Field of Invention
The present invention relates to an artificial DNA construct having an unique bidirectional
genetic promoter.
BACKGROUND OF THE INVENTION:
The process of gene expression involves two major steps; Transcription and Translation.
Transcription is the first step where gene expression is regulated during which RNA is formed.
A sequence of DNA at the 5' end of the gene is called "promoter", the key regulatory element
that controls the expression and function of the gene to which it is attached. Genetic engineering
based approaches involving transgene expression requires a promoter directing its transcription.
A number of promoters from different sources have been isolated, characterized and tested in
different systems (Odell et al, 1985, Lawton et al, 1987, Maiti et al., 1997, 1998, Bhattacharya et
al., 2002 Petrzik et al., 1998, Pattanaik et al., 2004, Richard- Poggeler and Shepherd 1997, Dey
and Maiti 1999a, 1999b, 2003). Some of these promoters like CaMV35S promoter (Grison et
al., 1996), and FMV35S (Gao et al., 2000) have been used successfully for transgene expression.
For effective metabolic engineering multiple genes need to be expressed. At such instances
conventional iterative strategies, co-transformation or sequential transformation with multiple
gene constructs followed by selection of desired occurrence based on multiple selection criteria
is adopted. Besides tedious and time consuming, the significant disadvantages of these strategies
are 1. The transgenes may not link and can segregate apart in subsequent generations, 2. Multiple
genes cannot be coordinately expressed. 3. Accumulations of selectable marker genes hurdles in
public acceptance (Halpin et al. 2005). One of the approaches to overcome the problems is, the
use of bidirectional promoter, which can regulate expression of two genes simultaneously. A
number of naturally occurring bidirectional promoters have been tested (Velten & Schell 1985,
Mitra et al. 2009). Synthetic bidirectional promoters for transgene expression in plants are
reported (Barfield & Pua, 1991; Xie et al, 2001). A bidirectional promoter was constructed by

placing a double enhancer in between two similar core promoters (Li et al. 2004). A synthetic
bidirectional expression module had been developed by placing a computationally designed
minimal promoter sequence on the 5' and 3' sides of a transcription activation module
(Chaturvedi et al. 2005). A plant transformation binary vector using bidirectional promoter for
dual gene expression in plants was constructed and tested (Zhang et al. 2007). A vascular
specific bidirectional promoter was constructed (Lv et al. 2009). Recently a methyl jasmonate
inducible bidirectional promoter was constructed and tested by Agrobacterium mediated
transient approach (Zheng et al. 2011). As more and more genomes are being sequenced,
putative bidirectional promoters are identified following bioinformatics based approaches and
tested (Wang et al. 2009, Williams and Bowles 2004; Krom and Ramakrishna 2008. Dhadi et
al.2009). The current invention describes a strategy for construction of bidirectional promoter.
The functionality of the promoter in transient systems such as onion epidermal cell and tobacco
protoplast system were tested. Further the functionality was tested in transgenic tobacco plant.
The strategy described here can be used for construction of bidirectional promoter for a wide
variety of systems.
OBJECTS OF THE INVENTION
An object of this invention is to propose an artificial DNA construct having a bidirectional
promoter.
Another object of this invention is to propose a unique pararetrovirus based bidirectional
promoter DNA fragment.
Still another object of this invention is to propose a pararetrovirus based bidirectional promoter
which is useful for simultaneous expression of two genes in eukaryotes and prokaryotes.

BRIEF DESCRIPTION OF THE INVENTION;
According to this invention there is provided an artificial DNA construct comprising a
bidirectional promoter having a functional promoter (FS5), hybrid promoter, FUAS35SCP at
each end of the bidirectional promoter operably linked to a polynucleotide (gene) coding for a
polypeptide (protein).
The present invention provides to a strategy for constructing a bidirectional promoter construct
by fusing one functional promoter upstream of another functional promoter in reverse orientation
which can be used successfully for simultaneous expression of two genes.
The present invention provides a bidirectional promoter (FS5FUAS35SCPBD) developed by
ligating the promoter fragment between the co-ordinates -170 to +31 of Figwort mosaic virus
sub-genomic transcript promoter (FS5, Bhattacharya et al. 2002) upstream of FUAS35SCP (a
hybrid promoter developed by ligating the upstream activation sequence (FUAS) of Figwort
mosaic virus full length transcript promoter with the TATA box containing core promoter region
of Cauliflower mosaic virus 35S promoter (35SCP)) in reverse orientation.
First the upstream activation sequence (FUAS) of F20 was fused with 35SCP to create a hybrid
promoter FUAS35SCP. The FS5 was fused in reverse orientation with the hybrid promoter
FUAS35SCP to generate a bidirectional promoter FS5FUAS35SCPBD.,
The present invention also provides methods to transfer nucleic acid into a plant cell.
** This promoter DNA clone PBSFS5FUAS35SCPBD is submitted to Microbial Type Culture
Collection and Gene Bank (MTCC). Chandigarh, India under Budapest treaty.
MTCC clone depositary number: MTCC 5668

BRIEF DESCRIPTIONS OF THE ACCOMPANYING DRAWINGS:
Figure 1: An illustration of the strategy for constructing a bidirectional promoter.
(A) Schematic diagram of a GENE EXPRESSION CASSETTE-1 containing PROMOTER-1,
GENE-1 and TERMINATOR-1 in 5' to 3' orientation.
(B) Schematic diagram of a GENE EXPRESSION CASSETTE-2 containing PROMOTER-2,
GENE-2 and TERMINATOR-2 in 5' to 3' orientation.'
(C) The schematic diagram of a unique bidirectional promoter where GENE EXPRESSION
CASSETTE-2 was fused in reverse orientation with the GENE EXPRESSION CASSETTE-1.
Minimal promoter regions (containing TATA element) in both of the gene constructs are
demarcated. The direction of gene expression is indicated by an arrow.
Figure 2: Construction strategy of bidirectional promoter FS5FUAS35SCPBD.
(A) Schematic representation of the FS5GFPrbcSE9 containing FS5 promoter, GFP reporter
gene and rbcSE9 terminator in 5' to 3' orientation.
(B) Schematic representation of the FUAS35SCPGUSrbcSE9 containing FUAS35SCP promoter,
GUS reporter gene and rbcSE9 terminator in 5' to 3' orientation.
(C) The schematic diagram of the bidirectional promoter construct rbcSE9-GFP-FS5-
FUAS35SCP-GUS-rbcSE9 where FS5GFPrbcSE9 was fused in reverse orientation with the
FUAS3 5 SCPGf/SrbcSE9.

Figure 3: Nucleotide sequence of the bidirectional promoter, FS5FUAS35SCPBD along with its
component promoters FS5 and FUAS35SCP in 5' to 3' orientation. TATA element of these
sequences is shown in bold and italics. In bidirectional promoter FS5FUAS35SCPBD sequence
the sequences of FS5 is linked with the FUAS35SCP promoter by a GAATTC sequence.
(A) Sequence for FS5 promoter.
(B) Sequence of FUAS35SCP promoter.
(C) Sequence of the bidirectional promoter FS5FUAS35SCPBD.
Figure 4: Simultaneous transient assay of two reporter genes; GFP and GUS under the control of
FS5FUAS35SCPBD in tobacco protoplast.
(A) Expression of GFP as green fluorescence was obtained under the control of one of the
component promoter FS5 of bidirectional promoter, FS5FUAS35SCPBD. GFP fluorescence
was captured under a Confocal Leica microscope as described by Sahoo et al. 2009.
(B) Activity assay of GUS reporter gene driven by the FUAS35SCP promoter, another promoter-
component of the bidirectional promoter, FS5FUAS35SCPBD as described earlier.
Figure 5: Simultaneous transient assay of two reporter genes; GFP and GUS under control of the
FS5FUAS35SCPBD in onion epidermal cell.
(A) Schematic diagram of the said bidirectional promoter construct rbcSE9-GFP-FS5-
FUAS35SCP-GUS'-rbcSE9 used in gold particle bombardment experiment.
(B) i. Expression of the GFP reporter gene as green fluorescence in onion epidermal cell under
the control of one of the component promoter FS5 of bidirectional promoter,
FS5FUAS35SCPBD. GFP fluorescence was captured under a Confocal Leica microscope as
described by Sahoo et al. 2009.

ii. The same experiment was performed with control vector (without GFP reporter gene)
using onion epidermal cell.
(C) i. Histochemical staining using X-gluc of onion epidermal cell expressing GUS reporter gene
(marked by an arrow) under control of FUAS35SCP promoter a component- promoter of the said
bidirectional promoter FS5FUAS35SCPBD.
ii. The same experiment was performed with control vector (without GUS reporter gene)
using onion epidermal cell.
Figure 6: Transgenic assay of GFP and GUS reporter gene expressed simultaneously under
control of said bidirectional promoter in transgenic tobacco plant lines.
(A) Schematic diagram of the said bidirectional promoter construct rbcSE9-GFP-FS5-
FUAS35SCP-GUS-rbcSE9 in context of plant transformation vector expression cassette.
(B) Analysis of GFP fluorescence from 11 independent transgenic plants expressing bidirectional
promoter FS5FUAS35SCPBD. GFP expression analysis was carried out using
spectrophotometer (VARIOSKAN FLASH, Thermo scientific).
(C) Analysis of GUS activity from the same 11 independent transgenic plants expressing
bidirectional promoter FS5FUAS35SCPBD. Biochemical GUS assay was carried out according
to standard protocol (Jefferson et al. 1987)
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel strategy for construction of bidirectional promoter; and
one such promoter developed by combining genetic elements from Figwort mosaic virus sub-
genomic transcript, Figwort mosaic virus full-length transcript and Cauliflower mosaic virus 35S

transcript promoters. This invention also describes the method of using the said bidirectional
promoter in plant system for simultaneous expression of two transgenes. The salient features of
this invention are:
■ Molecular cloning of FS5 (FMV genomic coordinates 5163 to 5363, FSgt promoter
coordinates -170 to +31) from Figwort mosaic virus sub-genomic transcript (FMV-Sgt)
promoter.
■ Molecular cloning of upstream activation sequence (FMV genomic coordinates 6690 to
6886, F20 promoter coordinates -259 to -54 from TSS) from Figwort mosaic virus full-
length transcript (F20) promoter.
■ Molecular cloning of TATA element containing core promoter sequence (CaMV
genomic coordinates 7092 to 7435, CaMV35S promoter coordinates -343 to +1) from
Cauliflower mosaic virus 35S (CaMV35S) promoter.
■ Construction of hybrid promoter FUAS35SCP by intermolecular hybridization of UAS
sequence from Figwort mosaic virus full-length transcript promoter with x core promoter
sequence of Cauliflower mosaic virus 35S promoter containing TATA element.
■ Construction of bidirectional promoter FS5FUAS35SCP by cloning the FS5 promoter
upstream of FUAS35SCP in reverse orientation.
■ The functionality of the bidirectional promoter coupled to GUS (attached to the
FUAS35SCP in forward orientation) and GFP (attached to the FS5 in reverse orientation)
on either end was tested by transient (in onion epidermal cell and tobacco protoplast
system) and transgenic (in tobacco plant) assay.
■ The bidirectional promoter FS5FUAS35SCP was found to be active in both the transient
and transgenic systems.

EXAMPLE 1: Preparation of transformation vectors
a. Construction of plasmid with promoter in forward orientation
Construction of promoter, FUAS35SCP
The FUAS (upstream activation sequence of F20 promoter; coordinates -259 to -54) and
35SCP (TATA box containing core promoter region of CaMV35S promoter; coordinates -343 to
+1)) were PCR amplified using synthetic appropriately designed oligonucleotide pairs (#1 and 2;
# 3 and 4, respectively, Table 1) to generate EcoRl and Hindi at 5' end, and Smal and Hindlll at
3' end of PCR fragments using F20 plasmid clone (Maiti et al. 1997) and pUCPMAGUS (Dey
and Maiti 1999a) as templates, respectively. The PCR products were gel purified, digested with
EcoRI and Hind III and cloned individually into the corresponding sites of pUCl 19 to generate
pUCFUAS and pUC35SCP, respectively.
The 35SCP promoter fragment was isolated from pUC35SCP as Hindi and Hindlll
fragment, and sub-cloned into the Smal and Hindlll sites of pUCFUAS to generate
pUCFUAS35SCP.
Cloning ofFUAS35SCP into the protoplast expression vector pUCPMAGUS
The FUAS35SCP promoter was isolated as EcoRI and Hindlll fragment from the
pUCFUAS35SCP and sub-cloned into the corresponding sites of the protoplast expression vector
pUCPMAGUS (Dey and Maiti 1999a) replacing the CaMV35S promoter. The resulting plasmid
is designated as pUPFUAS35SCPGUS (general structure: 5'-EcoRI-FUAS35SCP promoter-
HindIII-XzoI-GUS'-SstI-XbaI-rbcSE9-terminator-ClaI-3' in pUCPMA).
Removal ofXbalsite from vUPFUAS35SCPGUS vector
The pUPFUASSSSCPGt/S vector (general structure S'-EcoXl-YUASlSSCP-Hindlll-Xhol-GUS-
SstI-^&aI-rbcSE9-terminator-ClaI-3') was digested with Xbal, end filled with T4 DNA
Polymerase I, religated and transformed into E.coli TBI. The clones indigestible with Xbal were
selected. The resulting clone is designated as pUPFUAS35SCPGUSSAXba I

Clonim ofFUAS35SCP-GUS-rbcSE9 in vBSK
The 5'-£coRI-FUAS35SCP-/f/ndIII-A7joI-GLW-SstI (A*Z>aI)-rbcSE9 polyA track-ClaI-3'
fragment was PCR amplified using pUPFUAS35SCPAA2>a I as template and appropriately
designed synthetic oligonucleotides (#1 and 5, Table 1) so as to generate EcoRL at the 5' end and
CM and Kpnl sites at the 3' end. The PCR product was gel purified, digested with EcoRl and
Kpnl and cloned into the corresponding sites of pBSK vector. The resulting clone is designated
as pBFUASSSSCPGt/S1 with general structure 5'-£coRI-FUAS35SCP promoter-^/ndIII-^7joI-
GUS-Sstl (AXbal) rbcSE9 terminator-ClaI-KpnI-3' inpBSK.
b. Construction ofplasmid to place the promoter in reverse direction
Clonins ofFSst5 in pBSK in reverse direction
The FSgt5 promoter fragment (-170 to +31) was PCR amplified using pUCFSgt3 [FSgt 3(-150 to
+31 in pUC119)] as template and appropriately designed synthetic oligonucleotides (#6 and 7,
Table 1) to generate EcoRI at 5'end and Pstl at 3' end. The PCR product was gel purified,
digested with EcoRI and Pstl and cloned into the corresponding sites of pBSK to generate
pBSFS5 (general structure: 5'-£c0RI-FSgt5-P.y/I-3').
Clonim ofGFP
The GFP gene was PCR amplified using pUCPMAGFP as template and synthetic
oligonucleotides (# 8 and 9, Tablel) having appropriately designed sequence to generate Pstl site
at 5' end and Ba/wHI site at 3' end. The PCR product was gel purified, digested with Pstl and
BamWl and cloned into the corresponding sites of pBSFS5. The resulting clone is designated as
pBSFS5-GFP (general structure: 5'-£coRI-FSgt5-PStI-GFP-5a»jHI-3').

Cloning ofrbcSE? volv-A terminator
The rbcSE9 polyA terminator sequence was PCR amplified using pUCPMAGt/S as template and
appropriately designed synthetic oligonucleotides (#10 and 11, Table 1) to generate BarriiW at the
5' end and Xbal site at the 3' end. The PCR product was gel purified, digested with BamHl and
Xbal and cloned into the corresponding sites of pBSFS5-GFP to construct pBSFS5-GFP-rbcSE9
(general structure: 5'-£coRI-FSgt5-P.rtI-GFP-.Ba/wHI-rbcSE9 -Xbal-3').
Construction ofpBFSUAS35SCPbdGG the Bidirectional promoter construct
The 5'-£coRI-FSgt5-PM-GFP-5awHI-rbcSE9-JftoI-3' was digested with EcoRl and Xbal and
subcloned into the corresponding sites of pBFUAS35SCPGt/S' to generate the bidirectional
promoter construct pBFS5FUAS35SCPBDGG, (general structure: 5'-J*aI-rbcSE9-5awHI-GFP-
P5rt-FSgt5-JEcoRI-FUAS35SCP-M«dIII-^ioI-GC/5-SstI-AZ&flI-rbcSE9-ClaI-KpnI-3') in
pBS(KS+).
EXAMPLE 2
Transient assay of the bidirectional promoter in tobacco protoplast system.
Protoplasts from tobacco leaves were isolated according to Duke et al. 1991 Ten ug aliquot of
pBSFS5FUAS35SCPBDGG was electroporated into 2 X 106 protoplasts at 200V, 965 uF
capacitance for 40-50 ms using the GenePulser II Apparatus (Bio-Rad) with Capacitance
Extender II (model 165-2107). Electroporated protoplasts were incubated at 28°C for 20 hours in
dark. For GUS expression analysis the protoplasts were harvested and biochemical GUS assay
was performed according to Jefferson et al. 1987. For GFP expression analysis the protoplasts
were visualized under a confocal laser scanning microscope and green fluorescent images were
captured as described by Sahoo et al. 2009.

EXAMPLE 3
Transient assay of the bidirectional promoter in onion epidermal cell
The inner epidermal layers of onion were peeled and placed inside up on petridish containing
MS media. The DNA coated gold particles were prepared according to manufacturer protocol
(Bio-rad); 25 ug of purified plasmid was precipitated onto gold particles (In in size) using lOOul
of 0.05M spermidine and lOOul of 1M CaCb. The DNA coated gold particles were washed with
100% ethanol and finally resuspended in ethanol. These were used to bombard onion epidermal
cell at 250 psi. The petridishes with the onion epidermal peel sample were incubated for 24hr
before assaying the GUS and GFP activity.
EXAMPLE 4
Transsenic assay of the bidirectional promoter
The bidirectional promoter construct as Xbal I Clal fragment was isolated from
pBFS5FUAS35SCPBDGG and subcloned into the corresponding sites of promoter less pKYLX
71 vector to construct pKFS5FUAS35SCPBDGG. The pKFS5FUAS35SCPbdGG was
introduced into Agrobacterium tumifaciens strain C587Cl:pGV3850 by freeze thaw method
(Hofgen and Willmitzer 1988). Transgenic Tobacco plants (Nicotiana tabacum cv samsun NN)
were raised using the engineered Agrobacterium according to Dey and Maiti 1999a. GUS
activity from leaves of 11 independent transgenic tobacco plant lines was carried out according
to Jefferson et al 1987. The GFP expression analysis was carried out according to Robic et
al.2009.

WE CLAIM:
1. An artificial DNA construct comprising a bidirectional promoter having a minimal promoter
(FS5), hybrid promoter, FUAS35SCP at each end of the bidirectional promoter operably linked
to a polynucleotide (gene) coding for a polypeptide (protein).
2. The artificial DNA construct as claimed in claim 1, wherein said bidirectional promoter is
constructed of a promoter developed from Figwort mosaic virus sub genomic transcript, Figwort
mosaic virus full length transcript and cauliflower mosaic virus 35S promoters.
3. The artificial DNA construct as claimed in claim 1, wherein said bidirectional promoter is
constructed by cloning the FS5 promoter upstream of FUAS35SCP in reverse orientation.
4. The artificial DNA construct as claimed in claim 1, wherein the said minimal promoter is
functionally linked to the said hybrid promoter in opposite orientation, in opposite orientation to
the said hybrid promoter and 5' to the said hybrid promoter.
5. The artificial DNA construct as claimed in claim 1, wherein the said minimal promoter is a
minimal promoter obtained from Figwort mosaic virus sub-genomic transcript promoter co-
ordinates between -170 to +31, wherein the said hybrid promoter, FUAS35SCP constructed by
fusing the upstream activation domain sequence from Figwort mosaic virus full length transcript
promoter co-ordinates between -249 to +54 with the core promoter domain from CaMV35S co-
ordinates between -343 to +1.

6. The artificial DNA construct, wherein the said hybrid promoter, FUAS35SCP constructed by
fusing the upstream activation domain sequence from Figwort mosaic virus full length transcript
promoter co-ordinates between -249 to +54 with the core promoter domain from CaMV35S co-
ordinates between -343 to +1.
7. The artificial nucleic acid construct as claimed in claim 1, wherein the said polynucleotide
may code for an agronomically and economically significant polypeptide.
8. The artificial DNA construct wherein the said bidirectional promoter FS5FUAS35SCP are
active both in the transient and transgenic systems.
9. A plant comprising the nucleic acid construct as claimed in claim 1.

An artificial DNA construct comprising a bidirectional promoter having a functional promoter
(FS5), hybrid promoter (FUAS35SCP) at each end of the bidirectional promoter operably linked
to a polynucleotide (gene) coding for a polypeptide (protein).