IP Library Granted Patent US 9,951,344
Granted Patent B2
US 9,951,344 · App. 14/879,946 · Granted Apr 24, 2018

Exogenous terminators for controlling fungal gene expression

Inventors: Hal Alper (Austin, TX); Kathleen Curran (Emeryville, CA)
Assignee: Board of Regents, The University of Texas System
C12N15/80
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Quick Facts
Patent No.
US 9,951,344
App. No.
14/879,946
Granted
Apr 24, 2018
Kind
B2
Abstract

Provided herein are exogenous terminator sequences for use in fungi cell transcriptional termination.

Claims (50)

1. A fungal cell comprising an exogenous fungal transcription terminating nucleic acid sequence, wherein said fungal transcription terminating nucleic acid sequence comprises:

(i) an efficiency nucleic acid sequence of the formula (TA) n1 , wherein n1 is an integer from 4 to 20;

(ii) a positioning nucleic acid sequence of the formula AAWAAA, wherein W is A or T; and

(iii) a polyadenylation site nucleic acid sequence comprising the sequence (N) n4 Y-A n2 , wherein

N is A, C, T, or G;

n4 is an integer from 0 to 10;

Y is a polyadenylation site nucleotide and is C or T; and

n2 is an integer from 3 to 50.

2. The exogenous fungal transcription terminating nucleic acid sequence of claim 1 , wherein n1 is 4-15.

3. The exogenous fungal transcription terminating nucleic acid sequence of claim 1 , wherein n1 is 10-14.

4. The exogenous fungal transcription terminating nucleic acid sequence of claim 1 comprising the sequence:

(Sequence I)

X 1 -(TA) n1 -L 1 -AAWAAA-L 2 -((N) n4 Y-A n2 ),

wherein n1 is an integer from 10-20;

X 1 is absent or an upstream nucleic acid sequence 2 to 20 nucleotides in length;

AAW AAA is said positioning nucleic acid sequence;

(N) n4 Y-A n2 is said polyadenylation site nucleic acid sequence;

L 1 is a first linking nucleic acid sequence linking said positioning nucleic acid sequence to said efficiency nucleic acid sequence and L 2 is a second linking nucleic acid sequence linking said positioning nucleic acid sequence to said polyadenylation site nucleic acid sequence, wherein said first linking nucleic acid sequence and said second linking nucleic acid sequence are independently 5 to 30 nucleotides in length.

5. The exogenous fungal transcription terminating nucleic acid sequence of claim 4 , wherein X 1 is an upstream nucleic acid sequence 6 to 12 nucleotides in length.

6. The exogenous fungal transcription terminating nucleic acid sequence of claim 4 , wherein n1 is 12.

7. The exogenous fungal transcription terminating nucleic acid sequence of claim 4 , wherein W is T.

8. The exogenous fungal transcription terminating nucleic acid sequence of claim 4 , wherein n4 is an integer from 1 to 4 nucleotides and n2 is an integer from 3 to 10.

9. A fungal cell comprising an exogenous fungal transcription terminating nucleic acid sequence, wherein said fungal transcription terminating nucleic acid sequence comprises:

(i) an efficiency nucleic acid sequence of the formula (TA) n1 , wherein n1 is an integer from 3 to 20;

(ii) a positioning nucleic acid sequence of the formula AAWAAA, wherein W is A or T;

(iii) a polyadenylation site nucleic acid sequence comprising the sequence (N) n4 Y-A n2 , wherein

N is A, C, T, or G;

n4 is an integer from 0 to 1 O;

Y is a polyadenylation site nucleotide and is C or T; and

n2 is an integer from 3 to 50; and

(iv) a first linking nucleic acid sequence linking said positioning nucleic acid sequence to said efficiency nucleic acid sequence or a second linking nucleic acid sequence linking said positioning nucleic acid sequence to said polyadenylation site nucleic acid sequence, wherein said first linking nucleic acid sequence and said second linking nucleic acid sequence are independently 3 to 30 nucleotides in length and consist of less than about 35% of cytosine and guanine.

10. The exogenous fungal transcription terminating nucleic acid sequence of claim 9 , wherein said first linking nucleic acid sequence or said second linking nucleic acid sequence consists of less than about 30% cytosine and guanine.

11. The exogenous fungal transcription terminating nucleic acid sequence of claim 9 , wherein said first linking nucleic acid sequence is about 10-25 nucleotides in length.

12. A fungal cell comprising an exogenous fungal transcription terminating nucleic acid sequence, wherein said fungal transcription terminating nucleic acid sequence comprises:

(i) an efficiency nucleic acid sequence of the formula (TA) n1 , wherein n1 is an integer from 3 to 20;

(ii) a positioning nucleic acid sequence of the formula AAWAAA, wherein W is A or T;

(iii) a polyadenylation site nucleic acid sequence comprising the sequence (N) n4 Y-A n2 , wherein

N is A, C, T, or G;

n4 is an integer from 0 to 10;

Y is a polyadenylation site nucleotide and is C or T; and

n2 is an integer from 3 to 50;

(iv) an upstream nucleic acid sequence 5′ to said efficiency nucleic acid sequence of the formula (T) n3 , wherein n3 is an integer from 2 to 20.

13. The exogenous fungal transcription terminating nucleic acid sequence of claim 12 , wherein n1 is an integer of 3 to 12.

14. The exogenous fungal transcription terminating nucleic acid sequence of claim 12 , wherein n3 is an integer from 4 to 12.

15. The exogenous fungal transcription terminating nucleic acid sequence of claim 12 , further comprising a first linking nucleic acid sequence linking said positioning nucleic acid sequence to said efficiency nucleic acid sequence.

16. The exogenous fungal transcription terminating nucleic acid sequence of claim 1 , wherein said exogenous fungal transcription terminating nucleic acid sequence includes a length of less than about 200 nucleotides.

17. An expression construct comprising an exogenous fungi transcription terminating nucleic acid sequence of claim 1 .

18. A method of expressing a gene in a fungal cell, said method comprising:

(i) transforming the fungal cell of claim 1 with an expression construct comprising a gene operably linked to the exogenous fungal transcription terminating nucleic acid sequence;

(ii) allowing said cell to express said expression construct, wherein said exogenous fungal transcription terminating nucleic acid sequence modulates a level of transcription of said gene, thereby expressing said gene in said fungal cell.

Assignments (2)
CONFIRMATORY LICENSE Recorded Dec 15, 2015
From: UNIVERSITY OF TEXAS, AUSTIN
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 037291/0408 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 23, 2015
From: ALPER, HAL; CURRAN, KATHLEEN
To: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM
Reel/Frame 036870/0517 →
Continuity (2)
Provisional Application 62062766 · Oct 10, 2014
Related Publication 20160102314A1 · Apr 14, 2016