# US 8,465,915 B2 · Method for isolation of transcription termination sequences

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- Number: US 8,465,915 B2
- Document ID: US8465915B2
- Status: Granted
- Kind: B2
- Application number: 11/659,126
- Filed: 2005-07-30
- Published: 2007-10-18
- Granted: 2013-06-18
- Earliest claimed: 2004-08-02
- Assignees: BASF Plant Science GmbH
- Applicants: Hee-Sook Song; Michael Kock; Jeffrey A. Brown; Linda Patricia Loyall; Liqun Xing; Hongmei Jia; John McMillan; Lesley Ireland
- Inventors: Hee-Sook Song; Michael Kock; Jeffrey A. Brown; Linda Patricia Loyall; Liqun Xing; Hongmei Jia; John McMillan; Lesley Ireland
- Web page: https://patentgrep.com/patent/US8465915B2

## CPC

- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- C12N 15/1051: {Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors}
- C12N 15/82: for plant cells {, e.g. plant artificial chromosomes (PACs)}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to efficient, high-throughput methods, systems, and DNA constructs for identification and isolation of transcription termination sequences. The invention relates further to specific terminator sequences identified by the methods isolated from rice.

## Claims

### Claim 1

A method for identification and isolation of transcription termination sequences from a plant comprising the steps of:
i) providing a screening construct or screening vector comprising
a) a promoter sequence,
b) one or more insertion sites for insertion of DNA sequences, and
c) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic,
wherein insertion of an efficient transcription terminator into said insertion site changes expression of said additional sequence by said promoter sequence in comparison to no insertion,
ii) providing one or more DNA sequences to be assessed for their transcription termination capability by
a) identifying intergenic regions between paired genes having an intergenic distance of about 400 to 3,000 base pairs from a plant,
b) identifying one or more DNA sequences which are flanked on both sides by genes having at least 5% of expression level of actin in said plant, and
c) isolating the one or more DNA sequences identified in step b),
iii) inserting one or more copies of said DNA sequences obtained from step ii) into said insertion site of said screening construct or screening vector,
iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence,
v) identifying and/or selecting screening construct or screening vector with a changed readily detectable characteristic in comparison to no insertion, and
vi) isolating the inserted DNA sequences from said identified and/or selected screening construct or screening vector for use as transcription termination sequences and, optionally, determining their sequence.

### Claim 2 (depends on 1)

The method of claim 1, wherein said insertion site is localized in relation to said additional sequences at a position selected from the group consisting of:
i) upstream of the additional sequences between said promoter and said additional sequences,
ii) downstream of the additional sequences, and
iii) in between said additional sequences.

### Claim 3 (depends on 1)

The method of claim 1, wherein said DNA sequences to be assessed for their transcription termination efficiency are inserted into said insertion site in form of an inverted repeat.

### Claim 4 (depends on 1)

The method of claim 1, wherein the method for identification and isolation of transcription termination sequences from a plant comprises the steps of:
i) providing a screening construct or screening vector comprising in 5′ to 3′ direction
a) a promoter sequence,
b) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, and
c) one or more insertion sites for insertion of DNA sequences,
ii) providing one or more DNA sequences to be assessed for their transcription termination capability by
a) identifying intergenic regions between paired genes having an intergenic distance of about 400 to 3,000 base pairs from a plant,
b) identifying one or more DNA sequences which are flanked on both sides by genes having at least 5% of expression level of actin in said plant, and
c) isolating the one or more DNA sequences identified in step b),
iii) inserting at least two copies of a specific DNA sequence of said DNA sequences obtained from step ii) in form of an inverted repeat into said insertion site of said screening construct or screening vector, wherein insertion of an inverted repeat of an efficient transcription terminator into said insertion site allows expression of said additional sequences by said promoter sequence in comparison to no insertion,
iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence,
v) identifying and/or selecting screening constructs or screening vectors with said readily detectable characteristic in comparison to no insertion, and
vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and, optionally, determining their sequence.

### Claim 5 (depends on 1)

The method of claim 1, wherein the method for identification and isolation of transcription termination sequences from a plant comprises the steps of:
i) providing a screening construct or screening vector comprising in 5′ to 3′ direction
a) a promoter sequence,
b) one or more insertion sites for insertion of DNA sequences, and
c) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic,
wherein insertion of an efficient transcription terminator into said insertion site suppresses expression of said additional sequences by said promoter sequence in comparison to no insertion,
ii) providing one or more DNA sequences to be assessed for their transcription termination capability by
a) identifying intergenic regions between paired genes having an intergenic distance of about 400 to 3,000 base pairs from a plant,
b) identifying one or more DNA sequences which are flanked on both sides by genes having at least 5% of expression level of actin in said plant, and
c) isolating the one or more DNA sequences identified in step b),
iii) inserting one or more copies of said DNA sequences obtained from step ii) into said insertion site of said screening construct or screening vector,
iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence,
v) identifying and/or selecting screening constructs or screening vectors with a changed readily detectable characteristic in comparison to no insertion, and
vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and, optionally, determining their sequence.

### Claim 6 (depends on 5)

The method of claim 5, wherein said additional sequence is an inverted repeat of a known transcription terminator sequence localized in a way the second copy of said repeat downstream from said promoter sequence is in an orientation to constitute a functional transcription terminator, and wherein said screening construct or screening vector comprises further sequences between said insertion site and said promoter which result in an readily detectable phenotype, and wherein absence of an efficient transcription terminator in said insertion site will lead to silencing of expression of said further sequences in consequence of expression of said inverted repeat of said known transcription terminator.

### Claim 7 (depends on 1)

The method of claim 1, wherein the method for identification and isolation of transcription termination sequences from a plant comprises the steps of:
i) providing a screening construct or screening vector comprising in 5′ to 3′ direction
a) a promoter sequence, and
b) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, and embedded into said additional sequences one or more insertion sites for insertion of DNA sequences, wherein insertion of an efficient transcription terminator into said insertion site suppresses full-length transcription of said additional sequences by said promoter sequence in comparison to no insertion,
ii) providing one or more DNA sequences to be assessed for their transcription termination capability by
a) identifying intergenic regions between paired genes having an intergenic distance of about 400 to 3,000 base pairs from a plant,
b) identifying one or more DNA sequences which are flanked on both sides by genes having at least 5% of expression level of actin in said plant, and
c) isolating the one or more DNA sequences identified in step b),
iii) inserting one or more copies of said DNA sequences obtained from step ii) into said insertion site of said screening construct or screening vector,
iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence,
v) identifying and/or selecting screening constructs or screening vectors with a changed readily detectable characteristic in comparison to no insertion, and
vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and, optionally, determining their sequence.

### Claim 8 (depends on 7)

The method of claim 7, wherein said insertion site is localized within an intron comprised in said additional sequence.

### Claim 9 (depends on 7)

The method of claim 7, wherein said additional sequence comprises a first 5′-part which is encoding for a marker protein and a second 3′-part which is not protein encoding and has less than 30% identity to sequences endogenous to said transcription system, and wherein said insertion site is localized between said 5′-part and said 3′-part, and wherein for the purpose of selection and/or identification, a second expression cassette is introduced into said transcription system comprising a promoter function in said transcription system and operably linked thereto a sequence encoding for an antisense or double-stranded RNA molecule complementary to said 3′-part.

### Claim 10 (depends on 1)

The method of claim 1, wherein said additional sequence is selected from the group consisting of positive selection marker, negative selection marker, counter selection marker, reporter genes, and toxic genes.

### Claim 11 (depends on 10)

The method of claim 10, wherein said toxic gene is a construct for gene silencing of an essential endogenous gene or a selectable marker.

### Claim 12 (depends on 1)

The method of claim 1, wherein said DNA sequence to be assessed for their transcription termination efficiency is provided by a method selected from the group consisting of:
i) a method of selecting sequences by amplification from a host genome,
ii) a method of selecting sequences from a library of sequences by fragmentation of a host genome, and
iii) a method of selecting sequences from a library of synthetic sequences.

### Claim 13 (depends on 1)

The method of claim 1, wherein said DNA sequences to be assessed for their transcription termination efficiency are inserted into said insertion site by a method selected from the group consisting of:
i) recombinational cloning, and
ii) insertion by sequence specific restriction and ligation.

### Claim 14

A method for identification and isolation of intergenic sequences with transcription termination potential from a plant comprising at least the steps of:
a) identifying intergenic regions between paired genes having an intergenic distance of about 400 to 3,000 base pairs from a plant,
b) identifying intergenic sequences which are flanked on both sides by genes having at least 5% of expression level of actin in said plant, and
c) isolating the intergenic sequences identified in step b).

### Claim 15 (depends on 14)

The method of claim 14 further comprises the steps of:
d) identifying and/or isolating intergenic sequences which are flanked on both sides by genes having an expression pattern which is independent from the expression pattern of the other paired gene, and
e) identifying and/or isolating intergenic sequences which are flanked on one or both sides by genes having a low variability in length of the mRNA transcript derived from said paired genes.

### Claim 16 (depends on 14)

The method of claim 14, wherein said intergenic region
a) is localized between genes which have a tail-to-tail localization, and/or
b) has a length measured from the respective stop codons of the flanking genes from about 700 to about 2000 base pairs.