Promoter (FLt) for the full-length transcript of peanut chlorotic streak caulimovirus (PCLSV) and expression of chimeric genes in plants

The isolation, modification and use of wild-type and modified viral FLt promoters of peanut chlorotic streak caulimovirus (PClSV) in the expression of chimeric genes in plant cells. The FLt promoter from PClSV has been modified to have duplicated enhancer domains.

TECHNICAL FIELD 
The present invention relates to the fields of plant genetic engineering 
and plant molecular biology. More particularly, the present invention 
relates to the isolation, modification and use of wild-type and modified 
viral FLt promoters of peanut chlorotic streak caulimovirus (PClSV) in the 
expression of chimeric genes in plant cells. The FLt promoter from PClSV 
has been identified by the present inventors and modified to have 
duplicated enhancer domains. 
The FLt promoter with its single or double enhancer domains when linked to 
heterologous coding sequences to form chimeric gene constructs showed high 
levels of expression of these genes in cells and transgenic plants. These 
chimeric genes have been shown to be well expressed in plant cells. The 
FLt promoter with its double enhancer domain gives better expression of 
genes compared to the FLt promoter with its single enhancer domain in 
transformed plants. However, both plasmids with enhancer domains show 
improved levels of expression over promoters without enhancer domains. 
This invention also includes plant cells, plant tissue, and differentiated 
plants and seeds under control of the FLt promoter of PClSV. 
The invention is particularly directed to plasmids such as pPCSV22CAT 
containing the full-length transcript promoter of the peanut chlorotic 
streak caulimovirus. The plasmid is used to express chimeric genes in 
plants. 
BACKGROUND ART 
A virus is a group of submicroscopic infective agents with double or single 
stranded DNA or RNA as core genetic material surrounded by a protein (and 
lipid in some cases) shell called a `capsid` or `coat`. It has no 
semipermeable membrane and it can multiply only in living cells using host 
cellular components. The short segment of the virus genetic material (FLt 
promoter) used in this invention can not infect plants or other organisms 
to cause disease. It is useful with selected foreign genes to obtain 
expression of these genes in other plants to confer useful properties to 
those transgenic plants. 
The caulimoviruses and their promoters 
The following is a description of caulimoviruses also called plant 
pararetroviruses. Caulimoviruses derived their name from cauliflower 
mosaic virus (CaMV), the type member of the group (for reviews see 
Shepherd, 1989; Covey and Hull, 1992). More than a dozen types of 
caulimoviruses have been described to date. All have small circular DNA 
molecules as their genetic material. The genomes of CaMV (Gardner, et al., 
1981) and four other members of this group, namely carnation etched ring 
virus (CERV), (Hull, et al., 1986), figwort mosaic virus (FMV), (Richins, 
et al.,1987) soybean chlorotic mottle virus (SOCMV), (Hasegawa, 1989), and 
peanut chlorotic streak virus (PClSV) (Richins, 1993) have been fully 
sequenced. CaMV is a circular double stranded DNA virus with a genome size 
of approximately 8 kb. It is organized into seven open reading frames 
(genes) and two intergenic regions. In the case of CaMV and by analogy 
PClSV, the polypeptides corresponding to the six genes (I to VI) have been 
detected in infected cells and their functions have been identified. The 
cell-to-cell movement function (Thomas, et al., 1993; Ducasse et al., 
1995), aphid-transmission factor (Daubert et al., 1983; Woolson, et al., 
1983), minor capsid protein (Giband, et al., 1986), major capsid protein 
(Daubert, et al., 1982), reverse transcriptase (Takatsui, et al., 1992), 
and post-transcriptional transactivator (Bonneville et al., 1989) (also 
the inclusion body protein, Odell and Howell, 1980) are associated with 
ORFs I to VI respectively. 
Gene VII protein was not detected in vivo (Wurch, et al., 1991); and its 
function is not clearly established. However a sequence located with this 
ORF of FMV is involved in translation of viral genes (Gowda, et al., 
1991). The viral genome is replicated through reverse transcription of the 
terminally redundant full-length transcript (Bonneville and Hohn, 1993) by 
a virus encoded reverse transcriptase. Two major viral transcripts, known 
as 35S RNA and 19S RNA are synthesized exclusively from the minus strand 
DNA by the host RNA polymerase II (Odell, et al., 1981; Howell and Hull, 
1978). 
The large intergenic region (L-IR) which resides between gene VI and VII, 
contains the promoter (35S) for the full-length transcript which spans the 
entire viral genome (Dixon and Hohn, 1984; Scholthof, et al., 1992). The 
35S RNA serves as template for minus strand DNA synthesis by viral gene V 
encoded reverse transcriptase (Gordon, et al., 1988). The small intergenic 
region (S-IR) residing between gene V and gene VI contains a promoter 
(19S) which transcribes gene VI only (Odell and Howell, 1980). The PClSV 
is apparently lacking the S-IR sequence, however both FMV (Scholthof, et 
al., 1992) and PClSV (Richins, 1993) have also been shown to have 
transcripts similar to the 19S and 35S RNA found in CaMV infected plant 
cells. 
Regulatory elements of the cauliflower mosaic virus 35S promoter 
The CaMV 35S promoter, which spans about 941 base pair (bp) upstream from 
the transcription start site, has been shown to be active in various 
monocot and dicot cells. The cis-regulatory elements that are involved in 
directing transcription initiation reside within this region. The CaMV 35S 
promoter has a modular construction with elements consisting of an 
enhancer (Lam, 1994, and references there in) similar to those of other 
promoters like that of SV40 in mammalian systems (Ondek, et al., 1987; 
Schirm, et al., 1987; Fromental, et al., 1988). The 5' deletion analysis 
of CaMV35S promoter, studied in transformed tobacco calli or a protoplasts 
transient assay system, indicates that a promoter fragment of 343 bp 
upstream from the transcription start site is sufficient for high promoter 
activity (Odell, et al., 1985, Ow, et al., 1987). 
The high promoter activity is the result of synergistic and combinatorial 
effects of enhancer elements residing in the -343 to -46 region upstream 
of the TATA element promoter (-46 to +8) (Fang, et al., 1989, Benfey, et 
al., 1989, Benfey and Chua, 1990, Benfey, et al., 1990a and Benfey et al., 
1990b). 
Sequence motifs and Trans-acting factors in the CaMV promoter 
Several protein binding sequence motifs have been identified in the 
enhancer region of the 35S promoter (Lam, et al., 1989; Lam and Chua, 
1989; Prat, et al., 1989; Bouchez, et al., 1989, Yanagisawa and Izui, 
1992). Identical or similar sequence motifs are also present in promoters 
of other caulimoviruses (Bouchez, et al., 1989; Sanger, et al., 1990; 
Cooke and Penon, 1990; Richins, et al., 1993). Two nuclear binding protein 
factors, known as Activating Sequence Factor-1 and -2 (ASF-1 and ASF-2) 
from tobacco have been well characterized. ASF-1 binds to the activating 
sequence as-1 (-82 to -62) region of 35S promoter. Two TGACG motifs within 
this site are essential for DNA-protein interaction (Lam, et al., 1989). 
The as-1 motif is also found in full-length transcript promoters from 
other caulimovirus including FMV (Sanger, et al., 1990, and present 
studies), PClSV (Richins, 1993) and MMV (Shepherd group, unpublished 
observation). 
Modification of Promoter with multiple copies of an enhancer domain 
Single or multiple copies of enhancer sequences from the CaMV 35S promoter 
can increase homo- and heterologous promoter activity in an 
orientation-independent manner (Kay, et al., 1987; Ow, et al., 1987: 
Odell, et al., 1988; Fang, et al., 1989; Driesen, et al., 1993; Omirulleh, 
et al., 1993). The enhancement of promoter activity was proportional to 
the copy number of the enhancer sequence (Kay, et al., 1987; Ow, et al., 
1987; Omirulleh, et al., 1993). Similar observation was made when single 
or multiple copies of the enhancer sequence was inserted upstream of the 
TATA element of the CaMV19S promoter (Ow, et al., 1987; Driesen, et al., 
1993), rbcS-3A promoter (Fang, et al., 1989), the nos promoter (Odel, et 
al., 1988) or the FMV FLt promoter (Maiti, et al.,1995,1996) 
The engineering of novel traits in plants and other crops promises to be an 
area of great agricultural importance (Maiti and Hunt, 1992; Wagner, 
1992). Plant genetic engineering techniques allow researchers to introduce 
heterologous genes of interest into plant cells to obtain the desired 
qualities in the plants of choice. Plant genetic engineering is leading to 
rapid progress in the production of economically valuable germplasm with 
improved characters or traits such as insect resistance, virus resistance, 
fungal resistance, herbicide resistance, bacterial or nematode pathogen 
resistance, cold or drought tolerance, improved nutritional value, seed 
oil modification, delayed ripening of fruits, and male sterility, to name 
a few. These germplasms provide enhanced developments in breeding programs 
for crops improvement as well as a better understanding of gene regulation 
and organization in transgenic plants. The expression of useful new traits 
in plants is a major focus in plant biotechnology. 
Plant metabolic engineering is the application of genetic engineering 
methods to modify the nature of chemical metabolites in plants. For 
metabolic engineering where multiple genes need to be inserted into one 
cell, the use of different strong constitutive promoters is desirable in 
order to avoid genetic instability caused by recombination between 
identical or closely related promoter sequences, for example those taken 
from plants themselves. Through use of these promoter sequences the 
introduced genes can be transcribed to messenger RNA and then translated 
to resultant proteins to exhibit new traits or characters. 
Besides developing useful traits in crops, transgenic plants lead to a 
further understanding of molecular pathways involved in disease 
development and secondary metabolism in plants. Moreover, by engineering 
plants with specific foreign genes, the responses of plants to abiotic and 
biotic stress and stress-related metabolism are analyzed. The invention 
described herein which develops gene vectors with newly defined promoters 
of the caulimoviruses advances this effort. 
A wide variety of well-characterized genes of animal, human, bacterial and 
of plant origin, including those of several viruses, are available for 
engineering plants. For the most effective expression of this wide 
selection of genes either constitutive or regulated, versatile gene 
expression vectors are required. At the University of Kentucky, Dr. Arthur 
Hunt and his colleagues have developed a series of plant expression 
vectors (Schardl, at al., 1987) with a constitutive 35S promoter from 
cauliflower mosaic virus (CaMV) which have been successfully used to 
produce transgenic plants (Maiti, et al., 1988, 1989, 1991, 1993, 1994, 
1995; Graybosh, et al., 1989; Berger, et al., 1989; Yeargan, et al., 1992; 
Liod, et al., 1992). 
The most widely used promoter for plant transformation, as described 
earlier, has been the 35S promoter of CaMV. It is active in a wide variety 
of plants and tissues. It also is the most thoroughly characterized 
promoter with respect to the sequence elements active in its 
transcriptional activity (Benfey and Chua, 1990. Kay, et al., 1987 showed 
that the transcriptional activity of the CaMV 35S promoter could be 
increased approximately tenfold by making a tandem duplication of 250 base 
pairs of upstream sequence. Similar observations have been made with other 
promoters (McNeall, et al., 1989). A similar construct has been tested 
with the FMV--and FLt promoters. 
Certain promoters have a specific modular sequence which makes them either 
tissue-specific, developmentally regulated or environmentally regulated 
for the selective expression of genes in cells. Promoters capable of 
directing RNA synthesis at higher rates compared to other promoters are 
desirable for many purposes. If these promoters are able to direct the 
expression of genes in most types of plant tissues, they are defined as 
constitutive promoters. Previous work had established that the CaMV 35S 
promoter is one of the strongest constitutive promoters. The 
transcriptional activity of the CaMV 35S promoter is the result of 
synergistic and combinatorial effects of enhancer elements residing 
upstream of the TATA element. Single or multiple copies of the enhancer 
sequences from the CaMV 35S promoter can also increase the activity of 
heterologous promoters in an orientation--independent manner. The 
enhancement of promoter activity has been found to be related to the copy 
number of the enhancer sequence. We have developed expression vectors with 
the PClSV promoter with its single and duplicated enhancer domains. The 
upstream enhancer elements of the strong constitutive promoter from the 
full-length transcript of FMV or PClSV has been doubled in a strategy to 
strengthen this promoter even further. 
Promoters from other caulimoviruses such as FMV, and MMV, as well as the 
better characterized CaMV 35S promoter are found to be useful for plant 
genetic engineering. The Monsanto Co. has recently patented the 35S and 
the 19S promoters of CaMV in USA, and the full-length transcript promoter 
from FMV in Europe. The inventors have now developed new promoters of 
equal or better strength. 
U.S. Pat. No. 5,306,862 to Chappell, et al., discloses a method and 
composition for increasing sterol accumulation in higher plants. Column 8, 
lines 44-46 discloses the cauliflower mosaic virus promoter 35S. 
U.S. Pat. No. 5,349,126 to Chappell et al., discloses a process and 
composition for increasing squalene and sterol accumulation in higher 
plants. Column 4, lines 20-24 describe the cauliflower mosaic virus 35S 
promoter. The patent describes the pKYLX71 recombinant plasmid. 
Chemical Abstracts, Volume 118, Abstract No. 120793n, 1993, discloses the 
gene I of peanut chlorotic streak virus. 
Chemical Abstracts, Volume 118, Abstract No. 251150y, 1993, discloses a 
physical map of the peanut chlorotic streak virus which is transmissible 
in plants of Leguminosae and Solanaceae. The virus was determined not to 
be related to the cauliflower mosaic virus and the figwort mosaic virus. 
Polypeptides were purified of the peanut chlorotic streak virus having 58 
and 51 kDa. The virus is found to have a 8.1 kilo base pair length. 
Chemical Abstracts, Volume 122, Abstract No. 232376t, 1995, discloses a 
molecular analysis of the essential and nonessential genetic elements of 
the peanut chlorotic streak caulimovirus. 
Chemical Abstracts, Volume 122, Abstract No. 235454c, 1995, discloses 
reduced accumulation of tobacco mosaic virus in upper of leaves and plants 
inoculated with the peanut chlorotic streak caulimovirus. 
Chemical Abstracts, Volume 123, Abstract No. 307774c, 1995, discloses gene 
I mutants of peanut chlorotic streak virus. Gene I is suspected of 
encoding a protein for virus movement. 
Chemical Abstracts, Volume 106, Abstract No. 171526yn, 1987, discloses 
properties of ribonucleic acid in coat protein of the peanut chlorotic 
ring model virus. 
Chemical Abstracts, Vol. 116, Abstract No. 1417s, 1992 discloses the 
regions of sequence variation in caulimovirus gene VI. The figwort mosaic 
virus is used as a comparison. 
Chemical Abstracts, Vol. 116, Abstract No. 3719r, 1992 discusses the 
disease syndrome associated with expression of gene VI and caulimoviruses. 
There is a correlation between the level of gene VI and coded protein 
found in the disease. 
Chemical Abstracts, Vol. 116, Abstract No. 249819p, 1992 discloses the 
full-length transcript of the caulimovirus as a polycistronic messenger 
RNA whose genes are transactivated by the product of gene VI. The results 
show that the genome of figwort mosaic virus contains two promoters. 
Chemical Abstracts, Vol. 117, Abstract No. 186002q, 1992 discloses 
regulation of caulimovirus gene expression and involvement of cis-acting 
elements on viral transcripts. 
Chemical Abstracts, Vol. 118, Abstract No. 230014s, 1993 discloses that a 
zinc finger of a caulimovirus is essential for infectivity but does not 
influence gene expression. 
Chemical Abstracts, Vol. 122, Abstract No. 75400b, 1995, discloses that 
eukaryotic RNAse H shares a conserved domain with caulimovirus protein. 
Chemical Abstracts, Vol. 122, Abstract No. 232376t, 1995 discloses a 
molecular analysis of essential and non-essential genetic elements in the 
peanut chlorotic streak caulimovirus. 
Chemical Abstracts, Vol. 123, Abstract No. 251578x, 1995 discloses 
regulatory elements involved in caulimoviral gene expression. 
Plant expression vectors with the constitutive FLt promoter from PClSV have 
been developed by the present inventors. 
The present inventors have overcome deficiencies in prior art concerning 
transgenic plant promoters, and have developed useful promoters from PClSV 
for high level expression of foreign genes, for example, in transgenic 
tobacco. These vectors are be useful for both direct DNA uptake by 
isolated protoplasts and Ti plasmid-mediated gene transfer. Enhanced 
levels of transcription via highly active promoters are essential for high 
levels of gene expression in transgenic plants. 
SUMMARY OF THE INVENTION 
These inventions are in general applicable to plant genetic engineering. 
Specifically, the present inventions relate to the promoters from peanut 
chlorotic streak virus (PClSV) and these promoters direct the expression 
of genes in plant cells. A conventional gene is composed of a promoter 
region, a sequence encoding a 5' non-translated leader sequence of the 
transcribed messenger RNA, the structural gene itself and a 3' 
polyadenylation sequence. The promoter is a DNA fragment composed of 
modular sequence which directs and regulates the transcription to 
messenger RNA, the first step in expression of a gene. 
The proper regulatory signals/enhancer elements should be present in a 
defined location in order to express the inserted gene first as RNA and 
then as a resultant protein via the process of translation. The 
3'-polyadenylation sequence is a non-translated region which signals the 
adenylation of the 3' end of the RNA in order to stabilize the RNA in the 
cytoplasm for subsequent translation into protein. 
An objective of the present invention is to define and document the strong 
constitutive FLt promoter of PClSV to be used for expression of chimeric 
genes in transgenic plants. An additional object is to describe a strategy 
to further strengthen the promoter from the full-length transcript of 
other members of the caulimoviruses including PClSV. 
Thus the present invention provides a plasmid comprising a chimeric gene 
comprising a full-length transcript (FLt) promoter from peanut chlorotic 
streak virus (PClSV) operably linked to at least one heterologous gene 
sequence which is heterologous to the promoter. 
The invention also provides for plant cells and transgenic plants which 
contain the plasmid of the invention. 
The above and other objects of the invention will become readily apparent 
to those of skill in the relevant art from the following detailed 
description and figures, wherein only the preferred embodiments of the 
invention are shown and described, simply by way of illustration of the 
best mode of carrying out the invention. As is readily recognized the 
invention is capable of modifications within the skill of the relevant art 
without departing from the spirit and scope of the invention.

STATEMENT OF DEPOSIT 
Plasmids pKLP6 and pKLP36 in E. coli TB1 have been deposited with the 
Agricultural Research Service (ARS) Patent Culture Collection (NRRL), 1815 
North University Street, Peoria, Ill., USA, 61604, under the terms of the 
Budapest Treaty on Jul. 25, 1996. The deposit will be maintained for the 
life of the patent as required by Treaty. 
DETAILED DESCRIPTION OF THE INVENTION 
The invention provides for a plasmid or transformation vector comprising a 
chimeric gene comprising a full-length transcript (FLt) promoter from 
peanut chlorotic streak virus (PClSV) operably linked to at least one 
heterologous gene sequence which is heterologous to the promoter. In a 
preferred embodiment the plasmid further comprises at least one PClSV 
enhancer domain. The plasmid may include a single or double enhancer 
domain. 
In an alternative embodiment of the plasmid, the promoter directs 
transcription of heterologous genes downstream from said promoter, in 
plants. The promoter preferably comprises nucleotides 5799 to 6150 of the 
3' portion of gene VI (SEQ ID NO:1) and a downstream intergenic region of 
the PClSV genome. The promoter may also comprise a 5' non-translated 
leader sequence from peanut chlorotic streak virus. 
In a more preferred embodiment of the invention the plasmid further 
comprises a region of homology to an Agrobacterium tumefaciens vector and 
a T-DNA border region from Agrobacterium tumefaciens, wherein said 
chimeric gene is located between the T-DNA border and the region of 
homology. Similarly this embodiment of the invention may possess at least 
one PClSV enhancer domain. The expression vector may further comprise a 
disarmed plant tumor-inducing plasmid of Agrobacterium tumefaciens. 
Preferred plasmids in accordance with the invention are plasmids selected 
from PKLP6, PKLP36 and PCSV22CAT. Methods for obtaining these plasmids and 
characteristics of these plasmids are described herein. For example, a 
plasmid of the invention may comprise, in the 5' to 3' direction, a) the 
PClSV FLt promoter with single enhancer; b) a 3' nontranslated 
polyadenylation sequence of rbcS E9 gene; and (c) a structural sequence 
encoding neomycin phosphotransferase II. 
The invention provides for a plant cell which comprises the plasmid of the 
invention. The plant cell may express the plasmid with at least one PClSV 
enhancer domain. 
More importantly, the invention provides for transgenic plants which 
express the plasmid of the invention. In a preferred embodiment the 
transgenic plant is selected from, but not limited to cotton, soy bean, 
alfalfa, oilseed rape, flax, tomato, sugar beet, sunflower, potato, 
tobacco, maize, wheat, rice, lettuce and banana plants. The transgenic 
plant may express a heterologous gene present in the plasmid of the 
invention in plant tissue selected from, but not limited to calyx, 
filament, pedicel, style, ovary, corolla, anther, stigma, embryo, seeds, 
leaf, stem and root tissues. 
In sum, the invention includes any DNA construct comprising a PClSV 
promoter, and preferably includes a PClSV FLt promoter isolated from a 
PClSV protein-encoding DNA sequence. The DNA construct may be expressed in 
plant cells. The DNA construct is transcribed and translated in plant 
cells, and includes promoters such as a PClSV FLt promoter region free of 
PClSV protein-encoding DNA sequence and a PClSV FLt promoter region with a 
DNA sequence which is heterologous with respect to the promoter. In a 
preferred embodiment the construct comprises a DNA sequence which is 
heterologous with respect to the promoter and a 3' non-translated 
polyadenylation signal sequence. 
Thus, the present invention includes the following: i) isolation of the 
promoter for the full-length transcript (FLt) of peanut chlorotic streak 
virus from a full-length viral DNA clone (Reddy, et al., 1993) as 
described below in Experimental Section. ii) The invention provides for 
modification of the PClSV promoter to include duplication or 
multimerization of the enhancer domain of the Flt promoter from PClSV. The 
FLt promoter sequence for PClSV is shown in FIG. 1. iii) The invention 
provides for use of PClSV promoter in a method for transforming plant 
cells, expression vectors including PClSV promoter, a chimeric gene 
including PClSV promoter sequence, and transgenic plants, plant cells and 
seeds incorporating the PClSV promoter in a chimeric gene. 
Experimental Procedures 
Peanut chlorotic streak virus (PClSV) is a newly described member of the 
caulimovirus group (Reddy, et al., 1993). It has been partially 
characterized in this laboratory (Richins et al., 1993; Reddy, et al., 
1993) These investigations provide the materials (DNA clones) for the 
invention described herein. 
EXAMPLE 1 
Construction of PClSV FLt promoter with its single and double enhancer 
elements and creation of plasmids pKLP6 and pKLP36 
The construction strategy for isolating the PClSV FLt promoter and its 
enhancer is shown in FIGS. 2 A and B. The basic FLt promoter of PClSV, 150 
bp (position 5852 to 6001 of the PClSV sequence) (SEQ ID NO:2) was 
isolated after amplification by PCR using oligonucleotides containing the 
appropriate sites to generate EcoRI-HincII sites at the 5' end and a 
HindIII site at the 3' end of the fragment. The promoter sequence was 
inserted as an EcoRI-HindIII fragment into the corresponding sites of the 
plant expression vector pKYLX71 (FIG. 3) and the plasmid pUC119. The 
resulting plasmids were designated pKLP6 (FIG. 4) and pUCPFLt6, 
respectively (FIG. 2A). The upstream sequence containing enhancer 
elements, a 78 bp (position 5852 to 5929) (SEQ ID NO:3) of the PClSV FLt 
promoter was amplified by PCR with oligonucleotides engineered for the 
EcoRI-HincII sites at the 5' end and the SmaI-HindIII sites at the 3' end 
of the fragment. 
The enhancer element fragment was cloned into EcoRI and HindIII sites of 
PUC119 and the plasmid designated as pUCP-enhancer (FIG. 2B). The PClSV 
FLt basic promoter fragment as a HincII-HindIII fragment (isolated from 
pUCPFLt6) was inserted into the pUCP-enhancer plasmid after digestion with 
SmaI and HindIII. The resulting plasmid designated as pUCPFLt36 contains 
two copies of the enhancer elements (FIG. 2B). The PClSVFLt promoter with 
its double enhancer domain was inserted into the plant expression vector 
pKYLX71 at its unique EcoRI and HindIII sites that flank the promoter. The 
resulting plasmid was designated as pKLP36 (FIG. 5). The PClSV basic FLt 
promoter and enhancer elements was amplified from a full-length clone of 
PClSV (Reddy et al., 1993). 
EXAMPLE 2 
Testing the Expression Vectors with a GUS reporter gene 
Stable transformation and analysis of transgenic plants. 
The reporter gene GUS was tailored by PCR to include just the coding 
sequence with the initiation and termination codons, flanked by a Xho I 
site at the 5' end and a Sst I site at the 3' end. The PCR isolated 
fragment for the reporter gene (GUS) was digested with Xho I and Sst I, 
gel purified and cloned into the corresponding sites of the plant 
expression vectors PKLP6 and pKLP36 and the resulting constructs pKLP6GUS 
and pKLP36GUS were introduced into Agrobacterium tumefaciens strain 
C58C1:pGV3850 by triparental mating. Tobacco (cv. Samsun NN) was 
transformed with the engineered Agrobacterium as described earlier (Maiti 
et al., 1993). To examine the integration of genes in transgenic plants, 
genomic DNA was isolated following the procedure (Thomson and Henry, 1993) 
for PCR analysis. 
The integration of reporter GUS gene in the genome of transgenic plants (R0 
and R1 progeny) was detected by PCR amplification using appropriately 
designed oligonucleotides specific for the GUS gene sequence. Specificity 
of each PCR product was tested by Southern hybridization with a GUS probe. 
Although the GUS gene was used in this example as the gene heterologous to 
the PClSV promoter, any gene heterologous to the PClSV promoter which is 
desired to be expressed in a transgenic plant may be included in the 
plasmid. One of skill in the art can readily substitute any heterologous 
gene into the plasmid of the invention using conventional genetic 
engineering techniques. 
Examples Heterologous Genes which may be used with PClSV FLt Promoter 
Plant genetic engineering techniques allow researchers to introduce 
heterologous genes of interest into plant cells to obtain the desired 
qualities. A strong constitutive promoter like PClSV promoter is useful to 
direct the any gene to be used for plant genetic engineering, a field of 
biotechnology which is leading a rapid progress in the production of 
economically valuable germplasm with improved characters or traits such 
as: 
1. Insect resistance, (developed with Bt toxin gene, .alpha.-amylase 
inhibitor gene). 
2. Virus resistance, (developed with CP, protease or replicase gene). 
3. Fungal resistance, (developed with chitinase gene, ribosome inhibiting 
protein gene, glucanase gene). 
4. Herbicide resistance, (developed with acetolactate synthase, 
phosophinothricin acetyl transferase or bar gene, nitrilase gene, or 
2,4-dichlorophenoxyacetate monooxygenase gene). 
5. Bacterial or nematode pathogen resistance, (developed with 
.alpha.-hordothionin gene, Bt toxin gene, beet cyst nematode resistant 
locus). 
6. Cold or drought tolerance. 
7. Improved nutritional value, (developed with seed storage protein genes). 
8. Seed oil modification, (developed by controlling chain length and 
saturation with fatty acid synthesis genes including stearoyl-ACP 
desaturase, oleoyl-ACP thioesterase, .beta.-ketoacyl-ACP synthase and 
acyl-ACP thioesterase). 
9. Delayed ripening of fruits, (developed by controlling ethylene producing 
genes, ACC oxidase gene). 
10. Male sterility. 
11. Modification of carbohydrate (developed with antisence gene of granule 
bound starch synthase, branching enzyme encoding genes, glgB). 
12. Protein/peptides controlling human disease (Therapeutic peptides, 
proteins such as RMP-7, AC137, antithrombin hirudin, growth hormone, 
interleukin could be produced in plant-based system) to name a few 
examples. 
The above heterologous genes, and other heterologous genes may be inserted 
into plasmids pKLP6 and pKLP36 by genetic engineering methods known in the 
art. These newly created germplasms can enhance breeding programs for crop 
improvement, as well providing as a better understanding of gene 
regulation and organization in transgenic plants. Plant metabolic 
engineering is the application of genetic engineering methods to modify 
the nature of chemical metabolites in plants. For metabolic engineering 
where multiple genes need to be inserted into one cell, the use of 
different, strong, constitutive promoters is desirable in order to avoid 
genetic instability caused by recombination between identical or closely 
related promoter sequences taken from plants themselves. Through use of 
the promoter sequences of the invention the introduced genes can be 
transcribed to messenger RNA and then RNA translated to resultant proteins 
that exhibit new traits or characters. The invention described herein, in 
developing gene vectors with newly defined promoters of the 
caulimoviruses, advances this effort. 
A wide variety of well-characterized genes of animal, human, bacterial and 
of plant origin, including those of several viruses, are available for 
engineering plants. For the most effective expression of this wide 
selection of genes either constitutive or regulated, versatile gene 
expression vectors are required. 
EXAMPLE 3 
Comparative functional analysis of the CaMV35S and the FMV FLt promoters in 
transient expression experiments using tobacco leaf protoplasts: 
In earlier studies (Maiti, et al., 1996) the relative strengths of the CaMV 
35S and the FMV FLt promoters were compared. Different vector constructs 
with the GUS gene in transient expression experiments in protoplasts of 
Nicotiana edwardisonii were tested. Later the expression of the FMV FLt 
promoter with the PClSV FLt promoter in transgenic plants was compared 
(see example 2). 
Isolation of protoplasts from Nicotiana edwardsonii cell suspension 
cultures and electroporation of protoplasts with supercoiled plasmid DNA 
containing GUS has been described (Gowda et al., 1989; Kiernan et al., 
1993). Fluorometric GUS assays to measure GUS activity of plant tissue 
extracts and histochemical GUS assays to determine the distribution of GUS 
activity in plants, embryos and seedlings, were performed according to 
published procedures (Jefferson et al. ,1987). Protein in plant extracts 
was estimated (Bradford 1976) using BSA as a standard. The fluorometric 
GUS assays was performed as described earlier (Maiti et al., 1996). 
The results from the transient expression experiments are shown in Table 1. 
The gene constructs with the wild-type FLt promoter (pFMV 20 GUS) with its 
single enhancer domain showed about 2.5 fold higher promoter activity than 
the CaMV 35S promoter-GUS construct (pGG1) in these assays. A control 
plasmid pc-GUS (CaMV 35S promoter-GUS-nos 3' terminator) contains an extra 
out of frame ATG codon (as Sph I site GCATGC) in the multiple cloning site 
of pKYLX 7. The presence of this ATG codon causes about 7-8 fold less GUS 
activity compared to pGG1. The duplication of FMV FLt promoter enhancer 
domain in plasmid pKLF2-GUS increased the level of GUS activity about 4 
fold as compared to pKLF-GUS with the single enhancer domain. This 
difference was also observed in a stably transformed system in intact 
tobacco plants (Maiti, et al., 1996). 
EXAMPLE 4 
Comparative functional analysis of FMV and PClSV FLt promoters in 
transgenic plants 
The constructs shown in FIG. 6 were introduced into tobacco plants via the 
Agrobacterium co-cultivation method as described earlier (Maiti et al., 
1988). Transformations were done using Nicotiana tabacum cv Samsun NN. 
Primary transformants of tobacco were selected for resistance to kanamycin 
(300 mg/ml) and these were grown to maturity in the greenhouse. At least 
8-10 independent lines were generated for each construct tested. The 
expression levels of the GUS reporter gene in independent transformants 
developed for pKLP6GUS, pKLP36GUS are shown in FIG. 7. The expression from 
the PClSV FLt promoter was compared with that from the FMV promoter. 
Individual plant lines generated from independent calli expressing the same 
gene showed variable GUS activity. Similar patterns of plant-to-plant 
variations in gene expression have been reported with many other plant 
promoters as pointed out earlier (Maiti et al., 1996). Most of the plant 
lines developed with pKLF36GUS showed more activity than any of the plants 
transformed with pKLP6GUS. On average, about 3 fold higher activity was 
exhibited by plants transformed with pKLP36GUS, which has a duplicated 
enhancer domain as compared to plants transformed with pKLP6GUS which has 
a single enhancer domain. Hence, the PClSV FLt promoter with a duplicated 
enhancer domain is more active than the FLt promoter with a single 
enhancer domain. These constitutive promoters developed from PClSV and FMV 
FLt promoter were comparable in respect to expression of reporter genes in 
transgenic plants. 
EXAMPLE 5 
Expression levels in seedlings (Rl progeny) and young tobacco plants 
In order to examine the promoter activity in various tissues during 
seedling development, the expression of the GUS reporter gene in seedlings 
(Rl progeny) transformed with pKLP6GUS, or pKLP36GUS was examined by 
flurometric assay of tissue extracts and by histochemical staining of 
transverse sections of leaves, stems and roots. The PClSV promoter 
activity was monitored in 15 day old seedlings grown aseptically on an 
MS-agar medium in the presence of kanamycin (300 .mu.g/ml) and 3% sucrose. 
Several independent lines for each construct were studied. 
Comparison of activities of the FLt promoter indicated a gradient of 
expression in the following order; the highest level of activity was found 
in roots followed by leaves and stems. The histochemical staining shown in 
FIG. 8 is representative of the staining patterns analyzed in plants 
expressing high levels of GUS activity. In seedlings and sections of young 
leaves stained for GUS, the intensity of staining was markedly greater in 
vascular tissues of young leaves, petioles, stems and roots. 
The intensity of GUS staining observed in vascular tissue was in the 
following order: roots&gt;leaves&gt;stems (FIG. 8). The histochemical GUS assay 
in leaves showed more activity in midribs, veins and other vascular 
tissue, and in trichomes, than in leaf mesophyll and palisade cells (data 
not shown). No GUS activity was detected in transgenic plants containing 
the construct pKLP36CAT gene (FIG. 8A). 
The disarmed Agrobacterium strain transformed with plant expression vectors 
containing chimeric genes of interest can be used to engineer plants 
including but not limited to cotton, soybean, alfalfa, oilseed rape, flax, 
tomoto, sugar beet, sunflower, potato, tobacco, maize, wheat, rice and 
lettuce, banana. The use of DNA fragments or vectors including the PClSV 
promoter sequence tailored with heterologous DNA sequence in the 
transformation of plants by electroporation or particle gun transformation 
is within the scope of this invention. These embodiments and examples are 
provided in order to evaluate the practice of the present invention. These 
examples serve mainly illustrative purposes, and are not intended to limit 
the scope of the invention. 
TABLE 1 
______________________________________ 
Relative 
GUS 
activity 
Constructs (%) 
______________________________________ 
Control (TE buffer) 
00 
pUC8 GUS (No 00 
promoter) 
pc-GUS (extra ATG) 
7 
PGG1 (CaMV35S) 35 
pFMV 20-GUS (FMV 100 
FLt) 
pKLF (FMV FLt 100 
modified) 
pKLF 2 (2 .times. Enh FMV 
410 
FLt) 
______________________________________ 
Table 1: Relative .beta.-glucuronidase (GUS) activity of GUS fusion 
constructs containing different promoters electroporated into tobacco 
protoplasts. The GUS assay was carried out 20 hrs after electroporation. 
Assays and conditions were as described in the Methods. Promoter strength 
is presented as percentage of GUS activity normalized to pFMV 20 GUS for 
pUC based constructs or pKLFGUS for pKYLX7 based constructs, and represent 
the mean of three samples from at least two independent experiments, 
variation was within 12% of the presented value. 
FIG. 7 shows a comparison of the FMV promoter with the PClSV. These results 
suggest that expression of the FMV and PClSV are comparable and that the 
PClSV promoter is stronger than the CaMV promoter. 
DETAILED DESCRIPTION OF THE FIGURES 
FIG. 1. The DNA sequence of the full-length transcript promoter from the 
peanut chlorotic caulimovirus, (PClSV), (Richins et al., 1993). The 
nucleotide sequence (PClSV coordinates 5799 to 6150, a 352 bp fragment) 
includes the 3' end of gene VI (SEQ ID NO:1), and part of the large 
intergenic region, presented from left to right in the 5' to 3' direction 
of the transcript. The TATA box, CCACT box are shown in bold. The 
transcription initiation site for the full-length FMV transcript is 
indicated as +1, (position 6078). Repeat sequence domains (1a, 1b; to 6a, 
6b as indicated) are under lined or overlined. These sequence motif may be 
important for the promoter function. 
FIGS. 2A and 2B show a construction strategy of PClSV FLt promoter with its 
single and double enhancer domains. Number in parenthesis indicate 
nucleotide position in PClSV genome. MCS, multiple cloning sites 
FIG. 3. Physical map of pKYLX71. 
FIG. 4. Physical map of pKLP6; MCS, multiple cloning sites. 
FIG. 5. Physical map of pKLP36; MCS, multiple cloning sites FIG. 6. 
Schematic representation of chimeric GUS constructs used for assaying 
PClSVFLt promoter expression activity in transgenic plants. The identity 
of the respective promoter is shown for each plasmid. GUS represents the 
gene for .beta.-glucuronidase of E. coli. The position of restriction 
sites XhoI,SacI, EcoRI, HindIII ClaI used to assemble these plasmids are 
shown. The position of the left and right T-DNA borders (LB and RB 
respectively) the rbcS polyadenylation signal (3' REGION) and the Kmr gene 
are illustrated. NT3' or RT3' represent the polyadenylation sequences from 
NOS or RbcS gene respectively. 
FIG. 7. PClSV FLt promoter activity in transgenic plants expressing GUS 
reporter gene. Comparative analysis of the PClSV FLt promoter activity in 
independent transgenic plants Nicotiana tabacum cv Samsun NN (2 week old 
seedlings, Rl progeny/second generation) expressing a GUS reporter gene. 
Independent transgenic lines were developed with PClSV FLt promoter in 
construct pKLP6GUS containing a single enhancer domain, and in construct 
pKLP36GUS containing a double enhancer domains. Seeds obtained from 
transgenic plants were germinated in presence of kanamycin (200 .mu.g/ml). 
GUS activity was determined in tissue extracts from whole seedlings. 
Independent plants were developed for constructs pKLP6GUS line # 1, 2, 5, 
6, 19, 20, 21, and 22; and for construct pKLP36 line # 3, 4, 7, 8, 9, 10, 
11, 12, 13, 14, 15, 16, 17 and 18. (C) Negative control, tissue extract 
from Samsun NN (wt). Positive control, one of the best expressing lines 
either from (T1) pKLF GUS line #11 with FMV FLt promoter with single 
enhancer domain or (T2) pKLF2GUS line #12 with double enhancer domain 
compared with PClSV FLt promoter. GUS activity are presented as % activity 
of the best expressing pKLF2GUS#12 line with FMV FLt double enhancer 
domain. The presented data is the mean of three samples from at least two 
independent experiments, variation was within 10% of the presented value. 
FIG. 8. Histochemical localization of GUS activity in developing transgenic 
tobacco. 
A. Transgenic tobacco seedlings (X4), (pKLP36CAT, R1 progeny) with CAT 
gene, no GUS activity was detected. 
B. Seedling (X4), (pKLP6 GUS #21, R1 progeny) 14DAI; GUS activity was 
localized in root and leaves. 
C. Seedling (X4), (pKLP36 GUS #3, R1 progeny) 14DAI; GUS activity was 
localized in roots, roots hairs and leaves. 
D. Roots (3X) from six week old plants pKLP36CAT, R1 progeny, no GUS 
activity was detected. 
E. Roots (3X) from six week old plants pKLP6GUS #21, R1 progeny, GUS 
activity was detected in roots. 
F. Roots (X3) from six week old plants pKLP36GUS #3, R1 progeny, GUS 
activity was detected in roots. 
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The purpose of the above description and examples is to illustrate some 
embodiments of the present invention without implying any limitation. It 
will be apparent to those of skill in the art that various modifications 
and variations may be made to the composition and method of the present 
invention without departing from the spirit or scope of the invention. All 
patents and publications cited herein are incorporated by reference in 
their entireties. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 352 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ACAGAGGGATTTCTCTGAAGATCATGTTTGCCAGCTATGCGAACAATCATCGGGAGATCT60 
TGAGCCAATCAAAGAGGAGTGATGTAGACCTAAAGCAATAATGGAGCCATGACGTAAGGG120 
CTTACGCCATTACGAAATAATTAAAGGCTGATGTGACCTGTCGGTCTCTCAGAACCTTTA180 
CTTTTTATATTTGGCGTGTATTTTTAAATTTCCACGGCAATGACGATGTGACCTGTGCAT240 
CCGCTTTGCCTATAAATAAGTTTTAGTTTGTATTGATCGACACGATCGAGAAGACACGGC300 
CATTTGGACGATCATTTGAGAGTCTAAAAGAACGAGTCTTGTAATATGTTTT352 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 150 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GAGATCTTGAGCCAATCAAAGAGGAGTGATGTAGACCTAAAGCAATAATGGAGCCATGAC60 
GTAAGGGCTTACGCCATTACGAAATAATTAAAGGCTGATGTGACCTGTCGGTCTCTCAGA120 
ACCTTTACTTTTTATATTTGGCGTGTATTT150 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 78 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAGATCTTGAGCCAATCAAAGAGGAGTGATGTAGACCTAAAGCAATAATGGAGCCATGAC60 
GTAAGGGCTTACGCCATT78 
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