The first patent family

Definition of promoter and some of its elements

In the first patent family, promoters are defined in functional and structural terms. They are described as the transcription control units that contain the signals for RNA polymerase to begin transcription so that protein synthesis can occur. Promoters are located in the 5′ flanking or upstream region of the transcribed gene. The most common motifs present in promoters are:

the TATA element, which is the site where the TATA-binding protein (TBP) binds. This protein is part of a complex of polypeptides that recruit RNA polymerase II to begin transcription;
the transcription start site; and
the CCAAT consensus sequence.

A core promoter or minimal promoter contains the TATA box and the transcription start site. This core promoter may or may not have detectable activity in the absence of sequence(s) that enhance this activity or confer tissue- specific activity.

Other elements of promoter regions include:

INR, sequences near the transcription start site of some genes that provide an alternate site for binding factors to activate transcription;
enhancers; and
upstream elements.

The enhancers are not classified as upstream elements by the inventors. The upstream elements disclosed, unlike enhancers, are position and orientation dependent, interact with specific binding factors and are less common. The upstream elements also may be exchanged with other elements while maintaining their characteristic control over gene expression. In contrast, enhancers can increase the efficiency of transcription regardless of their distance and orientation to the transcription start site.

Approximate scope of protection

The synthetic promoters that are the subject of the Pioneer Hi-Bred’s inventions contain:

a TATA motif;
a GC-rich region (at least 64% GC); and
a transcription start site.

According to the inventors, the GC-rich region located between the TATA motif and the transcription start site in plant promoters acts as a very strong inducer of constitutive expression. It increases transcriptional activation efficiency. Plant-expressible promoters contain a region of about 40% GC, while a 64% or greater GC content is characteristic of animal promoters. The maize ubiquitin 1 gene (Ubi-1) core promoter, which produces high levels of activity in monocots, has a GC content more similar to animal promoters (64%). The GC content of promoters covered by the claims is at least 64%.

Both the U.S. and the Australian patents claim a synthetic promoter as described. Expression cassettes containing a structural gene linked to the promoter and a poly(A) signal are also part of the claimed inventions.

In addition, both patents claim:

A synthetic upstream element comprising at least 3 octopine synthase (OCS) binding motifs (TGACG) with an intervening sequence. This motif has been identified from several opine synthase genes, i.e., octopine, nopaline, mannopine, and from other genes such as histone genes.
Expression cassettes containing the synthetic upstream element linked to the synthetic promoter.
Expression cassettes where the synthetic upstream element is linked to a promoter which does not necessarily have the structural elements described above.

The U.S. patent also claims a promoter construct having a core promoter and the upstream activating region (UAR) of the Ubi-1 gene. An expression cassette containing these elements, a structural gene linked to the promoter and a poly(A) signal is also claimed.

A summary of the independent claims of the granted patents and some bibliographic data are shown in the following table.

Patent number

Title, Independent Claims and Summary of Claims

Assignee

US 6072050

Earliest priority – 24 February 1998
Filed – 24 February 1998
Granted – 6 June 2000
Expected expiry – 24 February 2018

Title – Synthetic promoters

Claim 1
A synthetic DNA promoter sequence functional in a plant cell, said promoter sequence comprising:a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich;
wherein said region is not a region between a TATA motif and a transcription start site of native maize
ubiquitin promoter, and
wherein said promoter sequence is set forth in SEQ ID NO:10.

Claim 2
A synthetic DNA promoter sequence functional in a plant cell, said promoter sequence comprising:a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich;
wherein said region is not a region between a TATA motif and a transcription start site of native maize ubiquitin promoter, and
wherein said promoter sequence is set forth in SEQ ID NO:1.

Claim 3
An expression cassette comprisinga synthetic promoter comprising:
a TATA motif,
a transcription start site and
a “region” between said TATA motif and said start site that is at least 64% GC rich,

a structural gene operatively linked to said promoter, and

a transcription end site polyadenylation signal;

wherein said “region” is not a region between a TATA motif and a transcription start site of native maize ubiquitin promoter, and

wherein sequence of said promoter is set forth in SEQ ID NO:1.

Claim 4
An expression cassette comprisinga synthetic promoter comprising:
a TATA motif,
a transcription start site and
a region between said TATA motif and said start site that is at least 64% GC rich,
a structural gene operatively linked to said promoter, and
a transcription end site polyadenylation signal;
wherein said region is not a region between a TATA motif and a transcription start site of native maize ubiquitin promoter, and
wherein sequence of said promoter is set forth in SEQ ID NO:10.

Claim 5
An expression cassette comprisinga synthetic promoter comprising:
a TATA motif,
a transcription start site and
a region between said TATA motif and said start site that is at least 64% GC rich,
a structural gene operatively linked to said promoter,
a transcription end site polyadenylation signal, and
an upstream element operatively linked to said promoter so that transcription is enhanced;
wherein said region is not a region between a TATA motif and a transcription start site of native maize ubiquitin promoter; and
wherein sequence of said upstream element is set forth in SEQ ID NO:2.

Claim 7A synthetic upstream element having a sequence set forth in SEQ ID NO:2.

Claim 8
An expression cassette comprising:a promoter sequence;
a structural gene operatively linked to said promoter sequence;
a polyadenylation signal; and
a synthetic upstream element comprising SEQ ID NO:2 operatively linked to said promoter so that expression is enhanced.

Claim 13An isolated nucleotide sequence comprising a DNA enhancer sequence comprising the nucleotide sequence set forth in SEQ ID No: 5.

Claim 14A nucleotide sequence comprising a promoter construct,
said construct comprising in operable linkage
a core promoter sequence and
a Ubi-1 UAR,
wherein said Ubi-1 UAR is a maize Ubi UAR comprising the sequence set forth in SEQ ID No:13.

Claim 15
An expression cassette comprising in operable linkage:a core promoter sequence,
a Ubi UAR operably linked upstream to said core promoter to form a synthetic promoter construct,
a nucleotide sequence of interest operably linked to said synthetic promoter, and
a polyadenylation signal;
wherein said Ubi-1 UAR comprises the sequence set forth in SEQ ID No:13.

The claims are directed to:

Synthetic promoters functional in plants comprising a TATA motif, a region at least 64% GC-rich, and a transcription start site. The DNA sequence of the promoters are either as defined in SEQ ID NO:10 or SEQ ID NO:1 (claims 1 and 2). The sequences of both are fairly similar, except that one includes G/C transversions.
Expression cassettes comprising these synthetic promoters and a structural gene linked to the promoter and a poly (A) signal (see claims 3-5).
A synthetic upstream element (SEQ ID NO:2).
An expression cassette as described having in addition the above mentioned upstream element linked to a promoter (claim 8). To be covered by the claim language, the promoter in this case does not necessarily have the structural elements (TATA motif, GC-richness, or transcription start site) described above, but must enhance the transcription process.
An isolated DNA sequence comprising a DNA enhancer sequence comprising the nucleotide sequence TGACG (SEQ ID No: 5).
A nucleotide sequence comprising a promoter construct comprising a core promoter sequence and a maize Ubi-1 UAR.
An expression cassette comprising a Ubi-1 UAR linked upstream to a core promoter, a nucleotide sequence of interest linked to the promoter and a poly (A) signal.

According to the specification, 64% or greater GC content from TATA to start of transcription is generally characteristic of animal promoters. Thus, claims 1-5 are potentially targeting the use of animal promoters in plant systems.

‘Having’ in claim 7 is ambiguous; would an infringing sequence contain only this, or this and other elements?

The isolated nucleotide sequence as an enhancer in claim 13 can have nucleotides in addition to the TGACG motif. Using bigger sequences containing TGACG could still infrige the claim.

The Ubi-1 UAR (upstream activating region) claimed in claims 14 and 15 comprises the sequence disclosed in SEQ ID No:13. The Ubi-1 UAR can be used in both directions and in multiple copies. However, the Ubi-1 UAR sequence disclosed here is 813 bp and use only portions of the sequence would not be covered by these claims.

Pioneer Hi-Bred International Inc.

US 6555673

Earliest priority – 24 February 1998
Filed – 21 April 2000
Granted – 29 April 2003
Expected expiry – 21 April 2020

Title – Synthetic promoters

Claim 1
A promoter construct comprising:
a) a synthetic core promoter functional in a plant cell,
wherein said synthetic core promoter has a sequence comprising
a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich,
wherein said sequence is set forth in SEQ ID NO:10;
b) a synthetic upstream element of SEQ ID NO:2 operatively linked to said synthetic core promoter so that control of transcription from said synthetic core promoter is enhanced; and
c) at least one upstream activating region.

Claim 14
A promoter construct comprising:
a) a synthetic core promoter functional in a plant cell,
wherein said synthetic core promoter has a sequence comprising
a TATA motif, a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich,
wherein said sequence is set forth in SEQ ID NO:1;
b) a synthetic upstream element of SEQ ID NO:2 operatively linked to said core promoter so that control of transcription from said core promoter is enhanced; and
c) at least one upstream activating region.

Claim 15
A promoter construct comprising:
a) a synthetic core promoter functional in a plant cell,
wherein said synthetic core promoter has a sequence comprising
a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich,
wherein said sequence is set forth inSEQ ID NO:1;
b) a synthetic upstream element of SEQ ID NO:2 operatively linked to said core promoter so that control of transcription from said core promoter is enhanced; and
c) at least one upstream activating region, wherein at least one of said upstream activating region(s) is selected from the groupconsisting of Ubi-1 UAR and CaMV 35S UAR.

Claim 16
A promoter construct comprising:
a) a synthetic core promoter functional in a plant cell,
wherein said synthetic core promoter has a sequence comprising
a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich,
wherein said sequence is set forth in SEQ ID NO:10;
b) a synthetic upstream element of SEQ ID NO:2 operatively linked to said core promoter so that control of transcription from said core promoter is enhanced; and
c) at least one upstream activating region, wherein at least one of said upstream activating region(s) is selected from the groupconsisting of Ubi-1 UAR and CaMV 35S UAR.

Claim 17
A promoter construct comprising:
a) a synthetic core promoter functional in a plant cell,
wherein said synthetic core promoter has a sequence comprising
a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich,
and wherein said core promoter is a synthetic core promoter comprising the sequence set forth in SEQ ID NO:1; and
b) at least two upstream activating regions, wherein at least one of said upstream activating regions is selected from the groupconsisting of Ubi-1 UAR and CaMV 35S UAR.

Claim 18
A promoter construct comprising:
a) a synthetic core promoter functional in a plant cell,
wherein said synthetic core promoter has a sequence comprising
a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich,
and wherein said core promoter is a synthetic core promoter comprising the sequence set forth in SEQ ID NO:10; and
b) at least two upstream activating regions, wherein at least one of said upstream activating regions is selected from the groupconsisting of Ubi-1 UAR and CaMV 35S UAR.

Claim 19
A promoter construct comprising the sequence set forth in SEQ ID NO:12.

Claim 20
A promoter construct comprising the sequence set forth in SEQ ID NO:15.

Claim 21
A promoter construct comprising the sequence set forth in SEQ ID NO:16.

Claim 22
A promoter construct comprising the sequence set forth in SEQ ID NO:17.

Claim 23
A promoter construct comprising the sequence set forth in SEQ ID NO:18.

Claim 24
A promoter construct comprising a sequence having at least 95% sequence identity to the portion of SEQ ID NO:12, which is not a CaMV 35S UAR sequence wherein control of transcription from said promoter is enhanced in comparison to transcription from a promoter consisting of the synthetic core promoter sequence set forth in SEQ ID NO:1.

Claim 25
A promoter construct comprising a sequence having at least 95% sequence identity to the portion of SEQ ID NO:15 which is not a Ubi-1 UAR sequence, wherein control of transcription from said promoter is enhanced in comparison to transcription from a promoter consisting of the synthetic core promoter sequence set forth in SEQ ID NO:1.

Claim 26
A promoter construct comprising a sequence having at least 95% sequence identity to the portion of SEQ ID NO:16 which is not a Ubi-1 UAR sequence, wherein control of transcription from said promoter is enhanced in comparison to transcription from a promoter consisting of the synthetic core promoter sequence set forth in SEQ ID NO:1.

Claim 27
A promoter construct comprising a sequence having at least 95% sequence identity to the portion of SEQ ID NO:17 which is not a Ubi-1 UAR sequence, wherein control of transcription from said promoter is enhanced in comparison to transcription from a promoter consisting of the synthetic core promoter sequence set forth in SEQ ID NO:1.

Claim 28
A promoter construct comprising a sequence having at least 95% sequence identity to the portion of SEQ ID NO:18 which is not a Ubi-1 UAR sequence, wherein control of transcription from said promoter is enhanced in comparison to transcription from a promoter consisting of the synthetic core promoter sequence set forth in SEQ ID NO:1.

This patent is a Continuation of US 6072050.

The claims are drawn to:

1. Promoter constructs comprise:

a synthetic core promoter which has a sequence (SEQ ID NO:10 or SEQ ID NO:1) comprising a TATA motif, a transcription start site, and a region between the TATA motif and the start site that is at least 64% GC-rich
a synthetic upstream element (SEQ ID NO:2) linked to synthetic core promoter so that control of transcription from the synthetic core promoter is enhanced
at least one upstream activating region

2. Promoter constructs comprising any of the following sequences: SCP1 promoter, SEQ ID NO:12; UCP1 promoter, SEQ ID NO:15; UCP2 promoter, SEQ ID NO:16; UCP3 promoter, SEQ ID NO:17 and UCP4 promoter, SEQ ID NO:18). (see claims 19-23)

3. A promoter construct comprising a sequence having at least 95% sequence identity to the portion of the SCP1 promoter, which is not a CaMV 35S UAR sequence. This promoter can direct higher transcription than a promoter consisting of the synthetic core promoter sequence (SEQ ID NO:1) (see claim 24).

4. A promoter construct comprising a sequence having at least 95% sequence identity to the portion of one of the promoters: UCP1 promoter, UCP2 promoter, UCP3 promoter, and UCP4 promoter, which is not a Ubi-1 UAR sequence.These promoters can direct higher transcription than a promoter consisting of the synthetic core promoter sequence (SEQ ID NO:1) (see claims 25-28).

This patent claims promoter constructs comprising all the claim elements in US 6072050. In addition, promoters with at least 95% identity to the portion of promoters SCP1 (35S UAR linked to core promoter of SEQ ID NO: 1), UCP1 (one copy of Ubi1 UAR linked with the core promoter), UCP2 (2 copies of Ubi1 UAR linked with the core promoter), UCP3 (3 copies of Ubi1 UAR linked with the core promoter) and UCP4 (4 copies of Ubi1 UAR linked with the core promoter) are also covered.

According to the specification, upstream activating region (UAR) is typically a position or orientation dependent element that primarily directs tissue, cell type, or regulated expression. The UAR in claims 1 and 14 is not restricted to Ubi-1 UAR and CaMV 35S UAR and therefore could be from any organism.

AU 751402 B2

Earliest priority – 24 february 1998
Filed – 23 February 1999
Granted – 15 August 2002
Expected expiry -23 February 2019

Title – Synthetic promoters

Claim 1
A synthetic DNA plant promoter sequence, said sequence comprising:
a TATA motif;
a transcription start site;
a region between said TATA motif and said start site that is at least about 64% GC-rich,
an upstream element; and
one or more upstream activating regions;
wherein said promoter sequence comprises a sequence selected from the group consisting of:
a) a nucleotide sequeuce set forth in SEQ ID NO; 12,
b) a nucleotide sequence set forth in SEQ ID NO: 15,
c) a nucleotide sequence set forth in SEQ ID NO: 16,
d) a nucleotide sequence set forth in SEQ ID NO: 17, and
e) a nucleotide sequence set forth in SEQ ID NO: 18.

Claim 2
An expression cassette comprising:
a synthetic promoter comprising
a TATA motif;
a transcription start site and a region there between that is at least about 64% GC rich;
an upstream element; and
one or more upstream activating regions;
a structural gene operatively linked to said promoter; and
a transcription end site polyadenylation signal, wherein said promoter sequence comprises a sequence selected from the group consisting of:
a) a. nucleotide sequence set forth in SEQ ID NO: 12,
b) a nucleotide sequence set forth in SEQ ID NO: 15,
c) a nucleotide sequence set forth in SEQ ID NO: 16,
d) a nucleotide sequence set Forth in SEQ ID NO: 17, and
e) a nucleatide sequence set forth in SEQ ID NO: 18.

Claim 10
An expression cassette comprising:
a synthetic core promoter comprising the sequence set forth in SEQ ID NO:1 or SEQ ID NO:10,
a synthetic upstream element comprising the sequence set forth in SEQ ID NO:2, and
an upstream activating region comprising the sequence set forth in SEQ ID NO:12 or 13,
a structural gene operatively linked to said promoter; and
a transcription end site polyadenylation signal.

Claim 11
A DNA sequence comprising a promoter construct, said construct
comprising in operable linkage:
a core synthetic promoter sequence comprising
a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich,
wherein said region is not a region between a TATA motif and a transcription start site of native maize ubiquitin promoter;
a heterologous upstream element; and
a heterologous upstream activating region operably linked to said core synthetic promoter; wherein said upstream activating region is selected from the group consisting of CaMV 35S UAR and Ubi-1 UAR.

Claim 19
A method for controlling the level of expression of a transgenic nucleotide sequence in a dicotyledonous plant cell, said method comprising transforming said plant cell with an expression cassette comprising
a synthetic promoter comprising
a TATA motif;
a transcription start site and a region there between that is at least about 64% GC-rich;
an upstream element; and
one or more upstream activating regions;
a structural gene operatively linked to said promoter; and
a transcription end site polyadenylation signal,
wherein said promoter sequence comprises a sequence selected from the group consisting of:
a) a nucleotide sequeuce set forth in SEQ ID NO; 12,
b) a nucleotide sequence set forth in SEQ ID NO: 15,
c) a nucleotide sequence set forth in SEQ ID NO: 16,
d) a nucleotide sequence set forth in SEQ ID NO: 17, and
e) a nucleotide sequence set forth in SEQ ID NO: 18.

Claim 34
A method for controlling the level of expression of a transgenic nucleotide sequence in a dicotyledonous plant cell, substantially as hereinbefore described with reference to any one of the examples.

The Australian patent claims cover all the claim elements for the synthetic promoters, the upstream element and the UARs as claimed in US 6555673. Addition to these, it also claims for a method for controlling the level of expression of a transgenic nucleotide sequence in dicotyledonous plant cells comprising transforming a plant cell with an expression cassette comprising a synthetic promoter comprising the above claim elements.

Remarks

Related applications are pending in Europe (EP 1056875) and Canada (CA 2314598). A patent was also granted in New Zealand (NZ 506182).

AU 729929 B2

Earliest priority – 11 June 1996
Filed – 10 June 1997
Granted – 15 February 2001
Expected expiry – 10 June 2017

Title – A synthetic plant core promoter and upstream regulatory element

Claim 1
A synthetic DNA plant promoter sequence, said sequence comprising:a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich.

Claim 3
An expression cassette comprisinga synthetic promoter comprising
a TATA motif,
a transcription start site and
a region there between that is at least 65% GC rich,
a structural gene operatively linked to said promoter, and
a transcription end site polyadenylation signal.

Claim 17A synthetic upstream element having a sequence of SEQ ID NO:2.

Claim 18
An expression cassette comprising:
a promoter sequence;
a structural gene operatively linked to said promoter sequence;
a polyadenylation signal; and
a synthetic upstream element comprising SEQ ID NO:2 operatively linked to said promoter sequence so that expression is enhanced.

Claim 23
A synthetic DNA plant promoter sequence functional in a plant cell, said sequence comprising:a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich; wherein said promoter sequence is less than 1000 bp.

The claims are related to:

A synthetic promoter comprising a TATA motif, a region at least 64% GC-rich, and a transcription start site.
An expression cassette comprising a synthetic promoter and a structural gene linked to the promoter and a poly (A) signal.
A synthetic upstream element as claimed in the related U.S. patent (US 6072050).
An expression cassette as described having in addition the the above upstream element linked to the promoter. The element enhances the transcription process.
A synthetic promoter functional in plant cells. The elements are the same as described for the former synthetic promoter but this one is limited to maximum 1 kb in length.

The claims for the synthetic promoter in this Australian patent is very broad as comparing to the related US and Canadian patents.

EP 914454B1

Earliest priority – 11 June 1996
Filed – 10 June 1997
Granted – 9 March 2005
Expected expiry – 9 June 2017

Title – A synthetic plant core promoter and upstream regulatory element

Claim 1
A synthetic upstream element having a sequence of SEQ ID NO:2.

Claim 2
An expression cassette comprising:
a promoter;
a structural gene operatively linked to said promoter sequence;
a polyadenylation signal; and
a synthetic upstream element comprising SEQ ID NO:2 operatively linked to said promoter so that expression is enhanced.

Claim 6
A nucleic acid vector comprising
(i) a promoter operatively linked to a structural gene, wherein the promoter is a synthetic DNA plant promoter sequence which comprises:
a TATA motif;
a transcription start site; and
a region between the TATA motif and the start site that is at least 64% GC-rich; and
(ii) an upstream element operatively linked to said promoter comprising the sequence of SEQ ID NO:2.

CA 2257719

Earliest priority – 11 June 1996
Filed – 10 June 1997
Granted – 31 May 2005
Expected expiry – 9 June 2017

Title – A synthetic plant core promoter and upstream regulatory element

Claim 1
A synthetic DNA promoter functional in plant cells, said promoter comprising:a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich, wherein the sequence of said promoter is set forth in SEQ ID NO:10.

Claim 2
A synthetic DNA promoter functional in plant cells, said promoter comprising:a TATA motif,
a transcription start site, and
a region between said TATA motif and said start site that is at least 64% GC-rich, wherein the sequence of said promoter is set forth in SEQ ID NO:1.

Claim 3
An expression cassette comprising:
a synthetic promoter functional in plant cells, wherein the sequence of said promoter is set forth in SEQ ID NO:1;
a structural gene operatively linked to said promoter; and
a transcription end site polyadenylation signal.

Claim 4
An expression cassette comprising:
a synthetic promoter functional in plant cells, wherein the sequence of said promoter is set forth in SEQ ID NO:1;
a structural gene operatively linked to said promoter; and
a transcription end site polyadenylation signal.

Claim 16
A synthetic upstream element having a sequence of SEQ ID NO:2.

Claim 18An expression cassette comprising:
a promoter sequence;
a structural gene operatively linked to said promoter sequence;
a polyadenylation signal; and
a synthetic upstream element comprising SEQ ID NO:2 operatively linked to said promoter so that expression is enhanced.

Remarks

Application also filed in Brazil (BR 9710690 A)

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The first patent family

Definition of promoter and some of its elements

Approximate scope of protection