Exogenous terminators for controlling fungal gene expression

Patent metadata

Field	Value
Publication number	US-9951344-B2
Application number	US-201514879946-A
Country	US
Kind code	B2
Filing date	Oct 9, 2015
Priority date	Oct 10, 2014
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

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

First claim

What is claimed is:

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.

Assignees

• Univ Texas

Inventors

• Alper Hal
• Curran Kathleen

Classifications

• C12N15/80 Primary

  for fungi · CPC title