Note: Descriptions are shown in the official language in which they were submitted.
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W O 97127307 PCTrUS97/01275
RASPBERRY PROMOTERS FOR EXPRESSION
OF TRANSGENES IN PLANTS
F~eld of the Invention
'Ihe present invention relates to ~e ~ nfific~tion of promoters from raspberry which are
capable of providing col~liluli~e expression of heterologous plant genes, and to rhim~ric genes,
cassette vectors, kits, transgenic plants, and methods employing such promoters.
R~ ,t~s
Adarns, D.O., and Yang, S.F., Plant Physiology 70: 117-123 (1977).
Alcama, K. et al., Plant Cell Reports 14(7):450 154 (1995).
Ausubel, F.M., et al., in CURRENT PRarOCOLS lN MOLECULAR BlOLOGY, John Wiley andSons, Inc., Media PA (1992).
Bal~s, E., et al., Gene 19(3):239-249 (1982).
Beachy, R., et al., Annu. Rev. Phytopathol. 28:451-74 (1990).
Beck, et al., Gene 19:327 336 (1982).
Bellini, C., et al., Bio/Technol 7(5):503-508 (1989).
Benfey, P.N., et al., Science 250:959-966 (1990).
B~1lV~I1UlO, E., et al., XXIst Annual Meeting of the Italian Society ~or ~riclllhTr~ Genetics,
Como, Italy, September 30-October 2, 1987 Genet. Agrar. 42(1) (1988).
Bestwick, R.K., et al., PCT TntPrn~tion~i Publication No. WO 95/35387, published 28
December 1995.
Brunke, K.J. and Wilson, S.L., European Patent Publication No. 0 559 603 A2, p~bli~h~d
September 08, 1993.
Comai, L. and Coning, A.J., U.S. Patent No. 5,187,267, issued 16 February, 1993.~ordes, S., et al., Plant Cell 1:1025-1034 (1989). Dayhoff, M.O., in ATLAS OFP~OTEIN
SEOUENCE AND STRUCTURE Vol. 5, National Biom~ l Research Foun-l~tion, pp. 101-110, arld
Supplement 2 to this volume, pp. 1-10 (1972).
Deikman, J., et al., EMBO J. 7:3315 (1988).
Deikman, J., et al., Plant Physiol. 10p:2013 (1992).
Delanney, X., et al., Crop Science 35(5):1461-1467 (1995).
Ooyle, J.J., and Doyle, J.L., Focus 12:13 15 (1990).
Ferro, A., et al., U.S. Patent No. 5,416,250, issued 16 May 1995.
Fillatti, J., et al., Biotechnology 5:726-730 (1987).
Fraley, R.~ et al., U.S. Patent No. 5,352,605, issued on October 4, 1994.
Fry, J., et al., Plant Cell Reports _:321:325 (1987).
Gritz, L., et al ., Gene 25: 179-188 (1983) .
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W O 97/27307 PCT~US97/01275
Guilley, H., et al., Cell ~Q(3):763-773(1982).
Hood, E., et al., J. Bacteriol. 168:1291-1301(1986).
Hooykaas, P.J.J., and Schilperoot, R.A., TR~NDS INBIOCHEMICAL SCIENCES, Tntern~tic~n~l t
Union of Biochemistry and Elsevier Science Publishers, v. 10(8):307-309 (Aug. 1985).
Hughes, J.A., et al., J. Bact. 169:3625-3632(1987).
Je~erson, R.A., et al., EMBO J. 6:3901~1987a).
Je~rson, R.A., Plant Mol. Biol. Rep. 5:387 (1987b).
Jongedijk, E., et al., Euphytfca 85:173-180(1995).
Kawasaki, E.S., et al., in PCR TECHNOI,OGY: PRINCIPLES AND APPLICATIONS 0~ DNA
lQ AMPLIFICATION (H.A. Erlich, ed.) Stockton Press (1989).
R~ein, T.M., et al., PNAS QUSA) 85(22):8502-8505(1988).
T ~PmPlli, U.K., Nature 227:680-685(1970).
Lee, J.J., et al., Meth. of En~mol. 152:633-648 (1987).
Leisner, S.M., and Gelvin, S.B., Proc. Natl. Acad. Sci. USA ~(8):2553-2557(1988).
M~ni~ti~, T., et al., in MOLECULAR CLONING: A LABORATORY MANUAL~ Cold Spring
Harbor Laboldlo.y (1982).
Melchers, L.S., et al., Plant J. 5:469480(1994).
Miki, B.L.A., etal., PLANTDNA INFECnOUS AGENTS (Hohn, T., etal., Eds.) Springer-Verlag, Wien, Austria, pp.249-265(1987).
Mullis, K.B., U.S. Patent No. 4,683,202, issued 28July 1987.
Mullis, K.B., et al., U.S. Patent No. 4,683,195, issued 28 July 1987.
Nagel, R., et al., F~MS Microbiol. Lett. 67:325 (1990).
Ni, M., et al., Plant J. _:661-676(1995).
Odell, J.T., et al., Nature 313:810-812(1985).
Odell, J.T., et al., J. Cell Biochem. (SUPPI. 1 IB):60 (1987).
Odell, J.T., et al., Plant Mol Biol 1~(3):263-272(1988).
Ochman, et al., PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATTONS. XVIII,
,A~ lemic Press, Inc., San Diego CA, USA and T.on~ n, F.ngl~nl1 p. 219-227(1990).
Pearson, W.R., Methods in Enzymology 183:63-98(1990).
Pearson, W.R. and Lipman, D.J., PNAS 85:2444-2448(1988).
Picton, S., et al., Plant Physiology 103(4):1471-1472(1993).
Ponstein, A.S., et al., Plant Physiology tO4:109-118(1994).
Rogers, S., U.S. Patent 5,034,322, issued on July 23, 1991.
Rogers, S., U.S. Patent No. 5,378,619, issued on January 3,1995.
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Saikl, R.K., et al., Science ~ :487~91(1988).
Sarnbrook, J., et al., in MOLECULAR CLONING: A LABORATORY MANUAL~ Cold Spring
Harbor Laboratory Press, Vol. 2 t1989).
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Tinius, C.N., et al., Crop Science 35(5):1451-1461 (1995).
Toubart, P., et al., Plant J. 3:367-373 (1992).
l'omInerup, ~I., et al., Eur. Congr. Biotechnol. 5:916-918(1990).
~Jan Den Elzen, P.L.M., et al, "ViNs and Fungal ~P,i~lA~ e; From Laboratory to Field"
in: lkE PRODUCrlON AND USES OF GENE rlcALLy TRANSFORMED PLANTS (Bevan, M.W., et al.,
10 7;ds~, :Royal Society Discussion Meeting, ('h~pmAn and Hall Ltd., London Fngl~n(7 1994.
V~lnthAmhi, K., et al., J. Bacteriol. 170(4):1523-1532(1988).
~ang, A.M., et al. in PCR PROTOCOLS: A GUIDE TO MErHODS AND APPLICATIONS (M.A.
Innis, et al., Eds.) Ac~lçrnic Press (1990).
Woloshuk, C.P., et al., J. Plant Cell 3:619-628 (1991).
Yao, J.L., et al., Plant Cell Reports 14(7):407~12(1995).
Zhou, H., et al., Plant Cell Reports 15:159-163 (1995).
Zhu, Q., et al., Plant Cell 7:1681-1689(1995).
Ba~ u-ld oftheTnvention
Promoters that regulate gene e,L~.es:,ion in plants are ~ ntiAI elements of plant genetic
~ngin~Pring. Several examples of promoters useful for the expression of selected genes in plants
are now available (Zhu, et al., 1995; Ni, et al., 1995).
To be expressed in a cell, a gene must be operably linked to a pLo.llùler which is recognized
by certain ~l~ylllcS in the cell. The 5' non-coding regions of a gene (i.e., regions imm~li-At~ly
5' to lhe coding region), referred to as pLonlulel:~ or llans~ Liollal regulatory regions, initiate
l.~,liL~Iion of the gene to produ-~e a mRNA Ll~scli~l. The rnRNA is then tr~n.cl~tyl at the
ribosomes of the cell to yield an encoded polypeptide.
}~lul~l~le~ typically contain from about 500-1500 bases, and canprovide regulated expression
of genes under their control. A prulllolei used for e~ essillg a heterologous gene in plant cells
3~ may be ~,hala.iLe.iG~d as (i) a cons~iLuli~reprc~ ûL~l, that is, a promoter capable of causing similar
levels of gene expression in all or many plant tissues, or, (ii) a tissue selective plullluL~l, that is,
one which is capable of regulating gene expression to select tissues in a plant l~Ai~~ru, -- IAIII (e.g.,
leaves or fruit).
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~any such promoters have been characterized, including those derived from plant viruses,
~igrobacter~um genes, and a variety of plant genes. Considerable effort has gone into the isolation
and characterization of co~ ve promoters to drive the expression of a variety of heterologous
genes in plant systems.
Viral promoters (i.e., pLo.lluLel~ from viral genes) for e~ g selected genes in plants,
have been jtl~ntified in the caulimovirus family of viruses (a group of double-stranded DNA
viruses), and include the Cauliflower Mosaic Virus (CaMV) 35S (Balazs, et al, 1982; Guilley,
et~l., 1982; Odell, etal., 1985; Odell, etal., 1987; Odell, etal., 198~; Toll""~ , etal., 1990;
Jefferson, et al., 1987a; Jefferson, 1987b) and CAMV 19S prul,.oLel~ (Fraley, et al., 1994), and
the Figwort Mosaic Virus (FMV) (Rogers, 1995) p,u",oler. PlullloLe~ useful for regulating gene
~ ression in plants and obtained from bacterial sources, such as Agrobacterium-derived
p,ulllOl~.a, have been identified and isolated. Such promoters include those derived from
Agrobacterium T-DNA opine synthase genes, and include the nopaline synthase (nos) promoter
~Rogers, 1991), the octopine synthase (ocs) plul-loler (I eisner and Gelvin, 1988) and mannopine
synthase (mas) p~.JllwL~f.
Plant prurl,oLel~ (promoters derived from plant sources) effective to provide co~ ;ve
expression, are less well known, and include hsp80, Heat Shock Protein 80 from cauliflower,
(Brunke and Wilson, 1993), and the tomato ubiquitin p,ollloLer (Picton, et al., 1993). These
piOlllOL~l~ can be used to direct the CO..~ l iv~ expression of heterologous nucleic acid sequences
20 in l, ~ r~,. .. ,ed plant tissues. At present, a relatively small number of plant prol,loLe,~, particular}y
co~ (iv-e plant prol~lote.~, has been j~lrntifirA The use of such promoters in plant genetic
engineering has been rather limited to date, since gene expression in plants is, for the most part,
typically tissue, developmentally, or envho.. ~,li.lly-regulated.
S.-l.l.n~. y of the ~ ioll
The present invention is directed to raspberry promoters which separately and in combination
provide moderate-level, co~ .live expression of nucleic acid sequ~nf~rs placed under their
control. The plull~oLers of the invention can also confer con~ ve expression on heterologous,
non~on~ ivt; promoters.
The present invention is directed to a plullloler which, in a native raspberry genome, is
operably linked to the coding region of a drul gene. Chimeric genes of the present i-lvelllion
contain a DNA sequpnre encoding a product of interest under the transcriptional control of a
raspberry ~rl~l promoter. The DNA ~equenre is typically heterologous to the piollloleL and is
operably linked to the promoter to enable col~L~ ive expression of the product.
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s
In one embodiment, the product is a polypeptide that permits selection of ~ r.,... Pd plant
cells cont~ining the chimeric gene by rendering such cells resistant to an amount of an zntibiotic
that would be toxic to non-transformed cells. Exemplary products include, but are not lirnited to,
arninog,rlycoside phosphul~ r~lases, such as neomycin phosphul.~sÇeld~e and hy~Lulllycill
S phosphul~ Ç~lase. In one such embodiment, a chimPric gene of the invention contains an hpt
gene sequence encoding hy~lollly~;h~ phosphuLIal3~rel~se II under the transcriptional control of a
dr~l p.ru-~lùler. In an alternate embodiment, a chimeric gene of the i~lve~llio~ contains an np~l~
gene sequence encoding neomycin phosphol.~is~.ase under the tr~nc~ ;O~A1 control of a drul
~r~ o~
In another embodiment, the product is a polypeptide that confers herbicide~ - \ce to trans-
formed plant cells expressing the polypeptide. In one such embodiment, a chimeric gene of the
present invention contains a bxn gene encoding a b~u~ yllil-specific nitrilase under the l.ansc.i~-
tional control of a drul plUIIIU~ . T~ ru....P~ plants co.~ g this chimpric gene express a
bromoxynil-specific nitrilase and are les;s~a..l to the application of bLOI1~Y1I;I-CO~ g herbi-
15 cides. Other exemplary DNA se~uenrps encoding genes co~r~ . ;..g herbicide resiet~nre include
the 3~PSP synthase gene (encoding 5-enolpyruvylchikim~te-3-rhosIlh~te ~y-lll-ase enzyme), which
confers rPcict~nce to glyphosate; an acetol~ct~te synthase gene, which confers reCict~nre to the
hPrbicirle"GLEAN"; abialaphosresict~nregene(the bar gene)codingforphosphinnthricinacetyl-
Ç~I~se (PAT), and the glyphosate-tolerant genes, CP4 and GOX. Chimeric genes of the
20 il.vt;~i.on contain one or more of these herbicide-rpcict~nre genes, operationally linked to a drul
pLullwl~l.
In another embodiment, the DNA sequence or cDNA sequence encodes a viral coat protein,
such as alfalfa mosaic virus coat protein, cucumber mosaic virus coat protein, tobacco streak virus
coat protein, potato virus coat protein, tobacco rattle virus coat protein, and tobacco mosaic virus
25 coat protein. Acco.~lh~g to one such embodiment, a chimPric gene of the invention contains a viral
coat protein gene, such as Al MV, CMV, l~V, PVX, l~V, or l~V, under the tr~n~criptior~l con-
troi of a drul pro..,ùler. Al~L--alively, the DNA sequence col.ci~onds to a gene encoding a
domin~nt defective protein, such as mutant forms of the ETRl gene which confer ethylene
~ insensitivity. In yet another embodiment, the DNA sequence corresponds to a gene capable of
3~ altering a plant biochemical pathway, such as such as the ACCD gene. The ACCD gene forms
~ a product which degrades a precursor in the ethylene biosynthetic pall.~.y.
In anûther aspect, the invention includes an isolated DNA molecule co...~ i--g a co~ ive
plo--.a,~e. fromaraspberry drul gene. One ~em~ ry raspberry drul p.ul..ùl~. is the drullO pro-
moter" presented herein as SEQ ID NO:3. Ano~er exemplary con~tihltive raspberry plOlllUlel
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is the dru259 plo~oler~pLesented as SEQ ID NO:4. Additional fragments may be derived from
the sequence .e~res~ iug the fi~ length drul promoter, SEQ ID NO:2, where the smaller
fragments are effective to regulate conslilulive expression of a DNA sequence under their control.
The present invention also includes the use of any of the above chimeric genes, DNA
5 constructs, and isolated DNA sequences to generate a plant Liansfo~ alion vector. Such vectors
can be used in any plant cell t~ ro~ ;ion method, inrhlfling Agrob~ ;.. -based m~th~d~,
ele~L~opo.dlion, microinjection, and microprojectile bonlbaldl-lent. These vectors may also form
part of a plant Ll~ rul ~ ~ ion kit. Other components of the kit may include, but are not limited
to, reagents useful for plant cell ~ r(J, ..,~linn
In another embodiment, the present invention includes a plant cell, plant tissue, transgenic
plant, fruit cell, whole fruit, seeds or calli containing any of the above-described raspberry
plOlllOL~l~, chimeric genes or DNA constructs.
In another aspect of the present invention, the dru pl'OIllu~ described herein are employed
in a method for providing moderate expression of a heterologous gene, such as a selectable marker
1~ gene, in transgenic plants. In this method, a chimPric gene of the present invention containing a
DNA sequence encoding a selectable marker product (~e.g., a neomycin phospholldi~relcse or
LygLUIII~\Cin phOS~/hO~ld~lSrelaSe) iS inkoduced into progenitor cells of a plant. Transgenic plants
c~ g the ehimeric gene are selected by their ability to grow in the presence of an amount of
selective agent (e.g.,lly~sloll.ycill, geneticin or k~lalllycill) that is toxic to non-lLal~rulllled cells.
The lla.-~rul--.ed plant cells thus selected are then regenerated to provide a dirr~ idled plant,
followed by selection of a ll ."cr~" .I.~d plant which expresses the product.
Further, the invention includes a method for producing a lla~lsgellic fruit-bearing plant. In
is method the chimeric gene of the present invention, typically carried in an eA~I~s:~ioll vector
allowing selection in plant cells, is introduced into progenitor cells of selected plant. These
progenitor cells are ~ten grown to produce a l~ sgellic plant.
The method may further comprise isolation of a drul plùl~o~er (such as drullO or dru25!~
by the following steps:
(i) selecting a probe DNA molecule co~ g a sequPnce homologous to a region of
raspberry drul gene DNA,
(ii) cr."~ ;"g the probe with a plurality of target DNA molecules derived from a ~ybe~ly
genome under conditions favoring specific hybridization between the probe molecule and a target
molecule homologous to the probe molecule,
identifying a target molecule having a DNA sequence homologous to the raspberry drul
gene,
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(iv) isolating plol.loLel sequences associated with the target molecule, and
(V) evaluating one or more of the isolated sequences or portion thereof for its ability to
regulate con~Lilulive expression of a dow~ leal-l gene under its control.
The chim~ric genes, vectors, constructs, isolated DNA molecules, products and methods of
5 the present invention can be produced using the raspberry drul ~ olel sequences ~ onti~lly as
described above.
These and other objects and features of the invention will become more fully ~ llL when
the following detailed description is read in conjun~fion with the arco...l.~..yi~lg figures and
eY~mr~ c~
10 Brief I)escription of the Fi~ures
Fig. 1 is a schpm~tic diagram illustrating the creation of plasmid pAG~31 co"lahlillg an
exemplary raspberry drul p~ull~lei~ referred to herein as dru259 pro, and the nptII gene;
Fig. 2 is a flow chart ~ s~lllillg the steps followed in constructing vector pAG~21
cont~ining a chimeric drullO pro-nptII gene;
Fig. 3 outlines the steps involved in the construction of Agrobacterium binary vector pAG-
7242, containing dr~llIO pro fused to the nptII gene, from plasmids pAG-1542 and pAG~21;
Fig. 4 is a flow chart depicting the creation of Agrobacterium binary vector pAG-7342
co..~ ille a ~himPric dru259 pro-nptII gene;
Fig. 5 is a graph representing relative levels of nptII gene expression across 10 transgenic
events for three different p~ollw~l-npt II chimeric gene combins~tions;
Figs. 6A and 6B present the genomic DNA sequence of the drul gene. Tn~lic~ted in the
figures are a CAAT box, TATA box, ATG start codon, two exons, an intron, splicing sites, a stop
codon and poly-adenylation sites;
Figs. 7A and 7B present the DNA sequence of the full length drul plolllble~
Fig. 8 presents the DNA sequence of the drullO plon~oLeL,
Fig. 9 presents the DNA sequence of the dru259 pl~lllU~
Fig. 10 presents r~lcsel~ e results of polyaclyl~nide gel electrophoretic analysis of
raspbelry drupelet proteins;
Figs. llA and 11B schen~tit~ y ,~,~selll the reverse transcriptase-polymerase chain
reaction (RT-PC~; Kawasaki, et al., 1989; Wang, et al., 1990) cloning of the raspberry drul
gene;
Fig. 12 presents a s~ h~ tic reprels~nt~tinn of the gene Ol~ ion and protein structure of
dr~l;
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WO 97127307 PCTrUS97/01275
Fig. 13 presents a Kyte-Doolittle hydrophilicity plot of the coding sequence of drul . In the
figure, the hydrophilicity window size = 7;
Fig. 14 shows the results of RNA dot blot analysis of drul RNA expression in raspberry leaf
and lcG~l~rle- RNA was isolated from green, mature green, breaker & orange/ripe raspberries
5 (co..~onding to stages I, II, III, IV, respectively);
Fig. 15 shows the results of a RNA hybridization study evaluating the expression of drul
RNA in l~lJbell~ leaf and fruit;
Fig. 16 shows the results of polyacrylamide gel electrophoretic analysis of raspberry drupelet
proteins obtained from drupelets at various stages of ripening, and
Figs. 17A and 17B depict a flow chart sl~ll.. ll ~ ;, .l~g the construction of plasmid pAG-1542.
Detailed Description of the I~ ..lion
r~ iv-ls
The following terms, as used herein, have the ...~ as inrlir~t~A
"Chimeric gene" as defined herein refers to a non-naturally occurring gene which is
composed of parts of dirrt;lellL genes. A chimeric gene is typically composed of a pl~,l.loLe.
se~ r~ e operably linked to a "heterologous" DNA sequence. A typical chimeric gene of the
present h~vt;~llioll, for llansro.lllalion into a plant, will include a raspberry dru p-ullwl~f(e.g., a
drullO or dru259 prol-lotel), a heterologous structural DNA coding seqUçnce (e.g., the
aminoglycoside phosphotfa~l~refase (nptII) gene) and a 3' non-translated polyadenylation site.
A "coii~ ;ve" promoter refers to a promoter that directs RNA pro~ ction in many or all
tissues of a plant Llansrolll~a~l~, as opposed to a tissue-specific pLOmO~, which directs RNA
syn~Lhesis at higher levels in particular types of cells and tissues (e.g., fruit specific plUlllU~ such
as the tomato E4 or E8 ~ w~e- (Cordes, et al., 1989; Bestwick, et al., 1995).
By "pro-.lc"~." is meant a seqllen~e of DNA that directs l.allSC~ iOII of a dowllsLre
heterologous gene, and includes promoters derived by means of ligation with operator regions,
random or controlled mutagenesis, addition or duplication of e~ er seq~l~n~c, addition or
m~tlifi-~ion with synthetic linkers, and the like.
By "plant promoter" is meant a promoter (as defined above), which in its native form, is
derived from plant genomic DNA.
"Raspberry promoter" refers to a promoter (as defined above) which, in its native form, is
derived from a raspberry genome. For example, a drul promoter, such as drul l O or dru259, is
a non-coding regulatory region which is operably linked, in a native raspberry genome, to the
coding region of a drul gene.
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A raspberry promoter derived from a specified gene (e.g., a raspberry promoter derived ~rom
the dn~l gene, such as drullO or dru25~ includes a promoter in which at least one or more
regions of the promoter are derived from the specified raspberry gene. An exemplary plulllv~
of this type is one in which a region of the promoter (e.g., a dru259promoter) is replaced by one
S or more sequPn~ Ps derived from a different gene, without substantially reducing the expression of
the resulting chimeric gene in a host cell, or altering the function of the unaltered dru259
pro.nùler.
"Promoter strength" refers to the level of promoter-regulated (e.g, drullO, dru255~) expres-
sion of a heterologous gene in a plant tissue or tissues, relative tû a suitable standard (e.g., cauti-
10 movirus cassava mottle vein virus plulllu~l CAS or the hsp80 promoter). Expression levels can
be measured by linking the promoter to a suitable reporter gene such as GUS ~-glucuronidase),
dihydrofolate re.11-ct~ce, or nptII (neomycin phosphu~ .re.~.e). Expression of the reporter gene
can be easily measured by fluoro--lt;llic, spectropholoi~ellic or histor~hemir ~l assays aefferson,
et al., 1987a; Jefferson, 1987b~.
For the purposes of the present invention, a moderate promoter is one that drives expression
of a reporter gene at about 10-90% of the level obtained with a promoter such as hsp80.
A "heterologous" DNA coding sequence is a structural coding seqrlPnl~e that is not native to
the pl.mt being tl~ ror...P~ or a coding seq~l~n~e that has been Pngineered for i~ rov~d
characteristics of its protein product.
E[eterologous, with respect to the plull~o~r, refers to a coding sequ~n~e that does not exist
in nature in the same gene with the promoter to which it is ~,u~l~;lllly ~tt~hed.
h gene considered to share sequ~nce identity with the drul gene, or a particular region or
regions thereof, has at least about 60% or preferably 80% global sequence identity over a leng~
of polynucleotide seqllpn~ e cû~ ding to the raspberry drul polynucleotide sequences rlicclosed
herein ~e.g., SEQ ID NOs~
"Sequen- e identity" is det~rmined ~Pnt;~lly as follows. Two polynucleotide sequences of
the sarne leng~ (preferably, corresponding to the coding sequ~n~es of the gene) are considered
to be idPntic~l(i,e., homologous) to one another, if, when they are aligned using the ALIGN
program, over 60% or preferably 80% of the nucleic acids in the highest scoring ~lignm~nt are
i~Pnti~ily aligned using a ~up of }, the default parame~ers and the default PAM matrix (I:)ayhoff,
1972).
The ALIGN program is found in the FASTA version 1.7 suite of seq~en/~e col"~ ol-prograrns ~earson and Lipman, 1988; Pearson, 1990; program available from William R.
Pearson, Dt;p~l.ll~,..l of Biological (~hPmi~try, Box 440, Jordan Hall, Charlottesville, VA).
CA 02243850 1998-07-21
W O 97/27307 PCT~US97/01~75
Two nucleic acid fragments are considered to be "selectively hybridizable" to a polynucleo-
tide derived from a drul gene if they are capable of specifically hybridizing to the coding
sequences or a variants thereof or of specifically priming a polymerase chain amplification
reaction: (i) under typical hybridization and wash conditions, as described, for example, in
S ~ni~ti~, et al ., 1982, pages 320-328, and 382-389; (ii~ using reduced stringency wash conrlition~
that allow at most about 25-30% basepair micm~tches, for eY~mple: 2 x SSC (contains sodium
3.0 M NaCl and 0.3 M sodium citrate, at pH 7.0), 0.1% sodium dodecyl sulfate (SDS) solution,
room ~e~ el~lLule twice, 30 minutes each; then2 x SSC, 0.1% SDS, 37~C, once, for 30 mimlte~;
then 2 x SSC, at room temperature twice, for 10 minutes each, or (iii) s~lec~tin~ primers for use
10 in typical polymerase chain reactions (PCR) under standard conditions (for ~Y~mpie7 in Saiki, et
al., 1988), which result in specific amplification of sequences of the desired target sequence or
its variants.
Preferably, highly homologous nucleic acid strands contain }ess than 2040% basepair
"~ , even more plerel~ly less than 5-20% basepair "~i~;"~ c. These degrees of
15 homology (i.e., sequence identity) can be selected by using wash conditions of a~propliate strin-
gency for i~entific~tio~ of clones from gene libraries (or other sources of genetic material), as is
well known in the art.
A "drul encoded polypeptide" is defined herein as any polypeptide homologous to (i.e.,
having ec~enti~lly the same seq~len~e identity as) a drul encoded polypeptide. In one embodiment,
20 a polypeptide is homologous to a drul encoded polypeptide if it is encoded by nucleic acid that
selectively hybridizes to sequen~ s of drul or its variants.
In another embodiment, a polypeptide is homologous to a drul encoded polypeptide if it is
encoded by drul or its variants, as defined above. Polypeptides of this group are typically larger
than 15, preferable 25, or more preferable 35, contiguous amino acids. Further, for polypeptides
25 longer than about 60 amino acids, se~-upn~e co,ll~a-isolls for the purpose of dete. ,i~ g
"polypeptide homology" or "polypeptide sequçnce identity" are performPd using the local
nm~.nt program LAI,IGN. The polypeptide sequence is co--lpaled against the drul amino acid
se~lsenre or any of its variants, as defined above, using the LALIGN program with a ktup of 1,
default pd~ eLeL:l and the default PAM.
3Q Any polypeptide with an optimal ~lignm~ont longer than 60 amino acids and greater than 55%
or preferably 80% of identically aligned amino acids is considered to be a "homologous
polypeptide." The LALIGN program is found in the FASTA version 1.7 suite of sequPn~e com-
parison programs (Pearson and Lipman, 1988; Pearson, 1990; program available from William
R. Pearson, De~ of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, VA).
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A polynucleotide is "derived from" drulif it h~ the same or s~lbst~nti~lly the same b~epair
sequçnre as a region of the drul protein coding sequPnce, cDNA of drul or complements thereof,
or if it displays homology as defined above.
A polypeptide or polypeptide "fragment" is "derived from" drul if it is (i) encoded by a drul
5 gene, or ~ii) displays homology to drul encoded polypeptides as noted above.
]n the context of the present invention, the phrase "nucleic acid seq~ ces," when lt:J~LLillg
to sequences which encode a protein, polypeptide, or peptide, is meant to include degenerative
nucleic acid se~ui~nrçs which encode homologous protein, polypeptide or peptide se~llçnt~çs as well
as the dicr1os~l se~ n~e
As used herein, a "plant cell" refers to any cell derivetl from a plant, inrll~-lin~,~ u~1tli
tiated tissue (e.g., callus) as well as plant seetls, pollen, progagules and e",l)1yus.
II. Identification and Isolation of a Raspberry drul Promoter
The present invention relates, in one aspect, to a p1ulllolei which, in a native r~pberry
genome, (i) is operably linked to the coding region of a drul gene, and (ii) fim~tionc as a
moderate strength, col~LiLu~ivc~ prullluL~,. . This aspect of the invention is based upon the diseu v ~,.y
of the drul gene in r~pberries, which is e~ esscd at very high levels in ripening Eruit. Expres-
sion directed by the full length drul plullluLei is fruit specific, and active during fruit ripening.
3n contr~t to the ru-~cLio11al activity of the drul plv~lloter, it has unexpectedly been
disco~ered that two new pLo1--o~e1s, both derived from the full length raspberry dn~l plUllll;)le~,
i) funt:tion as cu11~lilulive p1u1llol~.s (i.e., are not fruit-specific), and (ii) drive expression of genes
under their control at mod~r~te levels, as will be described in more detail below.
The klentific~tion ûf the drul gene from raspberries, as well as the isol~tion of two
~x~om~ ry drul p.o---ûLe.;, of the present invention, drullO and dru259, will now be described.
A. drul Protein Identification. Purification and Sequence D~le.1l1i,.d~ion
]Protein(s~ produced by ripening fruit, such as those produced by raspberry, are typically
analyzed by gel electrophoresis. A coomassie blue-stained SDS polyacrylamide gel of soluble
drupelet prote;ns is shown in Fig. lû (Examples lA-B). As can be seen from Fig. lO, twû highly
~un~n~ proteins isolable from raspberries are observed at a~,~lox;",~ly 17 and 15 kd, and are
referred to herein a~s drupel and drupe2, respectively. The amount of drupel and drupe2 relative
to the tûtal amount of soluble protein can be det~rmin~-d, for çY~mrle~ by sC~nning dç ,~i~"~ , y.
Sc~nning densilu.-le~ly analysis of the gel illustrated in Fig. lû in(ii(~t~$ that drupel and drupe2
ice ~L,p~ ;""~y 23 and 37%, respectively, of the total soluble protein in raspberry
drupelets. As a result of this det~rmin~tion (i.e., the high levels of drupel and drupe2), purifica-
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tion and sequencing of drupel and drupe2 can be carried out, for example, by using a direct
western blot a~oach.
In carrying out a western blot analysis, total drupelet proteins are western blotted to PDVF
membrane (Example lB) and the regions corresponding to drupel and drupe2 are subjected to
5 N-terminal amino acid sequence analysis. The drupel sample yields a thirty arnino acid
N-terminal sequence (Example lB). The amino terminal drupel sequence is presented herein as
SEQ ID NO:11.
B. Clonin~ drul Encodin~ Sequences
1. RT-PCR and Clonin~ of a drul cDNA Clone. The entire procedure for cloning
drlll, from cDNA synthesis to inverse PCR of a genomic copy of the gene, is shown s~hçm~ti-~lly
in Figures 1 lA and 1 lB.
In carrying out the cloning procedure, mature green raspberry drupelet mRNA is prepared
as described in F~mple 2A and 2B and used as template in a cDNA synthesis reaction. The
reaction is primed using the dTRANDOM primer (SEQ ID NO: 12) shown in Figures llA and
15 1 lB. The resulting cDNA (Example 2C) is subjected to a standard PCR reaction using primers
COL~ Ondi11g to a portion of the dTRANDOM pr;mer and a 512-fold degenerate primer (Drupe
20) based on the drupel amino terminal sequçnce (Exarnple 3).
The PCR amplific~tion products are then analyzed. Products from the above PCR reaction
include a 710 bp product that is agarose gel purified and subcloned into vector pCRII (FY~mrle
20 3). Subsequent sequence analysis of several of these clones allows i~1entific~tion of those clones
whose sequence encodes a protein m~t--hing the amino terminal sequPnce of drupel.
2. Inverse PCR Clonin~ of a Genomic Copy of the drul Gene. In this approach
to cloning the drul gene, genomic ~ be..y DNA is used in a PCR reaction using primers
intern~l to the cDNA sequence obtained as described above (Example 4). This reaction produces
a genomic clone of the drul gene cont~ining most of the protein coding region. A single intron
was i-lPntifiecl from the subsequent sequ~n- e analysis of this clone (Figure ~B). An inverse PCR
strategy may be employed to cl-a.a~,L~.~e and seqnence the 5' region of the gene COIlt~ lg the
dr~l plolllu~el (Example 5). Figures llA and 11B show s~h~m~tic~lly how this may be accom-
plished.
In ch~ r-i~ing the 5' flan~ing region of drul genomic DNA utilizing inverse PCR
te~hniqu~s, raspber~y genomic DNA is digested with NsiI and ligated under dilute cnnrliti- nc to
allow circula~alioll of the restriction rla~ ~. The ligated DNA is then subjected to PCR
~mr~lifi~tk~n using primers internal to the dr~l coding sequence and oriented in opposite dil~cLiolls
from each other. This produces a PCR reaction product cont~ining part of the first exon and 1.35
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kb of lhe promoter. Subsequent sequence analysis of this clone in combination with sequenre
il~ro, I.,:.(ion from the previously described clones produces the complete drul sequence.
3. Sequence Del~ ,a~ion and Evaluation of Gene Expression Patterns. The drul
gene ~igs. 6A, 6B) encodes a protein with the predicted amino acid sequ~nre presented as S~Q
5 ID NO:20. The predicted molecular weight for this protein is 17,088, which agrees closely with
the 17kd molecular weight detPrmined by gel electrophoresis (see Figure 10) of total drupelet
protein. The drul protein is relatively acidic with a predicted pI of 4.8. Nucleic acid and protein
homology searches of the current seq~l~nre databases can be carried out to look for signifi.~nt
matches. For drul, nucleic acid and protein homology searches of the current sequence .lh~ b~
10 produced no .~i~nific~nt matches. This result supports the original observation made with the
arnino terminal sequence of the protein that drupel is a novel protein.
The gene expression pattern of drul can be also be evaluated at the RNA and protein levels
to confirm the tissue specificity of the full length plul.lole.. Northern dot blots, Figs. 14 and 15,
of total RNA from raspberry leaf and receptacles at dirre ~llL ripening stages indicate a tissue and
15 stage specific gene expression pattern. This can be confirmPd by Co~ isoll of northern blots
of total RNA from various other plant tissues. The tissue and stage specific gene expression
pattern of drul was confirmed on northern blots of total RNA from leaf, receptacles, and drupelets
(see Figs. 14 and 15). In both cases, no drul expression is observed in leaf RNA. The RNA
~;A~ ion pattern in lec~ acles is temporally regulated while in drupelets it is fully expressed at
20 the ~wo stages ~i.e., green and ripe) analyzed.
A protein gel of drupelet lysates from dirrelèlll ripening stages can also be carried out to
filrther support stage specific expression of drul. As illustrated in Fig. 16, electrophoretic
analysis of raspberry drupelet proteins obtained from drupelets at various stages of ripening (i.e.,
green, mature green, breaker, orange, and ripe) further S~olL~ a stage specific e~A~ressioll pattern
25 in dNIpelets (Fig. 16).
C. Isolation of the Full Length drul P,0,-,~3ler
~ 'h~ )n of the drul genomic clone allows i~ol~tion of the drul pror,wLer. The
nucleatide sequence of an exemplary full length drul promoter is presented as SEQ ID NO:2.
30 III. Isolation of Plolllo~el ~ drullO and dru259
Two representative raspberry promoters of the invention, drullO and dru259, were isolated
from the full length transcript of the drul plol")ler, which has been charactPri7P~d as a stage and
firuit-specificploll~)~el. Surprisingly, these two new drul-derived pl~lllo~ have been found to
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14
function as moderate level, constitutive promoters when fused to heterologous genes and evaluated
for ~ulLa~lL patterns of expression in l1AI~rUI Ill~d plants.
The t~U~I~ aLed dru promoters, drullO and dru259, can be obtained from the full leng~ drul
promoter as described in Examples 7 and 8.
In utilizing this approach, a PCR reaction product containing part of the first exon and 1.35
kb of the drul ~loll-olel (as described in section II.B.2 above) is ligated into plasmid pCRII
(Invitrogen, ~rlcbacl, CA) to form a subclone, pAG-310, cont~ining the full length drul pro-
moter, as shown in Figs. 1 and 2. A 1.3 kb DNA r".~ "l from pAG-3 10 is then PCR ~mplifi~
under standard contlitinn~ using primers DrupeUp (~' primer, SEQ ID NO:7) and DrupeLow (3-
10 primer, SEQ ID NO:8).
~ ecovery of the ~mplified DNA is typically carried out by addition of solvent to the reactionmixture, followed by centrifugation, recovery of the aqueous phase, and precipitation with
sodium
acetate. The iecov~l~d DNA is then typically purified by centrifugation and repeated washing,
followed by drying of the recovered pellet.
The 1.3 kb DNA fragment is digested to cc-mpletion with restriction enzymes NsiI and XbaI,
followed by purification and ligation into plant expression vector, p3~S-GFP (Clontech, Palo Alto,
CA), which has been digested with XbaI and PstI. Restriction ~yllles, Psd ~used to digest p35S-
GFP) and NsiI (used to digest the drul PCR product), both generate the same 3' ovPrh~ngin~
cohesive ends CIGCA), so that upon lig~tinn, neither restriction site is iecol~L.I~cted. The re-
20 sulting int~rm~ te pl~mi-~ Ae~i~n~ted pAG-155, is represented srhPm~t~ ly in Figs. 1 and 2.
~ $ol~tion of the raspberry dru259 promoleL is aCCQmp1i~h~i by digesting plasmid pAG-155
with reStriCtiQn el~yl,les, SnaBI and EcoR V, which are both blunt end cutters, to release a 259
bp drul pL~JllnJlelrl~ elll, referred to herein as dru259.
Isolation of the raspberry drullO plo~ lis achieved by amplifying a 166 bp Çla~ l.L of
drul carried in plasmid pAG- 155 using primers drul-11 8H3 (SEQ IO NO:9) and GFPStartR (SEQ
ID NQ: 10) under standard PCR reaction conditions. The amplified product is then l~icoveled from
the reaction mixture, and purified as described above, followed by digestion of the 166 bp product
with ~indlII and EcoR V to produce the 112 bp promoter referred to herein as drullO.
The raspberry pLollloLe.~, drullO and dru259, can be used to regulate expression of
30 heterologous genes. Exemplary dru p~ollloler, dru259, has the nucleotide sequence pLesellLed
herein as SEQ ID NO:4. Fxemplary dru promoter, drullO, has the nucleotide sequence p~es~ ..led
as SEQ ID NO:3.
The construction of illustrative subclones, pAG-431 and pAG~21, containing nucleotide
sequ~nces co~ Jonding to dru259 and drullO, respectively, is presented in Figs. 1 and 2.
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IV. Identification of a Plant drul Promoter
T he present invention also provides a method for iden~iryi~.g and isolating a d~l plUIIIUle~,
e.g. dl~llO and dru259, from a variety of plant sources, e.g. raspberry. Such E~UIIIULe~l are
useful for the generation of vector constructs cn"l~i"i~l~ heterologous genes, such as s~lect~hle
5 marker genes, or genes conre~ g herbicide reci~t~n~e
Southern blot experiments are used to dellloll~lLAle the presence of DNA molecules having
~i~nTfjc~nt sec~uen~e identity (i.e., typically greater than 55%, more preferably greater than 80%
identity using standard sequence colllL,alison programs) with the raspberry drul gene in, for
example, strawberry, peach or plum. Similar Southern blot analyses may be pe~fonnP-d on other
10 fruit-bearing plants to identify additional drul genes.
Drul homologues are i~lPntifi~d in a Southern blot (Ausubel, et al., 1992) of the plant
genomic DNA, probed with a labelled DNA La~ el.l cu..l~i..;..g the coding se~ nce of the
raspberry ~rul gene.
The probe is typically selected to contain the coding sequence of dr~l, rather than the
1~ promoter sequence, because coding sequçn~ are typically more conserved from species to species
than are pr.~ olel sequences. Probe molecules are generated from raspberry genomic DNA using
primer-specific amplification ~Mullis, 1987; Mullis, et al., 1987). The oligonucleotide primers
~re selected such that the amplified region includes the entire coding se~lu~ e of the la~b-.ly
d~u7 gene, as provided herein. Primers may also be selected to amplify only a selected region of
20 the raspberry drul gene.
A~ llaLively~ a probe can be made by isolating restriction-digest r a~ co..~ ;..g the
s~qnç~re of interest from plasmid DNA.
The probe is labeled with a detectable moiety to enable subsequent identific~tion of
homologous target molecules. Exemplary labeling moieties include radioactive nucleotides, such
2~ as 32P-labeled nucleotides, digo~-y~llhl-labeled nucleotides, biotinylated nucleotides, and the like,
available from co~l""e,cial sources.
In the case of a primer-amplified probe, labeled mlcleotifi~ may be directly incorporated into
the prûbe during the amplification process. Probe molecules derived from DNA that has already
been isolated, such as restriction-digest ï~ from plasmid DNA, are typically end-labeled
30 ~Ausubel, et al., 1992).
Target molecules, such as HindIII DNA fragments from the genomes of the above-listed
plants, are electrophoresed on a gel, bloKed, and immobilized onto a nylon or nitrocel~-llose filter.
Labe]ed probe mn~çcnle5 are then contacted with the target m~lecul~s under con-liti-)ns favoring
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16
specific hybriAi7~t;on between the probe molecules and target molecules homologous to the probe
molecules (~ni~ti~, et al., 1982; Sarnbrook, et al., 1989; Ausubel, et al., 1992).
Following the i-lPntifir~tinn of plants cont~ining drul genes, the DNA co~ the desired
genes, inrh~ling the promoter regions, may be isolated from the respective species, by, for
5 example, the methods described herein for the isolation of the raspberry drul gene. Generation
of truncated prol~lol~l . may be accomplished by, for example, 5' deletions such as those described
herein for the isolation of the drullO and dru259 promoters.
Variants of t'ne drul p~ol~wLe~ may be isolated from dirr~l~l-l raspberry cultivars and from
other plants by the methods described above. A reporter gene, such as GUS ~-glucuronidase),
10 can be used to test tissue for constitutive, moderate level expression regulated by such PLO11WI~. ..
Expression of GUS protein can be easily measured by fluoiome~-ic, spectrophotometric or histo-
rhPm;r~l assays (Jefferson, et al., 1987a; Jeffer~on 1987b).
Further, using chimeric genes cont~ining drul piullwL~f sequP-n~s operably linked to
reportOE gene sequPnres, DNA sequPnres corresponding to regulatory domains can be i(l~ntifiP~
15 using, for exarnple, deletion analysis (Benfey, et al., 199~). For example, the dru259 promoter
sequence presented as SEQ ID NO:4 can be fimrtiQn~llly linked to the GUS reporter gene. Dele-
tion analysis can then be carried out by standard methods (Ausubel, etal., 1992; l\~ni~ti~, etal.,
1982; Sambrook, et al., 1989). Alle..,a~ively, regions of the full length drul promoter se~uPnre
can be ~mplified using sequence-specific primers in PCR, as ~ lstr~ted in Figs. 1 and 2. These
~mrlified fragments can then be inserted 5' to the GUS coding se~nçnr~s and the resulting
expression patterns evaluated for moderate level, co~ uLi~e expression, which are features of the
~ ybtil~y promoters of the invention.
V. Plant Tldll~r~ aLion
In support of the present invention, eY~mpl~ry chimeric genes corlt~ining a ~asybe~l~r plant
p~ ole- sequence operably linked to a heterologous DNA sequence, were constructed.
FYf~mpl~ry rhimPric gene constructs include drullOpro:nprII ~xample 10) and dru259pro:np m
(Example 9). The protein ~y~essed by the nptII gene, neomycin phospho~ rrl~e, is an
aminoglycoside phospho~ldnsreldse~ which confers kallalllyci~l r~ t~nre to transgenic plants
~ si~g the product. This protein, as well as other selectable marker products, and products
~onrel~ g herbicide rçcict~nce~ may function more effi~içntly if expressed (i) con~liLuli~ely, and
(ii~ at moderate levels (rather than ~eing ov~ Le~ed) in Llansgellic plants. Accordingly,
elrp.mrl~ry pro...oLel~ drullO and dru259 represent ideal pr~lllolel:, for satisfying this objective.
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A. Construction of Agrobacterium Binary Plant T.dl,~rc,L",ation Vectors
Construction of Agrobacterium binary vectors, pAG-7242 and pAG-7342, contAinin~ the two
representative chimeric genes described above, can be performed as described in FyAmple 10 and
Examp]e 19 (sch~mAticaTly represented in Figs. 34, drullOpro:nptII, and dru259pro:npm,
S respectively). These binary vectors also contain a gene encoding SAMase, S-adenosylm~thionin.-
hydrol~.e (Ferro, et al., l 99S; Hughes, et al., 1987), which is i,l""aLe.ial to the present h~vellliol~.
1. Construction of a Binary Plant Tla~ .l.d~ion ~ector pAG-7342 Ccl~lai.li.l~ a
drl-259::nprlr Chimeric Gene. Binary plant llall~rulll~alion vector, pAG-7342, is constructed by
excising a 13 kb nos pro::nptII fragment from subclone pAG-1542 by digestion with ~indlII and
10 BamHI, followed by ligationto a 1.1 kb HindlII-BamHI r".~",~", from subcloningvector, pAG-
431, to insert a dru259 pro::nptI~ chimeric gene.
Plasmid pAG-1542 can be prepared using conventional cloning techniques known in the art
(Sambrook, et al., 1989). This illustrative subcloning binary vector contains a neomycin
phospho~ .re.ase II s~lectA~le marker gene (nptII~ gene under the control of the nos p~v~lwler
15 iocated near the left border, and the SAMase gene (Ferro, et al., 1995) driven by the tomato E8
promoter (Deikman, et al., 1988; Deikman, et al., 1992) located near the right border. As pre-
viously stated, the presence of the tomato E8:SAMase construct is illllllA~ ;Al to the e~ sio
resu}ts described herein.
ConstructionofsubclonepAG-431, cu~lAillillgthedru259::nptIIrhim~ricgene~ isdescribed
20 in FY~mple 7. Construction of binary plant ~ r~ A1ion vector pAG-7342 is depicted
S~ lly in Fig. 4 and detailed in Example 9.
A flow chart ~w~ a~i~ing the construction of plasmid pAG-1542 is p-~st~ ed in Fig. 17.
2. Construction of a Binary Plant Tl a-lsrul ...alion Vector Containing a drullO: :~ptlI
~himeric Gene. Utilizing a similar ay~lOdcl, binary plant ll~ rù~ A~ n vector, pAG-7242, is
25 constmcted by excising a 13 kb nos pro::nptII r.a~",-ell~ from subclone pAG-1542 by digestion
with ~ind~I and BamH~, followed by ligation to â 0.95 kb dru259::nptII fr~m~nt from pAG~21
to forrn ~e binary plant llall~.~ll,laLion vector pAG-7242.
C~onstruction of binary plant ~LAncrol~A~ n vector pAG-7242 is depicted sch~-mAticAlly in
- Fig. 3 and described in Example 10. Construction of subclone pAG~21, contAining the
30 drui'lO::nptII ~him~ric gene, is described in Example ~.
B. Methods of Tl dl,~ro, Illh~ Plants
l;~e above-described chim~ric genes can be inserted, for example, into p}ant cells.
Transgenic plants containing these exemplary chimeric genes, regulated by the raspberry prvlllole.
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W 097/27307 PCTrUS97/01275
of the invention, express neomycin phosphullall~reldse II, which confers to plants ~ Les5illg the
product, resistance to the antibiotic, k~ llycin.
In expe~ .e,.l~ performed in support ûf the invention, the chimeric genes were inserted into
tomato plant cells, and the resulting levels and patterns of expression of the nptII s~lectAhle marker
5 gene were p~tAmin~(l Although nptII was selected as an eYernrlAry marlcer gene to ill~1ctr~te the
ability of a raspberry plant piolllcJLel of the invention to regulate expression of a gene under its
control, it will be un(lerstûod that expression of any of a number of heterologous genes can be
directed by the plUl~10~ > of the present invention.
For example, npd and nptI~ are different and distinct enzymes, with differences in both their
10 amino acid sequences and ~ub~Lldl~ specificities (Beck, et al., 1982). The raspberry plUlllUh,~D
of the invention are suitable for directing expression of either of these neomycin phosphotrans-
ferases.
Plants suitable for Llall~rolllla~ion using the ld~llbelly plulllolel~ of the invention include but
are not limited to, raspberry, tomato, strawberry, banana, kiwi fruit, avocado, melon, mango,
15 papaya, apple, peach, soybean, cotton, alfalfa, oilseed rape, flax, sugar beet, sunflower, potato,
tobacco, maize, wheat, rice, and lettuce.
Chimeric genes contAining a raspberry promoter, e.g., drullO, and dru259, can beLlalls~l~ed to plant cells by any of a number of plant l~ rolllld~ion methodologies. One such
method, employed herein, involves the insertion of a rhim~ric gene into a T-DNA-less Ti plasmid
20 carried by A. tumefaciens, followed by co-cultivation of the A. tumefaciens cells with plant cells.
As provided in Example 11, Agrobacterium binary plant llal-~rollll~ion vectors, pAG-7242
and pAG-7342, are individually introduced into a (li~ArmPd strain of A. tumefaciens by
electroporation (Nagel, et al ., 1990), followed by co-cultivation with tomato plant cells, to transfer
the rhim~rjc genes into tomato plant cells.
In addition to Agrobacterium-based methods, AllfL. .. ~.~;ve methodologies may be employed to
elicit Llal~rullllc~lion of a plant host, such as leaf disk-based llal~ru~ LiOll~ ele~ ui)o-aliûll,
microinjection, and microprojectile bombardment (particle gun ~ ru. IIIA1 ;on). These mPthod~
are well known in the art (Fry, et al., 1987; Comai and Coning, 1993; Klein, et al., 1988; Miki,
e~ al., 1987; Bellini, et al., 1989) and provide the means to introduce selected DNA into plant
30 y,t:llollles. Such DNA may include a DNA cassette which consists of a raspberry plul~o~er (e.g.,
dr~ll10, drrl259) functionally adjacent a heterologous coding sequ~nee.
Ad~litionAlly, an iterative culture-selection methodology may be employed to generate plant
(Ul~ ';, and is particularly suited for Ll~llsrullllalion of woody species, such as raspberry.
CA 02243850 1998-07-21
W O 97127307 PCT~US97101275
19
This method is described in detail in International Publication No. WO 95/35388, entitled "Plant
Geneti;c Tldnsfol,.,ation Methods and Transgenic Plants", published on 28 December 1995.
In employing an iterative culture-selection ~ausru~ dlion methodology, a ~himerjc gene of
interest is inserted into cells of a target plant tissue explant, such as by co-culturing a target
5 explan,t in the presence of Agrobacterium cont~ining the vector of interest. Typically, the co-
culturing is carried out in liquid for from about 1 to about 3 days. The plant tissue explant can
be obtained from a variety of plant tissues inr~u~in~, but not limited to, leaf, cotyledon, petiole
and meristem.
T~ rul ...~d explant cells are then screened for their ability to be cultured in selective media
10 having â threshold concentration of selective agent. Explants that can grow on the selective media
are typically ll a~ d to a fresh supply of the same media and cultured again. The explants are
then cultured under regeneration con~iition~ to produce regenerated plant shoots. These
regenerated shoots are used to generate explants. These explants from selected, re~." ,al~d plant
shoots are then cultured on a higher concentr~tion of selective agent. This iterative culture method
15 is repeated until essentially pure transgenic explants are obtained.
Pure transgenic explants are i~l\onti~ed by dividing the regenelaled plant shoots into ~Yrl~ntc
culturiing the e~rpl~nt~ and ve,iryillg that the growth of all explants is ~esisLdl,l to the highest
conceJltration of selective agent used. That is, in the presence of selective agent there is no
necrosisor signific~nt bleachingoftheexplanttissue. Upon col.ri.ll-~ ionofproductionç~pnt~ y
20 pure lldlls~nic explants, transgenic plants are produced by L~ ~aLh~g plants from the pure
transgenic e~ n
C. Identification and Evaluation of Plant Tldnsl;.ll.lallL~
Transgenic plants are assayed for their ability to synthesize product mRNA, DNA, protein,
andlor for their r~sict~n-~e to an aminoglycoside antibiotic, e.g., k~lallly~ill. The assays are
25 typicâlly co~ cted using various plant tissue sources, e.g., leaves, stem, or fruit.
]_eaf-based assays are i,~rl~l"..live if the raspberry promoter driving the heterologous gene
~trans,gene) is at least somewhat active in leaf tissue, as is the case for ~YPrnpl~ry plo.lloL~
dr~l ~0 and drJ1259. In such cases, leaf-based assays are useful for initial screens of the expression
- level of a transgene, since they can be performed much earlier than fruit-based assays. Fruit-based
30 assays, on the other hand, provide more accurate data on Lla~ elle expression in a target tissue
itself su~h as fruit.
RNA-based assays can be carried out using, for example, an RNAase protection assay
{RPA,~. In carrying out such an assay, mRNA is typically ~rtr~ted from plant cells derived from
both ~ n~rur~ed plants and wild-type plants. ~NAse Protection Assays ~RPA) can be performed
CA 02243850 1998-07-21
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2Q
according to the m~mlf~ rer~s instructions using an "RPAII" kit from Ambion, ~nc. (~i~ h
FL~, as previously described by Lee, et al., 1987.
Gene expression patterns for transgenic plants containing chim~ric genes regulated by a
raspberry promoter can also be evaluated by conducting Northern dot blots (e.g., Example 6).
P.o,.,ole~ function (i.e., tissue and/or stage specific ~ ion, or con~LiLulive expression) canbe
evaluated by comparing northern blots of total RNA from leaf and fruit tissues at different Li~t;"ing
stages to northern blots of total RNA from various other plant tissues.
Experim~-ntc carried out in support of the invention indicate that the la~bt~ Llot~
drul~ and dru2~9, do not filnrtion as stage or tissue-specific ~.~."lole.~. This is somewhat
10 ~ULLJIi~h~g, since a tissue and stage specific gene expression pattern of drul, regulated by the full
length drul pLon,olei, was confirmed on northern blots of total RNA from leaf, lec~ ;les, and
drupelets.
As a further co~lr~ d~ion of expression of a duwll~LI~aLll heterologous gene regulated by a
raspberry promoter of the invention, a Western blot analysis can be carried out. In cl~n~lcting
1~ a typical Western blot experiment, total soluble protein is extracted from frozen plant tissue and
e~u-ed using, for example, the Coomassie Plus protein assay (Pierce, Rockford, IL). Known
qll~ntitiP~ of soluble protein, or Icnown qll~ntities of purified protein product (e.g., neomycin-
phosphoL.~reiase I~, positive control) are resolved on a polyacrylamide gel and LLal~re~ied to
nylon membranes. The bound proteins were then probed with a monoclonal antibody specific~lly
20 i~ o. ta~Li~e with the protein product.
In another appioacll for co..ri....illp gene expression directed by l~!be~.y pl.~llloteL of the
illve;ll~iOII, a Southern hybrirli7~tion analysis is performed. Typically, plant DNA is extracted by
~rinrlin~ frozen plant tissue in elrtr~rtion buffer, followed by cpntrifilg~tion~ seyaLaLioll of the
resulting supernatant, and precipitation with cesium rhlori(1e The resulting CsCI gradients are
25 then cPntrifilged for an e~tPn~lP~d period of time (e.g., 48 h), and the recovered DNA is dialyzed
and pLe~ ikLLed with ethanol. Upon Leco~el~ of plant DNA, the DNA is digested with suitable
re~l-.cLioll er~y",es to obtain DNA fr~gmPnt~, followed by electrophoretic separation on âgarose
gel. The resulting bands are L-~n~re.-ed to nitrocellulose (Southern, 1975), and the blots are then
probed with a labelled DNA La~ l containing the nucleotide sequence of the transgene, to
30 conf}rm the pfesellce of DNA corresponding to a raspberry proll'ole~-chimPric gene construct, as
desc~il,ed a~ove.
D. Co~ ald~ e Evaluation of C~ene Expression and P,o,..ol~. Stren~th
E~pe~ e.l~ performed in support of the invention d~lllol~llale the ll~L ,llllation of tomato
plants with rhimeric genes operably linked to a raspberry plolllol~r of the present invention (e.g.,
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drullO, dr~25g). As is evident from the results of these experiments, the LaslJbc.~y promoters
of the invention are capable of regulating expression of genes placed under their control, and
function as moderate level, col.sLilu~ e promoters.
Tomato plants were ~lan~rvlll.ed with plant transformation vectors, pAG-7242 and pAG-
7342, each containing a raspberry promoter operably l;nked to an nptII gene (Exarnple 11). As
detailed in sections V.A.1-2 above, plant L-d lsÇ~ alion vector pAG-7242 contains the
drullO::nptII gene; and construct pAG-7342 contains the dru259::nptII gene. Chimpric genes
CQI1~ 1;1Ig either the hsp80 promoter or the CAS promoter (caulimovirus cassava mottle vein virus
p~u~lwlel) fused to the nplII gene were also prepared and used to l~a~rullll eomato plants, to
10 provide a Co~ dlàLiv~ basis for evaluating perform~n~e of the raspberry piOIlwLrl~ of the
invention.
Results from ten separate transgenic events employing the constructs described above are
provided in Exarnple 12. To detect the presence of nptII ~l~yllldliC activity in plant IIAI .CrU. ~
protein extracts from leaf tissue of rooted plants available at the time of culture were assayed by
15 ELIS~. In some cases, only 1 plant was available for assay (e.g., Table 1, last two rows, column
I~7), while in other instances (e.g., Table 1, row 2, column IV), ten separate transgenic events
were available for analysis.
]~ referring now to transgenic plants co~ g a L~~beLly promoter of the invention (e.g.,
drl~llO, dru25g), as can be seen from the results in Table 1 (specifically, rows 3-7), nptII
20 enzyma~ic activity was detected in a high percentage of the plants assayed, with values ranging
from about 2û-100%, depending upon the conce--l.alion of selection agent used and the number
of rooted plants tested. These results are comparable to those obtained for transgenic plants
co,.~ known pLol--oL~ npdI constructs, and indicate that the raspberry plo-,-ole-~ of the
invention are effective to regulate expression of heterologous genes placed under their control.
Also provided in Table 1 is a coll,~d-ison of ~ ro",~ ion frequency, that is, the ratio of
the m~mber of tissue explants producing regenerated shoots that are capable of rooting in the
presence of selection agent to the total number of initial explants, expressed ~ a perct~nlage.
B~ed on the results in colurnn III, and I~Çe..il~g to plants cu..~ g a raspberry pio",ule, of the
invention, on average, at least about half of the plants l,~ d with a ld~be-,y plo--,oLe,-con-
30 taining construct survived selection with antibiotic, that is, they were capable of rooting in the pre-
sence of an amount of selection agent that would otherwise be toxic to non-l . ,...~ r~ ", ..~-d plant cells .
As in the case of the neomycin phosphotransferase assay ~ cl-~.ced above, these results are
CQ~ r..~l with those obtained with known plant promoters (hsp80, C~S), and further illll~tr~e
(i~ the capability of the ,d~be..y promoters of the invention to regulate expression of genes placed
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WO 97/27307 PCT~US97101275
under their control, and (ii) the formation and use of chimeric gene constructs and 1. A"~rO, ...AIion
vectors contAinin~ a raspberry plollloLer (drullO, dru259), for Lr~sro..llillg a plant host to form
a transgenic plant ~ e~ g a heterologous gene.
The raspberry ,uLolnoL~ of the invention provide cull~liLuli~e expression of heterologous
5 genes, as evidenced by the detection of npt~I activity in all tissues obtained from ~I;al~sgellic plants
L,A ,~rc" ",~od with exemplary plant ~lAul~rulllla~ion vectors pAG-7242 and pAG-7342.
Plu~lw~el-driven expression of the nptII gene was evaluated by determinin~ npdI enzyme
levels in 1, A .~rO. IllA~ . The results are presented in Table 2 and in Pig. 5. Protein levels for leaf
tissue obtained from ~ .,ru- ~ ~A~ containillg the CAS::nptII chimeric gene are not int~lutl~d in
10 either the table or the figure, since values from two CAS::nptII events assayed were in excess of
6000 pgJml, inrlicAtin~ the high level of gene expression regulated by the CAS prolllû~er (i.e., a
strong p~ulllu~er). While the drul promoters of the invention appear to direct transgene wcL~lessioll
at levels somewhat lower than those observed for the hsp80 promoter, both drullO and dru259
are considered to f~lncti~n as mnd~rAt~-level promoters.
In looking at the results for the first two transgenic events in Table 2, the average npdI
enzyme level for drullO (dru255~)::npdI plants was about 5-9% that ~letermin~d for CAS::nptII
p~ants.
In rYA~ g these same results, using the hsp80 p-(~ ùler as a basis for coll-y~ison~ the
sverage npt~ enzyme activity determined for drullO (dru25g)::npdI plants was about 40~0% of
20 the nptII enzyme activity determined for hsp80::nptII plants.
Thus, plUlllU~ derived from the drul gene, e.g., drullO and dru259, provide somewhat
lower levels of gene expression than the hsp~O promoter, but are also considered to function as
mnd,~r~t~ strength p~u~uLel:,. As ~u~po-~ed by the data described above, each of the eYempl~Ary
Lly piol"ole~ described herein is capable of directing col~liluliv~ expression of a l-~s~ne
25 at s~ cient levels to support its use in regulating e-.ples~ioll of any of a number of heterologous
gene products.
~ 7Aition~Ally7the~ r(~ Alionoftomatoplantsusingtheraspberrypromotersofthepresent
invention illustrates that a p-ul~o~er region derived from ~pbel~y can be used to p.o---ole
sioll of a gene within plant cells from a completely different genus, family, or species of
30 plant.
~i. Vectors of the Present Invention
Thepresentinventionprovides vectors suitableforthe llAI-~r " IIIAIi~ nofplants. Thevectors,
chimeric genes and DNA constructs of the present invention are also useful for the expression of
CA 02243850 1998-07-21
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heterologous genes. Transgenic plants carrying the ~~him~-ic genes of the present il~v~n~ioll may
be a useful source of recombinantly-expressed material.
In one embodiment, the chimeric genes of the present invention have two components: (i)
a col~LiLULi-~e p-o-llolei derived from a raspberry drul gene, and (ii) a heterologous DNA sequçn~ e
- 5 encoding a desirable product.
The vectors of the present invention may be constructed to carry an expression cassette
cont~;ning an insertion site for DNA coding sequences of interest. The transcription of such
inserted DNA is then under the control of a suitable raspberry p~ull~olel (e.g., drullOpro or
dru2S9pro) of the present invention.
Such expression CA~el ~~s may have single or multiple transcription t~ AI ;on signals at the
coding-3'-end of ~e DNA sequence being expressed. The expression cassette may also include,
for exarnple, DNA seqnenees encoding (i3 a leader seq~lenre (e.g., to allow secretion or vacuolar
targeting), and {ii) translation le..~h~ ion signals.
Fur~er, the vectors ûf the present invention may include selectable markers for use in plant
15 cells ~such as, a neomycin phosphoLldllsÇ~l_se }I gene (nptI~) or a neoll-yl h~ phosphoL,a-~r~a~,e
gene). The presence of the nptII gene cûnfers re~ict~n~e to the antibiotic, k~dl--ycill. Anûther
aminoglycoside resi~tAnce gene for use in vectors of the invention includes a gene encoding
hy~"ull~y~in phosphotransferase, i.e., an hpt gene (Gritz, et al., 1983). Plant cells cû..L~hlillg the
hpt gene are able to grow in the presence of the arninocyclitol antibiotic, hyglom~cill B. Other
20 selectable marker seqllenees for use in the present h~ve~Lioll include gly-phosate-tolerant CP4 and
COX genes (Zhou, et al., 1995). T~ sge.lic plants ~ g either of these genes exhibit
tolerance to glyphosate, which can be used in selection media to select for plant ~lA,I~ro~ I~IAI~
The vectors may also include sequences that allow their selection and plu~,aLiûll in a
secondary host, such as, sequences conLaillillg an origin of replication and a selectable marker.
2~ Typic.al seCon~l~Ary hosts include bacteria and yeast. In one embodim~-nt the secondary host is
Esc}zeAchia coli, the origin of replication is a colEl-type, and the sPlectAhle marker is a gene
encoding ampicillin reSi~tAnee. Such sequences are well known in the art and are also com-
mercial~y available (e.g., Clontech, Palo Alto, CA; SL.dL~g~lle, La Jolla, CA).
The vectors of the present invention may also be modified to i,~e~ e~ te plant transfor-
30 maticn plasmids that contain a region of homology to an Agrobacterium tumefaciens vector, a T-
DNA border region from Agrobacterium tumefaciens, and ~-himPrie genes or expression cA~ett~s
(desc;ribed above). Further, the vectors of the invention may comI-ri~e a ~ Armed plant tumor in-
ducing plasmid of Agrobacterium tumefaciens.
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24
The vectors of the present invention are useful for moderate level conctitl-tive expression of
nucleic acid coding sequences in plant cells. For exarnple, a selected peptide or polypeptide
coding sequ~nre can be inserted in an expression cassette of a vector of the present invention. The
vector is then ~i~..crl~, ..led into host cells, the host cells are cultured under conditions to allow the
5 expression of the protein coding seql~ences, and the expressed peptide or polypeptide is isolated
from the cells. Tld~L~L-lled progenitor cells can also be used to produce lrd.Lsgcllic plants bearing
fruit.
The vectors, chimeric genes and DNA constructs of the present invention can be sold
individually or in kits for use in plant cell lL~l~Çu-lllaLion and the sl~bsequent generation of
10 transgenic plants.
A. Heterologous Genes
The methods and results described herein demonstrate the ability of the r~pberry plulllolc-
~of the invention to provide con~ilulivc, moderate level gene expression in transgenic plants. A
raspberry l)rolllo~er of the present invention includes a region of DNA that promotes lrdllsc.i~lion
1~ of the ;--~ ely adjacent ~dowlL~Lc~ll) gene con~ ulively, in llUll~,.VU5 pllant tissues. Accord-
ing to methods of the present invention, heterologous genes are operably linked to a raspberry
pro,llolcr of the present invention.
Exemplary heterologous genes for the 1- ,...cr,~"--~1 ;on of plants include genes whose products
are effective to confer antibiotic r~Cict~nce Some of these genes, in~hllling the nptII gene, are
20 described above.
Other genes of interest that can be used in coniunction with a raspberry pl~lllOl~L of the
inYention (e.g., dr~l10, dru25~ include, but are not limitcd to, the following: genes capable of
conrcllillg fungal recict~n( e, such as the polygalacturonase inhibiting protein tPGIP) gene from
Phaseol~s vlllgaris ~oubart, et al., 1992) and mnrlified forms of plant gl~7r~n~ce, ~hitin~ce
~5 (Jongediik, e~ al., 1995) and other pathogenesis related ~PR) genes ~MelfhPr~, et al., 1994;
Ponstein, e~l., 1994; Woloshuk, etal., 1991). The,ce geneproductc (e.g., ~hitin~cPc orbeta-1,3-
c~ c) can, for example, enhance lesi~ e to fungi such as Fusarium, Sclerotiniasclero~ior~m, and ~izoctonia solani. Tlt.n~ro ...~d plants ~ Lessillg these products exhibit
increased resistance to diseases such as seedling darnping off, root rot disease, and the like. Other
30 representative genes for confer}ing both viral and fugal r~cict~n~e to transgenic plants are
described in VIRUS AND FUNGAL RESISTANCE: FROM LABORATORY TO FIELD" (Van Den Elzen,
et al., 19~4).
Additional exemplary heterologous genes for use with a raspberry promu~, of the present
invention include genes whose products are effective to confer herbicide-resistance to ~ rO. .,.~i
CA 02243850 1998-07-21
W O 97t27307 PCTWS97/01275
plant cells. Exemplary herbicide resistance genes include a bialaphos r~si~t~nce gene (bar) which
codes ror phosphinothricin ac~Lyl~Lall~relase (PAT) (Akama, etal., 1995). Transgenic plants con-
taining- this gene exhibit tolerance to the herbicide, "BASTA". This gene can also be used as a
selP~ct~1e marker gene, since explants carrying the bar gene are capable of growing on selective
- 5 mêdia containing phosphinothricin (PPT), which is an active component of bialaphos.
Addition~1 herbicide resistance genes include those con~ .g resi~t~n~e to glyphosate-
CV'II;1'll;'l~ herbicides. Glyphosate refers to N-phosphonomethyl glycine, in either its acidic or
anionic forms. Herbicides containing this active ingredient include "ROUNDUP" and "GLEAN".
Exemplary genes for hl~pa-lillg glyphosate le~i~L~.ce include an EPSP syl-Lllase gene (5-
10 enolpyruvyl-3-pho~ph-lc~hikim~tP synthase) (Delanney, et al., 1995; Tinius, et al., 1995), or an
~-~etol~ct~e synthase gene (Yao, et al., 1995).
Other el~Pmrl~ry DNA coding sequences inciude a bxn gene encoding a b.o-l.o~y"il-specific
nitrilase (Stalker, e~ al., 1988), under the L~ s~ ional control of a drul p~ol~lù~er. TraL~Çullll~d
plants con~ail-i-.g this chimPric gene express a l~lol.lo,.ynil-specific nitrilase and are resistant to the
15 application of blulllo~yllil-cont~ining herbicides.
Other gene products which may be useful to express using the promoters of the present
il~vellLiOll include genes encoding a viral coat protein, to enhance coat-protein m~Ai~tpd virus-
re~ a~ e in transgenic plants. Exemplary genes include genes coding for alfalfa mosaic virus coat
protein ~AlMV), cucumber mosaic virus coat protein (CMV), tobacco streak virus coat protein
(TSY), potato virus coat protein (PVY), tobacco rattle virus coat protein (TRV), and tobacco
mosaic virus coat protein (TMV) (Beachy, et al., 1990). Thus, a chimeric gene of ~he invention
will contain a viral coat protein gene, such as an ALMV, CMV, TSV, PVX, TRV, or TMV gene,
under the transcli~Lional control of a raspberry a drul plullwlei.
B. F~ression in Heterologous Plant Svstems
Exp~lhlltllL~ performed in support of the present invention d~llloll~LL~Le the versatility of the
chimeric gene constructs of the invention. The vector constructs of the present invention can be
used for Lld~Ç~ I ;on and expression of heterologous se~len~es in L~u~sg~llic plants independent
of the original plant source for the promoter sequence. For ~Y~mple~ the drullO::npdI and
dr~2~9::~ptII chirneric genes were succP,ssfillly introduced into tomato plant cells.
~leSe data suggest that the raspberry promoters of the invention (e.g., drullO, dru25g) are
usefill for prullluLil~g gene expression in heterologous plant systems, i.e., plant cells other than
~s~beLly, such as tomato. Further, the expression m~ ted by the plumoLel~ appears to be
co~cl;l ~1 lve even in heterologous plants. These findings support the n~efilln~ss of the vectors,
chimeric genes and DNA constructs of the present invention for ~lall~u~ aLion of plants.
CA 02243850 1998-07-21
W 097127307 PCT~US97/01275
26
VII. ~ilitv
Experiments performed in support of the present invention d~ or~L~a~e that the gene
expression patterns of nptII directed by a drul promoter, such as drul l O or dru259, are observed
in various plant tissues (e.g., leaf, stem, fruit, root). Accordingly, use of a raspberry pLVIll
5 of the invention allows constitutive expression of a foreign gene placed under its control.
The raspberry drul-derived promoters of the invention, drullO and dru259, can be cloned
as described above employing sequ~nçe in~ alion described herein. These raspberry plollloL~l~
can be used to express any heterologous gene whose function would be ~nh~nrerl or enabled by
a moderate level, cnl.c~ e p.ol.lo~er. F~remrl~ry genes are described above.
The use of these promoters cannot be considered limited to raspberries, particularly in view
of the S~ccç!;c~ sro-~ation of tomato using the raspberry plvlllole.~ of the invention. Since
raspberry is ~c~Pnti~lly a miniature drupe fruit, it is likely that the raspberry pL'OlllO~ will
function in other drupe fruits. The constructs and methods of the present invention are applicable
to all higher plants inrhl~ling, but not limited to, the following: Berry-like fruits, for example,
15 Vitis ~grapes), Fragaria (~ awbe~ies), Rubus (la~pbeies, blackberries, loganberries), Ribes (cur-
rants and gooseberries), V~ccinium, (blueberries, bilberries, whortleberries, cranberries), Actinida
~ciwifruit and Chinese gooseberry). Further, other drupe fruits, inf lu~iing, but not limited to,
Malus (apple), Pyrus (pears), most members of the Prunus genera, sapota, mango, avocado,
apricot, peaches, cherries, plums, and npct~rin~c. A(l(lition~l plant sources are described above.
~he present invention provides compositions and methods to regulate plant cell expression
of any gene in a co~ e manner. In one embodiment, the p.o,..vLc~.~ of the present invention
can be used to regulate expression of a selectable marker gene, such as nptII. Alternatively, the
~belly piollloLel~ can be used to promote expression of a herbicide-r~ t~nre gene, or to
regulate expression of a gene encoding a viral coat protein, to provide enh~n~ed virus rP~;~t~nce
The raspberry prvmote. :, of the invention can be used in chimeric genes, plant l. ,., .!~rv~ ;on
vectors, expression c~csettes7 kits, and the like, to p-v~l~vle t,~îu.,..aLion of plant cells.
The la~ybelly promoters described herein may also be employed in a method for providing
mnd~r~te level expression of a heterologous gene, such as a selectable marker gene, in a liai~sgenic
plant.
T~e following examples illustrate, but in no way are intpn~led to limit the scope of the present
invention.
Materials and Methods
Biological reagents were typically obtained from the following vendors: S' to 3- Prime,
Boulder, C0; New F.ngl~n(l Biolabs, Beverly, MA; Gibco/BRL, Gaithersburg, MD; Promega,
CA 02243850 1998-07-21
W O 97127307 PCT~US97/01275
Madison, WI; Clfnt~r~h, Palo Alto, CA; and Operon, .Alam~A~, CA. Standard recombinant DNA
terhnirllTP~s were employed in all constructions (Adams and Yang, 19?7; Ausubel, et ~I., 1992;
Hooykaas and Schilperoot 1985; Sambrook, et al., 1989; Wang, et al., 1990; Kawasaki, et al.,
1989; Veluthambi, et al., 1988; Benvenuto, et al., 1988).
- 5 Example 1
~aspberry Drupelet Protein Charac~erization
and Purification
A. Protein Lysate Preparation and Gel Electrophoresis
Using a mortar and pestle cont~inin~ liquid nitrogen, a raspberry protein sarnple was
pr~ed by ~rinrlin~ the frozen drupes of one whole berry into a fine powder. Sample buffer
(0.05 M Tris, pH 6.8, 1% SDS, 5% beta~ .,a~oethanol, 10% glycerol; T ~melli, 1970) was
added ~900 ~LIs) to the tissue and the sample mixed by vortexing. The sarnple was heated for 10
minutes at 90-95~C and centrifuged at 14K rpm, 4~C for 10 minutes. The S.~ aL~lL was
~t;lllOYed from the ingohlhle debris pellet and stored at -20~C.
Drupelet proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophore-
sis (SDS PA&E) combined with coomassie blue staining using ~la-~daLd procedures. A coomassie
blue-stained SDS polyacrylamide gel of soluble drupelet proteins is shown in Figure 10. In the
figure: lane 1, molecular weight markers (BioRad, Ri~hmon-l CA), lanes 2, 3 and 5 each contain
9 ~g c~f raspberry drupelet protein lysate prepared separately from individual fruit. Lane 4
c~ I a higher amount of lysate.
Two highly abundant proteins were observed at alJ~rox i, .".~ ~ly 17 and 15 kd and were named
dLupel and drupe2, respectively. In Fig. 10 these two proteins are indicated by arrows. Scanning
densi~c~ll-rLly analysis of this gel in(lieated drupel and drupe2 comprise a~lo~hllaLely 23 and 37%,
respec~ively, of the total soluble protein in raspberry drupelets. As a result, a direct western blot
25 ~ a~to purific~tion and sequencing of the protein was followed.
E,. Protein Blot For Sequencing
A protein blot (Applied Biosystems, Inc. User ~ulletin Number 58; Ausubel, et ~l., 1992)
was prepared using the raspberry protein Iysate described above. Varying amounts of raspberry
protei~ Iysate (12-36 ~g/well) were loaded on a 10 well lB% SDS-PAC~E minigel (1.5 mm thick)
~ 30 with 4 5% stacker and electrophoresed at 100 volts in 25 mM Tris, 192 mM glycine, 0.1 % SDS
buffer for 2-2.5 hours.
Plroteins weretransblotted onto Applied BioSystem's "PROBLOTI " polyvinylidene r~ oride
(P~ membrane in a 25 mM Tris, 192 mM glycine, 10% meth~nol buffer at 90 volts for 2
hours at 4~C. After protein transfer, the blot was Coomassie blue stained and the 15 and 17
kilot~ tnn (kd~ protein bands were located on the b~ot and cut out. N-terminal seq~lenring of the
CA 02243850 1998-07-21
W097t27307 PCT~US9710127
28
proteins was carried out at the W.M. Keck Foundation, Biotechnology Resource Laboratory in
New Haven, CT.
The drupel sample yielded a thirty amino acid N-terminal sequence. The drupe2 sample did
not yield useful sequence hl~llllaLiOn likely due to a blocked amino t~ ,.c. The amino terminal
5 drupel seqll~nre is presented as SEQ ID NO:ll. This 30 amino acid drupel se~uenre was
cull.~3~ed to the protein database using BLAST searching; no signific~n~ matches were found
in~iC~ing that drupel is a novel protein.
EXAMPLE 2
Re~ . ;n~ a cDNA Clone C~ ,ondin~ to the Drupel Protein
A. Drupelet Total RNA Preparation
RNA was extracted from mature green raspberry drupelets. Four mature green raspberry
fruit, which had been picked in season and stored at -80~C, were used to extract RNA. The
ect;m~t~ weight of the drupelets was 12 grams. In a cold mortar, which co~lA;I~Pd liquid
nitrogen, the whole berries were fractured by tapping them with a pestle. The drupelets were
1~ s~a aled from the receptacles. The receptacles were ~e~ ved from the mortar and discarded.
The drupelets were ground to a powder in the mortar, adding liquid nitrogen as necessary to keep
the tissue frozen. The seeds were purposefully left intact. Homogenization buffer, 2 ml/gram of
tissue, was used to extract the RNA. [Homogeni7~tinn buffer: 200 mM Tris-HCl pH 8.5, 30Q mM
LiCI, 10 mM Na2EDTA, 1% (w/v) sodium deoxycholate, l.S~o (W/V) sodium dodecyl sulfate
(SDS), 8.5% (wlv) insoluble polyvinylpolypyrollidone ~PVPP), 1% (v/v) NP~0, 1 mMa.~ icdll,oxylic acid (ATA), S mM thiourea, and 10 mM dithiothreitol (DTT); the last three
comron~n~ were added after autoclaving].
The frozen powdered drupelet tissue was added to the buffer in 3 to 5 portions, vo~ hlg
between additions until all tissue was moistened. The tissue plus buffer solution (referred to herein
as the pulp) was diluted 1:1 with sterile water and 0.75 volumes of homogenization buffer were
added to the diluted pulp. The sample was inr~lhated at 65~C for 10 to 15 minlltec~ followed by
cen~ tion in a swinging bucket rotor at 9000 g for 15 minutes at 4~C. The ~u~lllal~ll was
L a~çel~ed to a clean tube. Cesium chloride (CsCI) was added to the sul~e ..al~L at 0.2 g/ml.
The sample was mixed until the CsCl dissolved.
A 4 ml cushion was dispensed into a 13eckman 1 x 3.5 inch polyallomer ultracentrifuge tube
(cushion: 5.7 M CsCl, 10 mM Tris-HCl, pH 8.0, 1 mM Na2EDTA, pH 8.). The sample was
gently layered on top of the cushion. The sample was spun in a Beckman E8-80M ull-ac~ uge
with a SW 28 rotor at 23,000 rpm at 20~C for 20 hours. After l~ ving the sample from the
CA 02243850 l998-07-2l
W O 97t27307 PCTrUS97/01275
29
ultracentrifuge the sl ye~ld~al-l was pulled off the sample by using a drawn Pasteur pipette attached
to an aspirator. A clear lens-like pellet was visible in the bottom of the tube.The pellet was dissolved in 500 ,ul SSTE and ~Lal~L~ d to a microfuge tube (SSTE: 0.8 M
NaCl, 0.4% SDS, 10 mM Tris-HCl, pH 8.0 and 1 mM Na2EDTA, pH 8). The sample was
- 5 extracted twice with an equal volume of chloroform:isoarnyl alcohol (24:1). To pieci~iL~Le the
RNA, 2.5 volumes ethanol were added to the aqueous phase. The sample was col1ected by
centrifugation, washed two times with 75% ethanol and re~u~e.lded in 100 ~11 TE. The yield was
1.6 mg. The RNA was reprecipitated with lt9 volume 3 M sodium acetate and 3 volumes ethanol
for storage at -20~C.
~. Drupelet mRNA Preparation
The igt)l~tion of mRNA from mature green ras~be~ly dNpelet total RNA was performP~d
using the "STRAIGHT A'S" mRNA isolation system (Novagen, M~ on, WI) according to the
m~nnf~rtllrer~s instructions. mRNA was isolated from t-h-e 1.6 mg of total RNA extracted from
mature green raspberry drupelets described above. The yield of mRNA from this procedure was
15 6.6 ~g.
C. Preparation of cDNA From Green Raspberry Drupelet mRNA
The rnRNA from mature green raspberry drupelet RNA was used as the temrl~te for cDNA
synthesis. The primer for the cDNA reactions was dTRANDOM ~SEQ ID NO: 12; synth~i7ed
by Operon Technologies, Inc., ,Ai~m~ , CA). The oligo(dT) region hybridized to the poly(A)
20 region of the mRNA pool. The other 15 nucleotides created a 5' overhang that was used to
f~rilit~te PCR amplification at a later step in the cloning process.
Ihe following reaction mixture was ~sen hled for the cDNA synthesis reaction: H2O, 10.2
,~1; 250 ng rnRNA, 0.8 ,~1; 5 x BRL RT buffer (BRL, Bethesda, MD), 4.0 ~l; 100 mM DTT
~tlithio~h~eitol - BRL, Bethesda, MD), Q.2 ,ul; "RNAguard" (23.4 U/~ul; an RNase inhibitor from
25 ph~rm:~r;~l, Piscataway, NJ), 0.~ ,ul; dNTP's (2.5 mM each), 2.0 ,ul; 50 ,uM primer, 1.0 ~cl;
[32P]dC:TP ~3000 Cilmmol; DuPont/NEN, Boston, MA), 1.0 ~1; and AMV reverse-l, ~ se
(38 U~ll; Life Sciences, Inc., St. Prl~ buLg, Florida), 0.3 ~1. The cDNA reaction was p~ ed
by combining mRNA and water for the reaction and heating to 65~C for 3 minutes. The mixture
was cooled on ice and microfuged (to collect con~len~tion~. The rem~inin~ reaction components
30 were then added.
h~fter in~llh~tin~ at 42 ~C for 1 hour the cDNA reactions were moved to ice and stored at 4~C
prior to their use in PCR reactions.
CA 02243850 1998-07-21
W O 971273Q7 PCTrUS97/01275
FXAMPLE 3
P~R Amplification ~nd Clonin~ of the cDNA drul Fra~ment
A degenerate PCR primer, Drupe20, was designPd for the 5' end of the cDNA based on the
reverse tr~n~l~tion of the drul protein sequence. A section of the known amino acid seqnçn~e of
5 drr~l (SEQ ID NO:13) was chosen for its proximity to the amino a~-"~ C and for the relatively
low leYel of degelle-a, y in its reverse-translated sequence (SEQ ID NO:14; Drupe20). The
Drupe20 primer (i) is the 512-fold degenerate nucleotide sequence corresponding to the amino acid
sequence presented as SEQ ID NO:13, and (ii) was used as the 3'-primer.
l~he 5' PCR primer (DrupeRAN18, SEQ ID NO:15, co-r~i~ondil.g to the cDNA primer,10 dTRANDOM) was designed for the 3' end. Polymerase chain reaction (PCR; Perkin-Elmer
Cetus, Norwalk, CT; Mullis, 1987; Mullis, et al., 1987, was performed following the manu-
facturer's procedure using "AMPLITAQ" (Perkin Elmer Cetus), PCR buffer II (50.0 mM KCl,
10 mM Tris-HCl, pH 8.3), 2 mM MgCl2, 0.2 mM of each dNTP, mature green drupelet cDNA
and Drupe20 and Drupel~AN18 primers under the following ct~n-litions:
l cycle at 95~C, 1 minute,
35 cycles at 95~C for 1 minute, 42~C for 1 minute and 72~C for 1 minute,
1 cycle at 72~C for 5 mimltP~ and cooling to 5~C.
There were two major products of the amplific~tiQn reaction: a predominant product of
~fo~ ,àlely 700 bp and a less abundant product of a~plOX;l~A~ y ~00 bp. The 700 bp band was
20 isolated from a 1 % "SEAPLAQUE" agarose gel using ,B-agarase (New F.n~l~nfl Biolabs, Beverly,
MA~ according to the supplier's instructions. This fragment was then ligated to the vector pCRII,
the TA cloning vector from Invitrogen (San Diego, CA), following the m~ml~t tl-rer's instructions.
The cDNA clones of the drul gene were i~l~ntifi~ by screening plasmid lllhlil~l~ DNA
prepared from 1.6 ml of culture using the alkaline Iysis method (Ausubel, et ~l., 1992). The
25 double-stran~leA DNA was sequenced by the dideoxy chaint~. ".i~ )ll method using the
"SEQUENASE" ver.2 enzyme and kit components (United States Biorhemio~l, Cleveland, Ohio)
and t~-35S]~ATP (I)uPont/NEN). I'he reactions were primed with the M13 ulliveLsal forward and
reverse primers {New Fngl~nrl Biolabs, Beverly, MA). Sequencing ~ea-~ions were resolved on
an acrylamide gel ("LONG RANGER GFL," FMC, Rockland, Maine) and bands detected by
30 autoradiography.
The seq~ence was read from the autoradiograph and analyzed for its homology with the
reverse translated N-terminal protein sequence from drupel. The actual DNA sequence was
detPnnin~d as opposed to the degenerate DNA sequence obtained through reverse tr~ncl~tion of
the protein sequ~nce The correlation between the cDNA and the rem~in~ler of the N-terminal
CA 02243850 1998-07-21
W 097127307 PCT~US97/01275
31
protein sequence was confirmed. A clone (dçsign~ted pAG-301) was selected, following these
criteria, for further rh~racterization. The nucleic acid sequence of the drul cDNA insert of pAG-
, 301 is presented as SEQ ID NO:16.
~e entire drul cloning procedure from cDNA synthesis to inverse PCR of a genomic copy
- 5 of the gene is shown schern~tic~lly in Figs. 11A and 11B.
EXAMPLE 4
R~CG~ J the Genomic DNA Fra~ment Co. . ~,o.~-li--e to the drul cDNA
The "CTAB" ~exadecyl-trimethyl-ammonium bromide) method (Doyle and Doyle, 1990)
was used to e~tract DNA from raspberry leaves. PCR primers (DruGenS', SEQ ID NO:17;
DruGen3', S~Q ID NO:18) were d~,sign~d based upon the complete drul cDNA sequence.
"OLIGO," a multi-functional program from National 13i- sci~nres, Inc. (Plymouth, MN), was used
to f~cilit~te design of the primers. PCR was performed following the ~ r~Cl~ S procedure
using "AMPLITAQ" (I?erkin-Elmer Cetus), PCR buffer (50.0 mM KCI, 10 mM Tris-HCl pH 8.3,
1~ and 1.5 mM MgCl~), 0.2 mM of each dNTP, raspberry genomic DNA and DruGen~' and
DruGen3' primers under the following ("HOT START") conditions:
1 cycle of 97~C for 5 minllt~ after which the "AMPLlTAQ" was added,
Z cycles of 97~C for 1 minute, 52~C for 1 minute and 72~C for 1 minute,
25 cycles of 94~C for 1 minute, 52~C for 1 minute and 72~C for 1 minute,
1 cycle of 72~C for S minl~t~ and cooling to 5~C.
lrhis ~mplifir~tiQn reaction produced 3 major products: a predomin~nt product of 710 bp
and 2 less abundant products of 690 and 625 bp. The PCR reaction products were then ligated
to the vector pCRII, the TA cloning vector from Invitrogen (San Diego, CA), following the
m~nnf~lrer's instructions. A clone was selected with a 710 bp insert and d~ign~ted pAG-302.
]?lasmid l:~NA of pAG-302 was prepared from 1.6 ml of culture using the alkaline lysis
method (Ausubel, e~ al., 1992) and seq~lçnred by the dideoxy chain-1~ ;"~ n method using
"SEQUENASE" ver.2 enzyme and klt components (USB, Cleveland, Ohio) and ~x-35S]-dATP
~DuPontJNEN). The sequencing reactions were primed with the M13 u~ al forward and
reverse primers (New F.ngl ~nrl Biolabs, Beverly, MA). Further sequenring reactions were primed
with ~ tion~l internal primers. Sequencing reactions were resolved on an acrylamide gel and
detected through autoradiography.
The sequen~e of the drul genomic DNA insert in pAG-302 is presented as SEQ ID NO: 19.
The sequence of the clone demonstrated that a genomic DNA fragment col-Gs~onding to the
drul ~DNA had been isolated.
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W O 97127307 PCT~US97tO1275
EXAMPLE 5
Recoverin~ the 5' Flankin~ Re~ion of the d ul
Genomic DNA Throu~h Inverse PCR
Inverse PCR primers (dPsign~ted DruInvUp, SEQ ID NO:5, and DruInvLow, SEQ ID NO:6)
5 were desi~n~d based upon the genomic DNA sequence and optimized using OLIGO. Genomic
raspberry DNA was digested with restriction enzyme NsiI. NsiI was chosen because, based on
~e cDNA sequent e~ NsiI was known to cut in the 3'-untranslated region of the gene. A small
portion of the NsiI digested DNA was run on an analytical agarose gel and a Southern transfer was
performed (Ausubel, et al., 1992).
The Southern blot was probed with the cDNA fragment contained in pAG-302. ~he probe
i~lPntified a NsiI fragment of about 2-2.3 l~b: this fragment hybridized strongly with the genomic
clone. A second, smaller fragment hybridized to the probe as well but hybridized weakly with
the genomic clone.
The r~rn~inin~ NsiI-digested raspberry DNA was electrophoresed on a 1% "SEAPLAQUL"
15 agarose gel (FMC, Rockland, ME). Using a BstEII lambda size standard as a guide, the digested
DNA in the range of 2-2.3 kb was excised from the gel. The DNA was purified using ,B-agarase
(New England Biolabs, Beverly, MA) following the ~ ....rAe~ 's instructions. The DNA was
self ligated at a relatively dilute concentration (1 ~lg/ml) to bias the formation of circular ligation
reaction products (Ochman, et al., 1990).
Inverse PCR was sl~hseq~ently performed on the self-ligated, NsiI-digested, size-selected,
genomic raspberry DNA. "AMPT ITAQ" from Perkin Elmer Corporation/Applied Biosysterns
Division ~Foster City, CA) was used to amplify the DNA. The m~n--f~(~tl-rer's procedure was
followed using PCR buffer, 0.2 mM of each dNTP, raspberry genomic DNA (prepared as
described herein), and DruInvUp (SEQ ID NO:S) and DruInvLow primers ~SEQ ID NO:6). The
following ("HOT START") reaction conditions were employed:
One cycle at 97~C for 5 mimlteS, after which the "AMPLITAQ" was added,
2 cycles at 97~C for 1 minute, 58~C for 1 minute and 72~C for 1 minute,
25 cycles at 94~C for 1 minute, 58~C for 1 minute and 72~C for 1 minute,
1 cycle at 72~C for 5 minutes, and cooling to ~~C.
This reaction produced 2 major amplification products, one of 1.8 kb and one of 900 bp.
The 1.8 kb band was isolated from a 1% "SEAPLAQUE" agarose gel using ~-agarase. This
fr~m~nt was ligated to pCRII to give rise to pAG-310. A sçhPm~tic repr~osent~tinn of the
ion of subclone pAG3 10 is presented in Figs. 1 and 2.
The pAG310 insert was sequenced in its entirety (SEQ ID NO:1) and the drul insert
sequence was found to be identic~l to the cDNA clone (SE(2 ID NO:16) and the genomic clone
CA 02243850 1998-07-21
W O 97127307 PCTrUS97/0127S
(SEQ ID NO:l9) in the regions where sequence was shared. The normal elements of plant genes
and their regulatory components were itlPntif;ed (E~igs. 6A and 6B) incl~ ing a CAAT box, TATA
box, ATG start codon, two exons, an intron, splicing sites, a stop codon and poly-adenylation
sites.
- 5 The gene org~ni~tion and protein structure of drul is schPrn~ti- ~lly displayed in Figure 12.
The gene encodes a protein having the predicted amino acid sequence presented as SEQ ID
NO:20. The predicted protein has a calculated molecul~lr weight of 17,087.64 and an e~ t~d
pI of 4.80. A Kyte-Doolittle hydrophobicity plot of the dml protein is presented as Fig. 13.
EXAMPLE 6
Chsf~ dtion of drul Gene Expression
A. RNA Dot Blots
RNA dot blots were pr~dled using 5,ug of total raspberry leaf RNA and 5 ,ug each of total
receptacle RNA from green, mature green, breaker, and orange/ripe raspberries (co~ ollding
to stages I, II, III, IV, respectively, in Figure 14). The blots were probed with the drul cDNA
fr~gmPnt, labeled with [32-P3dCTP (> 3000 Cilmmole3 by the random primed method (Boeringer
M5-nnheim Bio-hçmic~l~, Random Primed reaction kit, Tn(li~n~polis, IN).
The blots were allowed to hybridize overnight at 45~C in "HYBRISOL I" (Oncor,
~;thPI sburg~ MD). A probe concentration of 1.2 x 107 DPM/ml was used. The blot was
washed after the overnight hybridization with a final wash using 0.1 x SSC at 42~C for l hour.
I~e hybridizing probe was detected through standard ~utor~liographic methods. The ~ o;.~lr~
of ~he blot to film was for 4 hours and 10 minutes with an h-l~sifyi~g screen at -80~C.
Ihe results of this analysis are shown in Fig. 14. In the figure the RNA dots are,
Le,~e~tiYely from left to right, leaf RNA and ~ec~acle RNA from green (Fig. 14, "I"), mature
green ~ig. 14, "II"), breaker (Fig. 14, "III") and orange/ripe raspberries (Fig. 14, "IV").
B. ~:urther RNA ~ybridization Analysis
A~ plant RNA extraction method (Chang, et al., 1993) was used for receptacles and leaves.
'rhe raspberry drupelet RNA extraction method described above was used for the drupelets and
slld~be-ly fruit.
A Northern blot was prepared using 5 ~g/lane of each sample RNA. The RNA samples were
as follows: raspberry leaf (Fig. 15, lane 1), mature green raspberry receptacles ~Fig. 15, lane 2),
orangelripe raspberry receptacles (Fig. 15, lane 3), mature green raspberry drupelets (Fig. 15,
lane 4'1, and orange/ripe raspberry drupelets (Fig. 15, lane 5).
CA 02243850 1998-07-21
W O 97/27307 PCT~US97101275
34
The blot was probed with the drul cDNA fragment, labeled with [32P~dCTP (>3000
Cilmmole) by random primed reactions. Hyhri~1i7~tion was carried out overnight at 45~C in
"HYBRISOL I" (Oncor, Gaithersburg, MD). A probe concentration of 4.2 x 106 DPM/ml was
used. The blot was washed after the overnight hybritli7~tion with a final wash using 0.1 x SSC
5 $ 50~C for 30 minutes. The hybridizing probe was detected through standard ~utor~ )graphic
methods. The exposure of the blot to film was for 1 hour at room temperature without an
h~le.~iryi.,g screen.
The results of this analysis are presented in Fig. 15 and support a stage specific .3,.~?r~.sio
pattern in drupelets.
C. Protein Expression Analysis
Protein Iysates were prepared (as described in F~r~mple 1) from r~pberry drupelets at
~arious stages of ripening. The Iysates were si~e-fractionated by PAGE and the gel stained with
Coomaise blue (50% MeOH, 10 mM Tris-HCl pH 8.3, 1.5 mM MgC12). The results are
p~CS~llL~d in Fig. 16. In the f~gure the lysates in the lanes were as follows: lane 1, green
drupelet; lane 2, mature green drupelet; lane 3, breaker drupelet; lane 4, orange drupelet; and lane
5, ripe drupelet. The results of this analysis supports a stage specific expression pattern in
drupelets.
E~AMPLE 7
Creation of Subclone pAG431 Con~ the dru259 Promoter
Creation of subclone pAG-310 containing the full-length drul promoter is described in
Example 5 above.
A DNA fragment cont~ining the drul promoter was PCR amplified from subclone pAG-3 10
using primers, 5' primer, DrupeUp (SEQ ID NO:7) and 3' primer, DrupeLow (SEQ ID NO:8)
under standard PCR reaction conditions. The PCR reaction mixture contained the following
components: 79.0 ,Ibl water, 10.0 ,ul 10X Vent buffer, 1.0 ~1 DrupeUp primer (50~LM solution),
1.0 ~I DrupeLow primer (50 ,uM solution), 8.0 ,ul dNPTs (2.5 mM each), 1.0 ,ul temrl~te DNA
~100 ng). The PCR reaction conditions employed were as follows:
1 cycle at 97~C, 4 min-ltec, after which AMPLITAQ was added;
25 cycles at 94~C for 1 minute, 49~C for 1 minute, and 72~C for 1 minute,
1 cycle at 72~C for 5 min~ltes> followed by cooling to 5~C.
The amplification reaction produced a 1.3 kb rlag-l.elll product as illustrated in the top
portion of Figs. 1 and 2.
This r a~",-e--l was purified from the reaction mixture as follows. The PCR reaction mixture
was ~ ,s~.ed to a light Phase Lock Gel tube (5 Prime to 3 Prime, Boulder, CO.). The
CA 02243850 1998-07-21
W O 97/27307 PCTrUS97/0127~
following solvent combination, phenol:chloroform:isoamyl alcohol (25:24:1), was added to this
tube at a volume equal to the PCR reaction volume. The tube was spun in a microcenfrifllge
following the m~mlf~tllrer7s instructions. The upper aqueous phase was tLal~r~,,ed to a Select,
G-50 spin column (5 Prime to 3 Prime, Boulder, CO.) and the DNA was centrifuged through the
5 column according to the m~nllf~rer's instructions. To ~e eluant was added 1/10 volume of 3M
sodium acetate and 2.5 volumes of ethanol, in order to precipitate the DNA. The sample was
inf ub~ted on ice for a period of no less than 10 minntes, and then microcentrif~lged at 4~C for 30
minutes at 14,000 rpm. The supernatant was der~nt~d from the tube and the pellet washed twice
with 75% ethanol. The pellet was allowed to dry, and then resuspended in 25 ~1 1/2 strength TE
10 (5 m~I Tris HCl, 0.5 mM EDTA, pH 8). The DNA fr~gm~nt was digested to completion with
restriction enzymes NsiI and XbaI to produce a drul plolllol~l fragment. This fr~gm~nt was puri-
fied in the same manner as was the PCR product described above.
The non-integrating plant expression vector p35S-GFP (Clontech Labor~tories Palo Alto,
CA) was digested with XbaI and PstI. The digested plasmid was run on a 1% low melting point
15 agarose gel (SeaPlaque, FMC BioProducts, Rock}and, ME). The gel region co~ g the 3.7
kb fragment was cut from ~e body of the gel. The DNA La~ e~ll was then purified away from
the gel using ,~-agarase from New Fngl~n(l Biolabs (Beverly, MA), following the m~nllf~ rer~s
instructions. The gel region containing the 0.85 kb 35S p.~l,.oLe~ was discarded. The 3.7 kb
r.,.~ "( from p35S-GFP2 and the 1.3 kb drul plolllole. fragment were comhin~d in a ligation
20 reaction, using GibcolBRL's T4 ONA ligase, following the ",~".,r~ t;l's instructions, to form
the ;.~I~",.~li~te plasmid pAG-155. The resulting plasmid co..l~;.,;"g the raspberry drul prol,.ole~
was design~ted pAG-155, as illu~llal~:d in Figs. 1 and 2.
Plasmid pAG-155 was digested to completion with SnaBI and EcoRV, both blunt cutters,
releasing the 259 bp drul plo-l~oler fragment, design~ted herein as dru259, where nucleotide
25 number one is imm~li~t~1y 5' of the ATG start codon.The digested plasmid was run on a 1% low
melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME). The gel region
coll~ g the 259 bp drul promoter fragment was cut from the body of the gel. The DNA
Lla~ was ~en purified away from the gel using ~-agarase (New F.ngl~n~l Biolabs, Beverly,
MA), following the m~mlf~tnrer's instructions. The gel region cont~ining the rçm~inf~çr of the
30 plasmid was discarded.
Subclone pAG~11 co~t~ining the nos::nptII cassette was p-~)aled as follows. Cloning vector
pGE~I03Zf(+) (Promega, Madison, WI) was digested with XbaI and BamHI. The digested
plasmid was run on a 1% low melting point agarose gel ~SeaPlaque, FMC BioProducts, Rockland,
ME). The gel region cont~ining the 3.2 kb fragment was cut from the body of the gel. The DNA
CA 02243850 1998-07-21
W 097/27307 PCT~US97/01275
36
fragment was then purified away from the gel using ~-agarase from New Fngl~n~ Biolabs
(Beverly, MA), following the m~nnf~rtllrer's instructions.
The plant binary tLdl~s~oLLLla~ion vector pGPTV kan (Max-Planclc Institut, Koln, Germany)
was digested with XbaI and BamHI. The digested plasmid was run on a 1% low melting point
5 agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME). The gel region Cont~ining the 1.48
kb nos: :nptII fragment was cut from the body of the gel. The DNA fragment was then purified
away from the gel using ~-agarase (New England Biolabs, Beverly, MA), following the
.,.~...,r~ L s instructions. The gel region COllldillillg the 13.3 kb fragment was discarded.
The 3.2 kb fragment from pGEMC93Zf(+) and the 1.48 kb nos::nptII rLa~llltllL were
10 combined in a ligation reaction, using Gibco/BRL's T4 DNA ligase, following m~n..f~rh-rer-s
instructions to form the intermediate plasmid pAG-411.
Plasmid pAG-411 was digested to completion with HincII and PshAI, botb blunt cutters,
releasing the 636 bp nos promoter fr~m~-nt The digested plasmid was run on a 1% low melting
point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME). The gel region CO~ i--g the
15 4 kb fragment was cut from the body of the gel. The DNA fidglllellt was then purified away from
the gel using ~-agarase (New Fngl~nr~ Biolabs, Beverly, MA), following the ...~ .'s in-
~LLU~,Li~11S. The gel region containing the 636 bp nos piOlllO~. fragment was discarded.
The 4 kb fragment from pAG-411 and ~e 259 bp drul promoter fragment from pAG-155were combined in a ligation reaction, using Gibco/BRL's T4 DNA ligase, following m~n-lf~c-
20 tnrer's instructions, to form the intermediate vector pAG~31. The nucleotide sequence for ~e
~ncated Ç~lVlllU~t:l dru259 is presented herein as SEQ ID NO:4.
EXAMPLE 8
Creation of Subclone pA~421 Cont~inin~ the drull O Promoter
Construction of plasmid pAG-155, cont~ining the full-length drul promoter is described in
25 ~l~mrle 7.
A DNA fragment cont~inin~ 166 bp of the drul promoter was PCR ~mr~lified from sulL)clone
pAG-155 using primers Drul-118H3 ~SEQ ID NO:9) and GFPStartR (SEQ ID NO:10) under the
foliowing PCR reaction con-lition~.
One cycle at 97aC for 3 min--t~s, after which the AMPLITAQ was added;
Two cycles at 97~C for 1 minute, 47~C for 1 minute and 72~C for 1 minute;
25 cycles at 94~C for 1 minute, 47~C for 1 minute and 72~C for 1 minute;
One cycle at 72~C for 5 minutes, followed by cooling to 5~C.
The 166 bp of the ~rul promoter fragment was then purified as follows. The PCR reaction
mixture was Ll;al~r~LL~d to a light Phase Lock Gel tube (5 Prime to 3 Prime, Boulder, CO.). A
CA 02243850 1998-07-21
W O 97/27307 PCTrUS97/01275
mixed solvent system containing phenol:chloroform:isoanyl alcohol (25:24:1) was added to this
tube at a volume equal to the PCR reaction volume. The tube was then spun in a microcentrifuge
followirlg the m~mlf~tnrer~s instructions. The upper, aqueous phase was l~ r~l~d to a Select,
G-50 spin column (5 Prime to 3 Prime, Boulder, CO.) and the was DNA cPnt-;filged through the
5 column following the m~nl~f~ct~rer's instructions. To the eluant 1/10 volume of ~M sodium
acetate and 2.5 volumes of ethanol were added, to precipitate the DNA. The sample was
incubated on ice for a period of no less than lO minutes. Following inc~ batic-~, the sample was
microcentrifuged at 4~C for 30 minutes at 14,000 rpm. The supernatant was dec~nted from ~e
tube and the pellet washed twice wit'n 75% ethanol. The pellet was allowed to dry, followed by
10 resuspension in 31.6 ,ul H2O. This fragment was digested to completion with restriction e.~y~
Hiru~II and EcoRV to produce a 112 bp dru~ ploll~Le~ fragment. This rl~ll~en, was purified in
the same manner as the PCR product described above.
Creation of subclone pAG-411 containing the nos::nptII cassette is described in Example 7
above.
Plasmid pAG~Lll was digested to completion with HindIII and PshAI, releasing a 620 bp
nos ~)lUlllOLt;~ fragment. The digested plasmid was run on a 1 % low melting point agarose gel
(SeaPlaque, FMC BioProducts, Rockland, ME). The gel region cont~ining the 4 kb fragment was
cut from the body of the gel. The DNA rla~ L was then purified away from the gel using ,~-
agarase from New England Biolabs (13everly, MA), following the m~m~f~c~rer's instructions. The
20 gel region cont~ining the 420 bp nos promoter fragment was discarded.
The 4 kb ~L~I~nltll~ from pAG-411 and the 112 bp drul prOlnOlt~f fragment derived from
pAG-155 were combined in a ligation reaction, using Gibco/BRL's T4 DNA ligase, following
m~n~lf~ctllrer's instructions to form the int~ te. vector pAG-421. The steps followed in
constructing plasmid pAG-421 are represented srh~-m~tir~lly in Fig. 2.
Example 9
COn~lru~liO~l of a Binary Plant Tr~ ru} ..lslion
Vectûr pA~7342 Con~ining a dru259::nptll Cl-h-.~ . ;c Gene
A. Construction of Plasmid pAG-1542
A flow chart 5.-lllll.~.;~i--g the construction ûf plasmid pAG-1542 is illn~tr~ted in Fig. 17.
~ 30 Plasmicl pAG-1542 was constructed using conventional cloning techniques known in the art
(Sambrook, et al., 1989). Subcloning binary vector pAG-1542 cont~in~d the npt~ marlcer gene
- under t]tle control of the nos promoter located near the left border and the SAMase gene (Ferro,
et al., 19953 driven by the tomato E8 promoter (Deikman, et al., 1988; Deilcman, et al., 1992)
located near the right border.
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38
B. Construction of Binary Plant Transformation Vector. pAG-7342
Construction of subclone pAG-431, contAining the dru259: :nptII chimeric gene, is described
in F~Amrle ~.
Plasmid pAG-1542 was digested with Hinc~II and BamIII. The digested plasmid was ~un
5 on a 1 % low melting point agarose gel (SeaPlaque, PMC BioProducts, Rockland, ME). The gel
region contAinin~ the 13 kb fragment was cut from the body of the gel. The DNA r~ e..L was
then purified away from the gel using ~-agarase from New F.ngl~nfl Biolabs (Beverly, MA),
following the m~Amlf~rtllrer's instructions. The gel region Co~ g the 1.46 kb nos::nptII
fragment was discarded.
Plasmid pAG-431 was digested with HindlII and BamHI. The digested plasmid w~ run on
a 1% low melting point agarose gel (SeaPlaque, l:;MC BioProducts, Rockland, ME). The gel
region containing the 1.1 kb dru259: :nptII fragment was cut from the body of the gel. The DNA
Lagllle~l~ was then purified away from the gel using ~B-agarase from New England Biolabs
~Beverly, MA), following the m~Am~f~Açtllrer's instructions. The gel region CO.,IA;~ the
15 rem~AinrlPr of the plasmid was discarded.
The 13 kb fragment from pAG-1542 and the 1.1 kb dru259::npdI fragment from pAG-431
were combined in a ligation reaction, using Gibco/BRL's T4 DNA ligase, following m~Amlf~A--
turer's instructions to form the binary plant ~ Çu~ Liol- vector pAG-7342.
Construction ûf binary plant l.,...~l'u...,AIinn vector pAG-7342 is depicted srh~mAtir,~lly in
Fig. 4.
Example 10
Construction of a Binary Plant Transformation Vector
~onl~ a drullO::nptll Chimeric Gene
Construction of plasmid pAG-1542 is described in Example 9A.
Construction of subclone pAG~21, conlail~ g the drullO::nptII chimeric gene, is described
in Example 8.
Plasmid pAG-1542 was digested with HindIII and BamHI. The digested pl~mid was run
on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME). The gel
region contAining the 13 kb fragment was cut from the body of the gel. The DNA fragment was
then purified away from the gel using ,~-agarase from New FnglAn~l Biolabs (Beverly, MA),
following the mAnnf~rtllre~s instructions. The gel region COI~IA~ g the 1.46 kb nos::nptII
r,~~ was discarded. Plasmid pAG-421 was digested wi~ HindlII and BamHI. The digested
plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC l3ioProducts, Rockland,
ME). The gel region cont~Aining the 0.95 kb dru259: :nptII fragment was cut from the body of the
gel. The DNA rLagll~elll was then purified away from the gel using ,B-agarase from New FnglAn~l
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39
Biolabs (Beverly, MA), following the m~nl~f~ctllrer's instructions. The gel region conl~ .g the
r~m,tinAer of the plasmid was discarded.
1'he 13 kb fragment from pAG-1542 and the 0.95 kb drullO::nptII fragment from pAG~21
were combined in a ligation {eaction, using Gibco/BRL's T4 DNA ligase, following m~m~f~c-
S turer's instructions to form the binary plant ~ 7ro~ dlion vector pAG-7242. Construction of
binary plant ~lall~.folllldLion vector pAG-7242 is depicted schçm~tir~lly in Fig. 3.
Exam~le 11
Plant Transform~fion Using Binary Vectors pA~7242 and pAG-7342
Agrobacterium-based plant ~ansro~md~ion using binary vectors pAG-7242 and pAG-7342
10 co.~ chimeric drullO::nptII and dru254::nptII genes, respectively, was carried out using
tomato cotyledons as described below for exemplary plasmid pAG-7242.
A cherry tomato line (CH3) obtained from Sunseeds Co. (Morgan Hill, CA) was used as the
target for plant lla~ l&Lion experiments. Tldllsr(~ ation was carried out using a standard
cotyledon-based Agrobacterium co-cultivation method (Fillatti, et al., 1987).
Agrobacteril~m tumefaciens strain EHA101 (Hood, et al., 1986), a Ai~rmed deliva~ivt; of
Agrobacterium tumefaciens strain C58, was used to introduce coding sequences into plants. This
strain contains a T-DNA-less Ti plasmid. The pAG-7242 plasmid was L~a~sre~ied into EHA101
using electroporation P~c~nti~lly as described by Nagel7 et al. (1990). Briefly, an Agrobacterium
tumefaciens culture was grown to mid-log phase (OD 600 0.5 to 1.0) in MG/L agar media~0 co~ i,.;..g tryptone (5 g/l), yeast extract (2.5 g/1), NaCl (S g/l), mannitol (S g/l), sodium
e (1.17 g/l), K2HPO4 (0.25 g/l), MgSO4 (0.1 g/l) and biotin (2 ~g/l), adjusted to pE~ 7.2
by addition of sodium hydroxide.
After cutting off from each end approxi,l~aLely one third of the tomato cotyledon, the middle
third was used as the tissue explant. Cotyledon explants were pre-conAitioned overnight on
25 tobacco feeder plates (Fillatti, et al., 1987). The pre-c~mtliti-n~d explants were innoculated by
placing them in a 20 ml overnight culture of EHA105/pAG-7242 for 15 minutes. The explants
were then co-cultivated with E~A105/pAG-7242 for 2 days on tobacco feeder plates as described
by Fillatti, et al., (1987).
The explants were grown in tissue culture media col~l;.it~ g 2Z media (Fillatti, etal., 1987),
30 Murisheegee and Skoog (MS) salts, Nitsch and Nitsch vitarnins, 3 % sucrose, 2 mg/l seatin, 500
mg/l carbenicillin, 60-200 mg/l kandnly~;ill, and 0.7% agar. The explants were grown in tissue
culture for 8 to 10 weeks. The carbenicillin tre~tm~nt~ were kept in place for 2 to 3 months in
all me,dia. 'rhe explants and plants were kept on carbenicillin until ~ey were potted in soil as a
counter-selection to rid the plants of viable Agrobacterium tumefaciens cells.
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Table 1 presents a ~u~ al y of the plant ll~Çullllation experiments, inr~ in~ concentrations
of selecti~n agent utilized, and Lld~rolllla~ion frequçn~ . Results obtained for plant
l"~"~ru~"~ on experiments using the novel raspberry pl~,lllolel~ of the present invention are
compared to those obtained using binary vectors containing two different strong co"~ ;ve
5 pronwL~ a caulimovirus plolllOteL, the cassava mottle vein virus pL~llln)tt:l (CAS) and the hsp80
promoter. The CAS promoter was obtained from The Scripps Research Institute (La Jolla, CA).
Isolation of the hsp80 promoter, its nucleotide sequence, as well as vector constructions and
expression levels of transgenes containing the hsp80 promoter have been described (Brunke and
Wilson, 1993).
A com~aricon of the relative strength of nptII expression across 10 transgenic events from
c~, l",."l~; produced using four of the prolllo~el-nptII chimeric gene combinations described
above is presented in Fig. 5.
Example 12
~elative Expression of the nptll Marker Gene in Tr~.~s~.;c Plants
Cvl~a;.. ;.~ ol~lot~.s Derived From Raspberry
Leaf tissues from l0 separate Llallsgellic events employing vectors pAG7242 and pAG-7342,
CQIl~ raspberry promoters drullO and dru254, respectively, were assayed by ELISA to
de~P "li,le nptII expression levels, according to the m~3nllf~rt~rer's (5'-3', Inc., Boulder, CO)
reoomm~n~P~l protocols for (i) protein extraction and (ii) ~ inn of nptII expression levels.
20 Results from the ~ sro~ ion experiments are provided in Tables l and 2 below.The np~II assay was carried out with a few samples using rooted plants which were available
in culture at the time of testing. Thus, not all rooted plants were tested for nptII expression. The
results of the ELISA assay are presented in column (IV) of Table l below.
Table 1: Transformation Results
2~ (1) ~Il) (1ll) ~Iv
~romoterSelection Conc. of kanamy- T~ npt~
cin (mg/l) Expression
CAS 200 S0% 100% (10/10
go 60% 67% (4/6)
30dru259
200 55% 75% (3/4)
63% 1~% (1/6)
dn~llO 90 47% 67% (213)
200 30% 10~% (1/1)
50% 63% (~/8)
hsp80 9O 27% 100% (1/1)
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a) (Il) (111) ({V)
Pr~moterSelection Conc. of k~mamy- Tr ~r~,~ Frequency np~ll cill (mg/l) Expression
200 607~ 100% (1/13
In referring to ~e data presented in Table 1, L~ rO, ., ~linn frequency is defimed as ~e ratio
of the number of tissue exp}ants producing regenerated shoots that are capable of rooting in the
p~e~ ce of selection agent (kanamycin) to the total number of initial tissue PYri~ntC, t:~-yressed
5 as a pe~ee~-lage. NptII expression level, expressed as a peLce.l~age, is the ratio of nptrI positive
plants to the total number of rooted plants tested for nptII, based upon the results of the ELISA
assay described in Example 12. A positive npdI result is an E~ISA value greater than
bac~l uu~d. For example, the first entry under column (IV) inrlir~t~s that out of 10 events tested
for npt}I, 10 ~yhii~it~d positive ELISA results.
Relative expression levels of nptII are presented in Table 2. The data from l.~u-sgel~ic plants
c~ the CAS::nptlI construct are not inf l~lded in Table 2 due to the high ~A~ sioll levels
obsenred in llAn:~rQI 1l~ ; containing the CAS promoter. Values from the two CAS::nptII events
assayed were in excess of 6000 pg/ml of nptII.
The range of expression across events presented in Table 2 below as well as iilllcfr~ted
15 graphiically in ~ig. 5 is typical for transgene expression in plants.
Table 2: E~ ion of nptlI (p~/ml~
T~ nic
Event hsp80dru259drul l O
1 1223 523.66 518.1
2 748.74S4.84 324.6
3 687.3231.64 174
4 332.1222.34 151.7
294.9144.22 51.22
6 207.5107.02 49.36
7 194.4 103.3 34.48
8 79.12 64.24 30.76
9 21.46 51.22 17.74
10.3 38.2 12.16
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The data above indicate that the exemplary dru259 and drullO promoters direct lower
level expression of genes placed under their control than does the hsp80 promoter. However,
these two exemplary raspberry dru promoters are both capable of ~A~ressil~g sufficient levels of
nptII to allow selection of transgenic plants.
While the invention has been described with reference to specific methods and em-
bo-lim~ntc, it will be appreciated that various modifications and changes may be made without
departing from the invention.
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43
SEQUENCE LISTING
~N'~R~T INFOPI~TION:
s
(i) APPLICANT: Agritope, Inc.
~ii) TITLE OF lNv~N~lON: RASPBERRY PROMOTERS FOR EXPRESSION OF
TRANSGENES IN PLANTS
(iLi~ NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A~ ADDRESSEE: Dehlinger & Associates
(B) STREET: 350 Cam~ridge Avenue, Suite 250
(C) CITY: Palo Alto
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94306
(v~ COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compati~le
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D~ SOFTWARE: PatentIn Release #1.0, Version #1.25
~vi) ~UKX~Nl~ APPLICATION DATA:
~A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
~vii~ PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/592,936
(B) FILING DATE: 29-JAN-1996
(viii) AL 1~RN~Y /AGENT INFORMATION:
(A~ NAME: Evans, Susan T.
(B) REG}STRATION NUMBER: 38,443
~C) RE~KEN-CE/DOCKET NUMBER: 4257-0015
) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 324-0880
~B) TELEFAX: (415) 324-0960
4~
CA 02243850 l998-07-2l
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44
t2~ INFOR~ATION FOR SEQ ID NO:1:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGT~: 2213 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ili) nY~Oln~LICAL: NO
(iv~ ANTI-SENSE: NO
( Yi ~ ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: pAG310 in~ert sequence (dru 1 gene)
20 ~ xi ) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATGCATATCA ACAACTACGA ATAA~GAr~AT CAGC~l~'CC GTATCTGGTG GAl~lllGAG 60
l~CG~ATGA CCATCTAATT AAAr~AAr-~A r~AA~AATTAT ACATATTGTG GAC~ CCC~A 120
TATATAATTC TTATCATCTT TGTTACTGCC ATTATGATTA TA~AATGATA TTAAAGGGAT 180
GGTGTACCGT GTACTAATCA AATATCTACC TGATCTTATT GATTTGAAAG AT~ATPAAA~ 240
30GAAATTAAAA LL~ll~AAPA TAAACCCCTA GAATTATATA TAGTTCATTA AGTTCAAATT 300
AAL~ iG A~AC~l~lLA AGCAACCCTA CAACGTACTA AGCACCCTAG ~LCC~lGC 360
~ ~GGC&G TAAGAGGAGA TATCCTCAGT CGAATTATGA GCCGATCGAG GA~AGCTCGA 420
3~ .
TCAGTTGGAA AA~ l TCTTATGGCC AAGlL~lllC A~A~AA~A~A TTGAATTATT 480
GACTCTTAGC AACTTAAGTT T~AAACCGTG ACr~ACr-~PT AP~ATTTGAC AAATTAATCA 540
40CTTTAAGTGC CTAGTGGATC AGCGTCTAGG TTGGGAACCC CTCTACCTGC GTTTGATTCA 600
CCAAGCTATC A~AATGGTCA GACACTGTGC TGCAATGCAC AATTGGAGCA TTTCACATGC 660
GTTGCATGAA TTAllC~ll~ GGTTAGGAAA CCTTTGAAAT ACCTTGACTA AGGTAAAAA~ 720
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AAAAACTTGA CAAATTAATA AATATTAATA TTGATTTTGT ACGTACACGA CTTA~CrAA~ 780
CTCTCA~TGA TTTATTGATT TCTAATATAT ATATTAATAA CGTACGTCTA ATTGGATCAT 840
STCATGATCTA CAGCCATCAC ATCTCAGATG ATTTTCTTGC AATGAATTGC CTAAGCTGGC 900
GTTATT~TCT 111LL1CATA ATACAGTTTT AA~ rGGT ACGTATTGGA GCTGGTGATG 960
ACTTCTTAAG AAArP~r~AA TTAACGCCAT AGCTATTTGA TTT~TATATC CAAAAGGAGA 1020
AAATGTATAA GATCGTTGCT TACTTAATTT GCAGGCTAGG TTAATTGACA T~AAATAA'rT 1080
GAAGAGTACG TAGGGCCAAT GTTGCTGAGA TCTAGCATCA ATAAT~GGAT TTGGCTTGTC 1140
15GATCGATCAT CTTTATTTAA TTGAGAGGTA TGTATCCATA I~71111~LGA AATTAAA~TA 1200
TTACCT~,ATA ATTGAGCTGA AACTGTAGTG AATTTAACCT TTTCTAAGTT CTGCCCATAT 1260
ATAA~P',''~rC ACATAGGTAG CTGATCGATC GAT~7~TATAT ATGTACTTAG GGTTCTGATC 1320
AGTATCAATA TCGATCACAA GTGCTGATAA TTAA~r7~TGG TTCTTCAAGG TAAGGTGGAG 1380
GCTGACATTG AAATCTCAGC ACCTGCTGAC AAGTTCTACA AC~.~11~AA GAGTGAGGCT 1440
25CACCACGTCC C~AAAArTTC TCAAACTGGC ACr~TAArCG GAGTTGCGGT GCATGAAGGA 1500
GACTGGGAAA CTGATGGCTC CATTAAGATT TGGAATTATG CAATAGGTAA GCCATTATGT 1560
TGTTAGATTG TTAATTTAGA TTATTAACCA AAGCTGGCTT TGAATCACTA r~ATATAT~T 1620
TAGGGCACGC CAGTArAr-Arr 'l"l'L~L~L L 1A TAA~ LGl L 1C AGTGATTATT TTCTTACAAA 1680
TAT~r-AGGGC GAAGTGGGAA CATTCAAGGA GA~AGTAGAG CTAGACGATG Tr7AAcpAr-7Gc 1740
35AATAATTCTG AATGGGTTGG AAGGAGATGT GTTCCAGTAT TA~AA~AGCT TCAAGCCCGT 1800
CTATCAI~TTC ACTCA-A-AAGA ATGATGGCAG CAGCATTGCC AAAGTGTCCA TTGAATATGA 1860
GAAACTGAGT GAGGAAGTTG CAGATCCAAA TAAGTACATT CGCTTGATGA CTAATATCGT 1920
r~Ar7rA'~CTT GATGCCCACT TCATCAAGGC ATA~AAGGGA TATTATAATA AATCAAGCAT 1980
ATGAAACACG ATGAA~Ar-~T AGCTAGCCAC TATCTACTGC TGGTTTATAA GTTTAAArAT 2040
4~AATCATGTGA AC~ r1AAT GCATGCTTTG TTTGGTTACT 1CGL111AAT G1~11~11~T 2100
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GCACTAATAC CGTCAGTGTA ATAAAAGCTA GTGTGAAAGG ATCTGATATA TTGTGATGTA 2160
TCATGTATTC AACTACCAAC TAT~T~TGGT ATCATATTTA TATATCAAAT AAA 2213
(2) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE CHARACT3RISTICS:
(A) LENGTH: 1356 ba~e pairs
0 (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
1~
~GI~hiICAL: NO
~iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
~C) INDIVIDUAL ISOLATE: Exemplary full length drul promoter
Re~[uence
~xi~ SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATGCATATCA ACAACTACGA AT~r7~ T CAGCCTTTCC GTATCTGGTG GATGTTTGAG 60
TCGGTGATGA CCATCTAATT AAAG~7~Ar~ GAAAAATTAT ACATATTGTG GAC~LCCC~A 120
3Q
T~T~TTC TTATCATCTT TGTTACTGCC ATTATGATTA TA~AATGATA TTAAAGGGAT 180
GGTGTaccGT GTACTAATCA AATATCTACC TGATCTTATT GATTTGAAAG ATr7~TA~ 240
35GA~ATTAAAA l~'AAAA TAAACCCCTA GAATTATATA TAGTTCATTA AGTTCAAATT 300
AA~L~lllG A~ACGL~llA AGCAACCCTA CAACGTACTA AGCACCCTAG ~lCC~ll~C 360
CTCTCGGCGG TAAGAGGAGA TATCCTCAGT CGAATTATGA GCCGATCGAG GAAAGCTCGA 420
TQGTTGGAA AATCTTTCTT TCTTATGGCC AAc7~LL~lllC AA~ TATA TTGAATTATT 480
GACTCTTAGC AACTTAAGTT TCAAACCGTG ACGAACCAAT AAAATTTGAC AAATTAATCA 540
45CTTTAA&TGC CTAGTGGATC AGCGTCTAGG TTGGGAACCC CTCTACCTGC GTTTGATTCA 600
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CCAAGCTATC A~AATGGTCA GACACTGTGC TGCAATGCAC AATTGGAGCA TTTCACATGC 660
GTTGCATGAA TTA1 ~C~llG GGTTAG~.AAA CCTTTGAAAT ACCTTGACTA AGGTAAAAAA 720
~AAAACTTGA CAAATTAATA AATATTAATA TTGATTTTGT ACGTACACGA CTTAACCAAA 780
CTCTCAATGA TTTATTGATT TCTAATATAT ATATTAATAA CGTACGTCTA ATTGGATCAT 840
TCATGATCTA CAGCCATCAC ATCTCAGATG A11L1~OL1GC AATGAATTGC CTAAGCTGGC 900
GTTATTATCT ~LlLll l ~ATA ATACAGTTTT APA~GGT ACGTATTGGA GCTGGTGATG 960
ACTTCTTP~G AAA~AA~A~ TTAACGCCAT AGCTATTTGA TTTATATATC CAAAAGGAGA 1020
15AAATGTATAA GATCGTTGCT TACTTAATTT GCAGGCTAGG TTAATTGACA Tr~TAA~T 1080
GAAGAGTACG TAGGGCCAAT GTTGCTGAGA TCTAGCATCA ATA~TA~.GAT TTGGCTTGTC 1140
GATCGATCAT CTTTATTTAA TTGAGAGGTA TGTATCCATA 1~L1LLCTGA AATTAAAA~A 1200
;~0
TTACCTAATA ATTGAGCTGA AACTGTAGTG AATTTA~CCT TTTCTAAGTT CTGCCCATAT 1260
~TAAr~hCC ACATAGGTAG CTGATCGATC GATC~TAT ATGTACTTAG GGTTCTGATC 1320
25AGTATCA~TA TCGATCACAA GTGCTGATAA TTAAAC 1356
~2~ INFORMATION FOR SEQ ID NO: 3:
~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 base pair~
(B~ TYPE: nucleic acid
(C~ STRANnF.n~S: double
~D~ TOPOLOGY: 1 inear
(ii~ MOLECULE TYPE: DNA
~ ~Y~O1~LICAL: NO
~iv~ ANTI-SENS3: NO
( Yi ~ ORIGINAL SOURCE:
(C~ INDIVIDUAL ISOLATE: drullO promoter se~uence (minus 112
region ~rom start codon)
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48
(X1) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TAAGTTCTGC CCATATATAA CATACCACAT AGGTAGCTGA TCGATCGATC ~TA~TATGT 60
~ACTTAGGGTT CTGATCAGTA TCAATATCGA TCACAAGTGC TGATAATTAA AC 112
(2~ INFORMATION FOR SEQ ID NO:4:
U~N~L'i CHARACTERISTICS:
0 {A) LENGTH: 259 base pair~
~B~ TYPE: nucleic acid
~C1 STRANDEDNESS: double
~D~ TOPO10GY: linear
~ii) MOLECULE TYPE: DNA
~iii) ~Y~OL~L1CAL: NO
~1V) ANTI-SENSE: NO
2~
t~i~ ORIGINAL SOURCE:
tC) INDIVIDUAL ISOLATE: Dru259 promoter sequence (minu~ 259
reQLon from ~tart codon)
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:4
AATGTTGCTG AGATCTAGCA TCAATAATAG GATTTGGCTT GTCGATCGAT CATCTTTATT 60
30TAATTGAGAG GTATGTATCC ATAL~1L11C TGA~ATTAAA ATATTACCTA ATAATTGAGC 120
TGAAACTGTA GTGA~TTTAA C~1LL~1AA GTTCTGCCCA ~T~T~T ~C~GG 180
TAGCTGATCG ATCGATCATA TATATGTACT TAGGGTTCTG ATCAGTATCA ATATCGATCA 240
~5
CAAGTGCTGA TAATTAAAC 259
4~2~ INFORMATION FOR SEQ ID NO: 5:
(i} SEQUENCE CHARACTERISTICS:
~A~ LENGT~: 24 base pairs
(~ TYPE: nucleic acid
4~ tC~ STRANDEDNESS: single
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49
~D~ TOPOLOGY: linear
lii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
~vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: primer DruInvUp
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
TGA ATG GGT TGG AAG GAG ATG TGT 24
(2) INF~RMA~ION FOR SEQ ID NO:6:
(i) SEOUENCE CHARACTERISTICS:
~A) LENGT~: 24 base pairs
~3) TYPE: nucleic acid
~C~ STRANDEDNESS: 5 ingle
~D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
~iii; ~Y~l~hllCAL: NO
(iv; ANTI-SENSE: NO
~vi} ORIGINAL SOURCE:
~C~ INDIVIDUAL ISOLATE: primer DruInvLow
~Xi) S~QU~NC~ DESCRIPTION: SEQ ID NO:6:
35ATG GTG CCA GTT TGA GAA GTT TTG 24
~2~ INFORMAT~ON FOR SEQ ID NO:7:
~i~ SEQUENCE CHARACTERISTICS:
~A~ LENGTH: 23 base pairs
~B~ TYPE: nucleic acid
~C1 STRANDEDNESS: single
~D} TOPOLOGY: linear
45 ~ MOT ~CUT ~ TYPE: DNA
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~0
~iii) HYPOTHETICAL: NO
(iv3 ANTI-SENSE: NO
~vi) ORIGINAL SOURCE:
~C) INDIVIDUAL ISOLATE: 5' primer DrupeUp
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
10ccc GTC TAG ATA TCA GCA CTT GT 23
~2) INFORMATION FOR SEQ ID NO:8:
~i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
20 { li ) ~OLECULE TYPE: DNA
~iii) ~Y~OT~lIcAL: NO
(iv3 ANTI-SENSE: NO
tvi3 ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: 3' primer, DrupeLow
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TGA ATC ACG ATG AAA AGA G 19
(2~ INFORMATION FOR SEQ ID NO:9:
35 ( i ) s~Q~N~ CHARACTERISTICS:
~A) LENGTH: 18 base pair~
tB) TYPE: nucleic acid
(C) STRANDEDNESS: 9 ingle
(D) TOPOLOGY: linear
~ MOLECULE TYPE: DNA
ti~i) ~Y~OL~LlCAL: NO
tiv3 ANTI-SENSE: NO
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(vi~ ORIGINAL SOURCE:
~C~ INDIVIDUAL ISOLATE: PCR reaction primer Drul-118H3
~ (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:9:
s
CGC AAG CTT TTC TA~ GTT 18
t2) lN~-ORhATION FOR SEQ ID NO:10:
0 ~ i ~ S~UU~NC~ cHARACTERISTICS:
~A~ LENGTH: 21 base pairs
~B) TYPE: nuclelc acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: DNA
(iii~ ~O~ ICAL: NO
(iv~ ANTI-SENSE: NO
~vi~ ORIGINAL SOURCE:
(C~ INDIVIDUAL ISOLATE: PCR reaction primer, GFPStartR
(xi~ ~y~N~h DESCRTPTION: SEQ ID NO:10:
GTT CTT CTC CTT TAC TCA TCT 21
30(2~ lN~uR~ATION FOR SEO ID NO:ll:
~yU~NC~ CHARACTERISTICS:
(A~ LENGTH: 30 amino acids
~B~ TYPE: amino acid
~D~ TOPOLOGY: linear
MOLECULE TYPE: protein
~ ~Y~ CAL: NO
~vi~ ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: amino term~nAl drupel qe~uence
~xi~ ~QU~N~ DESCRIPTION: SEQ ID NO:ll:
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Val Leu Gln Gly Lys Val Glu Ala Asp Ile Glu Ile Ser Ala Pro Ala
l 5 l0 15
Ala Lya Phe Tyr Asn Leu Phe Ly5 Ser Glu Ala Xaa Trp Val
~2~ INFORMATION FOR SEQ ID NO:12:
ti~ SEQUENCE CHARACTERISTICS:
~A) LENGTH: 30 ~a~e pairs
~B) TYPE: nucleic acid
(C~ STRANDEDNESS: single
(D~ TOPOLOGY: linear
1~ (ii) MOLECULE TYPE: DNA
iii) ~Y~l~ CAL: NO
( iY) ANTI-SENSE: NO
~vi~ ORIGINAL SOURCE:
~C~ INDIVIDUAL ISOLATE: dTRANDOM primer
25 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:12:
TAGGCTCGTA GA~l~l LLl'l' 'L111'11'~111 3o
(2) INFORMATION FOR SEQ ID NO:13:
EQ~ ~:N~ CHARACTERISTICS:
~A) LENGTH: 7 amino acids
~B) TYPE: amino acid
~D) TOPOLOGY: linear
3~
MOLECULE TYPE: peptide
~iii; ~Y~O~ ICAL: NO
~) FRAGMENT TYPE: internal
~i) ORIGINAL SOURCE:
~C~ INDIVIDUAL ISOLATE: drul partial amino acid ~equence
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Gln Gly Lys Val Glu Ala Asp
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ~Y~Ol~llCAL: NO
(iv) ANTI-SENSE: NO
~vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: reverse translated sequence of SEQ ID
NO:1, 3' PCR primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CARGGNA~RG TNGARCGNGA 20
~2) INFORMATION FOR SEQ ID NO:15:
(L) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ~r~ CAL: NO
(iv) ANTI-SENSE: NO
(vi~ ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: 5' PCR primer, DrupeRAN18
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TAGGC$CGTA GACTCTTT 18
5(2) INFORMATION FOR SEQ ID NO:16:
(i) ~Q~N~ C~ARACTERISTICS:
(A) LENGT~: 751 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPG~ ICAL: NO
(Lv) ANTI-SENSE: NO
(~i) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: pAG301 insert, drul cDNA clone
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
25CAGGGAAAGG TGGAGGCTGA CATTGA~ATC TCAGCACCTG CTGACAAGTT CTArAACCTC 60
TTCAAGAGTG AGGCTCACCA CGlCCC~AAA ACTTCTCA~A CTGGCACCAT AACCGGAGTT 120
GCGGTGCATG AAGGAGACTG GGAAACTGAT GGCTCCATTA AGATTTGGAA TTATGCAATA 180
GAGGGCGAAG TGGGAACATT CAAGGAGAAA GTAGAGCTAG ACGATGTGAA CAAGGCAATA 240
ATTCTGAATG GGTTGGAAGG AGAL~l~llC CAGTATTACA AGAGCTTCAA GCCC~L'~AT 300
35CAATTCACTC AAA~-pATGA TGGCAGCAGC ATTGCCAAAG TGTCCATTGA ATATGAGAAA 360
CTGAGTGAGG AAGTTGCAGA TCr~AATA~G TACATTCGCT TGATGACTAA TATCGTCAAG 420
GATCTTGATG CCCACTTCAT CAAGGCATAA AAGGGATATT ATAAT~ATC AAGCATATGA 480
pAr~CGATGA A~G~GCT AGCCACTATC TACTGCTGGT TTATAAGTTT AAA~.~TAATC 540
ATGTGAACGT TGTAATGCAT G~l~ G GTTACTTCGT TTTAATGTCT TGTTATGCAC 600
45T~TACCGTC AGTGTAATAA AAGCTAGTGT GAAAGGATCT ~T~T~TTGT GATGTATCAT 660
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GTATTCAACT ACCAACTATA TATGGTATCA TATTTATATA Tr~AATAAAT TAATGTGAAA 720
AAAAAAAAAA A~A~A~AGAG TCTACGAGCC T 7Sl
5(2) INFORMATION FOR SEQ ID NO:17:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 18 base pairs
(B~ TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iLi) ~Y~O-~lICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: DruGen 5' primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
25AAGGTGGAGG CTGACATT 18
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 8 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ~Y~Ol~lICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: DruGen 3' primer
(xi) ~Q~N~'~ DESCRIPTION: SEQ ID NO:18:
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CTGACGGTAT TAGTGCATAA CA 22
(2) INFORMATION FOR SEQ ID NO:l9:
~ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 base pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: dou~le
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ~Y~O~llCAL: NO
~5 (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: pAG302, d~ul g~n~ic clone
~0
( Xi ) ~U~N~ DESCRIPTION: SEQ ID NO:l9:
AAGGTGGAGG CTGACATTGA AATCTCAGCA CCTGCTGACA AGTTCTACAA C~l~ll~AAG 60
25AGTGAGGCTC ACCACGTCCC CA~AACTTCT CAAACTGGCA Cr7~TAAccGG AGTTGCGGTG 120
CATGAAGGAG ACTGGGAAAC TGATGGCTCC ATTAAGATTT GGAATTATGC AATAGGTAAG 180
C QTTATGTT GTTAGATTGT TAATTTAGAT TATTAAc~AA AGCTGGCTTT GAATCACTAC 240
AATATAT~TT AGGGCACGCC AGTACAGATT ~ lAT AAll~lllUA GTGATTATTT 300
TCTT'~AA'~T ATAGAGGGCG AAGTGGGAAC ATTCAAGGAG AA'AGTAGAGC TAr-ACr,ATGT 360
35~.AArPA~GCA ATAATTCTGA ATGGGTTGGA AGGAGATGTG TTCCAGTATT ACAAGAGCTT 420
CAAGCCCGTC TATCAATTCA CTCAAAAGAA TGATGGCAGC AGCATTGCCA AA~L~C~AT 480
TGAATATGAG AAACTGAGTG AGGAAGTTGC AGATCCAAAT AAGTACATTC GCTTGATGAC 540
TAATATCGTC AAGGATCTTG ATGCCCACTT CATCAAGGCA TA~AAGGGAT ATTAT~ATAA 600
ATCAAGCATA TGPAP~PCr-A TGAAAAGAGA GCTAGCCACT ATCTACTGCT GGTTTATAAG 660
45TTT~Ar'~TA ATCATGTGAA ~ AATG CATGCTTTGT TTGGTTACTT C~l~lLAATG 720
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T~ ATG CACTAATACC GTCAG 745
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 152 amino acids
(B) TYPE: amino acid
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: predicted amino acid coding se~uence
of drul
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met VaL Leu Gln Gly Lys Val Glu Ala Asp Ile Glu Ile Ser Ala Pro
1 5 10 15
Ala Asp Lys Phe Tyr Asn Leu Phe Lys Ser Glu Ala His Hig Val Pro
20 25 30
Lys Thr Ser Gln Thr Gly Thr Ile Thr Gly Val Ala Val His Glu Gly
35 40 45
Asp Trp Glu Thr Asp Gly Ser Ile Lys Ile Trp Asn Tyr Ala Ile Glu
35 Gly Glu Val Gly Thr Phe Lys Glu LYQ Val Glu Leu Asp Asp Val Asn
Ly~; Ala Ile Ile Leu Asn Gly Leu Glu Gly Asp Val Phe Gln Tyr Tyr
Lys Ser Phe Lys Pro Val Tyr Gln Phe Thr Gln Lys Asn A~p Gly Ser
100 105 110
Ser Ile Ala Ly~ Val Ser Ile Glu Tyr Glu Lys Leu Ser Glu Glu Val
45 115 120 125
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Ala Asp Pro Asn Lys Tyr Ile Arg Leu Met Thr Asn Ile Val Lys Asp
130 135 140
Leu Asp Ala His Phe Ile Lys Ala
145 150
