
V
A
L
G
S
A
G
A
A
L
A
V
Domain V
S
A
E
E
E
E
E
E
E
E
E
R
R
E
E
E
E
E
E
E
E
E
*
G/S
GL
GI
GL
GI
GI
GL
GL
GI
GV
GL
GL
*
o
D
D
D
D
D
D
D
D
D
D
D
Domain VI
K
D
T
T
T
E
N
E
D
A
S
aa)
-
-
carpel factory in floral meristem determination 5239
C
Crn1
Rnt
Pac
Spo2
Ce12
Caf2
Ath
Eco
Crn2
Ce11
Spo1
Caf1
Q
R
Y
Q
G
S
R
A
H
S
Q
D
S
I
L
L
L
L
L
I
F
L
L
E
E
E
E
E
Q
E
D
I
K
A
K
E
E
R
K
R
E
S
E
N
I
L
T
K
A
I
K
R
D
H
L
R
K
I
I
L
L
L
L
I
F
I
A
E
G
G
K
N
G
G
G
R
S
R
Q
Y
Y
Y
Y
Y
I
L
L
Y
-VF
VF
SF
RF
EF
KF
TF
QF
GV
DC
PI
I
M
K
K
K
K
N
N
S
K
P
-HK
HI
NK
ER
EK
DK
HQ
NI
-P
-I
-T
S
S
K
A
G
S
E
R
C
D
S
M
T
R
L
Y
L
L
L
L
L
T
K
S
I
A
L
L
L
L
L
L
L
A
I
L
K
Y
H
V
V
L
Q
A
L
C
L
F
D
E
L
Q
E
K
Q
K
T
Q
E
N
K
A
A
A
A
A
A
A
A
A
IM
VY
IY
FI
FT
IT
FT
LT
FT
LT
LT
LT
K
L
P
H
H
H
D
H
R
T
S
A
G
S
N
P
A
A
A
R
R
S
A
A
T
G
Q
S
S
S
S
S
N
N
E
S
S
S
S
M
Y
R
Y
A
I
A
S
C
G
N
M
I
P
V
S
P
A
Q
Q
G
P
S
N
S
D
S
N
D
L
E
E
N
Q
K
N
G
N
T
P
E
E
Q
N
S
D
D
-
I
M
L
G
R
G
K
L
-
L
I
L
I
V
V
S
T
-
N
D
Y
T
K
-
-H
AH
IH
EN
GC
SC
ES
-H
GH
MS
FD
FC
N
N
N
Y
Y
Y
Y
N
N
L
Y
Y
E
E
E
Q
Q
Q
E
E
Q
E
D
E
R
R
R
Q
R
R
L
R
R
R
R
R
LE
LE
LE
LE
LE
LE
LE
LE
LE
FE
LE
AE
Y
F
F
F
F
F
L
F
W
T
F
L
L
L
L
L
L
V
L
L
L
I
Y
L
Crn1
Rnt
Pac
Spo2
Ce12
Caf2
Ath
Eco
Crn2
Ce11
Spo1
Caf1
A
N
K
F
A
A
V
T
S
K
K
Q
L
L
F
Y
L
A
N
L
L
E
I
M
V
V
V
V
V
V
V
V
V
V
I
V
Q
S
G
C
N
N
D
R
S
S
S
S
N
N
N
N
N
N
T
G
N
N
N
N
R
E
E
K
T
E
E
N
Q
C
C
M
N
Q
S
S
I
N
K
T
T
N
N
V
L
I
A
L
F
F
L
L
Q
L
L
L
AT
KQ
DK
SY
AS
AR
AR
AE
AV
YR
YK
YQ
L
W
F
I
L
V
V
L
V
L
V
F
AK
SI
AR
GF
AV
AV
AV
AR
CD
GK
AI
AL
N
M
L
V
K
K
N
E
D
K
D
V
C
Y
Y
L
F
H
H
F
L
L
C
K
R
N
G
N
E
K
Q
E
G
G
E
G
I
F
F
L
F
L
L
L
F
I
L
L
D
H
D
H
Q
H
Y
G
T
P
P
Q
E
E
K
K
K
L
S
E
E
Q
K
S
ML
KL
TL
YI
HF
YL
YL
CL
FV
LI
YA
YI
K
V
Q
I
R
R
R
I
V
L
Q
T
H
A
H
H
L
K
A
S
A
N
E
M
G
K
G
A
N
T
D
S
C
S
K
P
P
K
P
R
A
P
S
P
G
F
L
F
A
G
A
L
E
D
E
A
M
L
L
L
L
Y
A
I
P
C
Y
E
E
K
K
H
R
S
D
H
K
E
S
T
D
H
R
A
M
Q
Q
G
P
S
W
W
I
I
I
I
W
-
---FE
EK
RE
LE
--LP
---
Y
F
F
F
P
-
Q
V
V
V
C
-
E
K
K
E
Y
-
---LLC
----IP
---
S
T
-
E
C
C
S
Crn1
Rnt
Pac
Spo2
Ce12
Caf2
Ath
Eco
Crn2
Ce11
Spo1
Caf1
E
-
N
-
K
K
K
T
S
P
G
T
V
W
S
S
D
D
E
I
E
---------GG
-NS
D
D
---------VS
-VF
K
E
S
D
T
-
T
-
D
-
G
-
---------IE
---
T
-
I
-
T
-
F
-
P
-
K
-
Q
-
A
-
R
-
V
-
G
G
---------ND
-EM
D
E
I
D
S
G
---------PL
-EL
P
K
E
Y
C
G
DA
DS
DS
DA
DA
DA
DS
DS
DS
DS
DC
DA
V
I
F
V
V
V
I
I
V
F
F
Y
V
L
F
L
L
L
L
L
L
L
L
L
E
N
N
D
D
D
N
S
Q
K
K
K
L
S
L
Y
Y
H
M
Y
L
F
L
W
I
V
F
I
M
L
G
V
I
A
G
V
V
M
T
I
I
I
I
I
V
T
A
V
SH
TL
TR
VQ
TR
TR
IY
AN
SD
TD
SI
SR
H
I
I
Y
Y
H
D
A
F
Y
T
F
L
I
I
L
L
L
F
L
L
L
V
L
Y
Y
F
Y
F
F
I
Y
Y
Y
F
F
FM
NK
SK
KK
ED
FT
KL
HR
RR
HT
LK
LK
L
F
F
Y
S
Y
Y
F
F
L
F
Y
T
P
P
P
R
T
P
P
P
L
P
P
H
D
Q
N
Q
S
K
R
Y
D
D
Q
H
Y
M
A
Y
L
E
V
H
Q
T
K
F
S
D
T
S
P
A
D
H
H
Q
H
E
E
E
S
P
P
P
E
E
E
E
E
GG
GQ
GS
GE
GV
GR
GP
GD
GH
GK
YQ
GQ
L
L
L
L
L
L
L
M
M
L
L
L
A
S
S
T
T
T
T
S
S
S
H
T
T
T
K
D
D
D
K
R
L
F
F
R
YR
LR
LR
YK
LR
LR
LR
MR
LR
AR
NR
MR
T
M
A
S
S
A
A
A
T
S
K
Q
R
D
P
A
----NF
----FK
YG
PG
D
A
F
V
T
P
P
N
N
P
F
T
V
N
D
F
A
D
D
----I
EM
EV
AM
--AE
-ED
E
Y
Q
E
E
T
A
M
T
K
K
F
V
E
D
D
--F
-L
RE
TT
SS
---NE
-GG
Q
D
S
T
E
K
K
I
S
Y
L
Y
A
E
P
Y
E
S
S
K
S
S
E
G
P
R
F
H
D
A
E
I
F
L
I
F
G
E
E
N
D
N
H
L
D
A
N
K
P
E
S
S
D
E
DL
SN
DQ
WF
GQ
FG
NG
--GQ
-Q
EQ
I
F
L
W
E
L
L
V
-
N
Q
R
F
E
G
L
I
-
V
N
K
E
D
D
G
E
E
-
AE
GK
SQ
ID
IE
CK
-K
FR
LK
EK
---
P
-
N
R
D
---------LL
YA
LS
T
V
S
Q
L
Y
Q
Q
R
H
K
V
I
L
L
F
L
S
V
A
V
R
L
S
S
S
KH
KKPK
PK
PK
PK
ES
KD
DK
VK
SK
A
L
V
F
A
V
V
I
K
S
R
T
L
Y
I
I
M
L
L
L
I
I
L
A
A
A
S
G
G
A
A
A
A
A
A
NA
DV
DT
DT
DI
DI
DI
DT
DL
DA
DM
DV
F
F
F
L
F
V
V
V
V
V
V
V
E
E
E
E
E
E
E
E
E
E
E
E
A
A
A
A
S
S
S
A
A
A
A
A
V
Y
Y
M
V
I
T
L
F
L
S
L
M
I
L
I
A
A
I
I
I
I
I
I
A
G
G
C
G
G
G
G
G
G
G
G
AI
GL
AL
AI
AI
AI
AI
GV
AL
VH
AC
VY
Y
M
I
F
Y
F
F
F
Y
L
L
Y
L
E
L
L
L
L
M
L
V
L
L
V
D
D
D
D
D
D
D
D
D
T
D
E
GG
DP
GQ
SG
SG
SG
CSD
RG
LG
SG
GG
G
G
G
G
G
G
G
G
G
G
G
G
*
*
duplicated in CAF but not in
T K A K G D I E H K V Y Q L L K D Q G C E D F G T K C V I E E V K S S H K T L L N T E
D Spo2
C8A4.08C or K12H4.8. Lines
Ce12 S P I R E L M E - - - - - - - - - F E Q S K V R F - - - S K M E R I L E S G K V R V T
Caf2a H P V R E L Q E R C Q Q Q A E G L - - - - E Y K - - - - - - - - - A S R S G N T A T V
denote regions that show homology
Rnt E K T D K L D M N A K R Q L Y S L I G Y A S L R L H Y V T V K K P T A V D P N S I V E
to each other, but not to other known
Pac - P I D K L A K S K L F H K Y S T L G H I E Y R W - - - - - V D G A G G S A E G Y V I
sequences. The number of amino
Caf2b F T R Q T L N D I C L R K N W P - - - - - - M P S Y R C V K E G G P A H A K R F T F G
acids (aa) present in each domain is
Crn2 D A K S H L Q Q W C L A M R D P S S S E P D M P E Y R V L G I E G P T N N R I F K I A
Eco D P K T R L Q E Y L Q G R H L P - - - - - - L P T Y L V V Q V R G E A H D Q E F T I H
noted above each segment. Diagram
*
*
*
*
is drawn to scale. (B) The Nterminal helicase-like domain of
CAF (Caf; residues 239-779) was
Spo2 L H L T K Y Y G F S F F R H G N I V A Y G K S R K V A N A K Y I M K Q R L L K L L E D
aligned with similar domains
Ce12 V E V V N - - N M R F - - - - - - T G M G R N Y R I A K A T - - A A K R A L K Y L H Q
Caf2a E V F I D - - G V Q V - - - - - G V A Q N P Q K K M A Q K L - - A A R N A L A A L K E
present in the predicted C8A4.08C
Rnt C R V G D - - G T V L - - - - - G T G V G R N I K I A G I R - - A A E N A L R D K K M
protein of S. pombe (Spo; Accession
Pac A C I F N - - G K E V - - - - - A R A W G A N Q K D A G S R - - A A M Q A L E V L A K
no. Q09884, residues 2-479) and the
Caf2b V R V N T - - S D R G W T D E C I G E P M P S V K K A K D S - - A A V L L L E L L N K
predicted K12H4.8 protein of C.
Crn2 V Y Y K G - - - K R L - - - - - A S A A E S N V H K A E L R - - V A E L A L A N L E S
Eco C Q V S G - - L S E P - - - - - V V G T G S S R R K A E Q A - - A A E Q A L K K L E L
elegans (Ce1; Accession no.
*
*
*
* *
*
*
P34529; residues 3-509), and other
predicted helicase-like proteins from
Archaeoglobus fulgidus (Afu; Accession no. 2662588; residues 6-476), Methanococcus jannaschii (Mja; Accession no. 2127880; residues 11-471),
Methanobacterium thermoautotrophicum (Mth; Accession no. 2622527; residues 7-478), S. cerevisiae (Sce; Accession no. P40562; residues 77624), and C. elegans (Ce2; Accession no. 2662588; residues 283-822 and Ce3; Accession no. 1667297; residues 361-854). Also included in the
alignment are the Drosophila melanogaster protein VASA (Vas; Accession no. P09052; residues 258-597) and the mouse protein eiF-4A1 (Eif;
Accession no. FIMS4A; residues 29-364). A consensus sequence of seven domains (I-VI) present in these proteins is shown above the alignment
and numbered as in Gorbelenya et al. (1989). + denotes the hydrophobic residues (I,L,V,M,F,Y,W); o denotes the charged or polar residues
(S,T,D,E,N,Q,K,R). Asterisks mark positions where at least 10 of the 11 sequences matched the consensus. Identical residues are boxed in columns
where at least 3 of the residues are identical. The (20 aa) and (49 aa) notations denote either 20 or 49 amino acids, which were present in only one
protein and were removed from the alignment. (C) Similarity of CAF to the conserved regions of the catalytic domains of RNaseIII-like proteins.
The duplicate RNaseIII-like domains of CAF [(Caf1; residues 1345-1518)(Caf2; residues 1561-1707)], C8A4.08C [(Spo1; residues 9001038)(Spo2; residues 1085-1233)], K12H4.8 [(Ce11; residues 1295-1501)(Ce12; residues 1557-1717)], and RNC_CAEEL (Accession no. 001326)
[(Crn1; residues 1-110)(Crn2; residues 164-271)] were aligned with similar domains present in E. coli RNaseIII (Eco; Accession no. P05797;
residues 8-128), S. cerevisiae RNaseIII (Rnt; Accession no. Q02555; residues 206-331), the Pac1 gene of S. pombe (Pac; Accession no. P22192;
residues 138-262), and the predicted ATFCA3 protein from A. thaliana (Ath; Accession no. Z97338; residues 56-195). Identical residues are boxed
in columns where at least 3 of the residues are identical. Asterisks mark conserved positions that are sites of inactivating point mutations in E. coli
RNaseIII (Court, 1993). (D) Double stranded RNA binding domains (dsRNAbds) of CAF. Two putative dsRNAbds of CAF [(caf2a; residues 17331796)(caf2b; residues 1831-1906)] were aligned with similar domains found in C8A4.08C (Spo2; residues 1256-1341), K12H4.8 (Ce12; residues
1745-1808), RNC_CAEEL (Crn2; residues 298-373), E. coli RNaseIII (Eco; residues 155-225), S. cerevisiae RNaseIII (Rnt; residues 361-437),
and the Pac1 gene of S. pombe (Pac; residues 286-356). Identical residues are boxed in columns where at least 3 of the residues are identical.
Asterisks denote positions where the residues are highly conserved in a variety of additional dsRNAbds (Kharrat et al., 1995). Arrow indicates the
position of the T-DNA insertion in the second dsRNAbd of CAF (caf2B). All alignments were performed using PILEUP and PRETTYPLOT
[Genetics Computer Group, (1991), Madison, Wisconsin], using a gap creation penalty of 3.0 and a gap extension penalty of 0.05.
5240 S. E. Jacobsen, M. P. Running and E. M. Meyerowitz
RNA binding proteins (Fig. 8D; Kharrat et al., 1995). CAF
contains a duplication of this dsRNAbd, which is not found in
any of the other known RNaseIII-like proteins (Fig. 8A,D). The
most C-terminal of these putative dsRNAbds is disrupted by
the insertion of the T-DNA in the caf mutant, indicating that
this sequence is necessary for wild-type CAF function.
CAF RNA expression
To test whether CAF is a single copy gene in Arabidopsis, or
is a member of a family of closely related genes, we performed
DNA blot analysis with probes from both the helicase and
RNaseIII domains. Under high stringency conditions, both
Fig. 9. CAF RNA species in wild-type and caf mutant plants. A
Northern blot containing 2 µg of poly(A) RNA from caf mutant
plants (caf) or wild-type plants (wt) was hybridized with a probe
from the 5′ end of the CAF RNA (helicase probe; see Materials and
Methods) and exposed for 11 days. The same blot was stripped,
exposed for 13 days to ensure that no signal remained, and then
hybridized with a probe from the 3′ end of the CAF RNA (RNaseIII
probe; see Materials and Methods), and exposed for 8 days. Size
markers shown are from an RNA ladder which was run on the same
gel (Boehringer Mannheim RNA Molecular marker I).
for the conserved residues DExH and DEAD, with x
representing any amino acid (Gorbalenya, et al., 1989)]. Fig.
8B shows an alignment of CAF and the related sequences
along with eIF-4A and VASA, two RNA helicase proteins of
the DEAD box type. CAF contains sequence similarity within
all seven of the most conserved domains present in these
DEAD/DExH proteins (Fig. 8B; Gorbelenya et al., 1989).
These include domains I and II which are present in a wide
variety of ATPases, and domains III through VI which are
specific to helicases. Mutation analysis has shown these
domains to be important for ATPase and/or helicase activity
(reviewed by Gorbalenya and Koonin, 1993).
The middle portion of the CAF protein (amino acids 7801344) shares dispersed sequence identity with the above
mentioned S. pombe (C8A4.08C; 21% identity) and C. elegans
(K12H4.8; 18% identity) sequences, but is not similar to other
known proteins. However, CAF and these two related proteins
contain a C-terminal domain with sequence similarity to each
other (average of 31% identity), to several bacterial and yeast
RNAse III proteins (Nashimoto and Uchida, 1985; Elela et al.,
1996; Iino et al., 1991; Xu et al., 1990) and to predicted
proteins from a number of species, including A. thaliana, S.
pombe and C. elegans (Fig. 8C). CAF, C8A4.08C, K12H4.8
and another predicted protein from C. elegans (RNC_CAEEL)
each contain adjacent duplicated domains with similarity to the
RNaseIII catalytic domain (Fig. 8A, C). These sequences
contain two absolutely conserved residues which are the sites
of inactivating point mutations in E. coli RNase III (Court,
1993). At the extreme C terminus, these proteins contain
domains similar to double stranded RNA binding domains
(dsRNAbds) found in RNaseIII and in a variety of additional
Fig. 10. CAF RNA expression pattern. Sections of inflorescences or
seedlings were hybridized with either a CAF antisense probe (A-D)
or a control CAF sense probe (E), and exposed for 30 days.
(A) Bright-field photograph of a longitudinal section of an
inflorescence meristem bearing flowers on its flanks. (B) Darkfield/bright-field double exposure of the same section showing the
expression pattern of CAF. Yellow spots represent silver grains
exposed by the 35S-labeled CAF probe. (C) Bright-field photograph
of a longitudinal section of a 7.5-day-old seedling showing the apical
meristem and developing leaves on its flanks. (D) Dark-field/brightfield double exposure of the same section. (E) Dark-field/bright-field
double exposure of a 7.5-day-old seedling showing the background
level of signal from the sense probe. All photographs were taken at
the same magnification.
carpel factory in floral meristem determination 5241
probes detected only the restriction fragments predicted by the
CAF genomic sequence (see Materials and Methods). These
results show that CAF is single copy in the Arabidopsis
genome, and that under these conditions, the probes used are
specific for the CAF gene.
To analyze the expression pattern of CAF, RNA blot analysis
was performed with probes to both the helicase and RNaseIII
domains. The helicase probe detected an RNA of
approximately 6.2 kb, consistent with predictions from the
cDNA and genomic sequences (Fig. 9A). However, the
RNaseIII domain detected two RNA species, a 6.2 kb RNA and
an additional 2.5 kb RNA (Fig. 9B). Thus CAF produces two
predominant RNA species, one apparently encoding the full
1909 amino acid protein containing both the helicase and
RNaseIII like domains, and one containing only the RNaseIII
domain. Neither the 6.2 kb nor the 2.5 kb RNA species were
detected in RNA extracted from the caf mutant. Instead, two
larger transcripts of approximately 4.8 kb and 8.7 kb were
present (Fig. 9A,B), presumably because transcription of the
mutant caf gene extends into the T-DNA and adds
approximately 2.4 kb to the size of each of the two major RNA
species. The abundance of these aberrant caf mutant RNAs is
similar to that of the wild-type CAF RNAs.
Results from RNA blot and RT-PCR experiments indicate
that these two transcripts are present in vegetative tissue (leaves
and stems) from 2-week old plants, and present in
inflorescence tips from 4-week old plants (data not shown),
suggesting that CAF RNA may be expressed ubiquitously
throughout the shoot. To confirm these results and to test
whether CAF could be expressed in a cell layer-specific
manner, in situ hybridization experiments were performed
(Fig. 10). A low level of CAF RNA was found in all cells of
the apical and floral meristems, and in the flowers, cauline
leaves and stems. Thus CAF RNA is expressed evenly
throughout most shoot tissues. This expression pattern is
consistent with the caf mutant phenotype, in that most shoot
tissues show some defect in the mutant.
DISCUSSION
The caf mutant was initially chosen for study because of its
dramatic defect in floral meristem determinacy. caf mutants
exhibit over-proliferation of the floral meristem, such that
flowers contain excess numbers of stamens and carpels. The caf
mutant phenotype is distinct from that of the ag mutants, which
also show reduced floral determinacy. Whereas ag mutations
cause a reiteration of the floral program so that flowers show a
repeating pattern of sepals and petals, caf flowers show
unregulated cell division in the center of the flower, but show
normal floral organ identity. caf mutants are also defective in
other aspects of plant development. caf mutant plants lack nearly
all of the axillary inflorescence meristems normally found in the
wild type, and possess abnormally shaped cotyledons, leaves,
sepals, stamens and carpels. Thus the wild-type CAF gene plays
a role in specifying the determinate growth of the floral
meristem, but also functions in the specification of axillary
meristems and in the morphogenesis of organs.
It seems possible that the morphogenesis defects seen in the
caf mutant could be explained as a secondary consequence of
the defect in caf apical and floral meristem structure, in which
case, one might expect that CAF activity could be localized to
the meristems. However, we find that CAF RNA is evenly
expressed throughout most shoot tissues, including apical and
floral meristems, floral organs, stems and leaves. CAF is
expressed in actively dividing cells at roughly the same level
as in differentiated cells. Thus it seems more likely that CAF
may act in flowers to pattern meristem determinacy, and act
separately in other tissues to promote the proper development
of axillary meristems, leaves and floral organs. However, it is
still possible that the different floral and vegetative
abnormalities found in caf are mechanistically related. For
instance, one could interpret many of the vegetative and floral
phenotypes as being losses in cell division control, with too
much cell division giving rise to the extra carpels and too little
cell division contributing to a lack axillary meristems and the
reduced size of leaves, flowers and anthers.
The mutant allele of caf that we have studied, which is the
only mutant allele known, seems unlikely to be a complete
loss-of-function mutation. First, northern blot experiments
show that the CAF RNA is expressed at a normal level in the
caf mutant. Second, the T-DNA insertion in caf is predicted to
prematurely truncate the CAF protein at amino acid number
1836, replacing the last 73 amino acids of CAF with eight
amino acids from the T-DNA. This would leave 96% of the
CAF protein intact in the mutant. Third, in several mutant
screens carried out in our laboratory for mutations affecting
floral structure, we have failed to find additional caf alleles.
Thus, it is possible that stronger alleles of caf might have a
lethal phenotype, or have a phenotype that would not have been
detected in our screens. Given that CAF homologues exist in
fungi and animals, it seems likely that CAF may carry out a
conserved cell biological function (since plants and animals
arrived independently at multicellularity), and therefore that
CAF could be an essential gene, strong mutations in which
might lead to a lethal phenotype. Thus the caf mutation
described here may be a weak allele which uncovers CAF′s
role in the regulation of cell division in floral meristems.
The genetic interactions of caf with other floral mutations
support a role for CAF in controlling floral meristem
proliferation. The double mutant phenotypes of caf combined
with the floral homeotic mutants ap2-1, pi-1, and ag-1 are
roughly additive. However, caf shows a synergistic interaction
in combinations with two other mutations, sup-1 and clv3-2,
which increase cell proliferation in floral meristems, but which
do not seem to directly control organ identity. Epistasis was
not found in the double mutant combinations of caf with sup
or clv3 mutations, suggesting that CAF may, at least in part,
control meristem activity through a mechanism somewhat
different than that of SUP or CLV3.
The caf mutant phenotype resembles that of the sup mutants,
in two ways. First both types of mutations cause an increase in
the number of stamens and carpels (Gaiser et al., 1995;
Jacobsen and Meyerowitz, 1997). Second, in an ag mutant
background, both mutations cause a conversion of the inner
whorl sepals to petals. The caf and sup mutations differ greatly
however in their effects outside of the flower. Whereas the caf
mutation affects the morphogenesis of most of the organs of
the shoot, the sup mutations have no detectable effect on the
development of non-floral tissues.
The N terminus of the predicted CAF protein is similar to
DEAD/DExH type helicase/ATPase proteins from a number of
organisms. CAF and these related proteins (all of unknown
5242 S. E. Jacobsen, M. P. Running and E. M. Meyerowitz
function) form a new family of related sequences within the
helicase superfamily II (Gorbalenya et al., 1989; Gorbalenya and
Koonin, 1993). eiF-4A is the founding member of the DEAD box
RNA helicase family and is thought to function in unwinding
mRNA during translational initiation (Nielsen et al., 1985; Rozen
et al., 1990). VASA is a Drosophila DEAD protein which is
required for the formation of posterior pole plasm (Lasko and
Ashburner, 1988; Hay et al., 1988). It acts as a positive regulator
of oskar RNA translation (Webster et al., 1997 and references
therein), and is required for proper localization and translation of
nanos RNA (Gavis et al., 1996). Both eif-4A (in combination
with another factor, eif-4B) and VASA have been shown to
possess ATP-dependent RNA helicase activity in vitro (Rozen et
al., 1990; Liang, et al., 1994). Other helicase-related proteins are
known to be involved in mRNA splicing (reviewed by Kramer,
1996). For instance, PRP2, a DEAH type protein, is thought to
activate the precatalytic spliceosome (Kim and Lin, 1996). RAD3
is a DEAH family helicase-like protein, which acts in DNA repair
and has DNA-dependent ATPase and DNA helicase activity
(Harosh and Deschavanne, 1991). Another DEAD protein
appears to regulate entry into mitosis in Schizosaccharomyces
pombe (Warbrick and Glover, 1994). Other functions ascribed to
helicase proteins include rRNA processing (Kressler et al., 1997),
mRNA stability (Iost and Dreyfus, 1994), and mRNA
degradation (Anderson and Parker, 1996). Given the diverse
functions of helicase proteins, it is difficult to predict a specific
function for the helicase domain of CAF, however, it is likely that
this domain participates in nucleic acid dependent ATPase
activity and may act to unwind RNA or DNA.
The C-terminal portion of CAF and the related S. pombe
(C8A4.08C) and C. elegans (K12H4.8) proteins contain
homology to RNaseIII proteins. Relative to the RNaseIII
proteins from bacteria and yeast, these proteins have a
duplication of the putative RNase catalytic domain, followed
by either one or two double stranded RNA binding domains
(dsRNAbds). RNaseIII proteins cleave specific regions of RNA
that form base-paired stem-loop structures (reviewed by Court,
1993). Though RNaseIII cleaves only particular RNAs, the
sequence requirements for this specific cleavage are unknown.
In E. coli, RNase III is involved in the processing of ribosomal
precursor RNAs and certain mRNA molecules (Court, 1993).
In S. cerevisiae, RNaseIII proteins are known to process rRNAs
and small nuclear RNAs (Elela et al., 1996; Chanfreau et al.,
1997; Rotondo et al., 1995). Unlike other known RNaseIII
proteins, including the homologous C. elegans and S. pombe
proteins, CAF contains a second putative dsRNAbd. This
putative dsRNAbd is essential for wild-type CAF function
since disruption of this sequence by the insertion of the T-DNA
results in the caf mutant phenotype. The altered structure of
CAF relative to RNaseIII, and the fact that the other RNaseIII
homologues known to be present in A. thaliana do not contain
helicase domains, suggest that CAF may have a different
function than the bacterial and yeast RNaseIII proteins.
Given its homology to both RNA helicase proteins and
RNaseIII proteins, it seems likely that CAF acts as an RNA
processing enzyme. Extrapolating from the known functions of
RNA helicase proteins and RNaseIII proteins, and considering
that CAF is predicted to reside in the nucleus, CAF could act
in processes such as mRNA stability, mRNA splicing, rRNA 3′
end maturation, or snRNA processing. The helicase and
RNaseIII domains may act together to process RNAs, with the
helicase possibly acting to unwind or facilitate the recognition
of RNAs which are then cleaved by RNaseIII. Indeed, precedent
for the association of helicase and RNAse proteins already
exists; the DEAD box protein Rh1B exists in a complex (the
degradosome) with the endonuclease RNase E and the 3′ to 5′
exonuclease polynucleotide phosphorylase (reviewed by
Anderson and Parker, 1996). Since CAF-like proteins are found
in the genomes of C. elegans and S. pombe, this suggests that
CAF may carry out a similar function in all three kingdoms.
It is not clear how the 2.5 kb and 6 kb transcripts of CAF
are derived. One possibility is alternative splicing. However, in
our PCR-RACE experiments we did not detect chimeric
transcripts containing upstream exons, as might be expected if
exon skipping were occurring. A second possibility is that the
full length RNA is cleaved, and that only the 3′ cleavage
product stably accumulates. Indeed it is tempting to think that
the CAF protein itself could participate in such a cleavage
reaction. However, analysis of the RNA structure in the caf
mutant suggests that the CAF RNA processing still occurs,
since both a large and a small transcript accumulate (Fig. 9).
Thus, if CAF does process its own transcript, the second
dsRNAbd probably does not play a role in this process, as this
is absent in the caf mutant. A third alternative is that the CAF
gene contains two promoters, one that initiates at the beginning
of the gene and another that initiates in the middle. This would
be consistent with the observation that smaller transcripts with
homology to the 5′ helicase domain are not detected.
Why does mutation of the putative CAF RNA processing
enzyme result in the observed loss of control of cell division
seen in caf mutant floral meristems? One possibility is that CAF
is an mRNA processing enzyme, and that the caf mutant
phenotype results from the misregulation of one or more
specific mRNAs, at least one of which plays a major role in the
control of floral meristem determinacy. CAF would likely affect
the processing of additional RNAs as well, which are important
in other processes throughout the plant, such as those needed
for the proper specification of the normal number of pollen sacs
in an anther, for the specification of the axillary inflorescence
meristems, and for the proper shape of most organs of the shoot.
The authors thank Ken Feldmann for providing the insertional
mutagenesis population from which the caf mutant was isolated, and
the Arabidopsis Biological Resource Center for materials. We also
thank Leonard Medrano for RFLP mapping the CAF gene, and Pam
Green for useful discussions. S. E. J. was supported by an NIH
postdoctoral fellowship (GM15964). M. P. R. was a Howard Hughes
Predoctoral Fellow. This work was supported by NIH grant GM45697
to E. M. M.
REFERENCES
Alvarez, J. and Smyth, D. R. (1994). Flower development in clavata3, a
mutation that produces enlarged floral meristems. In: Arabidopsis: An Atlas
of Morphology and Development. (ed. J. Bowman), pp. 254-257. New York:
Springer-Verlag.
Anderson, J. S., Parker, R. (1996) RNA turnover: The helicase story
unwinds. Curr. Biol. 6, 780-782.
Barton, M. K. and Poethig, R. S. (1993). Formation of the shoot apical
meristem in Arabidopsis thaliana: an analysis of development in the wild-type
and in the SHOOT MERISTEMLESS mutant. Development 119, 823-831.
Bechtold, N., Ellis, J. and Pelletier, G. (1993) In planta Agrobacteriummediated gene transfer by infiltration of adult Arabidopsis thaliana plants.
C. R. Acad. Sci. Paris, Sciences de la vie -Life Sciences 316, 1194-1199.
Bowman, J. L., Alvarez, J., Weigel, D., Meyerowitz, E. M. and Smyth, D.
carpel factory in floral meristem determination 5243
R. (1993). Control of flower development in Arabidopsis thaliana by
APETALA1 and interacting genes. Development 119, 721-743.
Bowman, J. L., Sakai, H., Jack, T., Weigel, D., Mayer, U. and Meyerowitz,
E. M. (1992). SUPERMAN, a regulator of floral homeotic genes in
Arabidopsis. Development 114, 599-615.
Bowman, J. L., Smyth, D. R. and Meyerowitz, E. M. (1989). Genes directing
flower development in Arabidopsis. Plant Cell 1, 37-52.
Bowman, J. L., Smyth, D. R. and Meyerowitz, E. M. (1991). Genetic
interactions among floral homeotic genes of Arabidopsis. Development 112,
1-20.
Chanfreau, G., Elela, S. A., Ares, M. and Guthrie, C. (1997). Alternative
3′-end processing of U5 snRNA by RNase III. Genes Dev. 11, 2741-2751.
Chang, C., Bowman, J. L., DeJohn, A. W., Lander, E. and Meyerowitz, E.
M. (1988). Restriction fragment length polymorphism linkage map for
Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 85, 6856.
Clark, S. E., Running, M. P. and Meyerowitz, E. M. (1993). CLAVATA1, a
regulator of meristem and flower development in Arabidopsis. Development
119, 397-418.
Clark, S. E., Running, M. P. and Meyerowitz, E. M. (1995). CLAVATA3 is
a specific regulator of shoot and floral meristem development affecting the
same processes as CLAVATA1. Development 121, 2057-2067.
Clark, S. E., Williams, R. W. and Meyerowitz, E. M. (1997). the CLAVATA1
gene encodes a putative receptor kinase that controls shoot and floral
meristem size in Arabidopsis. Cell 89, 575-585.
Court, D. (1993). RNA processing and degradation by RNaseIII. In Control of
messenger RNA Stability. (ed. J. Belasco), pp. 71-116. Academic Press, Inc.
Drews, G. N., Bowman, J. L. and Meyerowitz, E. M. (1991). Negative
regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2
product. Cell 65, 991-1002.
Elela, S. A., Igel, H. and Ares, M. (1996). RNaseIII cleaves eukaryotic
preribosomal RNBA at a U3 snoRNP-dependent site. Cell 85, 115-124.
Feldmann, K. A. (1992). T-DNA insertion mutagenesis in Arabidopsis: Seed
infection/transformation. In: Methods in Arabidopsis Research (ed. C.
Koncz, N.-H. Chua, and J. Schell), pp. 274-289. Singapore: World Scientific.
Fletcher, J. C., Brand, U., Running, M. P., Simon, R. and Meyerowitz, E.
M. (1999). Signaling of cell fate decisions by CLAVATA3 in Arabidopsis
shoot meristems. Science 283, 1911-1914.
Gaiser J., C., Robinson-Beers K. and Gasser C. S. (1995). The Arabidopsis
SUPERMAN gene mediates asymmetric growth of the outer integument of
ovules. Plant Cell 7, 333-345.
Gavis, E. R., Lunsford, L., Bergsten, S. E. and Lehmann, R. (1996). A
conserved 90 nucleotide element mediates translational repression of nanos
RNA. Development 122, 2791-2800.
Gorbalenya, A. E. and Koonin, E. V. (1993). Helicases: amino acid sequence
comparisons and structure-function relationships. Curr. Opin. Struct. Biol.
3, 419-429.
Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. and Blinov, V. M.
(1989). Two related superfamilies of putative helicses involved in
replication, recombination, repair and expression of DNA and RNA
genomes. Nucl. Acids Res. 17, 4713-4730.
Harosh, I. and Deschavanne, P. (1991). The RAD3 gene is a member of the
DEAH family RNA helicase-like protein. Nucl. Acids Res. 19, 6331.
Hay, B., Jan, L. Y. and Jan, Y. N. (1988). A Protein Component of
Drosophila polar granules is encoded by vasa and has extensive sequence
similarity to ATP-dependent helicases. Cell 55, 577-587.
Huala, E. and Sussex, I. M. (1992). LEAFY interacts with floral homeotic
genes to regulate Arabidopsis floral development. Plant Cell 4, 901-913.
Iino, Y., Sugimoto, A. and Yamamoto, M. (1991). S. pombe pac1+, whose
overexpression inhibits sexual development, encodes a ribonucease III-like
RNase. EMBO J. 10, 221-226.
Iost, I., Dreyfus, M. (1994). mRNAs can be stabilized by DEAD-box proteins.
Nature 372, 193-196.
Jacobsen, S. E. and Meyerowitz, E. M. (1997). Hypermethylated
SUPERMAN epigenetic alleles in Arabidopsis. Science 277, 1100-1103.
Kayes, J. M. and Clark, S. E. (1998). CLAVATA2, a regulator of meristem
and organ development in Arabidopsis. Development 125, 3843-3851.
Kharrat, A., Macias, M. J., Gibson, T. J., Nilges, M. and Pastore, A. (1995).
Structure of the dsRNA binding domain of E. coli RNase III. EMBO J. 14,
3572-3584.
Kim, S. H. and Lin, R. J. (1996). Spliceosome activation by PRP2 ATPase
prior to the first transesterification reaction of Pre-mRNA Splicing. Mol. Cell
Biol. 16, 6810-6819.
Kramer, A. (1996) The Structure and function of proteins involved in
mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367-409.
Kressler, D., De La Cruz, J., Rojo, M. and Linder, P. (1997) Fal1p is an
essential DEAD-Box protein involved in 40S-ribosomal-subunit biogenesis
in Saccharomyces cerevisiae. Mol. Cell Biol. 17, 7283-7294.
Lasko, P. F., Ashburner, M. (1988) The product of the Drosophila gene vasa
is very similar to eukaryotic initiation factor-4A. Nature 335, 611-617.
Laux, T., Meyer, K. F. X., Berger, K. and Jurgens, G. (1996). The
WUSCHEL gene is required for shoot and floral meristem integrity in
Arabidopsis. Development 122, 87-96.
Liang, L., Diehl-Jones, W. and Lasko, P. (1994) Localization of vasa protein
to the Drosophila pole plasm is independent of its RNA-binding and helicase
activities. Development 120, 1201-1211.
Long, J. A., Moan, E. I., Medford, J. I. and Barton, M. K. (1996). A
member of the KNOTTED class of homeodomain proteins encoded by the
STM gene of Arabidopsis. Nature 379, 66-69.
Mandel, A. M., Gustafson-Brown, C., Savidge, B. and Yanofsky, M. F.
(1992). Molecular characterization of the Arabidopsis floral homeotic gene
APETALA1. Nature 360, 273-277.
Mayer, K. F. X., Schoof, H., Haecker, A., Lenhard, M., Jurgens, G. and
Laux, T. (1998). Role of WUSCHEL in regulating stem cell fate in the
Arabidopsis shoot meristem. Cell 95, 805-815.
Nakai, K. and Kanehisa, M. (1992). A knowledge base for predicting protein
localization sites in eukaryotic cells. Genomics 14, 897-911.
Nashimoto, H. and Uchida, H. (1985). DNA sequencing of the Escherichia
coli ribonuclease III gene and its mutations. Mol. Gen. Genet. 201, 25-29.
Nielsen, P. J., McMaster, G. K. and Trachsel, H. (1985). Cloning of
eukaryotic protein synthesis initiation factor genes: isolation and
characterization of cDNA clones encoding factor eIF-4A. Nucl. Acids Res.
13, 6867-6880.
Olszewski, N. E., Martin, F. B. and Ausubel, F. M. (1988). Specialized
binary vector for plant transformation-expression of the Arabidopsis
thaliana AHAS gene in Nicotiana tabacum. Nucl. Acids Res. 16, 1075610782.
Parcy, F., Nilsson, O., Busch, M. A., Lee, I. and Weigel, D. (1998). A genetic
framework for floral patterning. Nature 395, 561-566.
Rotondo, G., Gillespie, M. and Frendewey, D. (1995). Rescue of the fission
yeast snRNA synthesis mutant snm1 by overexpression of the double-strandspecific Pac1 ribonuclease. Mol. Gen. Genet. 247, 698-708.
Rozen, F., Edery, I., Meerovitch, K., Dever, T. E., Merrick, W. C. and
Sonenberg, N. (1990). Bidirectional RNA helicase activity of eucaryotic
translation initiation factors 4A and 4F. Mol. Cell Biol. 10, 1134-1144.
Running, M. P., Clark, S. E. and Meyerowitz E. M. (1995). Confocal
microscopy of the shoot apex. In Methods in Cell Biology: Plant Cell
Biology, Vol. 49, (ed. D. W. Galbraith, D. P. Burque and H. J. Bohnert), pp.
215-227. Academic Press: San Diego.
Sakai, H., Medrano, L. J. and Meyerowitz, E. M. (1995). Role of
SUPERMAN in maintaining Arabidopsis floral whorl boundaries. Nature
378, 199-203.
Schultz, E. A., Pickett, F. B. and Haughn, G. W. (1991). The FLO10 gene
product regulates the expression domain of homeotic genes AP3 and PI in
Arabidopsis flowers. Plant Cell 3, 1221-1237.
Schulz, B., Bennett, M. J., Dilkes, B. P. and Feldmann, K. A. (1995). TDNA tagging in Arabidopsis thaliana: Cloning by gene disruption. In Plant.
Mol. Biol. Manual K3, pp. 1-17. Kluwer Academic Publishers, Dordrecht,
Netherlands.
Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M. (1990). Early flower
development in Arabidopsis. Plant Cell 2, 755-767.
Steeves, T. A. and Sussex, I. M. (1991). Patterns in Plant Development.
Cambridge, U.K.: Cambridge University Press.
Warbrick, E. and Glover, D. (1994). A Drosophila gene encoding a DEAD
box RNA helicase can suppress loss of wee1/mik1 function in
Schizosaccharomyces pombe. Mol Gen. Genet. 245, 654-657.
Webster, P. J., Liang, L., Berg, C. A., Lasko, P. and Macdonald, P. M.
(1997). Translational repressor bruno plays multiple roles in development
and is widely conserved. Genes Dev. 11, 2510-2521.
Weigel, D., Alvarez, J., Smyth, D. R., Yanofsky, M. F. and Meyerowitz, E.
M. (1992). LEAFY controls floral meristem identity in Arabidopsis. Cell 69,
843-859.
Xu, H. P., Riggs, M., Rodgers, L. and Wigler, M. (1990). A gene from S.
pombe with homology to E. coli RNase III blocks conjugation and
sporulation when overexpressed in wild-type cells. Nucl. Acids Res. 18, 5304.
Yanofsky, M. F., Ma., H., Bowman, J. L., Drews, G. N., Feldmann, K. A.
and Meyerowitz, E. M. (1990). The protein encoded by the Arabidopsis
homeotic gene AGAMOUS resembles transcription factors. Nature 346, 3539.